Foundations in microbiology

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Abbreviatlon

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millimeter micrometer nanometer

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Common Numerical prefixes

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ffi ffigher Edueation FOUNDATIONS IN MICROBIOLOGY, SEVENTH EDITION Published by McGraw-HilI, a business unit of The McGraw-Hill Companies, lnc., l22l Avenue of the Americas, New York, NY 10020. Copyright @ 2009 by The McGraw-Hill Connpanies, Inc. Al1 rights reserved. Previous editions @ 2008,2005,2002, 1999,1996, and 1993. No part of this publication may be reproduced or distributed in any fonn or by any means, or stored in a database or retrieval system, without the prioi written consent of The McGraw-Hill Companies, Inc., including but not limited to, in any network or other electrodc storage or tansmissiolr, or broadcast for distance learning. Some ancillaries, including electronic and print componelrts, may not be available to customers outside the

Unfted Stat€s.

This book is printed on acid-fiee p4er.

34567890DOWDOW0 rsBN 978{H)7-128445-5 MHID H7-12&95-l The crodits section for this book begins on page

www.mhhe.com

C-l

and is considered an extension of the copyright page.

r lfrr, '

'

book is dedicated to the devoted public health workers who introduce medical advances and treatments enjoyed. by the

industrialized world to all humans.

About the Author IQtWr* ?arfr, ToUro

is a microbiologist and educator at pasadena City coilege. A native of ldaho, she began her college education at ldaho State University in Pocatello. There she found a comfortable niche that fit her particular abilities and interests, spending part of her free time as a scientific illustrator and part as a teaching assistant. After graduation, she started graduate studies at Arizona State University, with an emphasis in the physiological ecology of desert organisms. Additional graduate work was spent participating in research expeditions to British Columbia with the Scripps Institution of Oceanography. Kathy continued to expand her background, first finishing a master's degree at Occidental College and later taking additional specialized coursework in microbiology at the California Institute of Technology and California State University. Kathy has been teaching medical microbiology and majors' biology courses for over 30 years. She has been involved in developing curricula and new laboratory exercises in microbiology, and she has served as an advisor to the school's medical professions club. Throughout her career Kathy has nurtured a passion for the microbial world and a desire to convey the importance of

that world to beginning students. She finds tremendous gratification in watching her students emerge from a budding awareness of microorganisms into a deeper understanding of their significance in natural phenomena. Kathy is a member of the American Society for Microbiology. She keeps active in self-study and research and continues to attend workhops and conferences to remain current in her field. She also has contributed to science outreach programs by bringing minicourses in microbiology to students from kindergarten to high school.

vl

Brief Contents CHAPTER:: The Main Themes of MicrobiologY

atOtl6P

1

.,::.

CHAPTER .: Host Defenses: Overview and Nonspecific Defenses 420

':

,.'

The Chemistry of

CHAPTER .,.1 Tools of the Laboratory: The Methods for Studying

Microorganisms 57

cnnprrn

)

..

':'

CHAPTER ": Disorders in lmmunity 481 -t

::

CHAPTER =: A Survey of Prokaryotic Cells and

.

CHAPTER .. Adaptive, Specific lmmunity and lmmunization 447

Biology 27

Microorganisms 88

":

CHAPTER Diagnosing' Infections 513 :i i'

:=-

C

..i..u

HAPTER

A Survey of Eukaryotic Cells and Microorganisms 121

The Cocci of Medical lmportance 535

cnRprrR *':'

CHAPTER : The Gram-Positive Bacilli of Medical lmportance 565

a

An lntroduction to Viruses 157 ..=

CHAPTER , Elements of Microbial Nutrition, Ecology, and Growth 185 CHAPTER

'''

:.'?..

CHAPTER = Microbial Cenetics 252

=:l-

r',

The Gram-Negative Bacilli of Medical lmportance 594 CHAPTER

.;..'J

An Introduction to Microbial Metabolism: The Chemical Crossroads of Life 216

C

cnRPteR

-

i'l

'-

;',

Miscellaneous Bacterial Agents of Disease 621 CHAPTER

.tj

,,ll

::..

T

The Fungi of Medical lmportance 656 '',,

CHAPTER ..,,

"1 '

The Parasites of Medical lmportance 687

HAPTER

Genetic Engineering: A Revolution in Molecular Biology 289 ::

t,

CHAPTER -== :' Physical and Chemical Agents for Microbial

Control 316 .:.,

'

CHAPTER .: . Drugs, Microbes, Host-The Elements of Chemotherapy 347 'i: "iri

1::,.'

CHAPTER Microbe-Human lnteractions: Infection and

Disease 382

tt, .

CHAPTER =

lntroduction to Viruses That Infect Humans: The DNA Viruses 724 t.,

,.

CHAPTER .-,,..

The RNA Viruses That Infect Humans 749 CHAPTER

;"i '

Environmental Microbiology 787 CHAPTER

' i:.

,..

' ',,

t

Applied and lndustrial Microbiology 809 vlt

Contents Preface xx Cuided

Tour

xxiv

CHAPTE

A Special Note to Students xxxii

The Chemistry of

2.'l CHAPTE

R

Flow

Bonds and Molecules 32 Solutions: Homogeneous Mixtures of Molecules 37

4

2.2

Microbiology

Life

41

Lipids: Fats, Phospholipids, and Waxes 45 Proteins: Shapers

oflife

47

INSIGHT 2.1. Discovery

13

Better Living through Bacteria? 44

The Development of the Microscope: "Seeing Is Believing" 13

Chapter Summary with Key Terms 53

Method 15 The Development of Medical Microbiology 15 The Discovery ofSpores and Sterilization 15

Multiple-ChoiceQuestions 54 Writing to Learn 54

The Establishment of the Scientific

Concept Mapping 55 Critical Thinking Questions 56

1.7 Taxonomy: Organizing, Classifying, and Naming

Visual Understanding 56

18

The Levels ofClassification

Macromolecules: Superstructures of

Carbohydrates: Sugars and Polysaccharides 4l

The Nucleic Acids: A Cell Computer and Its programs 48 The Double Helix of DNA 50

Microbial Dimensions: How Small Is Small? 1l Lifestyles of Microorganisms 12

Microorganisms

Characteristics 29

Roles in the

9

The HistoricalFoundations of

29

The Major Elements of Life and Their Primary

1.3 Human Use of Microorganisms 7 1.4 lnfectious Diseases and the Human Condition 7 1.5 The General Characteristics of Microorganisms 9

1.6

27

Atoms, Bonds, and Molecules: Fundamental Building Blocks 28 Properties

1

Earth's Environments 4 Microbial Involvement in Energy and Nutrient

Biology

Different Types of Atoms: Elements and Their

The Main Themes of Microbiology 1.1 The Scope of Microbiology 2 1.2 The Origins of Microorganisms and Their

CellularOrganization

R

lnternet Search Topics 56

18

Assigning Specific Names 18 The Origin and Evolution of Microorganisms 20 Systems of Presenting a Universal Tree of

Life

20

CHAPTER

INSIGHT l.L Dlscovery Astrobiology: The Search for Extraterrestrial

Life

Tools of the Laboratory: The Methods for Studying Microorganisms 57 3.1 Methods of Culturing Microorganisms-The

5

INSIGHT 1.2 llistofical

The More Things Change . . . 9

Five Inoculation: Producing a Culture 58 Isolation: Separating Microbes from One Another 58 Media: Providing Nutrients in the Laboratory 60

INSIGHT 1.3 llistotical The Fall of Superstition and the Rise of

Microbiology

12

Chapter Summary with Key Terms 22

Incubation and Inspection 68

Multiple-ChoiceQuestions 23 Writing to Learn 24

Identification

3.2

l,S

69

VisualUnderstanding 26

The Microscope: Window on an lnvisible Realm 70 Magnification and Microscope Design 7l Variations on the Optical Microscope 74 Electron Microscopy 77

lnternet Search Topics 26

Preparing Specimens for Optical Microscopes 78

Concept Mapping 25 Critical Thinking Questions 25

viii

58

'

Contents 3.1

INSIGHT

'

:"t''

ix

Writing to Learn 118

Dlscovery

Concept Mapping 119 Critical Thinking Questions 119

The Uncultured 65

INSIGHT 3.2 Medlcal Media" INSIGHT 3.3: Dlscovery

Animal Inoculation: "Living

Visual Understanding'l2O

69

fnternet Search Topics 120

The Evolution in Resolution: Probing Microscopes Fs

Chapter Summary with Key Terms 83 Multiple-Choice Questions 84

CHAPTER

Writing to Learn 85

A Survey of Eukaryotic Cells and

Concept Mapping 85 Critical Thinking Questions 86

Microorganisms ',2',

VisualUnderstanding

@"

llri:gti;[.ft.Wffi

*-

5.1 The History of Eukaryotes 122 5.2 Form and Function of the Eukaryotic

87

fnternet Search Topics 87

Flagella

Locomotor Appendages: Cilia and The

CHAPTER

€

4.2

Life?

5.3 89

Cell

90

External Structures 90

Mycoplasmas and Other Cell-Wall-Deficient

Cell Membrane Structure

Bacteria

Fungal

101

Fungi

131

134

134

Fungi

135

5.5

The

Protists

Biology ofthe Protozoa

142

Protists

143

145

5.6 The Parasitic Helminths Morphology

150 151

Life Cycles andReproduction

ll4

142

Industry

'143

The Algae: Photosynthetic

General Worm

Characteristics 112 Free-LivingNonpathogenicBacteria 112 115

Cultivation

The Roles ofFungi in Nature and

Survey of Prokaryotic Groups with Unusual

Bacteria

l4l

Fungal Identification and

Sizes 106 Classification Systems in the Prokaryotae 108 Bacterial Taxonomy Based on Bergey's Manual 109

97

Nutrition

FungalClassification

Bacterial Shapes, Arrangements, and

Stain

130

Reproductive Strategies and Spore Formation 135

104

The Gram Stain: A Grand

of the

Organization of Microscopic

Bacterial Internal Structure 1O2 Contentsofthe Cell Cytoplasm 102

4.8 Archaea: The Other Prokaryotes INSIGHT 4.1 Dlscovery Biofilms-The Glue of Life 95 INSIGHT 4.2 Dlscovery

131

Survey of Eukaryotic Microorganisms 133

101

Unusual Forms of Medically Significant

128

The Cytoskeleton: A Support Network 132

Bacterial Endospores: An Extremely Resistant Life

4.6 4,7

Cell

Chloroplasts: Photosynthesis Machines

5.4 The Kingdom

98

Cell

128

Mitochondria: Energy Generators of the Ribosomes: Protein Synthesizers

The Cell Envelope: The Boundary Layer of Bacteria 97

Walls

Machine

Golgi Apparatus: A Packaging

Prokaryotic Profiles: The Bacteria and Archaea 90

Form

127

Endoplasmic Reticulum: A Passageway in the

Differences in Cell Envelope Structure 97

4.5

Form and Function of the Eukaryotic Cell:

lnternal Structures 127 Center

89

Structure of Cell

126

The Nucleus: The Control

Appendages: Cell Extensions 90

4,4

124

125

BoundaryStructures

The Structure of a Generalized Bacterial

4.3

Glycocalyx

Form and Function ofthe Eukaryotic Cell:

,':: ,.:,:,1,:€,

A Survey of Prokaryotic Cells and Microorganisms 88 4.1 Characteristics of Cells and Life What Is

Cell:

External Structures 124

151

A Helminth Cycle: The Pinworm l5l Helminth Classification and Identification 152 Distribution and Importance of Parasitic Worms

152

INSIGHT 5.1 Hlstorical The Extraordinary Evolution of Eukaryotic Cells

INSIGHT 5.2 Discovery Fungi: A Force of Nature 138

INSIGHT 4.3 Dlscovery Redefining Bacterial Size "1"14

Chapter Summary with Key Terms 153 Multiple-Choice Questions 154

Chapter Summary with Key Terms 1 17

Writing to Learn '154

Multiple-Choice Questions 118

Concept

Mapping

155

'123

Contents

Critical Thinking Questions 155

The Diffusion of Water: Osmosis 193

Visual Understanding 156

Endocytosis: Eating and Drinking by

lnternet Search Topics 156

7.2 Environmental

Cells

Factors That Influence

197

Microbes

198

Adaptations to Temperature 198

CHAPTER

1,.,

,

Gas

,i,litili+;:{iiulMw

The Search for the Elusive

6.2 The Position 6.3 The General Size

Range

Viruses

6.4 6.5

of Viruses in the Biological

EcologicalAssociationsamongMicroorganisms 202

Spectrum

158

Structure of Viruses ',59

Interrelationships between Microbes and

7.3

159

Growth 206 The Population Growth Curve 208 Stages in the Normal Growth Curve

Named

Multiplication 168 Cycles in Animal Viruses

166

Modes of Viral

6.7 6.8

Using Cell (Tissue) Culture Techniques 176

Life in the Extremes "199

Viruses

Steps in a Viable Plate

Infectious Particles ',79

Count-Batch Culture Method

Chapter Summary with Key Terms Multiple-Choice Questions 212 Concept

Mapping

INSIGHT 6.2 Discovery

VisualUnderstanding 215 lnternet Search Topics 215

INSIGHT 6.3 Medicsl 'a79

Chapter Summary with Key Terms

'181

Multiple-ChoiceQuestions 18i Writing to Learn '182 Concept

Mapping

CHAPTER

,:::::, ,'iE=-:+i

Enzymes: Catalyzing the Chemical Reactions of Life 218 Regulation of Enzymatic Activity and Metabolic Pathways 224

,a:'

*;;ffiffi

:'

Elements of Microbial Nutrition, Ecology, and Growth 185 7;a Microbial Nutrition 186 Chemical Analysis of Cell Contents 187 Sources ofEssential Nutrients 188 How Microbes Feed: Nutritional Types

.

An Introduction to Microbial Metabolism: The Chemical Crossroads of Life 2'16 8.1 The Metabolism of Microbes 217

183

Critical Thinking Questions 183 VisualUnderstanding 184 lnternet Search Topics 184

CHAPTER

2"11

214

Artificial Viruses Created! 178

Obesity?

2O9

Writing to Learn 213

'159

Critical Thinking Questions 214

A Vaccine for

210

INSIGHT 7.3 Dlscovery Attraction 2O3 INSIGHT 7.4 Mlcroblology

179

An Alternate View of

Growth

A Mutual

178

Medical lmportance of Viruses 17a Detection and Treatment of Animal Viral

6.9 Prions and Other Nonviral INSIGHT 6.L Dlscovery

208

7.2 Dlscovery

INSIGHT

Infections

206

Dining with an Amoeba 187

Animal Viruses 176

Embryos 177 Inoculation

Fission

INSIGHT 7.1 Dlscovery

168

in Cultivating and ldentifying

Using Live Animal

204

206

Other Methods of Analyzing Population

The Multiplication Cycle in Bacteriophages 173

Using Bird

Growth

Humans

The Rate of Population

160

How Viruses Are Classified and

6.6 Techniques

The Study of Microbial

The Basis of Population Growth: Binary

Envelopes

Multiplication

201

Miscellaneous Environmental Factors 202

158

Viral Compoments: Capsids, Nucleic Acids, and

ofpH

Osmotic Pressure 202

An Introduction to Viruses 157

6;,

Requirements 200

Effects

8.2 The Pursuit and Utilization of Ce1l Energetic

8.3

s

Energy 226

226

Pathways of Bioenergetics 229 Catabolism: An Overview of Nutrient Breakdown and Energy Release 230 Energy Strategies in Microorganisms 230

189

Transport: Movement of Chemicals across the Cell Membrane 192

AerobicRespiration

231

Acid-A Central Metabolite 233 The Krebs Cycle-A Carbon and Energy Wheel Pyruvic

233

xl

Contents

The Respiratory Chain: Electron Transport and Oxidative

Genetic Regulation of Protein Synthesis and Metabolism 271

9.3

Phosphorylation 235

The Lactose Operon: A Model for Inducible Gene Regulation in Bacteria 272

Summary of Aerobic Respiration 238

AnaerobicRespiration 239 The Importance of Fermentation 240

8.4

WantNot

275

9.4 Mutations: Changes in the Genetic Code 276

Biosynthesis and the Crossing Pathways of Metabolism 24'l The Frugality of the Cell-Waste Not,

Causes of

Mutations 27'l

Categories of

Mutations

278

Repair of Mutations 278

242

Assembly of the

8.5

A Repressible Operon

Cell

The Ames

244

Photosynthesis: The Earth's Lifeline 244 Light-DependentReactions 245 Light-IndependentReactions 246 Other Mechanisms of Photosynthesis 246

Transmission of Genetic Material in

Bacteria

INSIGHT 9.L DlscovetY INSIGHT 9.2 Historical Deciphering the Structure of DNA

INSIGHT 8.2 DlscoverY 2"19

256

INSIGIIT 9.3 DlscovetY

INSIGHT 8.3 DlscoverY The Enzyme Name Game 222

Revising Some Rules of Genetics 267

INSIGHT 8.4 Hlstorical

INSIGHT 9.4 Med-lcal Connection

Pasteur and the Wine-to-Vinegar Chapter Summary with

KeY

Terms

241

Writing to Learn 287 Concept Mapping 287 Critical Thinking Questions 288 VisualUnderstanding 288 Internet Search Topics 288

Concept Mapping 250 Critical Thinking Questions 250 Visual Understanding 251 fnternet Search Topics 251

;-

€>

'

ffis

;:+liiiiffi

2s2

9.", lntroduction to Genetics and Genes: Unlocking the Secrets of HereditY 253 The Nature of the Genetic

Material

253

The Structure of DNA: A Double Helix with Its Ownlanguage 255 The Significance of DNA

Structure

258

DNA Replication: Preserving the Code and Passing It On 258

9.2

Applications of the DNA Code: Transcription and Translation 26'l The Gene-Protein Connection 261 The Major Participants in Transcription and

Translation

262

Transcription: The First Stage of Gene Expression 264 Translation: The Second Stage ofGene Expression 264 Eukaryotic Transcription and Translation: Similar yet

Different

CHAPTER

270

€.#

",ffi

Genetic Engineering: A Revolution in Molecular Biology 289 10.1

Basic Elements and Applications of Genetic Engineering 29O

10.2 Tools and Techniques of Genetic Engineering PracticalProperties ofDNA 290 10.3 Methods in Recombinant DNA Technology: How to lmitate Nature 297

29O

Technical Aspects of Recombinant DNA and Gene Cloning 299 a Recombinant, Insertion into a Cloning Host, and Genetic ExPression 300

Construction of

10.4 Biochemical Products of Recombinant DNA Technology 3O2 10.5 Genetically Modified Organisms 303 Recombinant Microbes: Modified Bacteria and

268

The Genetics of Animal Viruses

Replication Strategies in Animal Viruses 272 Chapter Summary with KeY Terms 285 Multiple-Choice Questions 286

247

Multiple-ChoiceQuestions 248 Writing to Learn 249

CHAPTER

280

The Packaging of DNA: Winding, Twisting, and Coiling 255

Levers 2",8

An Unconventional EnzYme

279

9.5 DNA Recombination Events 28O

INSIGHT 8.1 DlscovetY Enzymes as Biochemical

Test

Positive and Negative Effects of Mutations 279

Viruses

303

Recombination in Multicellular Organisms 305

xu

Contents

10.6 Genetic Treatments: Introducing DNA into the Body 3O7 GeneTherapy

Writing to Learn 345

Mapping 345 Critical Thinking Questions 345 Concept

307

DNA Technology as Genetic Medicine 308

VisualUnderstanding 346

10.7 Genome Analysis: Fingerprints and Genetic Testing 308

Internet Search Topics 346

DNA Fingerprinting: A Unique picture of a Genome

INSIGIITIO.l

Dlscovery

OK, the Genome's Sequenced-What,s

INSIGHT

CHAPTER

Next? 296

',.-:,

t0,2z Mlcrohtology

A Moment to

Think

3O4

'12.1 Principles of Antimicrobial Therapy 348 The Origins of Antimicrobial Drugs 348 12.2 Interactions between Drugs and Microbes 350 Mechanisms of Drug Action 351 12,3 Survey of Major Antimicrobial Drug Groups 355

Writing to Learn 313

Mapping

t,,:i. W

"nr,

of Chemotherapy 347

Chapter Summary with Key Terms 312 Multiple-Choice Questions 3'13 Concept

eru

=44

314

Critical Thinking Questions 314 Visual Understanding 315

Internet Search Topics 315

Antibacterial Drugs That Act on the Cell Wall 355 Antibiotics That Damage Bacterial Cell Membranes 359 Drugs That Act on DNA or RNA 359

CHAPTER

Drugs That Interfere with Protein Synthesis 359 Drugs That Block Metabolic Pathways 360

Physical and ChemicalAgents for Microbial Control 316 11.1 Controlling Microorganisms 317

Antiparasitic Chemotherapy 362

Agents to Treat Fungal

Control 317 Forms 317 Terminology and Methods of Microbial Control 319 What Is Microbial Death? 320 How Antimicrobial Agents Work Their Modes of Action 322 11.2 Methods of PhysicalControl 323 Heat as an Agent of Microbial Control 323 General Considerations in Microbial

How Does Drug Resistance Develop? 366 Specific Mechanisms of Drug Resistance 366 Natural Selection and Drug Resistance 368

12.5 Interactions between Drugs and Hosts 369 Toxicity to Organs 369

Allergic Responses to Drugs 371 Suppression and Alteration of Microflora by

Antimicrobials

The Effects of Cold and Dessication 327 Radiation as a Microbial Control Agent 327

Drug

330

Chemical

Testing for the Drug Susceptibility

INSIGHTll.L

of

Times

318

INSIGHT 11.2 Mlcroblology Pathogen Paranoia: "The Only Good Microbe ls a

Microbe" 331 INSIGHT 11.3 Medlcal The Quest for Sterile Skin 340 Dead

Chapter Summary with Key Terms 343 Multiple-Choice Questions 344

374

INSIGHTT2.l Histortca,l Group

Hlstorlcal

Microbial Control in Ancient

of

Microorganisms 373 The MIC and the Therapeutic Index

331

Germicidal Categories According to Chemical

373

IdentifyingtheAgent 373

11.3 ChemicalAgents in MicrobialControl 330 Choosing a Microbicidal

371

12.6 Considerations in Selecting an Antimicrobial

Sterilization by Filtration: Techniques for Removing

Factors That Affect the Germicidal Activity Chemicals 332

361

'12.4 Interactions between Microbes and Drugs: The Acquisition of Drug Resistance 366

Relative Resistance of Microbial

Microbes

Infections

333

Drugs 349 INSIGHT 12.2 Dlscovety A Modern Quest for Designer Drugs 357 INSIGHT 12.3 Dlscovery New Perspectives in Antimicrobial Therapy 365 INSIGHT 12.4 Medlcsl From Witchcraft to Wonder

The Rise of Drug Resistance 37O Chapter Summary with Key Terms 377 Multiple-ChoiceQuestions 378

Writing to Learn 379

xlll

Contents

INSIGHT L3.4 Medlcal

Concept Mapping 380 Critical Thinking Questions 380

A Quick Guide to the Terminology of Infection

and Disease 4O'l

Visual Understanding 381

INSIGHT 1,3.5 Medical

Internet Search Topics 381

Koch's Postulates: Solving the Puzzle of New Diseases 414

CHAPTER

.:: :..., tl*==ailly,ilii=

Writing to Learn 416

uman I nteractions: lnfection and Disease 382 13.1 We Are Not Alone 383

Microbe-

H

Concept MaPping 418 Critical Thinking Questions 418

VisualUnderstanding

Contact, Colonization, Infection, Disease 383 Resident Flora: The Human as a

Habitat

Indigenous Flora of Specific Regions Flora of the Human

Skin

Chapter Summary with KeY Terms 415 Multiple-Choice Questions 416

419

lnternet Search ToPics 4'19

383

385

386

Tract 387 Tract 388 Flora ofthe Genitourinary Tract 388 '13.2 Maior Factors in the Development of an Flora ofthe Gastrointestinal Flora ofthe Respiratory

lnfection

of

Dose

Perspective

394

Becoming Established: Step Three-Surviving Host Defenses 395 How Virulence Factors Contribute to Tissue Damage

Disease 396

The Portal of Exit: Vacating the

Host

13.3

Sources and Transmission of

Microbes

Persist

4O3

Response 432

Immunity

of

13.4 Epidemiology: The Study of Disease in Populations 4',O Who, When, and Where? Tracking Disease in the

INSIGHT

l3.L

DlscoverY

Life without

Flora

390

INSIGHT

L3.2 Medlcal

437

Stimulants 440

407

410

433

Interferon: Antiviral Cytokines and Immune

Universal Blood and Body Fluid Precautions 408

Population

Inflammation

Phagocytosis: Partner to Inflammation and

405

Nosocomial Infections: The Hospital as a Source

Disease

Recognition: Activation of the Innate Immunologic 401

403

The Acquisition and Transmission of Infectious

Agents

The Communicating Body Compartments 424

The Stages of

403

Reservoirs: Where Pathogens

14.2 Structure and Function of the Organs of Defense and lmmunitY 423

The Inflammatory Response: A Complex Concert of Reactions to InjurY 432

402

The Persistence of Microbes and Pathologic

Conditions

421

14.3 Actions of the Second Line of Defense 432

Disease 399 and Symptoms: Warning Signals of Disease

The Process of Infection and Signs

,

Barriers at the Portal ofEntry: An Inborn First Line of Defense 421

Becoming Established: Step TWo-Attaching to the Host 394

and

--

-

14.1 Defense Mechanisms of the Host in

391

The Requirement for an Infectious

..

Host Defenses: Overview and Nonspecific Defenses 420

389

Becoming Established: Step One-Portals

Entry

CHAPTER

Complement: A Versatile Backup

System

440

Overall Stages in the Complement Cascade 441 Summary of Host Defenses 442

INSIGHT L4.1 Medical When Inflammation Gets Out of

INSIGHT L4.2 Med.lca.l The Dynamics of Inflammatory

Hand

Mediators 435

INSIGHT 14.3 Medlcal Some Facts About

Fever 437

Laboratory Biosafety Levels and Classes of Pathogens 392

Chapter Summary with Key Terms 443 Multiple-Choice Questions 444

INSIGHT L3.3 Hlstorlcal Human Guinea Pigs 395

Writing to Learn 444 Concept

Mapping 445

434

:.:l*,

]$,1

xiv

Contents

Critical Thinking Questions 445

4F':

cnnprrn

VisualUnderstanding 446

€ffE

lnternet Search Topics 446

CHAPTER

"is*r"*rr|*rnunity

'16.'l The lmmune Response: A Two-Sided Coin 482

Wg

-$--=

tp*f,. *rn*-t

^t"p-"", mmunization

447

f

;ffim

Overreactions to Antigens: Allergy/Hypersensitivity 482

'|6.2

"*

A General Scheme for Classifying Immunities 449 An Overview of Specific Immune Responses 451

'15.2 Development of the lmmune Response System 4S', Markers on Cell Surfaces Involved in Recognition of Self

Nonself

451

Diagnosis of

15.3 Lymphocyte

Responses and Antigens 456 Specific Events in B-Cell Maturation 456

457

4Sg

The Role ofAntigen Processing and Presentation 458

B-Cell Responses 459 Activation of B Lymphocytes: Clonal Expansion and

AntibodyProduction 459 Products ofB Lymphocytes: Antibody Structure and Functions 461 Monoclonal Antibodies: Useful Products from Cancer

Cells

466

15.6 T-Cell Responses 466 Cell-Mediated Immunity (CMI) 466 15.7 lmmunization: Methods of Manipulating tmmunity for Therapeutic Purposes 469 Passivelmmunization 469

Artificial Active Immunity: Vaccination Development of New Vaccines 473

INSIGHT 1.5.1 Hlstorlcal Antibodies 450

Limit

INSIGHT 15.3 Historlcal The Lively History of Active lmmunization 47O Chapter Summary with Key Terms 476

Multiple-ChoiceQuestions 477 Writing to Learn 478 Concept Mapping 479 Critical Thinking Questions 479

VisualUnderstanding 480 Internet Search Topics 480

'16.4 Type lll Hypersensitivities: lmmune Complex

Reactions 496 Mechanisms of Immune Complex Disease 496 Types of Immune Complex Disease 496

16.5 lmmunopathologies Involving

T

Cells

497

Type IV Delayed-Type Hypersensitivity 497 T Cells and Their Role in Organ Transplantation 499 Types

ofTransplants

501

16.6 Autoimmune Diseases-An Attack on Self

501

Genetic and Gender Correlation in Autoimmune Disease 502

'16.7 lmmunodeficiency Diseases: Compromised lmmune Responses 505 PrimarylmmunodeficiencyDiseases 505 SecondarylmmunodeficiencyDiseases 507

16.8 The Function of the lmmune

INSIGHT 15.2 Medlcal Monoclonal Antibodies: Variety without

4g2

Antibodies against A and B Antigens 493 The Rh Factor and Its Clinical Importance 494 Other RBC Antigens 496

The Origins of Autoimmune Disease 502 Examples of Autoimmune Disease 503

469

Routes of Administration and Side Effects of Vaccines 474 To Vaccinate: Why, Whom, and When? 474

Breast Feeding: The Gift of

490

Allergy 490 16.3 Type ll Hypersensitivities: Reactions That Lyse Foreign Cells 492 The Basis of Human ABO Antigens and Blood Types

456 Entrance and Processing ofAntigens and Clonal

15.5

489

Allergy

Treatment and Prevention of

Maturation

15.4 Cooperation in lmmune Reactions to Antigens

Cytokines, Target Organs, and Allergic Symptoms 486 Specific Diseases Associated with IgE- and Mast-CellMediated Allergy 488

Allergens

Response 453

Selection

Modes of Contact with Allergens 484 The Nature of Allergens and Their Portals of Entry 484 Mechanisms of Type I Allergy: Sensitization and Provocation 484

Anaphylaxis: A Powerful Systemic Reaction to

The Origin of Diversity and Specificity in the Immune

Specific Events in T-Cell

Type I Allergic Reactions: Atopy and

Anaphylaxis 484

15.1 Specific lmmunity: The Adaptive Line of Defense 44g

and

'r.,,r,lfffi

48'l

465

System in

Cancer 508

INSIGIIT r.6.r. Medlcal Of What Value ls

Allergy? 488

INSIGIIT 1"6.2 Medlcal Why Doesn't a Mother Reject Her Fetus? 495

INSIGIIT L6.3 Medical Pretty, Pesky, Poisonous

Plants 49a

INSIGHT 16.4 Medlcal The Gift of Life: Bone Marrow Transplantation 5Ol

INSIGHT L6.5 Dlscovery David

An Answer to the Mystery of

5O7

Contents

Chapter Summary with

KeY

Terms

509

CHAPTER

Multiple-ChoiceQuestions 510 Writing to Learn 5'10

.=

.-,. lmportance

The Cocci of Medical

Concept Mapping 511 Critical Thinking Questions 511

535

18.1 General Characteristics of the Staphylococci 536 Growth and Physiological Characteristics of

VisualUnderstanding 512

Staphylococcusaureus 536

Internet Search ToPics 512

The Scope ofClinical Staphylococcal Disease 538 Host Defenses against S.

aureus

Other Important Staphylococci

540 541

Identification of Staphylococcas Isolates in Clinical .:.

CHAPTER

Samples

i, :.::]i.1::{leslitl*i:€

18.2 General Characteristics of the Streptococci and Related Genera 543

Diagnosing lnfections 513 "17.1 Preparation for the Survey of Microbial

Beta-Hemolytic Streptococci: Streptococcus pyogenes 544

Diseases 514 Phenotypic Methods 514 Genotypic Methods 514 ImmunologicMethods

Group B: Streptococcus

Streptococcallnfections

515

5'17

Immediate Direct Examination of Specimen 517

18.3 The Family Neisseriaceae: Gram-Negative Cocci 554

Cultivation of SPecimen 518

17.4 Genotypic Methods

'

52O

Neisseria gonorrhoeae: The Gonococcus 554 Neisseria meningitidis: The Meningococcus 557

Differentiating Pathogenic from Nonpathogenic

DNA Analysis Using Genetic Probes 520

Neisseria

Roles of the Polymerase Chain Reaction and Ribosomal RNA in Identification 520

'17.5 lmmunologic Methods 52'l General Features of Immune Testing

559

Other Genera of Gram-Negative Cocci and

Coccobacilli Tampons and

The Western Blot for Detecting Proteins 524

TSS 539

INSIGHT 18.2 Medlcal f

ComplementFixation 525 MiscellaneousSerologicalTests 526

nvasive Group A Streps and "Flesh-Eating" Syndrome 547

INSIGHT 1.8.3 Medical Pelvic Inflammatory Disease and

Fluorescent Antibody and Immunofluorescent

Chapter Summary with

526

Immunoassays: Tests of Great

Sensitivity

Tests That Differentiate T Cells and B

527

Cells

529

KeY

Infertility

555

Terms 560

Multiple-Choice Questions 56'l

Writing to Learn 562 Concept Mapping 563 Critical Thinking Questions 563 VisualUnderstanding 564

In Vivo Testing 529 A Viral Example 529

INSIGHT 1.7.1 Medical New Guidelines for Enteric

560

INSIGHT 18.1 Medical 521

AgglutinationandPrecipitationReactions 523

Testing

550

Alpha-Hemolytic Streptococci: The Viridans Group 551 Streptococcus pneumoniae: The Pneumococcus 551

Overview ofLaboratory Techniques 516

'17.3 Phenotypic Methods

549

Laboratory Identification Techniques 549 Treatment and Prevention of Group A, B, and D

515

Collection

agalactiae

Group D Enterococci and Groups C and G Streptococci 549

"17.2 On the Track of the Infectious Agent: Specimen

541

Clinical Concerns in Staphylococcal Infections 542

Cultures

518

lnternet Search Topics 564

INSIGIIT 17.2 Medlcal When Positive ls Negative: How to lnterpret Serological Test Results 523 Chapter SummarY with

KeY

Terms

Multiple-Choice Questions 532

Writing to Learn 532 Concept Mapping 533 Critical Thinking Questions 533 Visual Understanding 534 lnternet Search Topics 534

531

CHAPTER :

r

The Gram-Positive Bacilli of Medical lmportance 565 19.1 Medically lmportant Gram-Positive Bacilli 19.2 Gram-Positive Spore-Forming Bacilli 566 General Characteristics of the Genvs The Genus Clostridium

568

Bacillus

566

566

ir

,-

xvi

Contents

19.3 Gram-Positive Regular Non-Spore-Forming Bacilli 576

Oxidase-Positive Nonenteric Pathogens in Family

An Emerging Food-Borne Pathogen: Listeria

Pasteurellaceae 615

monocytogenes 576 Erysipelothrix rhusiopathiae.' A Zoonotic pathogen 577

',9.4 Gram-Positive lrregularNon-Spore-Forming Bacilli diphtheriae

Corynebacterium

Haemophilus: The Blood-Loving

578

Gram-Negative Sepsis and Endotoxic

Shock

597

INSIGIIT 2O.2 Medlcal Diarrheal Disease 603

19.5 Mycobacteria: Acid-Fast Bacilli 580 Mycobacterium tuberculosis: The Tubercle Bacillus 580 Mycobacterium leprae: The Leprosy Bacillus 586 Infections by Nontuberculosis Mycobacteria (NTM) 5gg

INSIGHT 2O.3 Mlcrobiology Avoiding Gastrointestinal lnfections

611

Chapter Summary with Key Terms 617

Multiple-ChoiceQuestions 617 Writing to Learn 618

19.6 Actinomycetes: Filamentous Bacilli 589 Actinomycosis 589 Nocardiosis 589

Concept

Mapping

619

Critical Thinking Questions 6'19 Visual Understanding 620 Internet Search Topics 620

INSIGHTl9.l Historlcal Us 569 INSIGHT I"9.2 Medlcal

A Bacillus That Could Kill

BOTOX: No Wrinkles. No Headaches. No

Worries? 575

INSIGIIT L9.3 Medlcal

..ru

CHAPTER

The Threat from Tuberculosis Sg2

s g-A

ijt=:;.ffi Disease 62',

Chapter Summary with Key Terms 590 Multiple-Choice Questions 591

2",."1 The Spirochetes 622

Writing to Learn 591

Mapping

615

INSIGIIT 2O.1 Medlcal S7g

The Genus Propionibacterium 580

Concept

Bacilli

Treponemes: Members of the Genus Treponema 622 LeptospiraandLeptospirosis 628 Borrelia: Arthropod-Borne Spirochetes 629

592

Critical Thinking Questions 592 Visual Understanding 593

2"1.2 Curviform Gram-Negative Bacteria and Enteric Diseases 632 The Biology ofVibrio cholerae 632

lnternet Search Topics 593

Vibrio parahaemolyticus and Vibrio vulnificus: pathogens

,&

CHAPTER gq-F tt* lmportance ",***g*ve 594 d'"s.

Carriedby Seafood 633 CampylobacterYibrios 635

j+,,,:ffi Bacilli of Medical

20.', Aerobic Gram-Negative Nonenterlc Bacilli

Diseases of the

Helicobacter pylori: Gastric Pathogen 636

21.3 Medically lmportant and Biology 637

FamilyRickettsiaceae 63'7 SpecificRickettsioses 638 EmergingRickettsioses 641

595

Pseudomonas.'ThePseudomonads 595

20.2

Related Gram-Negative Aerobic

Rods 598

Coxiella and, Bartonella; Other Vector-Borne pathogens 641 Other Obligate Parasitic Bacteria: The Chlamydiaceae 642

Brucella and Brucellosis 599 Francisella tularensis and Tularemia 599

21.4 Mollicutes and Other Cell-Wall-Deficient Bacteria 645

Bordetella pertrzssls and Relatives 600

Biological Characteristics of the Mycoplasmas 645 Bacteria That Have Lost Their Cell Walls 646

Legionella and Legionellosis 601

20.3 ldentification and Differential Enterobacteriaceae 602

Characteristics of Family

Antigenic Structures and Virulence Factors 606

20.4 Coliform

Organisms and Diseases 607 Escherichia coli: The Most Prevalent Enteric Bacillus 607 Miscellaneouslnfections 608 Other

Coliforms

608

20.5 Noncoliform Lactose-Negative Enterics

609

Shigella

Nonenteric Yersinia pestisandPlague 613

21.5

Bacteria in Dental Disease 647 The Structure of Teeth and Associated Tissues 64i Hard-Tissue Disease: Dental Caries 648

Formation 648 Soft:Tissue and Periodontal Disease 649 Factors in Dental Disease 650 Plaque and Dental Caries

INSIGIIT 21.1 Hlstorlcal The Disease Named for a Town 630

Opportunists: ProteusandltsRelatives 609 True Enteric Pathogens: Salmonella and

Bacteria of Unique Morphology

609

INSIGIIT 21.2 Medlcal Oral Rehydration Therapy 635

Contents

INSIGIIT 2L.3 Medlcal Atherosclerosis and Blood

Writing to Learn 684

Infection

Concept Mapping 685 Critical Thinking Questions 685 VisualUnderstanding 686

645

Chapter Summary with Key Terms 651 Multiple-Choice Questions 652

Internet Search Topics 686

Writing to Learn 653 Concept Mapping 654 Critical Thinking Questions 654

'rury

,:i:l:=!,,ffi

€J

VisualUnderstanding 655

CHAPTER

lnternet Search Topics 655

t* **ta"t "t tedical lmportance 687 23.'l The Parasites of Humans 688

CHAPTER

G3 Gffi,

*tical

ttt"

,.r:;:irmilfrirffi lmportance 656

Fungi as"Infectious Agents 22.'l "-tt

23.3 Apicomplexan Parasites 698 Plasmodium: The Agent of Malaria

Epidemiology of the Mycoses 659

Fungi

General Life and Transmission

Elements of Diagnosis and

Fever 664 Valley Fever 665

Histoplasmosis: Ohio Valley

Control

Blastomycosis 668

The Trematodes, or

Flukes

713

23.5 The Arthropod Vectors of Infectious Disease 716

Paracoccidioidomycosis 669

INSIGHT 23.1 Dlscovery

Subcutaneous Mycoses 669

The Power of Parasites 699

The Natural History of Sporotrichosis: Rose-Gardener's Disease 669

Chromoblastomycosis and Phaeohyphomycosis: Diseases 670

INSIGHT

23.2 Mlcroblology

When Parasites Attack the "Wrong"

Fish

Waiter, There's a Worm in My

Cutaneous Mycoses 671 Characteristics ofDermatophytes 672

Chapter Summary with Key Terms 718 Multiple-Choice Questions 720

715

Concept Mapping 722 Critical Thinking Questions 722

Infections by Candida: Candidiasis 675 Cryptococcus neoformans and Cryptococcosis 677

Pneumonia

71O

Writing to Learn 72'l

22.5 Superficial Mycoses 674 22.6 Opportunistic Mycoses 675

ar,Ld

Host

INSIGHT 23.3 Medlcal

Mycetoma: A Complex Disfiguring Syndrome 671

Pneumocystis (carinii) jiroveci

707

Nematode (Roundworm) Infestations 708

Biology of Blastomyces dermatitidis: North American

22.4

705

Pathology of Helminth Infestation 705

Systemic Infections by True Pathogens 663

ofPigmentedFungi

7O5

Cycles

General Epidemiology of Helminth Diseases 705

Organization of Fungal Diseases 662

22.3

698

23.4 A Survey of Helminth Parasites

Infections 660 Control of Mycotic Infections 661

Coccidioidomycosis:

692

CoccidianParasites 702

660

Diagnosis of Mycotic

22.2

coli

TheFlagellates(Mastigophorans) 692 Hemoflagellates: Vector-Borne BloodParasites 694

Emerging Fungal Pathogens 658

ofthe

Infective Amoebas 689 The Intestinal Ciliate: Balantidium

657

Primary orTrue Fungal Pathogens 658

Pathogenesis

23.2 Typical Protozoan Pathogens 688

Pneumocystis

VisualUnderstanding 723 Internet Search Topics 723

678

Aspergillosis: Diseases of the Genus I spergillus 679

Zygomycosis 680 MiscellaneousOpportunists 680

22.7 FungalAllergies and Intoxications INSIGHT 22.1 Mlcroblology The Keratin Lovers 672

INSIGHT 22.2 Medlcal

681

CHAPTER

Multiple-ChoiceQuestions 683

,o;ffi

lntroduction to Viruses That Infect Humans: The DNA Viruses 724

24] 24.2

Viruses in Human Infections and Diseases 725 Important Medical Considerations in Viral Diseases 726 Survey of DNA Virus Groups Involved in Human

lnfections 727

Poisonous Fungi in Foods 679 Chapter Summary with Key Terms 682

-4

24.3

Enveloped DNA

Viruses 727

Poxviruses: Classification and Structure 727

xvll

xYiii

Contents

The Herpesviruses: Common, Persistent Human Viruses 730 The Viral Agents of

Hepatitis

24.4 Nonenveloped

Preventing HIV

738

DNA The Adenoviruses '/41

Viruses

Papilloma and Polyoma

Viruses

Infection

Treating HIV Infection and

772

AIDS

772

25.6 Other Retroviruses: Human T-Cell Lymphotropic Viruses 773 25.7 Nonenveloped Nonsegmented Single-Stranded RNA

741

Viruses: Picornaviruses and Caliciviruses 774

742

24.5 Nonenveloped Single-Stranded DNA Viruses:

Poliovirus and Poliomyelitis 774

The Parvoviruses 744

NonpolioEnteroviruses 777

INSIGHT24.1 Ilistorical

Caliciviruses

'779

25.8 Nonenveloped Segmented Double-Stranded

Smallpox: An Ancient Scourge Revisited 728

25.9

Herpesviruses: A Connection to Chronic Fatique

Prions and Spongiform Encephalopathies 7aO

ofCJD

Syndrome? 736

Pathogenesis andEffects

INSIGHT 24.3 Medlcal

Transmission and Epidemiology 781

The Cfinical Connection in Hepatitis

B

Prevention and/or Treatment 781

INSIGHT 25.1 Dlscovery Life in the Hot Zone 755

Writing to Learn 746

Mapping

747

INSIGHT 25.2 Medical

Critical Thinking Questions 747

A Long Way from Egypt: West Nile Virus in the United States 765

VisualUnderstanding 748 lnternet Search Topics 748

INSIGHT 25.3 Medlcal DS-Defining lllnesses (ADls)

Af

CnnPffn

' ,,

77'l

INSIGHT 25.4 Medical

jl

:;#.

The RNA Viruses That Infect Humans 749 25.1 Enveloped Segmented Single-Stranded Viruses 750 The Biology of Orthomyxoviruses:

25.2

780

Culture and Diagnosis 781

74O

Chapter Summary with Key Terms 744 Multiple-Choice Questions 745 Concept

RNA

Viruses:Reoviruses 779

INSIGHT 24.2 Medical

Influenza

RNA

Uncommon Facts about the €ommon

778

Chapter Summary with Key Terms 782 Multiple-Choice Questions 783

Writing to Learn 784 Concept

750

Cold

Mapping 785

Bunyaviruses andArenaviruses 754

Critical Thinking Questions 785 VisualUnderstanding 786

Enveloped Nonsegmented Single-Stranded RNA

lnternet Search Topics 786

Viruses 756 Paramyxoviruses 756 Rhabdoviruses 759

25.3 Other Enveloped

RNA Viruses: Coronaviruses, Togaviruses, and Flaviviruses 761 Coronaviruses 761

Rubivirus: The Agent of Rubella 762 Hepatitis C Virus 762

25.4 Arboviruses: Viruses Spread Vectors 763

by Arthropod

Epidemiology of Arbovirus Disease 763 General Characteristics of Arbovirus Infections 763 Diagnosis, Treatment, and Control of Arbovirus

Infection

25.5

766

Agent

AIDS 766

The Organization of Ecosystems 788 Energy and Nutritional Flow in Ecosystems 789 Ecological Interactions between Organisms in a

Community

Infection

791

26.2 The Natural Recycling of Bioelements Cycles Cycles

792 796

26.3 Microbes on Land and in Water 797

Infection

Stages, Signs, and Symptoms of AIDS 770

Diasnosis of HIV

Environmental Microbiology 787 26.1 Ecology: The Interconnecting Web of Life

Sedimentary

766

Epidemiology of HIV

l

,,,,,

Atmospheric

HIV Infection and Causative

CHAPTER

Soil Microbiology: The Composition of the

766

HIV Infection

772

and

Lithosphere 797 AquaticMicrobiology 800 The Structure of Aquatic Ecosystems 801

792

788

.^ li ir

Contents

27.3

INSIGHT 26.1, DlscoverY

General Concepts in Industrial

Microbiology 822

Greenhouse Gases, Fossil Fuels, Cows, Termites, and Global Warming 794

From Microbial Factories to Industrial Factories 824

INSIGIIT 26.2 MlcroblologY

SubstanceProduction 825

Bioremediation: The Pollution Solution? 798

INSIGHT 27.1 DiscoverY

INSIGHT 26.3 MlcroblologY

Baby Food and

Meningitis 818

The Waning Days of a Classic Test? 804

INSIGIfT 27.2 DiscovetY

Chapter Summary with KeY Terms 806 Multiple-Choice Questions 806

The Bioterrorism/ Biotechnology Connection 827

Writing to Learn 807

Multiple-ChoiceQuestions 828 Writing to Learn 829

Chapter Summary with

Concept Mapping 807 Critical Thinking Questions 808 VisualUnderstanding 808

Terms 828

Concept Mapping 829 Critical Thinking Questions 829

VisualUnderstanding 830

Internet Search ToPics 808

lnternet Search Topics 830

,ry,= cltAPttR *i* F

',"::::::,:.ii,.ffi

Applied and lndustrial Microbiology 809 27.1 Applied Microbiology and Biotechnology 810 Microorganisms in Water and Wastewater Treatment 810

27.2 Microorganisms and Food

KeY

8'12

Microbial Fermentations in Food Products from Plants 812 Microbes in Milk and Dairy Products 816 Microorganisms as Food 817 Microbial Involvement in Food-Borne Diseases 8l'7 Prevention Measures for Food Poisoning and Spoilage 819

A A_1 APPENDIX B B-1 APPENDIX C C_l APPENDIX D D-l APPENDIX E E-I APPENDIX F F-l APPENDIX

Glossary G-1 Credits C-l

lndex l-l

xtx

,-: fi

.

,,

:::-:::

Preface There are a Million Stories in the Microbe f ungle One measure of a subject's impact on the everyday lives of people is how often it is mentioned in the popular press. By this measure,

it

seems that microbiology has really come of age. Consider some

of the "buzz words" creeping into the tabloids of late: MRSA, C. dtff, killer cold viruses, bacterial cultures in yogurt, the bird flu, biofilms, caneer vaccines, designer bacteria, and personal gene chips, just to name a few. If a quick glance at some of the latest headlines has inspired you to enhance your understanding ofthese topics and hundreds ofothers, this book is a good place to start. Inquiring minds want to know! It is true that a substantial portion of discoveries in science right now are emerging from the realm of microbiology. In fact, microbiology has entered a new "golden age" that is generating information at a rapid rate. Much of it relates to genetics and infectious disease, but a lot ofit comes from discoveries about the activities of microbes in the natural environment. Because microbes are so small, widespread, and largely nonvisible, there will always be some places that have not yet been thoroughly explored where microbes are living and doing their thing. As greater attention is focused on the rainforest, oceans, bedrock, or even the human body, and advanced tools are used in probing these environments, our perspectives on the microbial world are expanded to new and greater dimensions.

multidimensional art pieces complement self-contained conceptspecific narrative; it is not necessary to read page content surrounding the artwork to grasp concepts being illustrated. Development of the art in this manner further enhances learning and helps to build a solid foundation ofunderstanding. This seventh edition has given us the opportunity to hone and improve the art even more. In addition to many new and revised figures, the Process Figures are now clearly defined as such and include colored steps that correlate the art to step-by-step explanations. Art has also been pulled into special Visual Understanding study tools to help students make comections between concepts presented in different chapters.

Early Survey of Microbial Groups and Taxonomy A unique feature of this text's format is the early survey of micro-

bial groups and their taxonomy (chapters 4, 5, and 6). By using general and specific names for microbes from the very beginning students develop a working background that eases them into the later chapters. I have always felt that microbes are the "stars of the show," and that students have a far greater appreciation for

later topics of nutrition, metabolism, genetics, and microbial control if they rccognize the main characters-bacteria, viruses, and eukaryotic microorganisms-and already know significant facts about them.

Relevant, Up-to-Date Disease Chapters

What Sets This Book Apart? Engaging, Straightforward Presentation The primary aims of this book have been 1) to present guiding principles in a straightforward and readable style that is neither too wordy nor too simplistic, and 2) to explain complex topics clearly and vividly. I have continued to organize the content in a logical order that builds foundations from early chapters to later ones. The text is backed up with numerous tables, flow charts, and other support features. Having many different levels and cognitive styles for students increases retention, understanding, and success in learning.

Unique among microbiology textbooks, chapter 17, "Diagnosing Infections" brings together in one place the current methods used to diagnose infectious diseases. The chapter starts with collecting samples from the patient and details the biochemical, serological, and molecular methods used to identiff causative microbes. The pathogen chapters (18-27) are organized by microbial group (taxonomy) because many users feel this orientation has greater coherence and concentrates more on infectious agents than anatomy and physiology. All characteristics of the microorganism and its diseases may be presented simultaneously. This system will not only be familiar to students from their laboratory work, but it helps them maintain a more distinct separation between microbial groups and their diseases.

A Vivid, Self-Explanatory Art Program My experiences as a teacher, microbiologist, and illustrator

Pedagogy Designed for the Way Students Learn

have helped me to visualize abstract concepts and transform them into scientifically accurate and altr activ e illustrations. Vivi{

Foundations in Microbiology makes learning easier through its carefully crafted pedagogical system. Following is a closer look

xx

Preface

previous chapters and pose queries that require students to combine knowledge from the current chapter with the material

at some of the key features that our students have taught us are useful.

o All

with Case File mysteries to solve. These real-world case studies help students appreciate and underchapters open

stand how microbiology impacts our lives on a daily basis. The

they already have learned from previous chapters.

o Concept Mapping Exercises ask students to organize information in more meaningful forms than just simple lists.

solutions appear later in the chapter, after the necessary eleat the beginning of each chapter pro-

vides students with a framework from which to begin their study

ofa chapter.

o In chapters 1-16 and26-27, major sections of the chapter

. .

are followed by Checkpoints that repeat and summarize the concepts ofthat section. In the disease chapters (18-25) the Checkpoints are in the form ofthe disease tables described

earlier.

o Insight

readings allow students to delve into material that goes

mations with quizzes are also on the website.

o Study on the Fly Content-now

students have access to downloadable chapter summaries and animations so they can study anywhere, anytime.

beyond the chapter concepts and consider the application of those concepts. The Insight readings are divided into four categories: Discovery Historical, Medical, and Microbiology.

r All chapters end with a summary and a comprehensive

o

r

array

of end-of-chapter questions that are not just multiple-choice, but also questions that require writing and critical thinking about topics in the chapter. Considering and answering these questions, and even better, discussing them with fellow students, can make the difference between temporary (or limited) learning and true knowledge of the concepts. Visual Understanding questions incorporate art to help shrdents connect important concepts from chapter to chapter. Concept Mapping assists in retention as well as contextual organization.

Up-To-Date Content o The single chapter (26) that covered

o

.

What's New with This Edition?

o

Since the science of microbiology is constantly changing and advancing, the textbook must also change and advance to stay curent and continue to be useful and relevant. With each edition we will continue to create a current, well-organized and scientifically accurate book, and provide an active learning opportunity

for students.

I

have been fortunate to have my colleague Barry Chess, Pasadena City College and Kelly Cowan of Miami University

of of

Ohio continue in their capacity as significant contributors. They have helped write new sections and Insight boxes, suggested ideas for new and improved figures, edited and updated text, and improved chapter overviews, summaries, and questions. Kelly is instrumental in developing case files and both she and Barry have constructed some of the active learning features in the endof-chapter sections. Many additions and innovations were done at the request ofreviewers and users, whose input continues to be invaluable.

Active Learning Experience

.

New Visual Understanding Questions supply a photo or a graphic that students have already seen, along with a thoughtprovoking question. Many of these questions use images from

Three different types of concepts maps are used throughout A new Appendix introduces students to concept mapping. Process figures now have matching numbered steps for easy to see explanations ofcomplex processes. Special icons correlate over 100 total animations to figures in the text. When students see the icon next to a figure legend, they'Il know to check out the accompanying website for a helpful animation to actively illustrate the concept. Additional ani-

the text.

ments have been presented.

o A Chapter Overview

xxt

both environmental and applied microbiology has been split into two separate chapters (26 and 27). Chapter 26 now focuses on ecological principles and the interactions of microbes with the environment, and chapter 27 examines the use of microbes in industry and biotechnology. Chapter 9 introduces some of the newer concepts in genetics that have emerged from genome analysis studies. The most significant discovery involves the role of special types of RNA in regulating genes and their expression. Applications of regulatory RNA in biotechnology and engineering oftransgenic animals have been added to chapter 10. To consolidate and streamline the section on chemical con-

trol of microorganisms in chapter 11, we have compiled

r o o o

r

o

several new tables that summarize and illustrate common applications. Now that probiotics have become more widely used and understood, their coverage has been updated and enlarged in chapters 12 and 13. Throughout the book there is much more emphasis on polymicrobial infections and biofilms. In chapter 17 we have included a more detailed table of specimen collection and increased coverage of PCR technology in diagnosis of infections. After much consideration and a number of requests, the spelling of prokaryote and eukaryote and related terms has been revised to the form with a "k" instead of a "c" throughout all chapters.

The section on photosynthesis that was originally covered in chapter 26 in Ihe section on environmental microbiology has been moved into chapter 8 along with other metabolic and bioenergy concepts. Overall, we have added a number of new case studies (called case files), photographs, figures, notes, and boxes.

For a complete listing of chapter-by-chapter changes, please visit the textb AMS website.

xxlt

Preface

Acknowledgments My involvement in this textbook

goes back over twenty-five yeaxs. Throughout this active and fulfilling time, I have had the good fortune to be supported by the best publishing staffin the business. I have collaborated with dozens of top-notch editors, researchers, production staff, illustrators, and designers. It has been clear to me that, from the very beginning, the textbook teams have shared my love for the project, and have brought their own expertise and commitment to maintaining a high quality product. This seventh edition has carried on this tradition. Several key people made significant contributions to this edition. First, I wish to commend my senior developmental editor, Kathleen Loewenberg, for her enthusiastic support and suggestions. Her experience, and thoughtful comments have been a real asset, and she is an awesome "figure wrangler," bringing a fresh perspective and keen eye to the art program. I greatly appreciate the contributions of the editorial coordinator Ashley Zellmer, who cheerfully takes on the sometimes tedious work of preparing and processing manuscript and keeping track of the numerous revisions in text and figures. I am indebted to senior sponsoring editor Jim Connely, who keeps us laughing when we need it, and whose advice "If you put something in, you'Il need to take something out" has been a useful guide for many a decision about content, length, and new features. I have received much helpful input from publisher Michelle Watnick, another experienced and well-informed member of the book team. I admire her ability to grasp "the big picture" ofbook creation. Senior project manager, Jayne Klein, has done a first-rate job of overseeing the minutiae of production. I especially appreciate her flexibility in considering changes I feel strongly about and the detailed efforts from her team. They can actually find an italicized period in a footnotejust to give you an idea ofthe level ofscrutiny this book receives! Other gifted and dedicated personnel that I would like to thank include the photo research coordinatoq Carrie Burger; photo researcher, Danny Meldung at Photo Affairs; Jeanne Patterson, the copy editor; and the book designer, Michelle Whitaker. No list of acknowledgments would be complete without mentioning senior marketing manager Tami Petsche, who has to wear several hats, including having to take a crash course in microbiology with each new edition. Just like the living world, this textbook is evolving. A major force behind this hend relates to the constant discoveries happening in microbiology that must be addressed and updated. But another undeniable force for change is the feedback that we get from users and reviewers. I want to make special mention of Dr. Wan H. Ooi and his colleagues Pramilla Sen, Marsha Turell, and Donna Wiersema offlo uston C ommunity College, and Dr. Reza Marvdashti of San Jacinto College for their insights in several chapters. Other reviewers who have provided substantive comments on content and accuracy are Melissa Rubin, Kelly Gridley, Dana Nayduch, and Davis Prichett. Our team of reviewers for the seventh edition has contributed valuable ideas for new figures, boxes, and coverage. They have helped to fine tune language, terminology, headings, Checkpoints, andpedagogy. These reviewers teach the subject and are interpreters of it to beginning students. It is obvious that

they share a passion for knowledge and wish to impart the excitement of microbiology to their classes. We commend you for your dedication. For the users ofthis book, we hope that you enjoy ourjourney into the world of microbiology and nurture a long-term interest in this fascinating science. Though many elaborate steps are taken to weed out errors, the very nature of an evolving book means that "mutations" may slip in without notice. If you detect any missing or misspelled words, missing labels, mistakes in content, or other errata, do not hesitate to contact the publisher, representative, or the author ([email protected]).

Reviewers Arden Aspedon, Southwestern OHahoma State University

DennisA. Bazylinski, University of Nevada, Las Donald

P.

Vegas

Breakwell, BrighamYoung University

Hoi Huen Chan, Hong Kong Community College, The Hong Kong Polltechnic University James

K. Collins, University ofArizona

Don C. Dailey, Austin Peay State University Cheryl G. Davts, Tuskegee University Susan

A. Gibson, South Dakota State University

Kelly Gridley, Santa Fe Community College Janelle Hare, Morehead State University

JenniferY. Harper, Coastal Georgia Community College Brenda B. Honeycutt, Armstrong Atlantic State University

Robert Iwan, lzver Hills Community College Harry W. Kestler, Lorain County Community College Si Wouk

Kim, Chosun University

Robert Klein, Owens State Community College Peter S. Kourtev, Central Michigan University Susan Forrest,

Butler Community College

Julie C. Matheny, Owens State Community College Dana Nayduch, Georgia Southern University

M. Theresa Pavlovitch, Pasadena City College Jack Pennington, Forest Park College

Mary Lou Percy, Navarro College Davis W. Pritchett, University of Louisiana at Monroe Wesley Rakestraw, Wallace State Community College

Michael W. Reeves, Georgia Perimeter College Melissa Rttbin, Kent State University Michael W. Ruhl, Vernon College Gail A. Stewart, Camden County College John R. Stevenson,

Miami University

Coe Vander Zee, Austin Community College

Preface

Symposium Attendees Chad Brooks,

lustin

Peay State University

Barry Chess, Pasadena City Cotlege Erin Christensen, Middlesex County College John Dahl, Washington State

(Iiiversity

Alison Davis, East Los Angeles College Susan Finazzo, Broward Community College

Clifton Franklun{ Ferris State University Edwin Gines-Candalaria, Miami Dade

Judy Haber,

Califtrnia State University Fresno

Eunice Kamunge, Essex County College Amine Kidane, Columbus State Community College Tracey Mills, Iry kch Community College Madhura Pradhan, Ohio State University Louise Thai, tJniversity of Missouri Delon Washo-Krupps, Arizona State University Samia Williams, Santa Fe Community College

xxlll

lnstructional Art Program Clarifies Concepts Foundations in Microhiology provides powerful artwork that paints conceptual pictures for students. The art combines vivid colors, multi-dimensionality, and self-contained narrative to help students study the challenging concepts of microbiology from a visual perspective-a proven study technique. Art is often coupled with photographs to enhance visualization and comprehension.

New! Text Art Correlated to Animations

ft fnt

ft Figure 11.4

symbol indicates to readers that the material presented in the text is also accompanied by an animation on the book's website. Students may view the animation on their computers or download it to their portable player and watch it on the fly!

ochomoraris

by

phasose

Modes of action affecting protein

function.

(a) The native (functional) state is maintained by bonds that create active sites to fit the substrate. Some agents denature the protein by breaking all or some secondary and tertiary bonds. Results are (b) complete unfolding or (c) random bonding and incorrect folding. (d) Some agents reactwith functional groups on the active site and interfere with bondino.

Process Figures Foundations in Microbiology illustrates many difficult microbiological concepts in steps that students find easy to follow. Each step is clearly marked with a yellow, numbered circle and correlated to accompanying narrative to benefit all types oflearners. Process Figures are now identified next to the figure number. The accompanying legend provides additional explanation.

@ Phasosm€

d @

4l

?rocers Flgure 14.18 The sequential evenb in phagocytosh. (l) Pha9ocyt€ 5 attra(red to baclera. (2) Cose-upview oJ proce$rhowing bacterla adhef ng to 5peca receptob by thet PAMPT. (3) Vacuore 6lormed around ba.teria during e.9! rmenr (a) Phagosom€ digenlveva.uoe resulc. (s) Lyso5ome5 i!sewith phagosone, iorming a phaqoysosome. (6) E.zymes and toric oxygen prodL.E kil and digen bacteria (7) Undigened partces are released. Inret: Scanning eectron mi.bgfaph oia macrophage actively €nqaged ln devouring bacterk (10,000:).

W enaso yosome

t

@

Ki inq and

dosrud$

d

!

c

;:*:%6,.6

Combination Figures Line drawings combined with photos give students two perspectives: the realism ofphotos and the explanatory clarity ofillustrations. The authors chose this method of presentation to help students comprehend difficult concepts.

XXIY

Flgure 9,16 Speeding up the protein

assembly line in bacteria.

(a) The mRNA tanscript enco!nte6 ribosoma parts immediately as t leaves the DNA. (b) The ribosomal factories a$embte a ong th€ mRNA in a chain, each ribosome reading the me$age and ranjating it into protein. Many producb will thus be well along the synthetic pathway before tanscription has even terminated. (c) Photomicrograph of a polyribosomal .omplex in action. Not€ that the protein ,1aih,, vary in tength dep€odin9 on the *age of tandation (30,000x).

Clinical Photos Color photos ofindividuals affected by disease provide students with a real-life, clinical view of how microorganisms manifest themselves in the human body.

Overview Figures Many challenging concepts of microbiology consist of numerous interrelated activities. Foundations in Microbiology visually summarizes these concepts to help students piece the activities together for a complete, conceptual picture. (a)Setional view ot a boil orfuruncle, a singl€ pustuleihai develops in a hatiollicle orgland and isthe classic lesion ofthe species. The inflamed inleclion sil€ b€comos abscessed when masses ol phagocyt€s, bacleria, and lluid are walled off by tibiln.

rffiM

@% E;. E==I

-

el

rhF nF.k.

ll:l:l:l Caftunctes are massve deeP lesions hal ,--,, ,,Dm mJnipte, '-'-'_--necl

(b) Afuruncle on the back of the hand. This lesion toms in a single lollicle.

no lutuncr6s. Swelling and rupture inlo the sutrounding lissues can b6 mafted,

Flgure 18.3 cutaneous lesions of Stophylococcus aurcus Fundamentally all are skin abrcesses thatvaryin size, depth, and deqree of tissue involv€ment.

clsr

Pedagogical Aids Promote Active Learning Foundations in Microbiology organizes every chapter with consistent pedagogical tools. These visual and contentrelated elements enable students to develop a consistent learning strategy and learn in more than one way, creating a higher retention rate. Let's look at the pedagogical features within each chapter:

Case Files All

chapters open with a real-world case file to help

students appreciate and understand how microbiology impacts lives on a dally basis. The solution to the case file appears later in the chapter, near where relevant material is

being discussed. These relevant "stories" pique interest and help students understand just how important it is to learn and retain the chaoter's content.

rrre 7

th€ micbhr found by the re5earche6 are extemophils. They "ld€" bi.g in €.vLonmentar conditbn! on snh thar irc6 the limiB or hot, a.. o-* #" #* .g- _

u"€

3-6i

*=

Flgure 2.12 Hydration

dil

spheres formed around ions

in solution. In this example, a sodium cation attracts the negatively charged region of water molecules, and a chloride anion attracts the positively charged region of water molecules. In both cases, the ions become surrounded by spherical layers of specific numbers and arrangements of water molecules.

that attract water to their surface are termed hydrophilic.* Nonpolar molecules, such as benzene, that repel water are considered hydrophobic.* A third class of molecules, such as the phospholipids in cell membraneso are considered amphipathic* because they have both hydrophilic and hydrophobic properties. Because most biological activities take place in aqueous (waterbased) solutions, the concentration of these solutions can be very important (see chapter 7). The concentration ofa solution expresses the amount of solute dissolved in a certain amou:rt of solvent. It can be figured by percentage or molarity. Percentage is the ratio ofsolute in solution expressed as some combination of weight or volume' A common way to calculate concentration by percentage is to use the weight of the solute, measured in grams (g), dissolved in a specified volume of solvent, measured in milliliters (ml). For example, dissolving 3 g of NaCl in the amount of water to produce 100 ml of solution is a 3o/o solution; dissolving 30 g in water up to 100 ml of solution produces a 30% solution; and dissolving 3 g up

(l liter) produces a0.3o/o solution. A frequent way to express concentration ofbiological solutions is by its molar concentration, or molariry (M).A standard molar solution is obtained by dissolving one mole, defined as the molecular weight of the compound in grams, in 1 L (1,000 ml) of solution. To make a 1 M solution of sodium chloride, we would dissolve 58 g of NaCl to give 1 L of solution; a 0.1 M solution would require 5.8 e of NaCl in 1 L of solution.

to

1,000 ml

Acidity, Alkalinity, ond the pH Scole Another factor with far-reaching impact on living things is the concentration of acidic or basic solutions in their environment. To understand how solutions become acidic or basic, we must look again at

a

hydrophitic (hy-clroh-fil'-ik) Gr. hydros, water, and philos, to love.

* hydrcphobic ftry-droh-fob'-ik) Gr. phobos,

feat

* amphipathic (an'-fu-patn'-ik) Gr. amphi, both.

Chapter

38

2

The Chemistry of Biology

,.od .d.el

-.dt -."-t

#a

^...'"

^."

.."-')i$$$;;$"*$..-":;r,'.N"rS$-:$"'-"*-..:"1"":.*""."':."-S

t |t t t I l l I t l t t

pHo

t t t l

10

Acidic

[H+l

l1

tz

13

t

14

Neutral

Increasing acidity

Figure

2.13

The pH scale.

Shown are the relative degrees of acidity and basicity and the approximate pH readings for various substances.

the behavior of water molecules. Hydrogens and oxygen tend to remain bonded by covalent bonds, but under certain conditions, a sin-

gle hydrogen can break away as an ionic H*, or hydrogen ion, leaving the remainder of the molecule in the form of an OH , or hydroxide ion. The H+ ion is positively charged because it is essentially a hydrogen that has lost its electron; the OH is negatively charged because it remains in possession of that electron. Ionization of water is constantly occurring, but in pure water containing no other ions, H+ and OH- are produced in equal amounts, and the solution re-

mains neutral. By one definition, a solution is considered acidic when one ofits components (an acid) releases excess hydrogen ions.6 A solution is basic when a component (a base) releases excess hy-

droxide ions, so that there is no longer a balance between the fwo ions. Another term used interchangeably with basic is alkaline. To measure the acid and base concentrations of solutions, scientists use the pH scale, a graduated numerical scale that ranges from 0 (the most acidic) to 14 (the most basic). This scale is a useful standard for rating the relative acid or base content ofa substance. Use figure 2.13 to famlliarize yourself with the pH readings of some common substances. It is not an arbitrary scale but actually a mathematical derivation based on the negative logarithm (reviewed in appendix B) of the concentration of H* ions in moles per liter (symbolized as [H*]) in a solution, represented as;

pU

: -log[H*]

a

exponents:

c pH 2 has an [H-] of I0-2 moles. o pH 14 has an [H+] of l0-14 moles (table

tenfold reduction:

' t',

t-

Hydrogen lon and Hydroxide lon Concentrations at a Given pH

Moles/L of Hydrogen lons 1.0 0.1 0.01

0.001

0.00001 0.000001 0.0000001 0.00000001

o pH 1 contains [0.] moles H*/L]. o pH 2 contains [0.01 moles H* lL]. o Continuing in the same manner up to pH

0.000000001 0.0000000001

14, which contains

[0.00000000000001 moles H* lL1.

0.00000000001 0.00000000000 I 0.0000000000001

6. Actually, it forms tne notatlon ot H

a hydronium ion (H3O+), but

for simplicity,s sake, we will use

2.2).

It is evident that the pH units are derived from the exponent itself. Even though the basis for the pH scale is [H+]. it is important to note that, as the [H+] in a solution decreases, the [OH ] increases in direct proportion. At midpoint-pH 7 or neutrality-the concentrations are exactly equal and neither predominates, this being the pH of pure water previously mentioned. In summary, the pH scale can be used to rate or determine the degree of acidity or alkalinity of a solution. On this scale, a pH

0.0001

Acidic solutions have a greater concentration of H+ than OH , starting with: pH 0, which contains 1.0 moles H"/liter (L). Each of the subsequent whole-number readings in the scale changes in [U+] by

These same concentrations can be represented more manageably by

0.0000000000000 I

Logarithm

r0-" 10t 102 10-3 10-4 105 106 l0 / 10-8 10-e lo-ro 10 rr lo-r2 10-13 lo-14

pH 0

I 2 3

4

Moles/L of OH14

l0-

I0-'' '' 10il l0 l0-

10

0-e

5

I

6

10-8

1

10-7

8

106 105

g

lo

ll

1

0-4

103 l0-2

D B

10'

A

1

0-u

2.1 Atoms, Bonds, and Molecules: Fundamental Building Blocks below 7 is acidic, and the lower the pH, the greater the acidity; a pH above 7 is basic, and the higher the pH, the greater the alkalinity' Incidentally, although pHs are given here in even whole numbers, more often, a pH reading exists in decimal form; for example, pH 4.5 or 6.8 (acidic) and pH '7.4 or 10.2 (basic). Because of the damaging effects ofvery concentrated acids or bases, most cells operate best under neutral, weakly acidic, or weakly basic conditions (see chapter 7). Aqueous solutions containing both acids and bases may be involved in neutralization reactions, which give rise to water and other neutral by-products. For example, when equal molar solutions

of hydrochloric acid (HCl) and sodium hydroxide (NaOH, a base) are mixed, the reaction proceeds as follows:

HCl+NaOH+HrO*NaCl Here the acid and base ionize to H+ and OH- ions, which form water, and other ions, Na* and Cl-, which form sodium chloride. Any product other than water that arises when acids and bases react is called a salt. Many of the organic acids (such as lactic and succinic acids) that function in metabolism* are available as the acid and the salt form (such as lactate, succinate), depending on the conditions in the cell (see chapter 8).

The Chemistry of Carbon and Orgonic Compounds So far, our main focus has been on the characteristics of atoms, ions, and small, simple substances that play diverse roles in the + metabolism (rtvh-tab'-ohJizrn) A general term referring to the totality chemical and physical processes occurring in the cell.

of

39

structure and function of living things. These substances are often lumped together in a category called inorganic chemicals. A chemical is usually inorganic if it does not contain both carbon and hydrogen. Examples of inorganic chemicals include HzO, o'2, NaCl (sodium chloride), Mg3(POa)2 (magnesium phosphate), CaCO, (calcium carbonate), and CO2 (carbon dioxide). In realiry however, most of the chemical reactions and structures of living things occur at the level of more complex molecules, termed organic chemicals. The minimum requirement for a compound to be considered organic is that it contains a basic framework of carbon bonded to hydrogens. Organic molecules vary in complexity from the simplest, methane (CHa; see fig:ute 2.4c), which has a molecular weight of 16, to certain antibody molecules (roduced by an immune reaction) that have a molecular weight of nearly I million and are among the most complex molecules on

earth. Most organic chemicals

in cells contain other

elements

such as oxygen, nitrogen, and phosphorus in addition to the carbon and hydrogen. The role of carbon as the fundamental element of life can best be understood if we look at its chemistry and bonding patterns. The valence of carbon makes it an ideal atomic building block to form the backbone of organic molecules; it has 4 electrons in its outer orbital to be shared with other atoms (including other carbons) through covalent bonding. As a result, it can form stable chains containing thousands of carbon atoms and still has bonding sites available for forming covalent bonds with numerous other atoms. The bonds that carbon forms are lineaq branche4 or ringe4 and it can form four single bonds, two double bonds, or one triple bond

(figure 2.14).

Linear

I

-e*

H

.6. aa

*.H

I

.6:n

Branched

-

C=o

lt *c-N l\

.i. *.it+ .6. aa

*.ir!+

tlllllllll *c*c*c*c*c*c*c*s*c*c* rlllllllll

lc::o';

tlltilll *c-c *c -c-c -c-c -crll:llll

|

-C-

.6:ri:

.C. +.C. +

\,/c=c ,/\

.i. *.i. +

a-aa

oaaa

.C:C.

\/

Ringed

\./

*t'"-J-

ll *f-r'l-

:c::c:

/\ -CE

_l_ I

ll * -c-c tl

N

-C-

.C. + N.+.C!::N:

(a)

Flgure 2.14 The versatility of bonding in carbon.

(b)

"c_

*lc'/ \;* \_/ /\

).1 t"-J i( ,/\ /\

made with other carbons, oxygen, and In most compounds, each carbon makes a total of four bonds. (a) Single, double, and triple bonds can be shared in these bonds' (b) Multiple bonding are electrons the how show models electron simple hydrogen. with made nitrogen; single bonds are -branched large and complex. are extraordinarily compounds, and ringed compounds, many of which of caibons can give rise to long chains,

Chapter

40

2

The Chemistry of Biology

Representative Functional Groups and Organic Compounds That Contain Them Formula of Functional Group Name R.

-io: Hl

Hydroxyl

R-ic

Alcohols,

CASE F'iLE

i

:

\i

:

indicates that the group attached at that site varies from one compound to another.

Can Be Found ln carbohydrates

o:

./t '/i

R-OH or R-NH2. The designation on a molecule is shorthand for remainder, and-R its placement in a formula such as

Carboxyl

2

Wrap-Up

Fatty acids, proteins,

organic acids

OH:

Bioremediation, the utilization of microbes to clean up pollution

or reduce waste, is based on unique metabolic activities of microbes.

H I

R_C-:NH;;

t"

Amino

Proteins, nucleic acids

The process developed by O'Connor and his colleagues involves heating recycled polystyrene to over 500.C to convert

the polystyrene into styrene oil and some other compounds. Pseudomonas putido acts on the styrene oil and gives off poly-

H

:o i// R-c

Ester

Lipids

Sulftydryl

Cysteine (amino acid), proteins

i\ : .....o-.R

i:

.ll R-C,l :H

SHI I

,

hydroxyalkanoate. Unlike polystyrene, which O,Connor calls a "dead-end product," PHA can be utilized to make other plastics and is biodegradable. P. putida and other relatives called pseudomonads are free-living organisms widespread in soil and aquatic areas. They have very versatile metabolic abilities and can decompose even human-made chemicals such as plastics, pesticides, and petroleum products. See: O'Connor, K. E., et al, 2006. A two-step chemo-biotechnologicol conversion of polystyrene to o biodegrodable thermoplostic. Environmental

Science & Technology 40 :2433-2437.

// R-C

l

:\

:

Carbonyl, terminal end

Aldehydes, polysaccharides

,H, iO

,lll R+Cil

C-

Carbonyl, internal

Ketones, polysaccharides

,r! Covalent bonds are chemical bonds in which electrons are shared between atoms. Equally distributed electrons form nonpolar covalent bonds, whereas unequally distributed electrons form polar covalent bonds.

Phosphate

DNA, RNA,ATP

r.: Ionic bonds are chemical bonds resulting from opposite charges. The outer electron shell either donates or receives electrons from another atom so that the outer shell of each atom is comoletelv

o tl R

-,O-

P

-OH

OH

,. *lil;r*

*The R designation on a molecule is shorthand for remainder, and its placement in a formula indicates that what is attached at that site varies fiom one comoound to another.

Functionol Groups of Orgonic Compounds One important advantage of carbon's serving as the molecular skeleton for living things is that it is free to bind with a variety of special molecular groups or accessory molecules called functional groups. Functional groups help define the chemical class

ofcertain groups oforganic compounds and confer unique reactive properties on the whole molecule (table 2.3). Because each type of functional group behaves in a distinctive manner, reactions of an organic compound can be predicted by knowing the kind of functional group or groups it carries. Many synthesis, decomposition, and transfer reactions rely upon functional groups

bonds are weak chemical attractions that form o.r*.." covalently bonded hydrogens and either oxygens or nitrogens on different molecules. r, Chemical equations express the chemical exchanges between atoms or molecules that occur during chemical reactions such as synthesis or decomposition. :: Solutions are mixtures of solutes and solvents that cannot be separated by filtration or settling. ":' The pH, ranging from a highly acidlc solution to highly basic solution, refers to the concentration ofhydrogen ions. It is expressed as a number from 0 to 14. ;: Biologists define organic molecules as those containing both carbon and hydrogen. '' Carbon is the backbone of biological compounds because of its ability to form single, double, or triple covalent bonds with itself and many different elements. ' Functional (R) groups are specific arrangements of organic molecules that confer distinct properties, including chemical reactivity, to organic compounds.

2.2 Macromolecules: Superstructures

iliii..

.ii,.r Macromolecules Macromolecule

of Life

4l

and Their Functions Description/Basic Structure

Examples/Notes

Monosaccharides

3- to 7-carbon sugars

Glucose, fluctose / Sugars involved in metabolic reactions; building block of disaccharides and polysaccharides

Disaccharides

Two monosaccharides

Maltose (malt sugar) / Composed of two glucoses; an important breakdown product of starch Lactose (milk sugar) i Composed of glucose and galactose

Carbohydrates

Sucrose (table sugar) / Composed ofglucose and fructose Polysaccharides

Starch, cellulose, glycogen

Chains of monosaccharides

i Cell wall, food

storage

Lipids Triglycerides

Fatty acids

*

Phospholipids

Fatty acids

+ glycerol *

Waxes

Fattv acids. alcohols

Steroids

Ringed structure

Mycolic acid / Cell wall of mycobacteria Cholesterol, ergosterol / Membranes of eukaryotes and some bacteria

Amino acids bound by peptide bonds

Enzyrnes; part of cell membrane, cell wall, ribosomes, antibodies / Metabolic reactions; structural components

Fats, oils / Major component of cell membranes; storage

glycerol

Membranes

PhosPhate

Proteins Polypeptides

Nucleic acids

Nucleotides. composed of pentose sugar * phosphate * nitrogenous base

Deoxyribonucleic acid (DNA)

Contains deoxyribose sugar and thymine, not uracil

Ribonucleic acid (RNA)

Contains ribose sugar and uracil, not tbmine

Superstructures of Life

The compounds of life fall into the realm of biochemistry. Biochemicals are organic compounds produced by (or components of) living things, and they include four main families: carbohydrates, lipids, proteins, and nucleic acids (table 2.4). The compounds in these groups are assembled from smaller molecular subunits, or building blocks, and because they are often very large compounds, they are termed macromolecules. All macromolecules except lipids are formed by polymerization, a process in which repeating subunits termed monomers* are bound into chains of various lengths termed polymers.* For example, amino acids (monomers), when arranged in a chain, form proteins (polymers). The large size and complex, three-dimensional shape of macromolecules enable them to function as structural components, molecular messengers, energy sources, enzymes (biochemical catalysts), nutrient stores, and sources ofgenetic information. In the following section and in later chapters, we consider numerTable 2.4

in

cells.

will alsobe auseful reference whenyou studymetabolism

in chaoter

fubosomes; mRNA, tRNA / Expression of genetic traits

Carbohydrates: Sugars and Polysaccharides

2.2 Macromolecules:

ous concepts relating to the roles of macromolecules

Purines: adenine, guanine;

Pyrimidines: cytosine, thymine, uracil Chromosomes; genetic material of viruses / Inheritance

8.

tilonorler (mahn'-oh-mur) Gr. mono' one, and meros' part' also the root for polysaccharide and pollpeptide.

The term carbohydrate originates from the way that most members of this chemical class resemble combinations of carbon and water. Carbohydrates can be generally represented by the formula (CH2O),, in which n indicates the number of units of this combination of atoms. The basic structure of a simple carbohydrate is a backbone of carbon bound to two or more hydroxyl groups. Because they also have either an aldehyde or a ketone group, they are

often designated as polyhydroxy aldehydes or ketones (figure 2.15). In simple terms a sugar such as glucose is an aldehyde with a terminal carbonyl group bonded to a hydrogen and another carbon. Fructose sugar is a ketone with a carbonyl group bonded between two carbons. Carbohydrates exist in a great variety of configurations. The common term sugar (saccharide*) refers to a simple carbohydrate such as a monosaccharide or a disaccharide that has a sweet taste. A monosaccharide is a simple polyhydroxy aldehyde or ketone molecule containing from 3 to 7 carbons; a disaccharide is a combination of two monosaccharides; and a polysaccharide is a polymer of five or more monosaccharides bound in linear or branched chain patterns (figure 2.15). Monosaccharides and disaccharides

't'

*

poltttet (pahl' -ee-mur) Gr. poly, many;

" sutthuridc (sak'-uh-ryd)

Gr. sakcharon, sweel

42

Chapter

2

The Chemistry of Biology

Aldehyde group

H-?'?-oH

Ho-?3-H H-?4-oH H-C-OH l5 H

-C-OH l6

H-?'?-oH

Ho-?3-H HO-C4-H

H-C-OH l5 H-C-OH

H

l6 H

ffi:""":"'

Ho-?3-H

H-f4-oH H-?5-oH H-C-OH l6

Figure 2.15 Common classes of carbohydrates. (a) Major saccharide groups, named for the number of sugar units each contains. (b) Three hexoses with the same molecular formula ring models are given. The linear form emphasizes aldehyde and ketone groups, although in solution the sugars exist in the ring form. Note that the carbons are numbered in red so as to keep track of reactions within and (Co Hrz 05) and different structural formulas' Both linear and

between monosaccharides,

are specified by combining a prefix that describes some characteristic of the sugar with the suffix -ose. For example, hexoses are composed of6 carbons, and pentoses contain 5 carbons. Glucose (Gr. sweet) is the most common and universally important hexose; fructose is named for fruit (one of its sources); and xylose, a pentose, derives its name from the Greek word for wood. Disaccharides are named similar$: lactose (L. milk) is an important component of milk; maltose means malt sugar; and sucrose (Fr. sugar) is common table sugar or cane sugar.

The Noture of Corbohydrate Bonds The subunits ofdisaccharides and polysaccharides are linked by means of glycosidic bonds, in which carbons (each is assigned a number) on adjacent sugar units are bonded to the same oxygen atom like links in a chain (figure 2.16). For example: Maltose is formed when the number I carbon on a glucose bonds to the oxygen on the number 4 carbon on a second glucose. Sucrose is formed when glucose and fructose bind oxygen between their number I and number 2 carbons. Lactose is formed when glucose and galactose connect by their number I and number 4 carbons.

In order to form this bond, one carbon gives up its OH group and the other (the one contributing the oxygen to the bond) loses the H from its OH group. Because a water molecule is produced, this reaction is known as dehydration synthesis, a process com_ mon to most polymerization reactions (see proteins, page 4g).

Three polysaccharides (starch, cellulose, and glycogen) are structurally and biochemically distinct, even though all are polymers of the same monosaccharide-glucose. The basis for their differences lies primarily in the exact way the glucoses are bound together, which greatly affects the characteristics of the

end product (figure 2.17). The synthesis and breakage of each type ofbond require a specialized catalyst called an enzyme (see chapter 8).

The Functions of Carbohydrates in Cells Carbohydrates are the most abundant biological molecules in nature. They play numerous roles in cell structure, adhesion, and

metabolism. Polysaccharides typically contribute to structural support and protection and serve as nutrient and energy stores. The cell walls in plants and many microscopic algae derive their strength

2.2 Macromolecules: Superstructures

,/a

of Life

43

H2O

+I Glucose

Glucose

(b)

+t I

H

Glucose

Fructose

(c)

+[

Galactose

Glucose

(d)

Slgure

2.16

Glycosidic bond in three common disaccharides'

dehydration synthesis. (b) Formation of the 1,4 bond between two c. glucoses to (aiGeneral scheme in the formation of a glycosidic bond by-between glucose and fructose to produce sucrose and water. (d) A 1,4 bond bond t,z of tt (c) Formation maltose and water.

iroauce

" between a galactose and glucose produces lactose'

and rigidity from cellulose' a long, fibrous polymer (figure 2.17a andlmsight 2.1). Because of this role, cellulose is probably one of the most common organic substances on the earth, yet it is digestible only by certain bacteria, fungi, and protozoa that produce the enzqe cellulase. These microbes, called decomposers, play an essential role in breaking down and recycling plant materials (see figwe 7.2). Some bacteria secrete slime layers of a glucose polymer called dextran. This substance causes a sticky layer to develop on teeth that leads to plaque, described later in chapter 21.

Other structural polysaccharides can be coqiugated (chemically bonded) to amino acids, nifiogen bases, lipids, or proteins- Agar, an indispensable polysaccharide in preparing solid culture media, is a natural component of certain seaweeds. It is a complex polymer of galactose and sulfur-containing carbohydrates. Chitin (ky-tun), a polymer of glucosamine (a sugar with an amino functional group) is a major compound in the cell walls of fungi and the exoskeletons of insects. Peptidoglycan* is one special class of compounds in which *

peptidoglycan (pep-tih-doh-gly'-kan).

44

Chapter

2

The Chemistry of Biology

Better Living through Bacteria? Probably the most prolific chemical factories on earth are not massive buildings with high-tech processing machinery but minute bacterial cells that are just doing what comes naturally. we learned in case File 2 at the

beginning of the chapter that microbes have the power ro decompose everything from garbage to toxic pollutants, but it turns out that they can also be used to synthesize a variety ofuseful organic chemicals. A recent discovery is a form ofcellulose synthesized by a common soil bacterium called Acetobacter xylinum. The composition of bacterial cellulose is similar to that from plants, being

a

polymer of glucose, except that bacte-

rial cellulose is composed of even finer fibers. This makes it an effective choice for a number ofmedical applications that require dense, strong materials. For example, it can double as a skin replacement fo, ,ru".I burn patients, and it could be adapted to repair smail brood vessels. It

polysaccharides (glycans) are linked to peptide fragments (a short chain of amino acids). This molecule provides the main source of structural support to the bacterial cell wall. The cell wall of gram_ negative bacteria also contains lipopolysaccharide, a complex of li_ pid and polysaccharide responsible for symptoms such as fever and shock (see chapters 4 and l3'). The outer surface of many cells has a delicate ,.sugar coating,'

composed of polysaccharides bound in various ways to proteins (the combination is called mucoprotein or glycoprotein). This struc_ ture, called the glycocalyxo* functions in attachment to other cells

or as a site for receptors-surface molecules that receive and " gl.t'cocah'x

(gb/'-koh-kay'Jix)

Gr.

glycos, sweet,

Cellulose Flgure 2.17 Polysaccharides. (a)

and calyx, covenng.

would also work well as a dressing impregnated with antibiotics and other drugs to deliver the medicines directly into an incision or wound. scientists at the university ofrexas are currently developing a method for mass production of this material. Actually, harnessing the chemical versatility of bacteria is nothing new For decades, the biotechnology industry has been using bacteria for synthesizing hundreds of common. everyday chemicals such as antibiotics, steroids, enzymes, vitamins, alcohols, and amino acids. Chapter 27 covers this aspect of applied microbiology.

Most bacteria exist as single cells that can make only a tiny amount of any one chemical. How does industry get these tiny organisms to make millions of pounds of these same chemicals? Answer availa

bl e a

t h ttp : //www.

m h he. com /t a I a

ro 7

respond to external stimuli. Small sugar molecules account for the differences in human blood types, and carbohyorates are a compo_

nent of large protein molecules called antibodies. Some viruies have glycoproteins on their surface for binding to and invading their host cells.

Polysaccharides are usually stored by cells

in the form of

glucose polymers such as starch (figure 2.17b) or glycogen that

are readily tapped as a source of energy and other metabolic needs. Because a water molecule is required for breaking the bond between two glucose molecules, digestion is also teimed hydrolysis.* Starch is the primary storage food of green plants, +

ht'drolr';is (hy-drol'-eye-sis) Gt. hydro, water,

and hydrein, to dissolve

(b) Starch

(a) Cellulose is composed of B glucose bonded in 1,4 bonds that produce linear, lengthy chains of polysaccharides that are H-bonded along their length' This is the typical structure of wood and cotton fibers. (b) starch is also composed of glucose polymers, in this case a glucose. The main structure is amylose bonded in a 1,4 pattern, with side branches of amylopectin bonded ty 1,6 bonds. The entire molecule is compact and granular.

2.2 Macromolecules: Superstructures microscopic algae, andsome fungi; glycogen (animal starch) is a stored carbohydrate for animals and certain groups of bacteria and protozoa.

Lipids: Fats, Phospholipids, and Waxes lipid, derived from the Greek word lipos, meaningfat,is not a chemical designation, but an operational term for a variety of The term

substances that are not soluble in polar solvents such as water (re-

will

The phospholipids serve as a major structural component ofcell membranes. Although phospholipids also contain glycerol and fatty acids, they differ significantly from triglycerides. Phospholipids contain only two fatty acids attached to the glycerol, and the third glycerol binding site holds a phosphate group. The phosphate is in turn bonded to an alcohol. which varies from one phospholipid to another (figure 2.19a). This class oflipids has a hydrophilic region from the charge on the phosphoric acid-alcohol "head" ofthe molecule and a hydrophobic region that corresponds to the long, uncharged "tail" (formed by the fatty acids). When exposed to an aqueous solution, the charged heads are athacted to the water phase, and the nonpolar tails are repelled from the water phase (figure 2.19b)-This property causes lipids to naturally assume single and double layers (bilayers), which contribute to their biological significance in membranes' When two single layers of polar lipids come together to form a double layer, the outer hydrophilic face of each single layer will orient itself toward the solution, and the hydrophobic portions will become immersed in the core ofthe bilayer. The structure oflipid bilayers confers characteristics on mernbranes such as selective permeability and fluid nature'

'l 'l 'l

HHHHHH

ffi

ble bond are considered unsaturated (figure 2.15b). Fats that contain such fatty acids are described with these terms as well. The structure of fatty acids is what gives fats and oils (liquid fats) their greasy, insoluble nature. In general, solid fats (such as beef tallow) are more saturate4 and oils (or liquid fats) are more unsaturated.

In most cells, triglYcerides

are

(a)

linkage is acted on by digestive enzymes called lipases, the fatty acids and glycerol are freed to be used in metabolism. Fatty acids are a superior source of energy, Yielding twice as much per gram as other storage molecules (starch). SoaPs are K- or Nasalts of fatty acids whose qualities make them excellent grease removers and cleaners (see chaPter I 1).

7. Alcohols are hydrocarbons containing functional group.

an

-OH

s

tI r--;;;;J

3lhl,

0

Triglyceride

l# I

Glycerolpon6

r--r----rrl

chain

OHHHHHH I ll illll

-c-c-c-c-c-c-c '1 '1 '1

,l 'l

'1

Triglycerides

Fatty acids 1

o.\ HHHHHHHHHHHHHHH iiirtltllllllll 'c-c-c-c-c-c-c-c-c-c-c-c-c-c-c-c-H -^/rtttlllllllllll iiHHHHHHHHHHHHH

stored in long-term concentrated form as droplets or globules. When the ester

'l

-c-c-c-c-?-t-:

hydrogens with single bonds. Fatty acids having at least one carbon-carbon dou-

45

MembfAne LipidS

dissolve in nonpolar property occurs beThis solvents such as benzene and chloroform. relatively long or contain lipids cause the substances we call and thus nonpolar are that chains complex C-H (hydrocarbon) as lipids classified compounds groups of hydrophobic. The main waxes' and steroids, are triglycerides, phospholipids, Important storage lipids are the triglycerides, a category that includes fats and oils. Triglycerides are composed of a single molecule ofglycerol bound to three fatty acids (figure 2.18)' Gtycerol is a 3-carbon alcoholT with three OH groups that serve as binding sites. Fatty acids are long-chain unbranched hydrocarbon molecules with a carboxyl grouP (COOH) at one FattY acid end that is free to bind to the glycerol. the The bond that forms between frt"--rt"--,"u,."**l."u,o*l -OH is defined as an chain acid group and the -COOH hydroThe Glycerol ester bond (figure 2.18a), OHHHHHH carbon portion ofa fatty acid can vary in I ll ll lll HO -C-C-C-C-C-C -C dependlength from 4 to 24 carbons and" ing on the fat, it maY be saturated or '1 'l unsaturated. A saturated fatty acid has OHHHHHH ll ll ll ll all ofthe carbons in the chain bonded to Ho

call that oil and water do not mix) but

of Life

Palmitic acid, a saturated fatty acid 2

HHHHHHHHHHHHHHHHH o.\'i | I I | C=C-C-C-H i i i r | | | | I | | C.b_C_C_C_C_C-C-C-C=C-C-C=CI

"/ttttlllHHHHXiHHHHH (b)

llll

Linolenic acid, an unsaturated fatty acid

Flgure 2.18 Synthesis and structure of a triglyceride'

bond, this is another form of dehydration lafBecause a water molecule is released at each ester hydrocarbon chains of the fatty acids, the represent R symbol jagged and lines The iynthesis. which are commonly very long. (b) Structural and three-dimensional models of fatty acids and triglycerides. (1) A saturated fatty acid has long, straight chains that readily pack together with and form solid fats (right). (2) An unsaturated fatty acid-here a polyunsaturated one (right). 3 double bonds-has bends in the chain that prevent packing and produce oils

46

Chapter

2

The Chemistry of Biology

Variable alcohol group

-f J

cn",s"o head

Polar lipid molecule Sllz-

7,{.=-

]ctycerot

Polar head

ffz-NonPolar

(

-",",-t1

(

taits

K

Site for --------> no ester bond with a CJ,"

Phospholipids in single layer

Cholesterol

fatty"-i-q", ,,i acro b_/_crr,

6 "tp-E' Cholesterol b"r{r.t

,,

nrb

--#;

-:

;=:(: _: -.:*-*:Fj_

Water- ss-.+€ - i!g- €':r:_ -+ai^

2

_

- ^J3c -Water -i=^;-Xr#s! l=+"" *a- _

Phospholipid bilayer

(b)

Figure

2.19

cHr-Q _

I

Hzl.. .

Figure 2.2O Cutaway view of a membrane with its bilayer of lipids. The primary lipid is phospholipidhowever, cholesterol is inserted in some membranes. Other structures are protein and glycolipid molecules. Cholesterol can become esterified with fatty acids at its OH group, imparting a polar quality similar to

that of phospholipids.

Phospholipids-membrane molecules.

(a) A model of a single molecule of a phospholipid. The phosphate-alcohol head lends a charge to one end of the molecule; its long, trailing hydrocarbon chain is uncharged.

(b) Phospholipids in water-based solutions become arranged

(1) in single layers called micelles, with the charged head oriented toward the water phase and the hydrophobic nonpolar tail buried away from the water phase, or (2) in double_layered systems with the hydrophobic tails sandwiched between two hydrophilic layers.

Miscelloneous Lipids Steroids are complex ringed compounds commonly found in cell membranes and animal hormones. The best known of these is the sterol (meaning a steroid with an oH group) called choresterol (figure 2.20). Cholesterol reinforces the structure of the cell mem_ brane in animal cells and in an unusual group of cell-wall-deficient bacteria called the mycoplasmas (see chapter 4).

The cell membranes of fungi also contain a unique sterol, called ergosterol. Prostaglandins are fatty acid derivatives found in trace amounts that function in inflammatory and allergic reac_

tions, blood clotting, and smooth muscle contraction. Chemi_

cally, a wax is an ester formed between a long-chain alcohol and a saturated fatty acid. The resulting material is typically pliable and soft when warmed but hard and water-resistant when cold (paraffin, for example). Among living things, fur, feathers, fruits, leaves, human skin, and insect exoskeletons are naturally water_ proofed with a coating of wax. Bacteria that cause tuberculosis

and leprosy produce a wax (wax D) that contributes to their pathogenicity.

:"-"acl

tn

Globular protein

\

A NOTE ABOUT MEMBRANES The word membrane appears frequently in descriptions of cells in chapters 4 and 5. The word itself can be used to indicate a lining or covering including such multicellular structures as the mucous membranes of the body. At the cellular level, however, a membrane is a thin sheet of molecules composed of phos_

pholipids and sterols (averaging about 40%o of membrane content) and proteins (averaging about 600lo). All cells have a membrane that completely encases the cytoplasm. Mem_ branes are also components of eukaryotic organelles such as nuclei, mitochondria, and chloroplasts, and they appear in internal pockets of certain prokaryotic cells. Even some viruses,

which are not cells, can have a membranous envelope. A standard model of membrane structure has the lipids forming a continuous bilayer. The polar lipid heads face to_ ward the aqueous phases and the nonpolar tails orient toward the center of the membrane. Embedded at numerous sites in this bilayer are various-sized globular proteins (figure 2.2O). Some proteins are situated only at the surface; others extend fully through the entire membrane. Membranes are dynamic and constantly changing because the lipid phase is in motion and many proteins can migrate freely about. This fluidity is essential to such activities as engulfment of food and discharge or secretion by cells. The structure of the li_ pid phase provides an impenetrable barrier that accounts for the selective permeability and transport of molecules. Membrane proteins function in receiving molecular signals (receptors), in binding and transporting nutrients, and as eizymes, topics to be discussed in chapters 7 and 8.

2.2 Macromolecules: Superstructures Proteins: ShaPers

47

contain' To each living thing are a consequence ofthe proteins they

of Life

best explalin the origin of the special properties and versatility of proteins, we must examine their general structure'

^Thebuildingblocksofproteinsareaminoacidsowhichexist in 20 different naturally occurring forms (table 2.5). Various

Thepredominantorganicmoleculesincellsareproteinsrafitting

prime' terrn-adopted from the Greek wordproteios, meaning first or qualities of unique and To a large extent, the structure, behavior,

ffiil.

of Life

Twenty Amino Acids and Thelr Abbreviations*

),p

+H3N-9-C.

i

'o-

l,o

H^

l)' +H3N-q-c.

+H3N-q-c\

lo-

lo-

|

i

i

i

d" g(r'cH.

lt-

HrC 'bn.

H.i

n.C'cn"

,-:,

Leucine (Leu)

Valine (Val)

l.p),o H3N-9-C\

*H3N-Q-C.

i

ll'o

iir rr

H

*HsN

o

,o

I

-c- c.'o-

SH

(Pro)

,o

-c\'o-

),o

+H3N-9-C.

lo

Cl-fu.

Clsteine (CYs)

-r.*-[-i(oto

Methionine

l,o I

Asparagine

f .o *H.N-c-ia -l'o-l'o i

A-

"\

'HgN

(Phe)

;

),o io-

-q-c.

-f-tt' i

f4=" o. H

c.

,:::,

6'

r:.H

b

Glutamine (Gln)

Lp

*Hrr.r-g-if

nctotc

cHc

:n"l*lanine

i

t-

(Asn)

'o-

i

E*,

,r*'

(M"t)

*HgN- c-c.

CHe

"rr\

'o-

"':.

i

i*,1

|

h \./

? Proline

l,o

rH3N-Q-c,

{*,

?", ?*''

?t, ',.'

,,.t-i

lsoleucine (lle)

I

I

/cH2 CHZ

H2C\

-n-

'V

E",

C*

^""

l,o

nH3N-q-c.

i

QHz |

!"'

-o'"b

l,pl,o+H3N-?-C\o

-Hsru-9-C.o

I CH,

ut9'"

+", ?t' , ?H' '

CHz

6*, ?H.'

?*'

NHg'

HrN"tNHr' Aspartic Acid (Asp) tjsil4Msr,,d14]i!#:rs.:]1rrsl!rrilixg{lll1(t1@

composition *The basic skeleton is in yellow; R groups are in purple, blue, or green, depending on the nature oftheir

Histidine (His)

: ;

48

Chapter

2

The Chemistry of Biology

combinations of these amino acids account for of proteins. Amino ac_

Bond formino

the nearly infinite variety

,.

|

_

t

idshaveabasicskeletonconsistingof acarbon i ,ro I t. ?, (#-r{f (called rhe q carbon) linked to an amino group "a_. ,*_q_"i,rrrrrr-N_c_c.r,,,,,uuil,

| ,.o H. T. _l{(,,rrr)^,_l_^r-"

"@"-i-\ H/ \ H)' l, I Jl,'Li.:1T:Y,,il::t';3,:,1+^:"#::: i. W t

among the amino acids occur at the R group, which is different in each amino acid and imparts the unique characteristics to the molecule und to

the proteins that contain it. A covalent bond a peptide bond forms between the amino

:il:ffiilTffiilT,'Irfl?'J#,?H:ff

called group "a

;X:

mation, it is possible to produce moiecules uuruinn in length from two amino ru durus acids to ru srarns chains contalnconiaing thousands of them.

/ "

u i

-

i-,'7- -i-"..;. "' i- t']"

Flgure 2,21

number

oi

Protein Structure ond Diversity The reason that proteins are so varied and specific is that they do not function in the form of a simple straighi chain of amino acids (called the primary structure). A protein has a natural tendencv to assume more complex levels of organization, called the secondlv. tertiary, and quaternary structures (figtre 2.22,y.

primary (1") structure of a protein is the fundamental chain of amino acids just described, but proteins vary extensively in the exact order, type, and number of amino acids. It is this aspect ihat gives rise to the unlimited diversity in protein form and function. A polypeptide does not remain in its primary state, but instead" it spontaneously arranges itself into a higher level of complexity called its secondary (2o) structure. The secondary state arises from numer_ ous hydrogen bonds occurring between the C:O and N_H groups ofpeptide bonds. This bonding causes the whole chain to coil or fold into regular patterns. The coiled spirar form is cailed the a helix and the folded, accordion form is called the B_pleated sheet. polypeptides ordinarily will contain both types of configurations. Once a chain has assumed the secondary structure, it goes on to form yet another level of folding and compacting_the tertiary (3) structure. This struch*e arises through additional intrachains forces and bonds between various parts ofthe o. helix and B_pleated sheets. The

The chief actions in creating the tertiary structure are additional

hydrogen bonds between charged functional groups, van der Waals forces between various parts ofthe polypepfide, and covalent disulfide

peptide @ep'-tyd)

Ctr.

ffi -i-\ ffi

I

{:

Rl

amino acids but usually has more than20 and'nspecified is often a smaller subunit of a protein. A protein is the largest of this class of comoounds and usually contains a minimum of 50 amino acids. It is common for the terms polypeptide and, protein to be used interchangeably, though not all polypeptides are large enough to be considered protelns. in chapter 9, we see that protein synthesis is notjust a random connec_ tion of amino acids; it is directed by information provided in DNA.

a

pepsrs, digestion.

8. Intrachain means within the chain; interchain would be between two chains.

I

I

,i,,,,*,

Various terms are used to denote the nature of compounds containing peptide bonds. peptide* usually refers to a molecule composed of short chains of amino acids, ,u"h u, a dipep_ tide (two amino acids), a hipeptide (three), and a tetrapeptide (four)

(figure 2.21). A polypeptide contains an

-oH

+

The formation of peptide bonds in a tetrapeptide.

bonds. The disulfide bonds occur between sulflir atoms on the amino acid cysteine,* and these bonds confer a high degree ofstability to the

overall protein structure. The result is a complex three-dimensional protein that is now the completed firnctional state in many cases. The most complexproteins assume a quaternary (4;) structure, rn which two or more polypeptides interact to form alarge,multiunii protein. The po$peptide units form loose associations based on weak van der Waals and other forces. The polypeptides in proteins with quaternary structure can be the same or diferent. The arrangement of these individual polypeptides tends to be ry,'rnmehical and wilr dictate the exact form ofthe finished protein (figure 2.22, step 4). The most

important outcome of bonding and folding is that each different fype ofprotein develops a unique shape, and its sur_ face displays a distinctive pattern ofpockets und brr-pr. As a result, a protein can react only with molecules that complernent or fit its particular surface features. Such a degree ofspeciiicity can provide

the functional diversity required for many thousands of differint cellular activities. Enrymes serve as the catalysts for all chemical reac_ tions in cells, and nearly every reaction requires a different enzyme

(see chapter 8). Antibodies are complex g$coproteins wlth specitc regions of attachment for bacteria, viruses, and other microorgan_ isms' certain bacterial toxins (poisonous products) react with onlv one specific organ or tissue. proteins embedded in the cell membrane have reactive sites restricted to a certain nutrient. Some proteins function as receptors to receive stimuli from the environmint. The functional, three-dimensional form of a protein is termed the native state, and if it is disrupted by some means, the protein is said to be denatured. Such agents as heat, acid, alcohol, and some disinfectants disrupt (and thus denature) the stabilizing inhachain bonds and cause the molecule to become nonfunctional, as de_ scribed in chapter 1 1.

The Nucleic Acids: A Cell Computer and lts programs The nucleic acids, deoxyribonucleicx acid (DNA) and ribonucreic* acid @NA), were originally isolated from the cell nucleus. Shortly *.c,v-steirte.(sis'-hsh-yeen) ". cle.oxyri

b on ucI

Gr. Kysrrs, sac. An amino acid first found in urine stones. elc (dee-ox,,-ee-ry,,-boh_noo_klay' _ik).

* ribonucleic (ry"-boh-noo-klay,-ik) It is easy to see why the abbreviations are used!

2.2 Macromolecules: Superstructures

fi) -

of Life

rne primary structure is a series of amino acids bound in a chain. Amino acids disPlaY small charged functional grouPs (red sYmbols).

Primary structure

o:(

The secondary structure develops when CO- and NHgroups on adjacent amino acids form hydrogen bonds. This action folds the chain into local configurations called the 0 helix and P-Pleated sheet. Most proteins have both types of secondary structures.

Secondary structure

N-H--'O:C.

)( 'C:O-N-

-

-H-N

)*' ).'..

r

,c:o

Detail of hydrogen bond

fO rn"

-

tertiary structure forms when portions ofthe secondary structure iurther interact by torming covalent disulfide bonds and additional interactions. From this emerges a stable three-dimensional molecule. Depending on the protein, this may be the final functional state.

Tertiary structure

The quaternary structure exists only in oroteins that consist of more than one polypeptide chain. Shown here is a model of the cholera toxin, composed of five separate polypeptides' each one shown in a ditterent color.

Quaternary structure

Process

iigure 2.22

Formation of structural levels in a protein.

Projected 3-dimensional shape (note grooves and projections)

49

Chapter

50

2

The Chemistry of Biology

*Hil' Phosphate

""ffi"

OHH

(a) A nucleotide, composed of a phosphate, a pentose sugar, and a nitrogen base (either A,T,C,G, or U) is the monomer of both DNA and RNA.

OH

ffi

Backbone

OH

(a) Pentose sugars

RNA ,

DNA

"\ N ..t

€{

,B {

'**.,

p

ryp

I

H

W

(b) Purine bases

H {

I

'H

,lp

I

o

o

H bonds (b) In DNA, the polymer is composed of alternating deoxyribose (D) and phosphate (P) with nitrogen bases (A,T,C,G) attached to the deoxyribose. DNA almost always exists in pairs of strands, oriented so that the bases are paired across the central axis of the molecule.

p

Figure

2.23

H

(c) In RNA, the polymer is composed of alternating ribose (R) and phosphate (P) attached to nitrogen bases (A,U,C,G), but it is usually a single strand.

The general structure of nucleic acids.

thereafteq they were also found in other parts ofnucleated cells, in cells with no nuclei (bacteria), and in viruses. The universal occur_ rence ofnucleic acids in all knor,vn cells and viruses emphasizes their important roles as informational molecules. DNA, the master computer ofcells, contains a special coded genetic program with detailed and specific instructions for each organism,s heredity. It tansfers the details of its program to RNA, "helper" molecules responsible for carrying out DNA's instructions and tanslating the DNA program into proteins that can perform life functions. For now, let us briefly consider the structure and some functions of DNA, RNA, and a close relative, adenosine triphosphate (MP). Both nucleic acids are polymers of repeating units called nucleotides,* each of which is composed of three smaller units: a nitrogen base, a pentose (5-carbon) sugar, and a phosphate (figure 2.23a). The nitrogen base is a cyclic compound that comes in two forms: purines (two rings) and pyrimidines (one ring). There are

two types of purines-adenine (A) and guanine

* nucleotide (noo'-klee-oh-tyd) From nucleus and acid.

(Gfand

three

ffi

"\ .r'

o

,W "To

:@

*l

N

H

H

(c) Pyrimidine bases

Figure 2.24 The sugars and nitrogen bases that up DNA and RNA.

make

(a) DNA contains deoxyribose, and RNA contains ribose. (b) A and G purine bases are found in both DNA and RNA. (c) pyrimidine bases are found in both DNA and RNA, but T is found only in DNA, and U is found only in RNA.

types of pyrimidines-thymine (T), cytosine (C), and uracit (U) (figure 2.24). Acharacteristic that differentiates DNA from RNA is that DNA contains all of the nitrogen bases except uracil, and RNA contains all of the nitrogen bases except thymine. The nitrogen base is covalently bonded to the sugar ribose in RNA and deoxyribose (because it has one less oxygen than ribose) in DNA. phosphate (POo'-), a derivative ofphosphoric acid (Hrpbo), provides the final covalent bridge that connects sugars in series. Thus, the backbone ofa nucleic acid strand is a chain ofalternating phosphate-sugar_ phosphate-sugar molecules, and the nitrogen bases branch oflthe side of this backbone (figure 2.23b, c).

The Double Helix of DNA DNA is a huge molecule formed by two very long polynucleotide strands linked along their length by hydrogen bonds between com_ plementary pairs ofnitrogen bases. The pairing ofthe nifrogen bases occurs according to a predictable pattern: Adenine ordinarily pairs

2.2

Macromolecules: Superstructures of Life

51

Cells Events in DNA RePlication

Events in Cell Division

Lffi ffi

H-bonding severed

trq pq

9J P=

ts4; F-* '

@G L-.1

;

.J

Itr:leGl

--.1.,.,".-rflfl

New bases

r*o ,lnst"

I I

.'k-=J---'.

tE: -i"

t\ 'H\

Base pairs

;

stlanos

kb4 ,b64€ e@4 r@e

Two double strands

,i

.."-..-.-\

Figure

2.26

H

Simplified view of DNA replication in cells'

The DNA in the cell's chromosome must be duplicated as the cell is dividing. This duplication is accomplished through the separation of the double DNA strand into two single strands. New strands are then synthesized using the original strands as guides to assemble the correct new comPlementarY bases.

usually extremely long, a feature that satisfies a requirement for storing genetic information in the sequence of base pairs the molecule contains. The hydrogen bonds between pairs can be disrupted when DNA is being copied and the fixed complementary base pairing is essential to maintain the genetic code.

Figure 2.25 A structural representation of the double helix of DNA. At the bottom are the details of hydrogen bonds between the nitrogen bases of the two strands.

Moking New DNA: Possing with thymine, and cytosine with guanine. The bases are attracted in this way because each pair shares oxygen, nitrogen, and hydrogen atoms exactly positioned to align perfectly for hydrogen bonds (figure 2.25). For ease in understanding the structure of DNA, it is sometimes compared to a laddeq with the sugar-phosphate backbone representing the rails and the paired nitrogen bases representing the steps. The flat ladder is useful for understanding basic components and orientation, but in realiry DNA exists in a three-dimensional arrangement called a double hetix. Abetter analogy may be a spiral staircase. In this model, the two strands (helixes) coil together, with the sugar-phosphate forming outer ribbons, and the bases embedded between them (figure 2.25). As is true of protein, the structure

of DNA is intimately related to its function. DNA molecules

are

On the Genetic Messoge The biological properties of cells and viruses are ultimately programmed by a master code composed of nucleic acids' This code is in the form of DNA in all cells and many viruses; a number of viruses are based on RNA alone. Regardless of the exact genetic makeup, both cells and viruses can continue to exist only if they can duplicate their genetic material and pass it on to subsequent generations. Figure 2.26 srtmmarizes the main steps in this process

in cells. During its division cycle, the cell has a mechanism for making a copy of its DNA by replication,* using the original strand as a * repliccttiott (reh"-plih-kay'-shun) A process that makes an exact copy'

1'

Chapter

2

The Chemistry of Biology

pattern (figure 2.26). Note that replication is guided by the double_ stranded nature ofDNA and the precise pairing ofbases that create the master code. Replication requires the separation of the double strand into two single strands by an enzyme that helps to split the

hydrogen bonds along the length of the molecule. This event ex_ poses the base code and makes it available for copying. Free nucle_ otides are used to synthesize matching strands that complement the bases in the code by adhering to the pairing requirements

and

C-G.

ofA-T

The end result is two separate double strands with the same order of bases as the original molecule. With this type of rep_ lication, each new double strand contains one ofthe original single strands from the starting DNA.

OH

OH

Adenosine Adenosine diphosphate (ADP)

RNA: Orgonizers of Protein Synthesis

Adenosine triphosphate (ATP)

Like DNA, RNA consists of a long chain of nucleotides. How_ ever, RNA is a single strand containing ribose sugar instead of

(a)

deoxyribose and uracil instead of thymine (see figure 2.23). Sev_ eral functional types of RNA are formed using the DNA template through a replicationlike process. Three major types of RNA are important for protein synthesis. Messenger RNA (mRNA) is a copy of a gene from DNA that provides instructions for the order of amino acids; transfer RNA (IRNA) is a carrier that delivers the correct amino acids during protein synthesis; and ribosomal RNA (rRNA) is a major component of ribosomes (described in chapter 4). More information on these important processes is presented in chapter 9.

ATP: The Energy Molecule of Cells A relative of RNA involved in an entirely different cell activity is adenosine triphosphate (ATP). ATp is a nucleotide containing adenine, ribose, and three phosphates rather than just one (figure 2.27). Itbe_ longs to a oategory of high-energy compounds (also including guano_ sine triphosphate, GTP) that give offenergy when the bond is broken between the second and third (outermost) phosphate. The presence of these high-energy bonds makes it possible forATp to release and store energy for cellular chemical reactions. Breakage ofthe bond ofthe terminal phosphate releases energy to do cellular work and also gener_ ates adenosine diphosphate (ADp). ADp can be converted back to

+

=

Macromolecules are very large organic molecules (polyners) built up by polymerization of smaller molecular subunits (monomers). Carbohydrates are biological molecules whose monomers are sim_ ple sugars (monosaccharides) linked together by glycosidic bonds

to form polysaccharides. Their main functions are protection and support (in organisms with cell walls) and also nutrient and energy stores.

,e Lipids are biological molecules such as fats that are insoluble in water and contain special ester linkages. Their main functions are cell components, cell secretions, and nutrient and energy stores. Proteins are biological molecules whose polymers are chains of = amino acid monomers linked together by peptide bonds. are called the "shapers of life', because ofthe many biological

i? Proteins

Figure 2.27 Modelof an ATp molecule, the chemical form of energy transfer in cells. (a) Structural formula: The wavy lines connecting the phosphates represent bonds that release large amounts of energy when broken.

(b) Ball-and-stick model shows the arrangement of atoms in three dimensions.

AIP when the third phosphate is restored, thereby serving

as an en_ ergy depot. Carriers for oxidation-reduction activities (nicotinamide adenine dinucleotide [NAD], for instance) are also derivatives of nucleotides (see chapter 8).

*i

Protein structure determines protein function. The primary struc_ ture is dictated by amino acid composition. proteins undergo in_ creased levels of folding and complexity, due to internal bonds, called the secondary, tertiary, and quaternary structures. The final level retains a particular shape that dictates its exact function. I'E Nucleic acids are biological molecules whose polymers are chains of nucleotide monomers linked together by phosphate-pentose sugar covalent bonds. Double-stranded nucleic acids are linked to_ gether by hydrogen bonds. Nucleic acids are information molecules that direct cell metabolism and reproduction. Nucleotides such as AIP also serve as energy transfer molecules in cells.

Chapter Summary with

KeY Terms

53

Chapter Summary with KeY Terms Bonds, and Molecules: Fundamental Building Blocks Atomic Structure and Elements 1. A11 matter in the universe is composed of minute particles called atoms-the simplest form of matter not divisible into a simpler substance by chemical means' Atoms are composed of smaller particles called Protons, neutrons, and electrons. 2. a. Protons are positively (+) charged, neutrons are without

2.1 Atoms,

A.

(-)

b. c.

B.

charged. charge, and electrons are negatively Protons and neutrons form the nucleus of the atom'

Electrons orbit the nucleus in energy shells' 3. Atoms that differ in numbers of the protons, neutrons, and electrons are elements. Elements can be described by mass number (MN), equal to the number of protons and neutrons it has, and atomic number (AN), the number ofprotons in the nucleus, and each is known by a distinct name and symbol. Elements may exist in variant forms called isotopes. The atomic mass or weight is equal to the average of the mass numbers. Bonds and Molecules 1. Atoms interact to form chemical bonds and molecules. If the atoms combining to make a molecule are different elements, then the substance is termed a compound. 2. The type of bond is dictated by the electron makeup of the outer orbitals (valence) of the atoms. Bond rypes include:

consist ofcarbon and hydrogen covalent$ bonded in various combinations. Inorganic compounds do not contain both cmbon and hydrogen in combination. B. Macromolecules are very large compounds and are generally assembled from single units called monomers by polymerization. C. Macromolecules of life fall into basic categories of carbohydrates, lipids, proteins, and nucleic acids. Carbohydrates are composed of carbon, hydrogen, and oxygen and contain aldehyde or ketone groups.

l.

a. Monosaccharides

b.

2.

such as glucose are the simplest carbohydrates with 3 to 7 carbons; these are the monomers of carbohYdrates. Disaccharides such as lactose consist oftwo

monosaccharides joined by glycosidic bonds. c. Polysaccharides such as starch and peptidoglycan are chains of five or more monosaccharides. Lipids contain long hydrocarbon chains and are not soluble in polar solvents such as water due to their

nonpolar, hydrophobic character. Common components of fats are fatty acids, elongate molecules with a carboxylic acid group. Examples are triglycerides,

phospholipids, sterols, and waxes. are highly complex macromolecules that are crucial in most, ifnot all, life processes. a. Amino acids are the basic building blocks of proteins. They all share a basic structure of an amino

3. Proteins

group, a carboxyl group, arr R group, and hydrogen bonded to a carbon atom. There are 20 different R groups, which define the basic set of 20 amino

a. Covalent bonds, with

shared electrons. The molecule shares the electrons; the balance of charge will be potar if unequal or nonpolar if equally

shared/electricallY neutral.

b. Ionic bonds, where electrons

c. d. e.

f.

C.

are transferred to an

atom that can come closer to filling up the outer orbital. Dissociation of these compounds leads to the formation ofcharged cations and anions. Ilydrogen bonds involve weak athactive forces

acids.

c.

between hydrogen and nearby oxygens and nitrogens. Van derWaals forces are also weak interactions between polarized zones ofmolecules such as proteins. Chemicals termed reactants can interact in a way to form different compounds termed products. Examples

ofreactions are synthesis and decomposition. An oxidation is a loss ofelectons and a reduction is a gain ofelectrons. A substance that causes an oxidation by taking elechons is called an oxidizing agent, and a substance that causes a reduction by giving electrons is

called a reducing agent. Solutions, Acids, Bases, and PH

l.

life forms. is a short chain of amino acids bound by peptide bonds: a protein contains at least 50 amino

acids, found in all

b. A peptide

A solution is a combination of

a solid,

incredible variation in shapes is the basis for the diverse roles proteins play as enzymes, antibodieso receptors, and structural components.

4. Nucleic

liquid or

solvent in natural systems.

the concentration ofH' such that a pH ofless than 7.0 is considered acidic, and a pH ofmore than that, indicating fewer H*, is considered basic (alkaline).

2,2 Macromolecules: Superstructures of Life

A.

Biochemistry studies those molecules that are found in living things. These are based on organic compounds' whichusually

acids

blocks ofnucleic acids. They are composed of a nitrogen base, a pentose sugar, and a phosphate. Nitrogen bases are ringed compounds: adenine (A), guanine (G)' cytosine (C), thymine (T), and uracil (U). Pentose sugars may be deoxyribose or ribose. Deoxyribonucleic acid (DNA) is a polymer of nucleotides that occurs as a double-stranded helix with hydrogen bonding in pairs between the helices.

a. Nucleotides are the building

gaseous chemical (the solute) dissolved in a liquid medium (the solvent). Water is the most common

2. Ionization of water leads to the release of hydrogen ions (H+) and hydroxyl (OH-) ions. The pII scale expresses

The structure of a protein is very important to the function it has. This is described by the primary structure (the chain of amino acids), the secondary structure (formation of a helixes and B-sheets due to hydrogen bonding within the chain), tertiary structure (cross-links, especially disulfide bonds' between secondary structures), and quaternary structure (formation of multisubunit proteins). The

b.

It has all ofthe bases except uracil, and the pentose sugar is deoxyribose. DNA is the master code for a cell's life processes and must be transmitted to the offspring through rePlication.

Chapter

54

c.

2

The Chemistry of Biology

Ribonucleic acid (RNA) is a polymer of nucleotides where the sugar is ribose and the uracil is used instead of thymine. It is almost always found single shanded and is used to express the DNA code into proteins.

d.

Adenosine triphosphate (ATP) contains a nucleotide and is involved in the transfer and storase ofenergy in cells.

Questions

e4ffMultiple-choice

Select the correct answer from the answers provided. For questions with blanks, choose the combination of answers that most accuratelv comnletes the statement.

1. The smallest unit of matter with unique characteristics is

a. an electron b. a molecule 2. The

c.

an atom

d.

a proton

charge of a proton is exactly balanced by the

_

charge

of a (an)

-

a. negative, positive,

electron

c. positive, negative, electron d. neutral, negative, electron

-. neutral, neutron b. positive,

3. Elechons move around the nucleus of an atom in pathways called a. shells c. circles b. orbitals d. rings

12. Bond formation in polysaccharides and polypeptides is accompanied by the removal of a a. hydrogen atom c. carbon atom b. hydroxyl ion d. water molecule 13. The monomer unit of polysaccharides such as starch and cellulose is a. fructose c. ribose b. glucose d. lactose 14. A phospholipid contains a. three fatty acids bound to glycerol

b. three fatty acids, a glycerol, and a phosphate c. two fatty acids and a phosphate bound to glycerol d. three cholesterol molecules bound to glycerol

4. Which part of an element does not vary in number? a. electron b. neutron

c. proton d. all ofthese vary

5. Proteins are synthesized by linking amino acids with a. disulfide c. peptide b. glycosidic d. ester

I

6. The amino acid that accounts for disulfide bonds in the tertiary

considered

a. a compound b. a monomer

6. Bonds in which atoms

c. a molecule d. organic share electrons are defined as

a. hydrogen b. ionic

_

bonds.

c. double

d. covalent 7. A hydrogen bond can form between _ adjacent to each other a. two hydrogen atoms

b. two oxygen atoms c. a hydrogen atom and an oxygen atom d, negative charges 8. An atom that can donate electrons during a reaction is called a. an oxidizing agent c. an ionic agent b. a reducing agent d. an electrolye 9. In a solution of NaCl and water, NaCl is the _ and water is the a. acid, base b. base, acid 10. A solution with a pH of 2

a. has less H*

b. I

_.

than a solution with a pH of 8.

-

has more H+

1. Fructose is a type of a. disaccharide

b. monosaccharide

W#writins

structure of proteins is a. tyrosine b. glycine

c. has more OH d. is less concentrated

bonds.

c. cystelne d. serine

17. DNA is a hereditary molecule that is composed a. deoxyribose, phosphate, and nitrogen bases b. deoxyribose, a pentose, and nucleic acids c. sugar, proteins, and thymine d. adenine, phosphate, and ribose 18. What is meant by the term

c. solute, solvent d. solvent, solute

_

I

5. If a substance contains two or more elements of different types, it is

of

DNI replication?

a. synthesis ofnucleotides b. cell division c. interpretation ofthe genetic code d. the exact copying of the DNA code into two new molecules 19. Proteins can function as

a. enzymes b. receptors

c. antibodies d. a, b, and

c

20. RNA plays an important role in what biological process? a. replication

b. protein synthesis c. polysaccharide d. amino acid

c. lipid metabolism d. water transport

to Learn

These questions are suggested as a writing-to-learn expeience. For each question, compose a one- or two-paragraph answer that includes the factual information needed to completely address the question. General page references for these topics are given in parentheses. I

. How

are the concepts of an atom and an element related? What

causes elements to

differ? (28,29)

2. a. How are mass number

and atomic number derived? What is the atomic mass or weight? b. Using data in table 2.1, give the electron number of nitrogen, sulfur, calcium, phosphorus, and iron.

c. What is distinctive about isotopes of elements, and why are they important? (29,30,31) 3. a. How are the concepts of molecules and compounds related? b. Explain why some elements are diatomic. c. Compute the molecular weight of oxygen and methane. (32)

f,f,

Concept Mapping

4. a. Why is an isolated

13.

atom neutral?

organic?

b. Describe the concept of the atomic nucleus, electron orbitals,

b. What characteristics of carbon make it ideal for the formation of

and shells. causes atoms to form chemical bonds? do some elements not bond readily? Why Draw the atomic structure of magnesium and predict what kinds

organic compounds? What are functional grouPs? d. Differentiate between a monomer and a polymer. How are polymers formed? f. Name several inorganic compounds. (39,40,41)

c. What d. e.

of bonds it will

5.

make. (28,31'32)

Distinguish between the general reactions in covalent, ionic' and hydrogen

bonds.

(32, 33, 35)

t4. b.

6. a. Which kinds of elements tend to make covalent bonds? b. Distinguish between a single and a double bond'

water? (32,33) 7. a. Which kinds of elements tend to make ionic bonds? b. Exactly what causes the charges to form on atoms in ionic bonds? c. Verify the proton and electron numbers for Na* and Cl-' d. Differentiate between an anion and a cation, using examples'

What is a glycosidic bond? d. What are some of the functions of polysaccharides

t5. a. Draw simple structural molecules of triglycerides and phospholipids to compare their differences and similarities' b. What is an ester bond? How are saturated and unsaturated fatty acids different? d. What characteristic of phospholipids makes them essential components of cell membranes? Why is the hydrophilic end of phospholipids attracted to

ofits valence? (33,34)

8. Differentiate between oxidation and reduction, and between an oxidizing agent and areducing agent, using examples' (35)

water? (45,46) t6. a.

are there?

10. a. Compare the three basic types of chemical formulas' b. Review the types of chemical reactions and the general ways they

11.

72.

c. What is a pePtide bond? d. Differentiate between a peptide,

b.

What determines whether a substance is an acid or a base? Briefly outline the PH scale. How can a neutral salt be formed from acids and

polypeptide' and a protein'

molecule.

a.

a.

a

e. Explain what causes the various levels ofstructure ofa protein

in equations. (36,37)

Define solution, solvent, and solute. b. What properties of water make it an effective biological solvent, and how does a molecule like NaCl become dissolved in it? How is the concentration of a solution determined? d. What is molarity? Tell how to make a 1 M solution of Mg3@Oo)2 and a 0.1 M solution ofCaSO+. (37)

Describe the basic structure of an amino acid.

b. What makes the amino acids distinctive, and how many of them

9. Why are hydrogen bonds relatively weak? (35)

can be expressed

f. What functions I

e.

a

MaPPins

Appendix E provides guidance for working with concept maps' l. Supply your own linking words or phrases in this concept map, and provide the missing concepts in the empty boxes'

are made ol

cell? (47,48'

49)

nucleotide and a polynucleotide, and compare and contrast the general stucture of DNA and RNA' Name the two purines and the three pyrimidines' Why is DNA called a double helix? What is the function of RNA? What is AIP, and what is its function in cells? (48' 50, 5l ' 52)

7. a. Describe b. c. d.

do proteins perform in a

bases? (37,38,39)

%#concept

in

cells? (41,42,43,44)

e. What kind of ion would you expect magnesium to make, on the basis

What characterizes the carbohydrates? Differentiate between mono-, di-, and polysaccharides, and give examples of each.

c. What

is polarity? d. Why are some covalent molecules polar and others nonpolar? e. What is an important consequence of the polarity of

a. What atoms must be present in a molecule for it to be considered

Chapter

56

2

The Chemistry of Biology

critieal'*.hinking euestions

ffi

Critical thinlcing is the ability to reason and solve problems using facts and concepts. These questions can be approached from a number of angles, and in rnost cases, they do not have a single correct answer. 1. a. The o'octet rule" in chemistry helps predict the types of bonds that atoms will form. In general, an atom will be most stable if fills its outer shell of 8 electrons. Atoms with fewer tfian 4 valence electrons tend to donate electrons and those with more

d. What is the pH of a solution with moles/ml (M) of H+? e. What is the pH of a solution with moles/ml (M) of OH*?

it

than 4 valence electrons tend to accept additional electrons; those

hydrophobic, and what makes them so?

c. Distinguish between polar and ionic compounds, using your own words.

usually bind together. c. What type of chemical reaction is occurring between Na andCl2? 2. Explain this statement: 'All compounds are molecules, but not all molecules are compounds." Give an example.

6. In what way are carbon-based compounds like children's Tinker Toys or Lego blocks?

7. Is galactose an aldehyde or a ketone sugar? 8. a. How many water molecules are released when a triglyceride is

3. Predict the kinds of bonds that occur in ammonium (NHr),

formed?

and magnesium chloride as those in figure 2.4.)

b. How many peptide bonds are in a tetrapeptide? 9. Looking at figure 2.25, canyou

see why adenine forms hydrogen bonds with thymine and why cytosine forms them with guanine?

4. Work out the following problems: a. What is the number of protons in helium? in iron? b. Will an H bond form between H3C-CH:O and HrO? Draw a simple figure to support your answer. c. Draw the following molecules and determine which are polar: cl2, NH3, cH4.

1

'

of 0.00001

anions.

b. Make simple drawings to show how the diatomic atoms N and Cl

(S-S),

a concentration

b. What kinds of substances will be expected to be hydrophilic and

atoms.)

(lvtgclr). (Use simple models such

of 0.00001

5. a. Describe how hydration spheres are formed around cations and

with exactly 4 cart do both. Using this rule, determine what category each of the following elements falls into: N, S, C, B O, H, Ca,Fe, and Mg. (You will need to work out the valence of the

phosphate (POa), disulfide

a concentration

I

0.

Saturated fats are solid at room temperature and unsaturated fats are not. Is butter an example ofa saturated or an unsaturated fat? Is olive oil an example ofa saturated or an unsaturated fat? Explain why sterols like cholesterol can add "stiftress" to membranes that contain them.

visuall understand i ng

ffI

Figures

and 2 ate both highly magnified views ofbiological substances. Using figure 2.17 from chapter 2 as your basis for comparison, speculate which molecules are shown and give the reasons for them having the microscopic upp"*un"" we see here.

(1) (200x)

@ffi

rt

nternet search.Topics

1. Go to: http://www.mhhe.com/talaro7, and click on chapter 2, access the URLs listed under Intemet Search Topics, and research the

following: a. Make a search for basic information on elements, using one or both of the websites listed in the Science Zone. Click on the icons for C, H, N, O, R and S and list the source, biological importance, and other useful information about these elements.

Use a search engine to explore protein structure. Type . protein database" and go to the molecule-of-the-month feature. Draw the

tlree-dimensional shape of five different molecules and discuss how their structure affects their functions. Go to this website to observe visuals and information about all known elements on earth: http ://www.chemicalforums.com/ index.php?page=periodictable

il ls

$Yi

Tools of the Labt The Methods for icroorqqnisms J '

Stu$ing

.3rm'

. 4,.

CASE FILE

3

94- year-old woman went to her local hospital emergency department about a month after the World Trade Center disaster in 2001 . She described a 5-day history of weakness, fever, nonproductive cough, and generalized myalgia (muscle aches). Otherwise, for a person her age she was fairly healthy, although she did have a history of chronic obstructive pulmonary disease, hypertension, and kidney failure' On physical examination, her heart rate was above normal and she had a fever of 102.3'F (39.1'C), but initial laboratory studies (blood cell count blood chemistries, and chest X ray) were normal. Because the early findings suggested an infection, the patient was admitted to the hospital. Samples of blood and urine were sent to the microbiology laboratory for further

testing. The next day, microscopic evaluation of a stained urine culture revealed red rod-shaped bacteria. A Gram stain of a blood culture revealed large purple rods. This finding in the blood was unusual, so a sample culture on appropriate solid media was sent to the state health department laboratory. Even after antibiotic therapy was adjusted to fit the microscopic findings, the patient's condition deteriorated. Her most serious symptoms localized to her chest, and she was transferred to the intensive care unit. Four days after admission, the health department announced that the bacteria found in the patient's blood were Bocillus onthracis. She was suffering from inhalation anthrax. Further testing showed these bacteria to be of the same strain that had been involved in the recent bioterrorist attack. Despite treatment, the patient died on the fifth day after admission, one of five fatalities associated with the release of anthrax spores.

I

Explain the techniques and equipment involved in identifying the bocteria by staining ond how these findings ore reported.

) )

Whot other microscopic tests would be useful in identificqtion? Outline some steps thot might be used to process o blood sample. Case File

3 Wrap-Up oppeors on page

81

.

57

58

Chapter

3

Tools of the Laboratory

CHAPTER OVERVIEW

Microbes are managed and characterized with the Five l's-inoculation, incubation, isolation, inspection, and identification. Cultures are made by removing a sample from a desired source and inoculating it into containers of media.

Media can be varied in chemical and physical form and functional purposes, depending on the intention. Growth and isolation of microbes lead to pure cultures that permit the study and testing of single species. Cultures can be used to provide information on microbial morphology, biochemistry, and genetic characteristics. F Unknown, unseen microbes can become known and visible. b. The microscope is a powerful toolfor magnifying and resolving cells and their parts. MF Microscopes exist in several forms, using light, radiation, and electrons to form images of varying magnification and appearance. M' Specimens and cultures are prepared for study in fresh (live) or fixed (dead) form. Staining procedures highlight cells and allow them to be described and identified. ffifh

3.1 Methods of Culturing Microorganisms-The Five l's Biologists studying large organisms such as animals and plants can, for the most part, immediately see and differentiate their experimental subjects from the surrounding environment and from one another. In fact, they can use their senses of sight, smell, hearing, and even touch to detect and evaluate identifying characteristics and to keep track of growth and developmental changes. Because microbiologists cannot rely as much as other scientists on senses other than sight, they are confronted by some unique problems. First, most habitats (such as the soil and the human mouth) harbor microbes in complex associations. It is often necessary to separate the organisms from one another so they can be identified and studied. Second to maintain and keep track of such small research subjects, microbiologists usually will need to grow them under artificial conditions. A third difficulty in working with microbes is that they are not visible to the naked eye and are widely distributed. Undesirable ones can be inconspicuously introduced into an experiment, where they may cause mlsleading results. These impediments motivated the development of techniques to conhol microbes and their growth, primarily sterile, aseptic, and pure culfure techniques.r

Microbiologists use five basic techniques to manipulate, grow, examine, and characterize microorganisms in the laboratory: inoculationo incubation, isolation, inspectiono and identification (the Five I's; figure 3.1). Some or all of these procedures are performed by microbiologists, whether the beginning laboratory student, the researcher attempting to isolate drug-producing bacteria from soil, or the clinical microbiologist working with a specimen from a patient's infection. These procedures make it possible to handle and

maintain microorganisms as discrete entities whose detailed biology can be studied and recorded. Even though these procedures are presented in a given order, keep in mind that the steps described overlap to some extent. For example, isolation requires a certain method of inoculation as well as incubation, and inspection may be a part of every stage of the process.

lnoculation: Producing a Culture To cultivate, or cultureo* microorganisms, one introduces a tiny sample (the inoculum) into a container of nutrient medium* (p1. media), which provides an environment in which they multiply. This process is called inoculation.* Any instrument used for sampling and inoculation must initially be sterile (see footnote l). The observable growth that appears in or on the medium is known as a culture. The nature of the sample being cultured depends on the objectives of the analysis. Clinical specimens for determining the cause of an infectious disease are obtained from body fluids (blood cerebrospinal fluid), discharges (sputum, urine, feces), or diseased tissue. Samples subject to microbiological analysis can include nearly any natural material. Some common ones are soil, water, sewage, foods, air, and, inanimate objects. Procedures for proper specimen collection are discussed in chapter 17.

lsolation: Separating Microbes from One Another Certain isolation techniques are based on the concept that ifan individual bacterial cell is separated from other cells and provided

* culture (kal'-chrtt) Gn cultus, to tend or cultivate. It

can be used as a verb or

a noun.

l.

Sterile means the complete absence of viable microbes: aseptic refers to prevention of infection; pure culture rcfers to growth ofa single species ofmicrobe.

a medium (mee'-dee-um) pl. media; L., middle.

* inoculation (in-ok"-yoo-lay'-shun) L. lz, andoculus,

eye.

An Overview of MajorTechniques Performed by Microbiologists to Locate, Grow, Observe, and Characterize Microorganisms

Specimen Collection: Nearly any object or material can serve as a source of microbes. Common ones are body fluids and tissues, foods, water, or soil. Specimens are removed by some form of sampling device: a swab, syringe, or a special transport system that holds, maintains, and preserves the microbes in the sample. Discussed on page 58.

URINEI '-1:

A GUIDE TO THE FIVE l's: How the Sample ls Processed and Profiled

# I

I

ffi^r,,l,l@i; $g

Streak plate

€'

Incubator Blood bottle

2 lncubation:

1 Inoculation:

The sample is placed into a coniainer of sterile medium containing appropriate nutrients to sustain growth. lnoculation involves spreading a sample on the surface of a solid medium or introducing a sample into a flask or tube. Selection of media with specialized functions can improve later steps of isolation and identification. Some microbes may require a live organism (animal, egg) as the growth medium.

An incubator creates the proper temperature and other conditions conducive to growth. This promotes multiplication of the microbes and usually takes a period of hours or days. Incubation gives rise to a culture-the visible growth oi the microbe in or on the medium. Further discussion on page 68.

Further discussion on pages 6G-67.

ffi

,."",,),ff ii,l

Biochemical tests

lsolated microbes may take the form of seoarate colonies on solid media, or turbidity (cloudiness) in broths. Further isolation (subculturing) involves taking a bit of growth from an isolated colony and inoculating a separate medium. This makes a Pure culture-one that contains onlY a single species of microbe. More deiail on pages 60, 61, 69.

Figure

3.1

\\

.a+-

, w sq

Subculture

One result of inoculation and incubation is isolation of the microbe,

ig i€ii#;

[_$

4*.

3 lsolation:

tW

tr'T

\w

w7

' i,.l

flrz-\ Fl

-,'*

,*,

a-

Microscopic morphology: shape, staining reactions

4 Inspection: The colonies or broth cultures are observed macroscopically for growlh characieristics (color, texture, size) that could be useful in analyzing the specimen contents. Slides are made to assess microscopic details such as cell shape, size, and motility. Staining techniques may be used to gather specific information on microscopic morphology. See pages 68, 69.

lmmunologic tests

DNA analysis

5 ldentification: A major purpose of the Five "l"s is to determine the type of microbe, usually to the level of soecies. Information used in identification can include relevant data already taken during initial inspection and additional tests that further describe and differentiate the microbes. These include biochemical tests to determine metabolic activities specilic to the microbe, immunologic tests, and genetic analysis. See pages 69, 70.

A summary of the general laboratory techniques carried out by microbiologists.

all the steps shown or to perform them exactly in this order, but all microbiologists participate in at least some of these activities. In some cases, one may proceed rightfrom the sample to inspection, and in others, only inoculation and incubation on special media are required. 59

It

ii not necessary to perform

60

Chapter

3

Tools of the Laboratory

adequate space on a nutrient surface, it

\ %M

will grow

into a discrete mound of cells called a colony (figure 3.2). Because it arises from a single cell or unit, a colony consists of just one species. Proper isolation requires that a small number of cells be inoculated into a relatively large volume or over an expansive area of medium. It gener-

ally requires the following materials: a medium that has a relatively firm surface (see description of agar on pages 62 and 63) contained in a clear, flat covered plate called a Petri dish, andinocu-

lating tools. In the streak plate method, a small droplet of sample is spread with a tool called an inoculating loop over the surface of the medium according to a pattern that gradually thins out the sample and separates the cells spatially over several sections ofthe plate (figure 3.34 6). Because of its ease and effectiveness, the streak plate is the method of choice for most applications. In the loop dilution, or pour plate, tech-

nique, the sample is inoculated, also with a loop, into a series of cooled but still liquid agar tubes so as to dilute the number of cells in each successive tube in the series (figure 3.3c, d).

/

o

I

'

/ws*\ \

:3'':"

Mixture or cells in

I

t"'o[l I

s"o",."tiolot

't rltilil#

I

3::'fi::?3J:1"-

f

*rffi-;T*'*i::#-] 'r lr

"ffi,

\1

tl Yl

become isolated l_ Macroscopic containing I Colony level cells.

Microbes visible as colonies millions of

I

view

I I

_l

Flgure

3.2

lsolation technique.

formation of an isolated colony, showing the microscopic events and the macroscopic result. Separatlon techniques such as streaking can be used to isolate single Stages in the

cells. After numerous cell divisions, a macroscopic mound of cells, or a colony, will be formed. This is a relatively simple yet successful way to separate different types of bacteria in a mixed sample.

Inoculated tubes are then plated out (poured) into sterile Petri dishes and are allowed to solidify (harden). The end result (usually in the second or third plate) is that the number of cells per volume is so decreased that cells have ample space to grow into separate colonies. One difference between this and the streak plate method is that in this technique some of the colonies will develop deep in the medium itself and not just on the surface. With the spread plate technique, a small volume of liquid, diluted sample is pipetted onto the surface of the medium and spread around evenly by a sterile spreading tool (sometimes called oohockey a stick"). Like the streak plate, cells are spread over separate areas on the surface so that they can form individual colonies (figure 3,3e,f\. Before we continue to cover information on the Five I's on page 68, we will take a side trip to look at media in more

inorganic and organic compounds. This tremendous diversity is evident in the types of media that can be prepared. At least 500 different types of media are used in culturing and identifying microorganisms. Culture media are contained in test tubes, flasks, or Petri dishes, and they are inoculated by such tools as loops, needles, pipettes, and swabs. Media are extremely varied in nutrient content and consistency and can be specially formulated for a particular purpose. For an experiment to be properly controlled, sterile technique is necessaxy. This means that the inoculation must start with a sterile medium and inoculating tools with sterile tips must be used. Measures must be taken to prevent introduction of nonsterile materials, such as room air and fingers, directly into the media.

detail.

Types of Media

Media: Providing Nutrients in the Laboratory A major stimulus to the rise of microbiology in the late

Most media discussed here are designed for bacteria and fungi, though algae and some protozoa can be propagated in media. Vinrses can only be cultivated in live cells (see Insight 3.2). 1800s

was the development of techniques for growing microbes out

of

their natural habitats and in pure form in the laboratory. This milestone enabled the close examination of a microbe and its morphology, physiology, and genetics. It was evident from the very first that for successful cultivation, the microorganisms being cultured had to be provided with all of their required nutrients in an artificial medium. Some microbes require only a very few simple inorganic compounds for growth; others need a complex list of specific

Media can be classified according to three properties (table 3.1):

1. physical state,

2. chemical composition, and 3. functional type.

Physicol Stotes of Media Liquid media are defined as water-based solutions that do not solidify at temperatures above freezing and that tend to flow freely

3.1

Methods of Culturing Microorganisms-The Five l's

6l

Note: This method only works if the spreading tool (usually an inoculating loop) is resterilized (llamed) after each of steps 1-4. Loop containing sample

\t^.

^a"-----

LJ[jL#\@W 12345

(a)

Steps in a Streak Plate; this one is a four-part or quadrant streak.

wg# 1

2

{

I

123

(c)

Steps in Loop Dilution; also called a pour plate or serial dilution

(e) Steps in a Spread Plate

Flgure

3.3

Methods for isolating bacteria.

(a) Steps in a quadrant streak plate and (b) resulting isolated colonies of bacteria. (c) Steps in the loop dilution method and (d) the appearance of plate 3. (e) Spread plate and (f) its result.

62

Chapter

ffi

3

Tools of the Laboratory

Three categorles of Media Classiflcation

Physical State

(Medium's Normal Consisteqlc )

Chemical Composition (Type of Chemicals Medium Contains)

Functional Type (Purpose of

Medium)*

At ordinary room temperature, semisolid media exhibit a clotlike consistency (figure 3.5) because they contain an amount

of solidifying

agent

(agar or gelatin) that thickens them but does not

produce a firm substrate. Semisolid media are used to determine the motility of bacteria and to localize 1. Liquid 1. Synthetic (chemically 1. Generalpurpose a reaction at a specific site. Motility test medium and 2. Semisolid defined) 2. Enriched 3. Solid(canbe 2. Nonrynthetic 3. Selective sulfur indole motility (SIM) medium both contain a (complex; not converted to 4. Differential small amount (0.3-0.5%) of agar. In both cases, the liquid) chemically defined) 5. Anaerobic growth medium is stabbed carefully in the center with an 4. Solid (cannot 6. Specimen hansport inoculating needle and later observed for the pattern be liquefied) 7. Assay of growth around the stab line. In addition to motil8. Enumeration iry SIM can test for physiological characteristics *Some media can serve more than one function. For example, a medium such as brain-heart infusion is used in identification (hydrogen sulfide production general purpose and enriched; mannitol salt agar is both selective and differential; and blood agar is both and indole reaction). enriched and differential. Solid media provide a firm surface on which cells can form discrete colonies (see figure 3.3) and when the container is tilted (figure 3.4). These media, termed brothso are advantageous for isolating and culturing bacteria and fungi. They milks, or infusions, are made by dissolving various solutes in distilled come in two forms: liquefiable and nonliquefiable. Liquefiable solid water. Growth occurs throughout the container and can then present media, sometimes called reversible solid media, contain a solidifying a dispersed" cloudy, or flaky appearance. A common laboratory meagent that changes its physical properties in response to temperature. dil;rm, nutrient broth, contains beef extract and peptone dissolved in By far the most widely used and effective of these agents is a ^gar,2 water. Methylene blue milk and litmus milk are opaque liquids containing whole milk and dyes. Fluid thioglycollate is a slightly viscous broth used for determining patterns of growth in oxygen. 2. This material was first employed by Dr. Hesse (see appendix B, 1881)

Figure

3.4

Sample liquid media.

(a) Liquid media tend to flow freely when the container

is tilted. (b) Urea broth is used to show a biochemical reaction in which the enzyme the pH of the solution and causes the dye to become increasingly pink. Left: uninoculated broth, pH 7; middle: weak positive, pH 7.5; right: strong positive, pH 8.0. (c) Presence-absence broth is a medium for detecting

urease digests urea and releases ammonium. This raises

the presence of coliform bacteria in water samples. It contains lactose and bromcresol purple dye. As coliforms use lactose, they release acidic substances. This lowers the pH and changes the dye from purple to yellow (right is Escherichio coli). Noncoliforms such as Pseudomonos grow but do not change the pH (purple color indicates neutral pH).

3.1

Figure 3.5

Methods of Culturing Microorganisms-The Five l's

63

Sample semisolid media.

(a) Semisolid media have more body than liquid media but less body than solid media. They do not flow freely and have a soft, clotlike consistency. (b) Sulfur indole motility (SlM) medium. (1) The medium is stabbed with an inoculum and incubated. Location of growth can be used to determine nonmotility (2) or motility (3). The medium reacts with any H2S gas to produce a black precipitate (4). polysaccharide isolated from the red alga Gelidium. The benefits of agr are numerous. It is solid at room temperature, and it melts (liquefies) at the boiling temperatue of water (100"C or 212'F). Once liquefie4 agar does not resolidiff until it cools to 42"C (108"F), so it can be inoculated and poured in liquid form at temperatures (45"C to 50"C) that will not harm the microbes or the handler (body temperature is about 37oC ot 98.6"F). Agar is flexible and moldable, and it provides a basic framework to hold moisture and nutrients. Another useful property is that it is not readily digestible and thus not a nutrient for most microorganisms. Any medium containing l% ro 5% agar usually has the word

(a)

agar in its name. Nutrient agar is a common one. Like nutrient broth, it contains beef extract and peptone, as well as l.5oh agar by weight. Many of the examples covered in the section on functional categories of media contain agar.

Although gelatin is not nearly as satisfactory as agaq it will create a reasonably solid surface in concentrations of l0Yoto l5oh. The main drawback for gelatin is that it can be digested by microbes and will melt at room and warmer temperatures, leaving a liquid. Agar and gelatin media are illustrated in figure 3.6. Nonliquefiable solid media have less versatile applications than agar media because they do not melt. They include materials such as rice grains (used to grow fungi), cooked meat media (good for anaerobes), and potato slices; all of these examples remain solid after heating. Other solid media containing egg or serum start out liquid and are permanently coagulated or hardened by moist heat.

(b)

Flgure

3.6

Solid media that are reversible to liquids.

(a) Media containing

1o/o-5o/o agar are solid enough to remain in place when containers are tilted or inverted. They are reversibly solid and can be liquefied with heat, poured into a different container, and resolidified. (b) Nutrient gelatin contains enough gelatin (12o/o)Io take on a solid consistency. The top tube shows it as a solid. The bottom tube indicates what happens when it is warmed or when microbial enzymes digest the gelatin and liquefy it.

64

Chapter

3

Tools of the Laboratory

Chemicol Content of Mediq Media with chemically defined compositions are termed synthetic. Such media contain pure chemical nutrients that vary little from one source to another and have a molecular content specified

by means of an exact formula. Synthetic media come in many forms. Some media, such as minimal media for fungi, contain nothing more than a few salts and amino acids dissolved in water. Others contain a variety of precisely measured ingredients (table 3.2A). Such standardized and reproducible media are most useful in research and cell culture. But they can only be used when the exact nutritional needs of the test organisms are known. Recently a defined medium was developed for the parasitic protozoan Leishmania that contains 75 different chemicals.

Chemically Defined Synthetic Medium

for Growth and Maintenance of Pathogenic ita phylococcus o u reu s

Acids

Each of These Amino Acids

Acids

Cystine

Arginine

Aspartic acid

Histidine

Glycine

Glutamic acid

Leucine

Isoleucine

Phenylalanine

Lysine

Proline

Methionine

0.25 Grams Each

0.5 Grams

of These Amino

Tryptophan

Serine

Tyrosine

Threonine

0.12 Grams Each

of These Amino

Valine

Additional ingredients 0.005 mole nicotinamide

1

0.005 mole thiamine o.oo5 mole

pyridoxine l-vitamins

0.5 micrograms

biotin _J sulfate

1.25 grams magnesium

1.25 grams dipotassium hydrogen

-1

phosphate

chloride 0.125 grams iron chloride 1.25 grams sodium

I

fSalts

)

Ingredients dissolved in 1,000 milliliters of distilled water and buffered to a final pH of 7.0.

Brain-Heart Infusion Broth: A Complex,

Nonsynthetic Medium for Growth and Maintenance of Pathogenic Staphylococcas oureus 27,5 grams brain-heart extract, peptone extract 2 grams glucose 5 grams sodium chloride

If even one component of a given medium is not chemically definable, the medium is a nonsynthetic, or complexr' medium. This type of medium cannot be represented by an exact chemical formula. Substances that can make it nonsynthetic are extracts from animals, plants, or yeasts, including such materials as ground-up cells, tissues, and secretions. Examples are blood serum, and meat extracts or infusions. Other nonsynthetic ingredients are milk, soybean digests, and peptone. Peptone is a partially digested protein, rich in amino acids,

that is often used as a carbon and nitrogen source. Nutrient broth, blood agar, and MacConkey agar, though different in fi.rnction and appearance, are all complex nonslmthetic media. They present a rich mixture of nutrients for microbes with complex nutritional needs. Table 3 .2 provides a practical comparison of the two categories, using a medium to grow Staphylococcus aureus. Every substance in medium A is known to a very precise degree. The dominant substances in medium B are macromolecules that contain dozens of un-

known (but potentially required) nutrients. Both

A

and

Media to Suit Every Function Microbiologists have many types of media at their disposal, with new ones being devised all the time. Depending upon what is adde4 a microbiologist can fine-tune a medium for nearly any purpose. Microbiologists have always been aware of microbes that could not be cultivated artificially. Newer DNA detection technologies have shown us that there are probably many times more microbes ofthis type than previously thought. Although discovery and identification of microorganisms have relied on our ability to grow them, now we can detect a single bacterium in its natural habitat (Insight 3.1). That said, it is highly unlikely that microbiologists will abandon culturing, simply because it provides a constant source of microbes for detailed study and research. General-purpose media are designed to grow a broad spectrum of microbes that do not have special growth requirements. As a rule, these media are nonsynthetic (complex) and contain a mixture of nutrients that could support the growth of a variety of bacteria and fungi. Examples include nutrient agar and broth, brain-heart infusion, and trypticase soy agar (TSA). TSA contains partially digested milk protein (casein), soybean digest, NaCl, and agar. An enriched medium contains complex organic substances such as blood serum, hemoglobin, or special growth factors that certain species must be provided in order to grow. These growth factors are organic compounds such as vitamins and amino acids that the microbes cannot slmthesize themselves. Bacteria that require growth factors and complex nutrients are termed fastidious. Blood agar, which is made by adding sterile animal blood (usually from sheep) to a sterile agar base (figure 3.7a) is widely employed to grow fastidious streptococci and other pathogens. Pathogenic Neisseria (one species causes gonorrhea) are grown on Thayer-Martin medium or chocolate agar, which is made by heating blood agar and does not contain chocolate-it just has that appearance (figure 3.70).

Selective and Differential Media Some of the cleverest and most inventive media recipes belong to the categories of selective

2.5 grams disodium hydrogen phosphate

Ingredients dissolved in 1,000 milliliters of distilled water and buffered to a final pH of7.0.

B will

satisfactorily grow the bacteria. (Which one would you rather make?)

3. Complex

means that the medium has large molecules such as proteins, polysaccharides, lipids, and other chemicals that can vary greatly.

3.1

Methods of Culturing Microorganisms-The Five l's

ol

The Uncultured For some time, microbiologists have suspected that culture-based methods are unable to identify many kinds of bacteria. This was first confirmed by environmental researchers, who came to believe that only about lo/o (and in some environments it was 0.001%) of microbes present in lakes, soil, and saltwater environments could be grown in laboratories by the usual methods and, ttrerefore, were unknown and unstudied. These microbes are termed viable but nonculturable' or VBNC. Scientists had spent several decades concocting recipes for media and having great success in growing all kinds ofbacteria from all kinds of environments. They had plenty to do, just in identiffing and studying those. But, by the 1990s, a mrmber of specific' nonculturing tools based on genetic testing had become widely available. When these methods were used to sample various environments, they revealed a vast 'Jungle" ofnew species that had never before been cultured. This discovery seemed reason-

able, because it may not yet be possible to exactly re-create the many correct media and conditions to grow such organisms in the lab' Medical microbiologists did not expect this same result, given how widely sampled and well understood the normal human flora have been. But it turns out that they were also missing a large proportion of microbes that are normal residents of the body. In 1 999, three Stanford University scientists applied these techniques to subgingival plaque harvested from one of their own mouths. They used small, known fragments of DNA or probes that can highJight microbes in specimens. Oral biologists had

previously recovered about 500 bacterial strains from this site; the Staniord scientists found 30 species that had never before been cultured or described. This discovery energized the medical world and led to increased investigation of normal human flora, using non-culture-based methods to

find VBNCs in the human body. The new realization that our bodies are hosts to a wide variety of un-l,crown microbes has several implications. As evolutionary

microbiologist

Fluorescent microscopic view of unusual coccus-shaped bacteria (pink) from the human oral cavity that have never been isolated in culture.

Paul Ewald has aske{ "What are all those microbes doing in there?" He points out that many oral microbes previously assumed to be innocuous are now associated with cancer and heart disease. Many ofthe diseases that we currently think of as noninfectious will likely be found to have an infectious cause once we continue to look forVBNCs. Ewald suggests that many ofour oral residents (and gastrointestinal flora), ineluding those that may turn out to be pathogeaic, may have been obtained from a very common

activity-kissing. Another intriguing speculation

about these unculturables

is that they help maintain the health and stability of the body.

Do you think that all of the VBNCs are really not culturable? Suggest some other possibilities. Answer available at http://www.mhhe.com/ talaroT

Colony with zone of beta hemolysis

(a)

Figure 3.7

(b)

Examples of enriched media. (a) Blood agar plate growing bacteria from the human throat. Note that this medium can also differentiate among colonies by the zones of (b) Culture of hemolysis they may show. Note that some colonies have clear zones (beta hemolysis) and others have less defined zones. Neisseiiasp. on chocolate agar. Chocolate agar gets its brownish colorfrom cooked blood and does not produce hemolysis.

Chapter

66

3

Tools of the Laboratory

ii

'+;a;"*pr"

Examples of Selective Media, Agents,

€J

and Functions

+3

Selective

Agent

Mueller tellurite

Potassium

tellurite

Enterococcus

Sodium azide, tetrazolium

Isolation offecal

Phenylethanol chloride

Isolation of staphylococci and streptococci

Tomato juice, acid

Isolation of lactobacilli

faecalisbroth

/\

Phenylethanol

(PEA) Tomato juice

agar

agar

Used For Isolation of Corynebacterium diphtheriae enterococcr

from saliva

(a)

General-purpose nonselective medium (All species grow.)

tvtixeO sampte'

Selective medium (One species grows.)

il -----t-

$ii

u,,

gii

j

MacConkey agar Eosin-methylene blue agar (EMB)

Bile, dyes

SalmonellalShigella

Bile, citrate, brilliant green

Isolation of Salmonella and Shigella

Malachite green

Isolation and

Lowenstein-Jensen

(LJ)

,/\ :r

(b) Flgure

of

coliform bacteria in specimens

dye

Sabouraud's agar

(r1:r=

ffi'

Isolation

maintenance

of

Mycobacterium

/\ ,

Isolation of gramnegative enterics

(SS) agar

$

Bile, crystal violet

(MAC)

t--

r:l

. ._=.=_..

pH of 5.6 (acid) inhibits bacteria

l'*,:'*"

.*

rr

General-purpose Differential medium medium (All three species grow but may similar show different reactions.)

nondifferential (All species have a appearance.)

3.8 Comparison of selective and differential media with general-purpose media.

(a) A mixed sample containing three different species is streaked onto plates of general-purpose nonselective medium and selective medium. Note the results. (b) Another mixed sample containing three different species is streaked onto plates of general-purpose nondifferential medium and differential medium. Note the results.

and differential media (figure 3.8). These media are designed for special microbial groups, and they have extensive applications in isolation and identification. They can permit, in a single step, the preliminary identification of a genus or even a species. A selective medium (table 3.3) contains one or more agents that inhibit the growth of a certain microbe or microbes (call them A, B, and C) but not another (D). This difference favors, or seleclg microbe D and allows it to grow by itself. Selective media are very important

inprimary isolation of a specific type of microorganism from samples containing mixtures of different species-for example, feces, saliva, skin, water, and soil. Theyhasten isolationby suppressingthe unwanted background organisms and allowing growth of the desired ones.

Mannitol salt agar (MSA) contains a high concentration of NaCl (75%) that is quite inhibitory to most human pathogens. One exception is the genus Staphylococcus, which grows well in this medium and consequently can be amplified in mixed samples (figure 3.9a). Bile salts, a component of feces, inhibit most gram-positive bacteria while permitting many gram-negative rods to grow. Media for isolating intestinal pathogens (MacConkey agar, Hektoen enteric [HE] agar) contain bile salts as a selective agent (figure 3.96). Dyes such as methylene blue and crystal violet also inhibit cerlain grampositive bacteria. Other agents that have selective properties are antimicrobial drugs and acid. Some selective media contain strongly inhibitory agents to favor the growth of a pathogen that would otherwise be overlooked because of its low numbers in a specimen. Selenite and brilliant green dye are used in media to isolate Salmonella from feces, and sodium azide is used to isolate enterococci from water and food.

Differential media grow several types of microorganisms but are designed to bring out visible differences among those microor-

ganisms. Differences show up as variations in colony size or color, in media color changes, or in the formation of gas bubbles and precipitates (table 3.4). These variations come from the types of chemicals contained in the media and the ways that microbes react to them. In general, when microbe X metabolizes a certain substance not used by organism Y, then X will cause a visible change

in the medium and Y will not. The simplest differential media show two reaction types such as the use or nonuse ofa particular nutrient or a color change in some colonies but not in others. Some

3.1

67

Methods of Culturing Microorganisms-The Five l's

of Differential Media Examoles I Substances That

Facilitate

Medium

Differentiation

Differentiates

Blood agar (BAP)

Intact red blood cells

\pes ofRBC

Mannitol, phenol

Species ofpathogenic Staplrylococcus from

Mannitol salt agar (MSA) Hektoen enteric

(HE) agar**

red, and 7.5% NaCl*

Brom thymol blue, acid fuchsin, sucrose, salicin, thiosulfate, ferric ammonium citrate,

damage (hemolysis)

nonpathogens

Salmonella, Shigella, and other lactose

nonfermenters from fermenters; H2S reactions are

also observable.

and bile Lactose, neutral red

Bacteria that ferment lactose (lowering the pH) from those that do not

Eosin-methylene blue (EMB)

Lactose, eosin,

Same as MacConkey

Urea broth

Urea, phenol red

MacConkey agar

(MAC)

agaf

methylene blue

Bacteria that hydrolYze wea to ammonia and increase the pH

sulnrr moole motility (SIM)

(b

Figure

3.9

Thiosulfate, iron

H2S gas prooucers;

motility; indole formation

Examples of media that are both selective

and differential. (a) Mannitol salt agar can selectively grow Staphylococcus species from clinical samples. lt contains 7.5olo sodium chloride, an amount of salt that is inhibitory to most bacteria and molds found in humans. lt is also differential because it contains a dye (phenol red) that changes color under variations in pH, and mannitol, a sugar that can be converted to acid. The left side shows S. epidermidis, a species that does not use mannitol (red); the right shows S. oureus, a pathogen that uses mannitol (yellow). (b) MacConkey agar differentiates between lactose-fermenting bacteria (indicated by a pink-red reaction in the center of the colony) and lactose-negative bacteria (indicated by an off-white colony with no dye reaction)'

media are sufficiently complex to show three or more different reactions (figure 3.10). Dyes are effective differential agents because many of them are pH indicators that change color in response to the production ofan acid or a base. For example, MacConkey agar contains neutral red' a dye that is yellow when neutral and pink or red when acidic' A common intestinal bacterium such as Escherichia colithat gives off acid when it metabolizes the lactose in the medium develops red to pink colonies, and one like Salmonetla that does not give off acid remains its natural color (off-white). Media shown in figure 3.9 (mannitol salt agar) and figure 3.11 (fermentation broths) contain phenol red dye that also changes color with pH-it is yellow in acid and red in neutral and basic conditions. A single medium can be classified in more than one category depending on the ingredients it contains. MacConkey and EMB media, for example, appear in table 3.3 (selective media) and table 3.4 (differential media). Blood agar is both enriched and differential'

iron (TSIA)

Triple-sugar agar

XLD agar

iron, Phenol dYe

Fermentation of

Triple sugars, and red

sugars, H2S

Production Can differentiate Enterobacter, Escherichia. Proteus,

Lysine, xylose, iron, thiosulfate, phenol red

Providencia, Salmonella, and Shigella Birdseed agar

Seeds

Cryptococcus neoformans and other fungi

from

thistle plant

*NaCl also inhibits the salt-sensitive species. **Contains dyes and bile to inhibit gram-positive bacteria'

Miscellaneous

Media A reducing medium

contains a sub-

stance (thioglycollic acid or cystine) that absorbs oxygen or slows the penetration of oxygen in a medium, thus reducing its availability. Reducing media are important for growing anaerobic bacteria or for determining oxygen requirements of isolates (described in chapter 7). Carbohydrate fermentation media contain sugars that can be fermented (converted to acids) and a pH indicator to show this reaction (see figure 3.9a and figure 3.1 l). Media for other biochemical reactions that provide the basis for identifying bacteria and fungi are presented in several later chapters. Transport media are used to maintain and preserve specimens that have to be held for a period of time before clinical analysis or to

sustain delicate species that die rapidly

if

not held under

stable

68

Chapter

3

Tools of the Laboratory

Gas bubble

Cloudiness indicating growth

Flgure

3.ll

Carbohydrate fermentation in broths.

This medium is designed to show fermentation (acid production) using phenol red broth and gas formation by means of a small,

inverted Durham tube for collecting gas bubbles. The tube on the left is an uninoculated negative control; the center tube is positive for acid (yellow) and gas (open space); the tube on the right shows growth but neither acid nor gas.

Flgure 3.lO Media that differentiate multiple characteristics of bacteria. (a) Triple sugar iron agar (TSIA) inoculated on the surface and stabbed into the thicker region at the bottom (butt). This medium contains three sugars, phenol red dye to indicate pH changes (bright yellow is acid, various shades of red, basic), and iron salt to show Hrs gas production. Reactions are (1) no growth; (2) growth with no acid production (sugars not used); (3) acid production in the butt only; (4) acid production in all areas of the medium; (5) acid and H25 production in butt (black precipitate). (b) A state-of-the_art medium developed for culturing and identifying the most common urinary pathogens. CHROMagar OrientationrM uses color-forming reactions to distinguish at least seven species and permits rapid identification and treatment. In the example, the bacteria were streaked so as to spell their own names. Which bacterium was probably used to write the name at the top?

conditions. Stuart's andAmieb transport media contain buffers and absorbants to prevent cell destruction but will not support growth. Assay media are used by technologists to test the effectiveness of antimicrobial drugs (see chapter 12) nd,by drug manufacturers to assess the efect of disinfectants, antiseptics, cosmetics, and pre_ servatives on the growth ofmicroorganisms. Enumeration media are used by industrial and environmental microbiologists to count the numbers of organisms in milk, water, food, soil, and other samples. A number of significant microbial groups (viruses, rickettsias, and a few bacteria) will only grow on live cells or animals. These obligate parasites have unique requirements that must be provided by hosts (Insight 3.2).

Incubation and Inspection Once a container of medium has been inoculated it is incubated, which means it is placed in a temperature-controlled chamber (incubator) to encouxage microbial $owth. Although microbes have adapted to growth at temperatures ranging from freezing to boiling, the usual temperatures used in laboratory propagation fall between 2AoC and 40oC. Incubators can also control the content of atmospheric gases such as oxygen and carbon dioxide that may be required for the growth of certain microbes. During the incubation period Ganging from a few hours to several weeks), the microbe multiplies and produces growth that is observable macroscopically. Microbial growth in a liquid medium materializes as cloudiness.

3.1

Methods of Culturing Microorganisms-The Five l's

69

Animal Inoculation: "Living Media" A great deal ofattention

has been focused on the uses

ofanimals in biol-

ogy and medicine. Animal rights activists are vocal about practically any experimentation with animals and have expressed their outrage quite forcefully. Certain kinds of animal testing may seem trivial and unnecesnecessary to use animals bred for pigs, mice' chickens, and even guinea experimental purposes' such as aid for studying, growindispensable an canbe armadillos. Such animals of animals involves use special One microorganisms. ing, and identiffing vaccines for influofbirds. (embryos) stages life early ofthe inoculation enza are currently produced in chicken embryos- The major rationales for sary, but many times

it is absolutely

5.

animal to distinguish between pathogenic or nonpathogenic strains of Listeria or Candida (a Yeast). Some microbes will not grow on artificial media but will grow in a suitable animal and can be recovered in a more or less pure form' These include animal viruses, the spirochete of syphilis, and the leprosy bacillus (grown in armadillos).

live animal inoculation can be summarized as follows: inoculation is an essential step intesting the effects of drugs and the effectiveness of vaccines before they are administered to humans' It makes progress toward prevention. ffeatment. and cwe possible' Researchers develop animal models for evaluating new diseases or for studying the cause or process ofa disease. Koch's postulates are

1. Animal

2.

of proofs to determine the causative agent of a disease and require a controlled experiment with an animal that can develop a typical case ofthe disease. Researchers have also created hundreds a series

of engineered animals to monitor the effects of genetic diseases and to study the actions of the immune system. 3. Animal are an importalt source of antibodies, antisera, antitoxins' and other immune products that can be used in therapy or testing' 4. Animals are sometimes required to determine the pathogenicity or toxicity ofcertain bacteria. one such test is the mouse neutralization test for the presence ofbotulism toxin in food. This test can help identify even very tiny amounts oftoxin and thereby can avert outbreaks of this disease. Occasionally, it is necessary to inoculate an

sediment, a surface ma| or colored pigment. Bacteria and fungi grow on solid media in the form of masses called colonies' In some ways, culturing microbes is analogous to gardening' Cultures are formed by "seeding" tiny plots (media) with microbial cells. Extreme care is taken to exclude weeds (contaminants)' A pure culture is a container of medium that grows only a single known species or type of microorganism (figure 3.12a). This tlpe of culture is most frequently used for laboratory study, because it allows the precise examination and control of one microorganism by itself. Instead of the termpure cultureo some microbiologists prefer the term axenic, meaning that the culture is free of other living things except for the one being studied. A standard method for preparing a pure culture is to subculture" or make a second-level culture from a well-isolated colony. A tiny bit of cells is transferred into a separate container of media and incubated (see figure 3. I , step 3). A mixed culture (figure 3.12b) is a container that holds two or more easily differentiated species of microorganisms, not unlike a garden plot containing both carrots and onions. A contaminated culture (figure 3.12c) has had contaminants (unwanted microbes of uncertain identity) introduced into it, like weeds into a garden'

Because contaminants have the potential for disrupting experiments and tests, special procedures have been developed to control them. as vou will no doubt witness in your own laboratory.

The nude or athymic mouse has genetic defects in hair formation and thymus development. lt is widely used to study cancer, immune function,

and infectious diseases.

Discuss some of the complications one may expect when growing microbes in live animals. Answer available at http://www.mhhe'com/ talaroT

ldentification How does one determine what sorts of microorganisms have been isolated in cultures? Certainly microscopic appearance can be valuable in differentiating the smaller, simpler prokaryotic cells from the larger, more complex eukaryotic cells. Appearance can often be used to identify eukaryotic microorganisms to the level of genus or species because of their more prominent, observable cells, Bacteria are generally not as readily identifiable by these methods because very different species may appear quite similar. For them, we must include other techniques, some of which characterize their cellular metabolism. These methods, called biochemical tests, can determine fundamental chemical characteristics such as nutrient requirements, products given offduring growth, presenae of enzgnes, and mechanisms for deriving energy. These tests are discussed in more depth in later chapters that cover identification of pathogens. Several modern diagnostic tools that analyze genetic characteristics can detect microbes based on their DNA' These DNA profiles can be extremely specific, even sufficient by themselves to identify some microbes (see chapter 10). Identification can also be accomplished by testing the isolate against known antibodies (immunologic

testing). In the case of certain pathogens, further information is ob-

tained by inoculating a suitable laboratory animal. By compiling

70

Chapter

3

Tools of the Laboratory

Manassas, Virginia, which maintains a voluminous alray of frozen

and freeze-dried fungal, bacterial, viral, and algal cultures. Clinical labs in particular use standard, known cultures in quality control and verification of pathogens.

€

The Five I's-inoculation, incubation, isolation, inspection, and identification-summarize the kinds of laboratory procedures used in microbiology.

€ Microorganisms can be cultured on

s s =

€ ee

a variety of laboratory media that provide them with all of their required nutrients. Media can be classifiedby ther physical state as liquid, semisolid, liquefiable solid, or nonliquefiable solid. Media can be classified by their chemical composition as either synthetic or nonsynthetic,depending on the precise content oftheir chemical composition. Media can be classified by theirfunction as either general-purpose media or media with one or more specific purposes. Enriched se_ lective, differential, transport, assay, and enumerating medi aare all examples ofmedia designed for specific purposes. Some microbes can be cultured only in living cells (animals, embryos, cell cultures).

During inoculation, a specimen is introduced into a container of medium. Inoculated me dia are incubated at a specified temperature to encourage growth and form a culture.

# Isolation occurs when colo nies oigtnate from single

cells. Colonies are composed of large numbers of cells massed together in visible

mounds.

€ A culture

e (c)

Figure 3.12

may exist in one of the following forms:. A pure culture contains only one species or type of microorganism. A mixed cul_ ture contains two or more known species. A contaminated culture contains both known and unknown (unwanted) microorganisms. During inspection, the cultures are examined and evaluated macro_ scopically for growth characteristics and microscopicallv for cel_

lular appearance.

Various conditions of cultures.

(a) Three tubes containing pure cultures of Escherichiq coll (white), Micrococcus /ufeas (yellow), and Serratia morcescens (red). (b) A mixed culture of M. luteus and E. colireadily differentiated by their colors. (c) This plate of S. mqrcescens was overexposed to room air, and it has developed a large, white colony. Because this intruder is not desirable and not identified, the culture is now contaminateo.

physiological testing results with both macroscopic and microscopic traits, a complete picture of the microbe is developed. Expertise in final identification comes from specialists, keys, manuals, and computerized programs. In chapter 17 and the pathogens chapters, we present more detailed examples of identification methods.

Maintenance ond Disposal of Cultures In most medical laboratories, the cultures and specimens constitute a potential hazard and require immediate and proper disposal. Both steam sterilizing (see autoclave, chapter I l) and incineration (burning) are used to destroy microorganisms. Many teaching and research laboratories maintain a line of stock cultures that represent "living catalogs" for study and experimentation. The largest culture collection can be found at the American Type Culture Collection in

w

€

Microorganisms are identified by means of their microscopic morphology, their macroscopic morphology, their biochemical reac_ tions, immunologic reactions, and their genetic characteristics.

Microbial cultures are usually disposed of in two ways: steam sterilization or incineration.

3.2 The Microscope: Window on an Invisible Realm Imagine Leeuwenhoekos excitement and wonder when he first viewed a drop of rainwater and glimpsed an amazingmicroscopic world teeming with unearthly creatures. Beginning microbiology students still experience this sensation, and even experienced microbiologists remember their first view. The microbial existence is indeed another world" but it would remain largely uncharted without an essential tool: the microscope. your efforts in exploring microbes will be more meaningful if you understand some essentials of microscopy* and specimen preparation. a microstr4;.v (mye-lcaw'-skuh-pee) techniques.

Gr. The science that studies microscoDe

3.2

The Microscope: Window on an Invisible Realm

7l

Magnification and Microscope Design The two key characteristics of a reliable microscope are magnification,* the ability to make objects appear enlarged, and resolving power, the ability to show detail. A discovery by ear$ microscopists that spurred the advancement of microbiology was that a cleag glass sphere could act as a lens to magfiry small objects. Magnification in most microscopes results from a complex interaction between visible light waves and

the curvature of the lens. When a beam or ray of light transmitted through air strikes and passes through the convex surface ofglass, it experiences some degree ofrefraction,* defined as the bending or change in the angle of the light ray as it passes through a medium such as a lens. The greater the difference in the composition of the two substances the light passes between, the more pronounced is the refraction. When an object is placed a certain distance from the spherical lens and illuminated with light, an optical replica, or image, of it is formed by the refracted light. Depending upon the size and curvature of the lens, the image appears enlarged to a particular degree, which is called its power of magnification and is usually identified with a number combined with x (read "times"). This behavior of light is evident if one looks through an everyday object such as a glass ball or a magniffing glass (figure 3.13). It is basic to the function of all optical, or light, microscopes, though many of them have additional features that define, refine, and increase the size of the image. The first microscopes were simple, meaning they contained just a single magrrifying lens and a few working parts. Examples ofthis type ofmicroscope are a magniffing glass, a hand lens' and Leeuwenhoek's

* magnification (mag'-nih-fih-kay''-shun) L. magnus,

grea,t,

andJic*e, to make'

a refmct, refraction (ree-frakt', ree-frak'-shun) L. reftingere, to break apart'

Flgure 3.14

Flgure 3.13

Effects of magnification.

Demonstration of the magnification and image-forming capacity of clear glass "lenses." Given a proper source of illumination, this magnifying glass and crystal ball magnify a ruler two to three times' Note the tendency for distortion of the image with simple lenses. basic little tool shornm earlier in figlure l'9a. Among the refinements that led to the development of today's compound (two-lens) microscope were the addition of a second magrrifying lens system, a lamp in the base to give offvisible light and illuminate* the specimen, and a special lens called the condenser that converges or focuses the rays of light to a single point on the object. The fundamental parts of a modern compound light microscope are illustrated in figure 3.14.

* illuminate (ill-oo'-mih-nay) L. illuminatus, to light up'

The Parts of a student

laboratory microscope. This microscope is a compound light microscope with two oculars (called binocular). It has four objective lenses, a mechanical stage to move the specimen, a condenser, an iris diaphragm, and a built-in lamP. Body Nosepiece

Objective lens (4) Mechanical stage I Substage condenser Aperture diaphragm control Base with light source Field diaPhragm lever

Light intensity control

Coarse adjustment knob Fine focus adjustment knob Stage adiustment knobs

72

Chapter

3

Tools of the Laboratory

Brain

Retina

t

the final image formed by the combined lenses is a product of the separate powers of the two lenses: Power

Eye

Virtual image

Objective lens

Specimen

of

l0x 10x 10x

Total

Magnification 40X 100x 400X

: :

1,000X

Microscopes are equipped with a nosepiece holding three or more objectives that can be rotated into position as needed. The power of the ocular usually remains constant for a given microscope. De_

{.o) Real image formed by objective lens

addition to magnification, a microscope must also have adequate resolution, or resolving porver. Resolution defines the capacity ofan

Condenser lens

Light source

Flgure 3.15 The pathway of light and the two stages in magnification of a compound microscope. As light passes through the condenser, it is gathered into a tight beam that is focused on the specimen. Light leaving the specimen enters the objective lens and is refracted so as to form an enlarged primary image, the real image. One does not see this image, but its degree of magnification is represented by the lower circle. The real image is projected through the ocular, and a second image, the virtual image, is formed by a similar process. The virtual image is the final magnified image that is received by the lens and retina of the eye and perceived by the brain. Notice that the lens systems cause the image to be reversed.

Principles of Light Microscopy To be most effective, a microscope should provide adequate magni-

fication, resolution, and clarity of image. Magnification of the object or specimen by a compound microscope occurs in two phases. The first lens in this system (the one closest to the specimen) is the objective lens, and the second (the one closest to the eye) is the ocular lens, or eyepiece (figure 3.15). The objective forms the in! tial image of the specimen, called the real image. When the real image is projected to the plane of the eyepiece, the ocular lens mag-

nifies it to produce a second image, the virtual image. The virtual

will

: Ocular : 10x

Usual Power

pending on the power of the ocular, the total magnification of standard light microscopes can vary from 40x with the lowest power objective (called the scanning objective) to 2,000X with the highest power objective (the oil immersion objective).

formed by ocular lens

image is the one that

x

4X scanning objective lOX low power objective 40X high dry objective l00X oil immersion obiective

Ocular lens

Light rays strike specimen

of

Objective

be received by the eye and converted to a

retinal and visual image. The magniS'ing power of the objective alone usually ranges from 4X to 100X, and the power of the ocular alone ranges from lOX to 20X. The total power of magnification of

Resolution: Distinguishing Magnified Objects

Clearly

In

optical system to distinguish or separate two adjacent objects or points from one another. For example, at a distance of l0 inches, the lins in the human eye can resolve two small objects as separate points just as long as tlre two objects are no closer than0.2mm apart. The eye examination given by optomehists is in fact a test ofthe resolving power of the human eye for various-size letters read at a distance of 20 feet. Because microorganisms are exhemely small and usually very close together, they will not be seen with clarity or any degree of detail un_ less the microscope's lenses can resolve them. A simple equation in the form of a fraction expresses the main

mathematical factors that influence the expression

of resolving

power.

Wavelength

Resolving power (RP)

:

of

light in nm

2 X Numerical aperture of obiective lens From this equation, it is evident that the resolving power is a function of the wavelength of light that forms the image, along with certain characteristics of the objective. The light source for optical microscopes consists of a band of colored wavelengths in the visible spec_ trum. The shortest visible wavelengths are in the violet-blue portion of

the spectrum (400 nm), and the longest are in the red portion (750 nm). Because the wavelength must pass between the objects that are being resolved, shorter wavelengths (in the 400-500 nm range) will provide better resolution (figure 3.16). Some microscopes have a special blue filterplaced over the lamp to limit the longer wavelengths of light from entering the specimen.

The other factor influencing resolution is the numerical aperture (NA), a mathematical constant derived from the physical struc_ ture ofthe lens. This number represents the angle oflight produced by refraction and is a measure of the quantity of light gathered by the lens. Each objective has a fixed numerical aperfure reading ranging from 0. I in the lowest power lens to approximately 1.25 in the highest power (oil immersion) lens. Lenses with higher NAs provide better resolving power because they increase the angle of refraction and widen the cone oflight entering the lens. For the oil immersion lens to

3.2

Flgure 3.16

The Microscope: Window on an Invisible Realm

73

Effect of wavelength on resolution'

A simple model demonstrates how the wavelength influences the resolving power of a microscope. Here an outline of a hand represents the obieci being illuminated, and two different-size circles represent the wavelengths of light. In (a), the longer waves are too large to penetrate

between the finer spaces and produce afuzzy, undetailed image' ln

(b),shorterwavesaresmallenoughtoentersmallspacesandproduce a much more detailed image that is recognizable as a hand'

Flgure

3.t8

Effect of magnification.

co-mparison of objects that would not be resolvable versus those that

wou|dbereso|vableunderoiIimmersionatl,000Xmagnification. Note that in addition to differentiating two adiacent things, good resolution also means being able to observe an obiect clearly'

Flgure 3.17 Workings of an oil immersion

lens'

(the one with To-maximize its resolving power, an oil immersion lens highest magnification) must have a drop of oil placed at its tip' This

foims a continuort medium to transmit a cone of light from the condenser to the obiective, thereby increasing the amount of light

and,consequently,thenumericalaperture'Withoutoil,someofthe peripheral light that passes through the specimen is scattered into ihe air or o.rto ttte glass slide; this scattering decreases resolution.

arrive at its maximum resolving capacity, a drop of oil must be inglass slide. serted between the tip of the lens and the specimen on the prevents refracglass, it Because oil has the same optical qualities as slide from the passes light tive loss that normally occurs as peripheral aperture numerical the into the air; this property effectively increases (figure 3.17). There is an absolute limitation to resolution in optical microscopes, which can be demonstated by calculating the resolution of the oil immersion lens using a blue-green wavelength of light: 500 nm

RP

:

2

x

1.25

200 nm (or 0.2 pm)

Given that a smaller resolving power means improved resolution, you will notice that having a shorter wavelength and a larger numerical aperture will both provide desired improvements' In practical terms, this means that the oil immersion lens can resolve any cell or cell part as long as it is at least 0.2 ;rrn in diameter and that it can resolve two adjacent objects as long as they are no closer than 0.2 pm (figure 3.18). In general, organisms that are 0'5 pm or more in diameter are readily seen. This includes frurgi and protozoa and some of their internal structures, and most bacteria' However, a few bacteria and most viruses are far too small to be resolved by the optical microscope and require electron microscopy (see figure 1'7 and figure 3.24). In summary then, the factor that most limits the

clarity of a microscope's image is its resolving power' Even

if

a

light microscope were designed to magnify several thousand times,

its resolving power could not be increase4 and the image it produced would simply be enlarged andfinzy. Other constaints to the formation of a clear image are the quality of the lens and light source and the lack of confrast in the specimen. No matter how carefirlly a lens is constructed, flaws remain' A typical problem is spherical aberratiorS a distortion in the image caused by irregularitiesin the lens, which creates a curved, rather than flat, imug"lr"" figure 3.13). Another is chromatic aberration, a rainbowlike iirage that is caused by the lens acting as a prism and separating visible light into its colored bands. Brightress and direction of illumination also affect image formation. Because too much light can reduce confast and burn out the image, an adjustable iris diaphragm on most microscopes controls the amount of light entering the condenser. The lack of contrast in cell components is compensated for by using special lenses (the phase-contast microscope) and by adding dyes'

Chapter

74

'-t.,, ,,,,.,,

3

Tools of the Laboratorv

':: comparisons of Types of Microscopy

Microscope

Maximum Practical

Magnification

Resolution

lmportant Features

Visible light as source of illumination

Bright-field

2.000x

0.2 pm (200

Dark-field

2,000x

0.2 pm

nm)

Common multipurpose microscope for live and preserved stained specimens; specimen is dark, field is white: provides fair cellular detail Best for observing live, unstained specimens; specimen is bright, field is black; provides outline specimen with reduced internal cellular detail

of

Phase-contrast

2,000x

0.2trtm

Differential interference

Used for live specimens; specimen is contrasted against gray background; excellent for internal cellular detail

2,000x

0.2 ym

Provides brightly colored, highly contrasting, three-dimensional images of live specimens

2,000X

0.2 pm

Specimens stained with fluorescent dyes or combined

Ultraviolet rays as source of illumination

Fluorescent

with

fluorescent antibodies emit visible light; specificity makes this microscope an excellent diagnostic tool Confocal

2,000x

0.2ptm

Specimens stained with fluorescent dyes are scanned by laser beam: multiple images (optical sections) are combined into three-dimensional image

by a computer; unstained specimens can be viewed using light reflected from specimen

Electron beam forms image of specimen

Transmissionelectronmicroscope(TEM)

100,000X

0.5 nm

Sections of specimen are viewed under very high

magnification; finest detailed structure ofceils and viruses is shown; used only on preserved material Scanning electron microscope (SEM)

650,000x

l0 nm

Atomically sharp tip probes surface of specimen Atomic force microscope (AFM)

100,000,000x

Scans and magnifies external surface

of specimen; produces striking three-dimensional imase

001Angstrom"'Hffi

.'$:r#;"i#il.iJ'"il,xf ;:#'lo**,

laser and translated to image Scaruring tunneling microscope

(STM)

100,000,000x

Variations on the Optical Microscope optical microscopes that use visible light can be described by the field, meaning the circurar area viewed throusl the ocular lens. With special adaptations in lenses, condenserJ, and light sources, four special types ofmicroscopes can be described: bright-field dark-field phase-contrast, and interference. A fifth type of optical microscope, the fluorescence microscope, uses ultraviolet radiation as the illuminating source, and a sixth, the nature of theft

confocal microscope, uses a raser beam. Each of these microscopes is adapted for viewing specimens in a particular way, as described in the next sections and summarized in table 3.5.

0.01

Angstroms

Tip moves over specimen while voltage is appliedgenerating cw:ent that is dependent on distance between trp artd surfac1l. atoms ca1 be move! wUn nO..,.

on this page with light reflected off the surface, a bright_field microscope forms its image when light is transmitted throush the specimen. The specimen, being denser and more opaque tian its surroundings, absorbs some of this light, and the rest of the lisht

is transmitted directly up through the ocular into the field. -As will produce an image that is darker than the surrounding brightly illuminated fierd. The bright-field microscope is a multipurpose instrument that can be used for both live, unstained material and preserved, stained material. The bright-field image is compared with that of other microscopes in figure 3.19. a result, the specimen

Bright- Field M icroscopy

Dork-Field Microscopy

The bright-field microscope is the most widely used type of light

A bright-field microscope can be

microscope. Although we ordinarily view objects like the words

adapted as a dark-field microscope by adding a special disc called a stop to the condenser. The

3.2 periphstop blocks all light from entering the objective lens except right that is reflected off the sides of the specimen itself. The "rur image is a particularly striking one: brightly illuminated resulting specimurs surroundedby a dark (black) field (figure 3'19b)' Some oi Leeuwenhoek's more successful microscopes probably operated with dark-field illumination. The most effective use of dark-field

microscopy is to visualize living cells that would be distorted by

t)

The Microscope: Window on an Invisible Realm

drying or heat or cannot be stained with the usual methods. It can the organism,s shape and permit rapid recognition of swimming cells that may appear in fresh specimens, but it does notreveal fine internal details.

o"tti*

Phase- Contrast

ond I nterference Microscopy

If similar objects made of clear glass, ice, and plastic are immersed in the same container of water, an observer would have difficulty telling them apart because they have similar optical properties. In

the same way, internal components of a live, unstained cell also lack sufficient contrast to distinguish readily. But cell structures do dif-

fer slightly in density, enough that they can alter the light that

tlrough them in subtle ways. The phase-contrast microscope Las been constructed to take advantage ofthis characteristic' This passes

microscope contains devices that transform the subtle changes in light waves passing through the specimen into differences in light iritensity. Foi example, thicker cell parts such as organelles alter the

pathwayoflighttoagreaterextentthanthinnerregionslikethe cytoptas-. Light patterns coming from these regions will vary in contrast. The amount of internal detail visible by this method is

greater than by either bright-field or dark-field methods. The phaseiontrast microscope is most useful forObserving intracellular structures such as bacterial spores' granules, and organelles, as well as

the locomotor structures of eukaryotic cells (figure 3'19c and figure 3.20a).

Likethephase-contrastmicroscope,thedifferentialinterfercontrastlolcl microscope provides a detailed view of unstained, live specimens by manipulating the light' But this

ence

microscope has additional refinements, including two prisms that add contrasting colors to the image and two beams of light rather than a single one. DIC microscopes produce extremely well-defined images that are vividly colored and appear three-dimensional

(figure 3.20b).

rescen ce M i crosco PY The fluorescence microscope is a specially modified compound microscope furnished with an ultraviolet (uv) radiation source and filters that protect the viewer's eye from injury by these danFIu o

gerous rays.

Thi name for this type of microscopy is based ol the

ise of certain dyes (acridine, fluorescein) and minerals that show fluorescence.Thismeansthatthedyesgiveoffvisiblelightwhen bombarded by shorter ultraviolet rays. For an image to be forme4

the specimen must first be coated or placed in contact with

a

,o*.i of fluorescence. Subsequent illumination by ultraviolet radiation causes the specimen to emit visible light, producing an intense blue, yellow, orange, or red image against a black

Flgure 3.19 A five cell

of

Three views of a basic cell'

Porameciumviewed with (a) bright-field

(400x), (b) dark-

fie|d(400x),and(c)phase-contrast(400X).Notethedifferenceinthe

method upp"urance of the fieiO and the degree of detail shown by each the cells (fine on hairs) cilia the are phase-contrast in Only oi microscopy. groove? noticeable. cun you see the nucleus in the examples? The oral

field'

Fluorescence microscopy has its most useful applicatioqs in diagnosing infections caused by specific bacteria' protozoans' and vinlses. A staining technique with fluorescent dyes is commonly used to delect Mycobacterium tuberculosii (the agent of tuberculosis) in patients' specimens (see figure 19.20)' In a number of diagnostic procedures, fluorescent dyes are boundto specific antibodies. These fluorescent antibodies can be used to detect the causative agents in such diseases as syphilis, chlamydiosis, trichomoniasis, h"tp"t, and influenza. A newer technology using fluorescent nucleic acid stains can differentiate between live and dead cells in mixfures (figure 3.21) or detect uncultured cells (see Insight 3'l)'

76

Chapter

3

Tools of the Laboratory

Figure 3.21 Fluorescent staining on a fresh sample of cheek scrapings from the oral cavit!. Cheek epithelial cells are the larger green cells with red nuclei. Bacteria appearing here are streptococci (red spheres in long chains) and tiny green rods. This particurar staining technique arso indicates whether cells are alive or dead; live cells fluoresce green, and dead cells fluoresce red (60x).

Flgure 3.2O

Visualizing internal structures.

(a) Phase-contrast micrograph of a bacterium containing spores. The relative density of the spores causes them to appear as bright,

shiny objects against the darker cell parts (600x). (b) Differential interference micrograph of Amoebo profeus, a common protozoan.

Note the outstanding internal detail, the depth of field, and the bright colors, which are not natural (160x).

A fluorescence microscope can be handy for locating microbes in complex mixtures because only those cells targeted by the technique will fluoresce. Optical microscopes may be unable to form a clear image at

higher magnifications, because samples are often too thick for con_ ventional lenses to focus all levels of cells simultaneously. This is especially true of larger cells with complex internal srructures. A newer type ofmicroscope that overcomes this impedimentiscalled,the scanning confocal microscope.This microscope uses a laser beam of lisht to scan various depths in the specimen and deliver a sharp image io_

cusing onjust a single plane. It is thus able to capture a highly focused view at any level, ranging from the surface to the middle of the cell. It

is most often used on fluorescently stained specimens, but it can also be used to visualize live unstained cells and tissues (figure 3.22).

Flgure

3.22

Confocal microscopy of a basic cell.

This Poramecium is stained with fluorescent dyes and visualized by a scanning confocal microscope. Note the degree of detail that may be observed at different depths of focus.

3.2

conventional light microscopes are our windows on the microscopic world then the electron microscope (EM) is our window on

If

the tiniest details of that world. Although this microscope was originally conceived and developed for studying nonbiological materials such as metals and small electronics parts, biologists immediately recognized the importance of the tool and began to use it in the early 1930s. One of the most impressive features of the electron microscope is the resolution it provides. Unlike light microscopes, the electron microscope forms an image with a beam of electrons lhat can be made to travel in wavelike patterns when accelerated to high speeds. These waves are 100,000 times shorter than the waves of visible light. Because resolving power is a function of wavelength, electrons can capfure the smallest structures. Indeed, it is possible to resolve atoms with an electron microscope, though the practical resolution for biological applications is approximately 0.5 nm. The degree of resolution Transmission Electron Microscope

Light Microscope

H ttr

=--\

Electron nrn

H

condenser,"..

s& -----€,#

.H* --+,e .i:1";:jl5::'i; f"

l'

l-

some applications. Its capacity for magnification and resolution makes the EM an invaluable tool for seeing the finest structure of cells and viruses. Ifnot for electron microscopes, our understanding of biological structure and function would still be in its early theoretical stages. In fundamental ways, the electron microscope is a derivative compound microscope. It employs components analogous the of to, but not necessarily the same as, those in light microscopy (figure 3.23). For instance, it magnifies in stages by means of two lens systems, and it has a condensing lens, a specimen holder, and a focusing apparatus. Otherwise, the two types have numerous differences (table 3.6). An electron gun aims its beam through a vacuum to ring-shaped electromagnets that focus this beam on the specimen. Specimens must be pretreated with chemicals or dyes to increase contrast and usually cannot be observed in a live state. The enlarged image is displayed on a viewing screen or photographed for further study rather than being observed directly through an eyepiece. Because images produced by elec-

trons lack color, electron micrographs (a micrograph is

Two general forms of EM are the transmission electron microscope (TEM) and the scanning electron microscope (SEM) (see table 3.5). Transmission electron microscopes are the method of choice for viewing the detailed structure of cells and viruses. This microscope produces its image by transmitting electrons through the specimen. Because electrons cannot readily

r,on, ,.uu.

penetrate thick preparations, the specimen must be stained or coated with metals that will increase image contrast and sectioned into extremely thin slices (20-100 nm thick). The electrons passing through the specimen travel to the fluorescent screen and display a pattern or image. The darker and lighter areas of the

_l

Specimen

of Light Microscopes and =_ ,.o .=, !.omnari191t

.:;?l,

-.,i,]

ocular lens

----l

.

ir

*" -t-";-:- ; .- -,.1-;;'--''F ''

t',',

i,'

Characteristic

Light or Optical

Electron (Transmission)

Useful magnification

2,000x

1,000,000x or more

Maximum resolution

200 nm

0.5 nm

Image produced by

Light rays

Electron beam

Image focused by

Glass objective lens

Electromagnetic objective lenses

Image viewed

Glass ocular lens

Fluorescent screen

Specimen placed on

Glass slide

Copper mesh

Specimen may be alive.

Yes

Usually not

Specimen requires special stains or treatment.

Depends on

Yes

Colored images formed

Yes

't,.'.

(a) Figure

EYe

3.23

through

(b)

viewing screen

Comparison of two microscopes.

(a) The light microscope and (b) one type of electron microscope (EM; transmission type). These diagrams are highly simplified, especially for the electron microscope, to indicate the common components. Note that the EM's image pathway is actually upside down compared with that of a light microscope. From Cell Ultrastructure 1 st edition by jensen/Park. @ 1967. Reprinted with permission of Brooks/Cole, a division of Thomson Learning: www.thomsonrights.

com. Fax 8O0 730-2215.

a

photograph of a microscopic object) are always shades of black, gray, and white. The color-enhanced micrographs used in this and other textbooks have computer-added color.

Electron oeam

{.-'=*'; i I ;

77

allows magnification to be extremely high-usually between 5,000 X and 1,000,000X for biological specimens and up to 5,000,000X in

Electron Microscopy

Lamp

The Microscope: Window on an Invisible Realm

technique

No

78

Chapter

Figure 3.24

3

Tools of the Laboratory

Transmission electron micrographs.

(a) Human parvoviruses (B-19) isolated from the serum of a patient with erythema infectiosum. This DNA virus and disease are covered in chapter 24. What is the average diameter in nanometers of a single virus? (b) A section through an infectious stage of Toxoplasmo gondii, the cause of a protozoan disease, toxoplasmosis. Labels indicate fine structures such as cell membrane (Pm), Golgi complex (Go), nucleus (Nu), mitochondrion (Mi), centrioles (Ce), and granules (Am, Dg).

image correspond to more and less dense parts on the specimen (figure 3,24).The TEM can also be used to produce negative images and shadow casts of whole microbes (see figure 6.3). The scanning electron microscope provides some of the most dramatic and realistic images in existence. This instrument is designed to create an extremely detailed three-dimensional view of all kinds of objects-from plaque on teeth to tapeworm heads. To produce its images, the SEM bombards the surface of a whole, metalcoated specimen with electrons while scanning back and forth over it. A shower of electrons deflected from the surface is picked up with great fidelity by a sophisticated detector, and the electron pattern is displayed as an image on a television screen. The contours of the specimens resolved with scanning electron micrography are very revealing and often surprising. Areas that look smooth and flat with the light microscope display intriguing surface features with the SEM (figure 3.25). Improved technology has continued to

Figure 3.25

Scanning electron micrographs.

(a) A false-color scanning electron micrograph (SEM) of Poramecium, covered in masses of fine hairs (100x). These are actually its locomotor and feeding structures-the cilia. Cells in the surrounding medium are bacteria that serve as the protozoan's "movable feast." Compare this with figure 3.19 to appreciate the outstanding three-dimensional detail shown by an SEM. (b) A fantastic ornamental alga called a coccolithophore displays a complex cell wall formed by calcium discs. This alga often blooms in the world's oceans (see chapter 1).

refine electron microscopes and to develop variations on the basic plan. One of the most inventive relatives of the EM is the scanning probe microscope (Insight 3.3).

Preparing Specimens for Optical Microscopes A specimen for optical microscopy is generally prepared by mounting a sample on a suitable glass slide that sits on the stage between the

3.2

The Microscope: Window on an Invisible Realm

79

The Evolution in Resolution: Probing Microscopes In the past, chemists. physicists, and biologists had to rely on indirect methods to provide information on the structures of the smallest molecules. But iechnological advances have created a new generation qf microscopes that "see" atomic structure by actual[y feeling it' Scanning probe microscopes operale with a minute needle tapered to a tip that can be as narrow as a single atom! This probe scans over the exposed surface of a material and records an image of its outer texfure. These revolutionary microscopes have such profound resolution that they have the potential to image single atoms and to magnify 100 million times. The scanning tunneling microscope ISTM ) was the first of these ,t rt."t t *g.,Jn prou. that trovers near the surface of an ;;;;;t;;; obiect and follows its topography while simultaneously giving off an electrical signal of its pathway, which is then imaged on a screen' The STM is used primarily for detecting defects on the surfaces ofelectrical conductors und .ornput", chips composed of silicon, but it has also orovided the first incredible close-up views of DNA, the genetic mate-

rial lsee Insight 9.2). "'";;;.t;r'.",

rn. atomic force microscope (AFM). genlly forces

a diamond and metal probe down onto the surface

of a specimen like a

needle on a record. As it moves along the surface, any deflection of the metal probe is detected by a sensitive device that relays the information

to an imager. The AFM is very useful in viewing the detailed functions of biolosical molecules such as antibodies and enzymes. The latest versions ofthese microscopes have recently increased the resolving power to around 0.5 A, which allowed technicians to image a pair of electrons! Such powerful tools for observing and positioning al oms have spawned a field called nanotechnology-the science of the "small." Scientists in this area use physics, chemistry, biology, and engineering to manipulate small molecules and atoms' Working at these dimensions, they are currently creating tiny molecular tools to miniaturize computers and other electronic devices. In

condenser and the objective lens. The manner in which a slide specimen, or mount, is prepared depends upon: (1) the condition of the

specimen, either in a living or preserved state; (2) the aims of the examiner, whether to observe overall structure' identify the microorganisms, or see movement; and (3) the type of microscopy available' whether it is bright-fiel4 dark-fiel4 phase-contrast, or fluorescence.

"Carbon monoxide man." This molecule was constructed from 28 single CO molecules (red spheres) and photographed by a scanning tunneling microscope. Each CO molecule is approximately 5 A wide. may be possible to use microstructures to deliver drugs' analyze DNA, and treat disease.

the future.

it

Looking back at figure 2.1, name some other chemical features that may become visible using these high-power microscopes. Ansu'er available at http : //wk w.mhhe.com/taloroT

adhesive or sealant, and a coverslip from which a tiny drop of sample is suspended. These types of short-term mounts provide a true assessment of the size, shape, arrangement, color, and motility of cells. Greater cellular detail can be observed with phasecontrast or interference microscopy.

coversrip

Hanging drop

lagi:*':t^^ *^-

Fresh, Living Preporations Live samples of microorganisms are placed in wet mounts or in hanging drop mounts so that they can be observed as near to their natural state as possible. The cells are suspended in a suitable fluid (wateq broth, saline) that temporarily maintains viability and provides space and a medium for locomotion. A wet mount consists of a drop or two of the culture placed on a slide and overlaid with a cover glass. Although this type of mount is quick and easy to prepare, it has certain disadvantages. The cover glass can damage larger cells, and the slide is very susceptible to drying and can contaminate the handler's fingers.

A more satisfactory alternative is the hanging drop slide (below) made with a special concave (depression) slide' an

Vaseline

Fixed, Stoined Smeors A more permanent mount for long-term study can be obtained by preparing fixed stained specimens. The smear technique' developed by Robert Koch more than 100 years ago, consists of spreading a thin film made from a liquid suspension of cells on a slide and airdrying it. Next, the air-dried smear is usually heated gently by a Drocess called heat fixation that simultaneously kills the specimen

80

Chapter

3

Tools of the Laboratory

and secures it to the slide. Another important action of fixation is to preserve various cellular components in a natural state with mini-

mal distortion. Fixation of some microbial cells is performed with chemicals such as alcohol and formalin.

Like images on undeveloped photographic film, the

un_

stained cells of a fixed smear are quite indistinct, no matter how great the magnification or how fine the resolving power of the microscope. The process of "developing,' a smear to create con_ trast and make inconspicuous features stand out requires staining techniques. Staining is any procedure that applies colored chem_ icals called dyes to specimens. Dyes impart a color to cells or cell parts by becoming affixed to them through a chemical reaction. In general, they are classified as basic (cationic) dyes, which have a positive charge, or acidic (anionic) dyes, which have a negative charge.

Negative versus Positive Staining Two basic types of stain_ ing technique are used, depending upon how a dye reacts with the specimen (summarized in table 3.7). Most procedures involve a positive stain, in which the dye actually sticks to cells and gives them color. A negative stain, on the other hand, isjust the reverse

(like a photographic negative). The dye does not stick to the specimen but dries around its outer boundary, forming a silhouette. Nigrosin (blue-black) and India ink (a black suspension of carbon particles) are the dyes most commonly used for negative staining. The cells themselves do not stain because these dyes are

negatively charged and are repelled by the negatively charged surface of the cells. The value of negative staining is its relative simplicity and the reduced shrinkage or distortion of cells, as the smear is not heat fixed. A quick assessment can thus be made regarding cellular size, shape, and arrangement. Negative staining

(a) (b)

A NOTE ABOUT DYES AND STAINING Because many microbial cells lack contrast, it is necessary to use dyes to observe their detailed structure and identifv

them. Dyes are colored compounds related to or derivei from the common organic solvent benzene. When certain double-bonded groups (C:O, C:N, N:N) are attached to complex ringed molecules, the resultant compound gives off a specific color. Most dyes are in the form of a sodium or chloride salt of an acidic or basic compound that ionizes when dissolved in a compatible solvent. The color-bearing ion, termed a chromophore, is charged and has an attrac_ tion for certain cell parts that are of the opposite charge

(flgure 1.26).

Basic dyes carry a positively charged chromophore and are attracted to negatively charged cell components (nucleic acids and proteins). Because bacteria contain large amounts of negatively charged substances, they stain readily with basic dyes, including methylene blue, crystal violet, fuchsin, malachite green, and safranin. Acidic dyes with a negatively charged chromophore bind to the positively charged molecules in some parts of eukaryotic cells. One example is eosin, a red dye used in staining blood cells. Because bacterial cells have numerous acidic substances and carry a slightly negative charge on their surface, they tend to repel acidic dyes. Acidic dyes such as nigrosin and lndia ink can still be used successfully for bacteria using the negative stain method (flgure 3.26c). With this, the dye settles around the cell and in this way creates an outline of it.

Positive-Type

(c)

(b)

ral JEq-sicoE

ktsdd@]

Chromophore

l"l @-'.

Staining

ov"

] grH, a_-1 z'# '\cH2-( --)J I ll \

/ \\ /\\.,/ 4\.v )-\

\:/

"<sor-\_( -"'X*.-cnr{)

Nisrosin

Culaway View of Cell

,"J" o-/

€6

ffi Figure

d#€ E-B

\e

oo o @i

ol

ool oo

--13

o-\:/ \7 \:,/

3.26

Staining reactions of dyes. (a) Basic dyes such as methylene blue are positively charged and react with negatively charged cell areas. (b) Acidic dyes such as eosin are negatively charged and react with positively charged cell areas. (c) Acidic dyes such as nigrosin can be used with negaiive staining to create

background around a cell.

a

3.2

The Microscope: Window on an Invisible Realm

CASE FILE

Appearance

ofcell

3

8l

Wrop-Up

Positive Staining

Negative Staining

The Cram stain is used to visualize and differentiate bacteria into

Colored by dye

Clear and colorless

broad categories. Purple-stained bacteria like B. onthrocis are called gram-positive, and red cells are gram-negative. These classifications relate information about the cell wall structure of each. Because bacteria are so small, the oil immersion lens (100x) on a bright-field compound microscope is used to distinguish cells and determine their color and shape. Other useful techniques would be a spore stain, capsule stain, and hanging drop slide.

Background

Not stained (generally white)

Dyes

employed

Subtypes

ofstains

Stained (dark gray or black)

Basic dyes: Crystal violet Methylene blue Safranin Malachite green

Acidic dyes: Nigrosin India ink

Several rypes: Simple stain Differential stains Gram stain Acid-fast stain

Few rypes: Capsule Spore

Spore stain Structural stains One tlpe of capsule

The blood sample is processed as follows: (1) inoculais aseptically obtained and placed into liquid medium; (2) incubation-the specimen is left overnight in appropriate conditions; (3) inspection-a Cram stain is prepared

tion-the blood

and viewed; (4) isolation-the culture growing in the liquid is transferred with proper technique to differential and selective solid media; (5) identification-the announcement of Bocillus onthrqcis. After isolation, further biotesting is performed to identify this bacterium. To further identify this particular strain as identical to those in other anthrax cases, a DNA study is also performed.

Negative stain of oral bacteria

See: CDC. 2001 . LJpdote: lnvestigotion of bioterrorism-reloted inhalotional anthrox-Connecticut, 2001. MMWR 50 :1 049-1 051.

Flagella SPore

endospore stains. Some staining techniques (spore, capsule) fall into more than one category. Gram staining is a 125-year-old method named for its de-

Granules Nucleic acid

uses dyes of contrasting color to clearly emphasize differences be-

veloper, Hans Christian Gram. Even today, it is an important diagnostic staining technique for bacteria. It permits ready differentiation ofmajor categories based upon the color reaction of the cells: gram-positive, which stain purple, and gram-negative, which stain red. This difference in staining quality is due to structural variations found in the cell walls of bacteria. The Gram stain is the basis of several important bacteriologic topics, including bacterial taxonomy, cell wall structure, and identification and diagnosis of infection; in some cases, it even guides the selection of the correct drug for an infection. Gram staining is discussed in greater detail in Insight 4.2. The acid-fast stain, like the Gram stain, is an important diagnostic stain that differentiates acid-fast bacteria (pink) from non-acid-fast bacteria (blue). This stain originated as a specific method to detect Mycobacterium tuberculosls in specimens. It was determined that these bacterial cells have a particularly impervious outer wall that holds fast (tightly or tenaciously) to the dye (carbol fuchsin) even when washed with a solution containing acid or acid alcohol. This stain is used for other medically important mycobacteria such as the Hansen's disease (leprosy) bacillus andfor Nocardia, an agent of lung or skin infections (see

tween two cell types or cell parts. Common combinations are red and purple, red and green, or pink and blue. Differential stains can also pinpoint other characteristics, such as the size, shape, and arrangement of cells. Typical examples include Gram, acid-fast, and

chapter l9). The endospore stain (spore stain) is similar to the acid-fast method in that a dye is forced by heat into resistant survival cells called spores or endospores. These are formed in response to

is also used to accentuate the capsule that surrounds certain bacteria and yeasts (figure 3.27). Simple versus Differential Staining Positive staining methods are classified as simple, differential, or structural (figwe 3.27). Whereas simple stains require only a single dye and an uncomplicated procedure, differential stains use two different-colored dyes, called the primary dye and the counterstalz, to distinguish between cell types or pafts. These staining techniques tend to be more complex and sometimes require additional chemical reagents to produce the desired reaction. Most simple staining techniques take advantage of the ready binding of bacterial cells to dyes like malachite green, crystal violet, basic fuchsin, and safranin. Simple stains cause all cells in a smear to appear more or less the same color, regardless of type, but they can still reveal bacterial characteristics such as shape, size, and arrangement.

Types

of Differential Stains An effective differential

stain

82

Chapter

3

Tools of the Laboratory

(a) Simple Stains

(b) Differential Stains

Crystal violet stain of Escherichia coli (1 ,000x)

Gram stain Purple cells are gram-positive. Red cells are gram-negative

Methylene blue stain of Corynebacterium

Acid-fast stain Red cells are acid{ast. Blue cells are non-acid{ast

(1,000x)

(c) Special Stains

India ink capsule stain of ryptococc u s n e olo r m a n s

C

(500x)

(900x).

Flagellar stain of Proteus vulgaris. A basic stain was used to build up the flagella

(1,s00x).

3.27

Types of microbiological Flgure stains. (a) Simple stains. (b) Differential stains: Cram, acid-fast, and spore. (c) Special stains: capsule and flagellar. The spore stain (bottom) is a method that fits two cateoories: differential and

S

special.

Spore stain, showing spores (green) and vegetative cells (red)

(1,000x)

adverse conditions and are not reproductive (see chapter 4). This stain is designed to distinguish between spores and the cells that make them-the so-called vegetative cells. Of significance in medical microbiology are the gram-positive, spore-forming members of the genus Bacillus (the cause of anthrax) and Clostridium (the cause of botulism and tetanus)-dramatic diseases of universal fascination that we consider in later chapters. Structural stains are used to emphasize special cell parts that are not revealed by conventional staining methods. Capsule staining is a method of observing the microbial capsule, an unstructured protective layer surrounding the cells of some bacteria and fungi. Because the capsule does not react with most stains, it is often negatively stained with India ink, or it may be demonstrated by

special positive stains. The fact that not all microbes exhibit capsules is a useful feature for identiffing pathogens. One example is Cryptococcus, which causes a serious firngal meningitis in AIDS patients.

Flagellar staining is a method of revealing flagella, the tiny, slender filaments used by bacteria for locomotion. Because the width of bacterial flagella lies beyond the resolving power of the light microscope, in order to be seen, they must be enlarged by depositing a coating on the outside of the filament and then staining it. This stain works best with fresh, young cultures, because flagella are delicate and can be lost or damaged on older cells. Their presence, numbeg and arrangement on a cell can be helpful in identification.

Chapter Summary with

Magnification, resolving power, lens quality, and illumination source all influence the clarity of specimens viewed through the optical microscope.

The maximum resolving power of the optical microscope is 200 nm, or 0.2 pm. This is sufficient to see the internal structures of eukaryotes and the morphology of most bacteria. There are six types ofoptical microscopes. Four types use visible light for illumination: brighffield, dark-fiel4 phase-contrast, and interference microscopes. The fluorescence microscope uses UV light for illumination, but it has the same resolving power as the other optical microscopes. The confocal microscope can use UV

light or visible light reflected from specimens.

KeY Terms

83

Electron microscopes (EMs) use electrons, not light waves, as an illumination source to provide high magnification (5,000x to 1,000,000x) and high resolution (0.5 nm). Electron microscopes can visualize cell ultrastructure (TEM) and three-dimensional images of cell and virus surface features (SEM). Specimens viewed through optical microscopes can be either alive or dea4 depending on the type ofspecimen preparation, but most EM specimens have been killed because they must be viewed in a vacuum. Stains are important diagnostic tools in microbiology because they can be designed to differentiate cell shape, structure, and other features of microscopic morphology.

Chapter Summary with Key Terms Five I's Microbiology as a science is very dependent on a number of specialized laboratory techniques. Laboratory steps routinely employed in microbiology are inoculation, incubation, isolation, inspection, and identification. 1. Initially, a specimen must be collected from a source, whether environmental or a patient. 2. Inoculation of a medium is the first step in obtaining a culture of the microorganisms present. 3. Isolation of the microorganisms, so that each microbial cell present is separated from the others and forms discrete colonies, is aided by inoculation techniques such as streak plates, pour plates, and spread plates. 4. Incubation of the medium with the microbes under the right conditions allows growth to visible colonies. Generally, isolated colonies would be subcultured for further testing at this point. The goal is a pure culture, in most cases, or a mixed culture' Contaminated cultures can ruin correct analysis and sfudy. 5. Inspection begins with macroscopic characteristics of the colonies and continues with microscopic analysis. 6. Identification correlates the various morphological, physiological, genetic, and serological traits as needed to be able to pinpoint the actual species or even strain of microbe. Media: Providing Nutrients in the Laboratory 1. Artificial media allow the growth and isolation of microorganisms in the laboratory and can be classified by their physical state, chemical composition, and fi.rnctional types. The nutritional requirements of microorganisms in the laboratory may be simple or complex. 2. Physical types of media include those that are liquid, such as broths and milk, those that are semisolid, and those that are so1id. Solid media may be liquefiable, containing a solidiffing agent such as agar or gelatin. 3. Chemical composition of a medium may be completely chemically defined, thus synthetic. Nonsynthetic, or complex, media contain ingredients that are not completely definable. 4. Functional tlpes of media serve different purposes, often allowing biochemical tests to be performed at the

same time. Types include

3.1 Methods of Culturing Microorganisms-The

A.

B.

5. 6.

general-purpose, enriched,

selective, and differential media. a. Enriched media contain growth factors required by microbes. b. Selective media permit the growth of desired microbes while inhibiting unwanted ones. c. Differential media bring out visible variations in microbial growth. d. Others include anaerobic (reducing), assay, and enumeration media. Transport media are important for conveying certain clinical specimens to the laboratory. In certain instances, microorganisms have to be grown in cell culfures or host animals. Cultures are maintained by large collection facilities such as the American Type Cultwe Collection located in Manassas. Virginia.

3.2 The Microscope: Window on an Invisible Realm A. Optical, or light, microscopy depends on lenses that refract light rays, drawing the rays to a focus to produce a magnified image. I . A simple microscope consists of a single magnif ing lens, whereas a compound microscope relies on two lenses: the ocular lens and the objective lens.

2.

The total power of magnification is calculated from the product ofthe ocular and objective magnifying powers.

3. Resolution,

or the resolving power, is a measure of a microscope's capacity to make clear images of very small objects. Resolution is improved with shorter wavelengths of illumination and with a higher numerical aperture of the lens. Light microscopes are limited by resolution to magnifications around

2,000x.

4.

B.

Modifications in the lighting or the lens system give rise

to the bright-fiel{ dark-fiel{ phase-contrast, interference, fluorescence, and confocal microscopes. Elechon microscopy depends on electromagnets that serve as lenses to focus electron beams. A transmission electron microscope (TEM) projects the electrons through prepared sections of the specimen, providing detailed structural images ofcells, cell parts, and viruses. A scanning electron

Chapter

84

3

Tools of the Laboratory

microscope (SEM) is more like dark-field microscopy, bouncing the elechons offthe surface of the specimen to

microbes are negatively charged and athact basic dyes. This is the basis of positive staining. In negative staining the microbe repels the dye and it stains the background. Dyes may be used

detectors. C.

Specimen preparation in optical microscopy is governed by the condition ofthe specimen, the purpose ofthe inspection, and the type ofmicroscope being used. 1. Wet mounts and hanging drop mounts permit examination ofthe characteristics oflive cells, such as

2.

alone and in combination.

1. Simple stains

just one dye and highlight cell

2. Differential

motility, shape, and arrangement. Fixed mounts are made by drying and heating a film of

3.

the specimen called a smear. This is then stained using dyes to permit visualization of cells or cell parts. D. Staining uses either basic (cationic) dyes with positive charges or acidic (anionic) dyes with negative charges. The surfaces of

e4f,M

use

morphology. stains require aprimary dye and a contrasting counterstain in order to distinguish cell types or parts. Important differential stains include the Gram stain, acid-fast stain, and the endospore stain. Structural stains are designed to bring out distinctive characteristics. Examples include capsule stains and

flagellar stains.

uhiple-choice euestions

Multiple-Choice Questions. Select the correct answer from the answers provided. For questions with blanks, choose the combination of answers that most accuratelv comDletes the statement.

1. Which of the followine is not one of the Five I's?

a. lnspectlon b. identification

1

d. incubation e. inoculation

c. induction

12. Bacteria tend to stain more readily with cationic (positively charged)

2. The term culture refers to the an incubator

growth of microorganisms in

-

"-"piO b. macroscopic, media

c. microscopic,

the body

d. artificial, colonies

3. A mixed culture is a. the same as a contaminated culture

dyes because bacteria a. contain large amounts b. contain large amounts c. are neutral d. have thick cell walls

ofalkaline substances ofacidic substances

13. The primary difference between a TEM and SEM is in

b. one that has been adequately stirred

a. magnification capability

c.

b. colored versus black-and-white images c. preparation of the specimen d. type oflenses

d.

4.

1. Motility is best observed with a a. hanging drop preparation c. streak plate b. negative stain d. flagellar stain

one that contains two or more known species a pond sample containing algae and protozoa

Agar is superior to gelatin as a solidifying agent because agar

a. does not melt at room temperature

b. solidifies at75oC c. is not usually decomposed by microorganisms d. both

a and c

5. The process that most accounts for magnification is a. a condenser c. illumination b. refraction oflight rays d. resolution 6. A subculture is a

a longer wavelength

t7. Multiple Matching. For each type of medium, select all of light.

c. not changed d. notpossible

8. A real image is produced by the a. ocular

c.

condenser

b. objective

d.

eye

9. A microscope that has a total magnification of 1,500X when using the oi1 immersion obiective has an ocular of what oower?

a. 150X b. 1.5x

15. What type of medium is used to maintain and preserve specimens before clinical analysis? a. selective medium c. enriched medium b. transport medium d. differential medium 16. Which of the following is NOT an optical microscope? a. dark-field c. atomic force b. confocal d. fluorescent

a. colony growing beneath the media surface b. culfure made from a contaminant c. culture made in an embryo d. culture made from an isolated colony

7. Resolution is with a. improved b. worsened

14. A fastidious organism must be grown on what type of medium? a. general-purpose medium c. synthetic medium b. differential medium d. enriched medium

c.15X d. 30x

10. The specimen for an electron microscope is always a. stained with dyes c. killed b. sliced into thin sections d. viewed directly

descriptions that fit. For media that fit more than one description, briefly explain why this is the case. mannitol salt agar a. selective medium chocolate agar b. differential medium - MacConkey agar c. chemically defined - nutrient broth (synthetic) medium - brain-heart infusion d. enriched medium - broth e. general-purpose medium - Sabouraud's agar f. complex medium triple-sugar iron agar g. transport medium - SIM medium

-

Concept Mapping

85

writins to Learn

Wf

These questions are suggested as awritingio-learn expetence. For each question, compose a one- or two-puagraph answer that includes the factual information needed to completely address the question. General page references for these topics are given in parentheses.

1. a. Describe briefly what is involved in the Five I's' (58, 59) b. Know the definitions of inoculation, growth, and

c. How does a value greater than worse?) (73) d. How does a value less than

contamination. (58, 68, 69) 2. a. Name two ways that pure, mixed, and contaminated cultures

e. What can be done to

are

similar and two ways that they differ from each other. (69)

3. a. Explain what is involved in isolating microorganisms and why it

this.

sample. (60,

61)

c. Describe how an isolated colony forms. (60) d. Explain why an isolated colony and a pure culture are not the same

thing.

(60)

4. a. Explain the two principal fimctions of dyes in media. (67) b. Differentiate among the ingredients and functions ofenriched, selective, and differential

media.

(64, 66, 67)

5. Differentiate between microscopic and macroscopic methods of observing microorganisms, citing a specific example of each

method. (59,60,69) 6. a. Differentiate between the concepts of magnification, refraction, and

resolution.

(7

I, 72, 73)

b. Briefly explain how an image is made and magnified. (7 |, 72) c. Trace the pathway oflight from its source to the eye, explaining what happens as it passes through the major parts ofthe microscope. (71,72) 7. a. On the basis of the formula for resolving power, explain why a smaller RP value is preferred to a larger one. (72,73) b. Explain what it means in practical terms if the resolving power is 1.0

%gf

pm.

(73)

compare? (73)

9. a. Compare and contrast the optical compound microscope with the electronmicroscope. (77) b. Why is the resolution so superior in the electron microscope? (77) c. What will you never see in an unretouched electron micrograph? (77) d. Compare the way that the image is formed in the TEM and sEM. (77,78)

(58, 59, 61)

b. Compare and contrast three common laboratory techniques for separating bacteria in a mixed

1.0 trrm

microscope to improve resolution? (73)

8. Compare bright-field, dark-field, phase-contrast, confocal, and fluorescence microscopy as to field appearance, specimen appearance, light source, and uses. (74,75,76)

b. What must be done to avoid contamination? (58, 69) is necessary to do

a

1.0 pm compare? (Is it better or

10. Evaluate the following preparations in terms of showing microbial size, shape, motiliry and differentiation: spore stain, negative stain, simple stain, hanging drop slide, and Gram stain. (79, 80, 81, 82) I

1. a. Itemize the various staining methods, and briefly chancterize

each. (80,81,82) b. For a stain to be considered a differential stain, what must it do? (81) c. Explain what happens in positive staining to

cause the reaction in the cell. (80) d. Explain what happens in negative staining that causes the final

result. (80,81) 12. a. Why are some bacteria difficult to grow in the laboratory? Relate this to what you know so far about metabolism. (64,65) b. What conditions are necessary to cultivate viruses in the laboratory? (69)

concept Mappins

Appendix E provides guidance for working with concept maps. l. Supply your own linking words or phrases in the concept map, and provide the missing concepts in the empty boxes.

Construct your own concept map using the following words as the concepts. Supply the linking words between each pair ofconcepts.

inoculation

qtaininq

isolation

biochemical tests

incubation

subculturing

inspection

source of microbes

identification

transport medium

medium

streak plate

multiplication

86

Chapter

3

Tools of the Laboratory

€ritical Thinking Questions

@ff

Critical thinking is the ability to reason and solve problems using facts and concepts. These questions can be approached from in most cases, they do not have a single correct answer. 1. Describe the steps you would take to isolate, cultivate, and identiff a microbial pathogen from a urine sample. (Hint: Look at the

Glucose Yeast extract Peptone KH2PO4

Distilled

water

has the

number of angles, and

other biochemical products. They have patented these animals and are selling them to researchers for study and experimentation. a. What do you think of creating new animals just for

Five I's.)

2. A certain medium

a

experimentation?

following composition:

b. Comment on the benefits, safery and ethics of this trend.

15 o

5o 5o

9. This is a test of your living optical system's resolving power. Prop your book against a wall about 20 inches away and determine the line in the illustration below that is no longer resolvable by your eye. See if you can determine your actual resolving power, using a millimeter ruler.

1,000 ml

a. Tell what chemical category this medium belongs to, and

explain

why this is true. b. Both A and B media in table 3.2have the necessary nutrients for S. aureus, yet they are very different. Suggest the sources ofmost required chemicals in B. c. How could you convert Staphylococcus medium (table 3.2A) into a nonsynthetic medium?

So, Naturalists obseffe,

3. a. Name four categories that biood agar fits. b. Name four differential reactions that TSIA shows.

aflea has smallerffi

c.

Observe figure 3.5. Suggest what causes the difference in growth pattern between nonmotile and motile bacteria. d. Explain what a medium that is both selective and differential does, using figure 3.8.

4. a. What kind of medium might you

make to selectively grow a bacterium that lives in the ocean? b. One that lives in the human stomach? c. What characteristic of dyes makes them useful in differential

fleas that on him prey;

media?

d. Why are intestinal bacteria able to grow on media containing

and these have smaller still

bile?

5. a. When buying a microscope, what features are most important to

lo bite'em; and so proceed,

check for? b. What is probably true of a $20 microscope that claims to magnify 1,000 x ?

adinfinit!m.

W ffi W R ft

Poem by Jonathan Swift.

6. How can one obtain 2,000x magnification with a l00x objective? 7. a. In what ways are dark-field microscopy and negative staining

10. Some human pathogenic bacteria are resistant to most antibiotics,

alike?

How would you prove a bacterium is resistant to antibiotics using laboratory culture techniques?

b. How is the dark-field microscope like the scanning electron microscooe?

8. Biotechnology companies have engineered hundreds of

different

types of mice, rats, pigs, goats, cattle, and rabbits to have genetic diseases similar to diseases of humans or to synthesize drugs and

1

I

. a.

Suggest some reasons that some microbes cannot be cultivated in

artificial media. b. What methods must be used to identify them?

Internet Search Topics

visual understandins

@ 1

87

. Figure 3.3a, b. If you were using the quadrant streak plate method to plate a very dilute broth culture (with many fewer bacteria than the broth used for 3b) would you expect to see single, isolated colonies in quadrant 2,3, or 4? Explain yow answer.

Loop containing sample

(\_*/-'-4--g-€ik /J( z\J (. ffi) W) (a)

Figure 1.6. Which of these photos from chapter Which is a TEM image?

gacle{um: E. coli

\n'

1

is an SEM image?

Fungus Thamnidium

Vitusi Herp6 simplq

""F - rrnet search Topics

Wrnt(

1. Search through several websites using the keywords "electron micrograph." Find examples ofTEM and SEM micrographs and their applications in science and technology.

2.

Search using the words "laboratory identification of anthrax" to make an outline of the basic techniques used in analysis of the microbe, under the headings of the Five I's.

3. Go to: http://wwwmhhe,com/talaro7,

and click on chapter 3, access the URLs listed under Internet Search Topics, and research the

following: a. Explore the website listed, which contains a broad base of information and images on microscopes and microscopy. Visit the photo gallery to compare different types of microscope images.

Use the interactive website listed to see clearly how the numerical aperture changes with magnification. Access this website to "play" with a scanning electron

microscope: http ://micro.magnet,fsu,edu/primer/j ava/

electronmicroscopy/magnifyl /index.html following website to experience interactive microscope games and access information on function and uses: http:// nobelprize.org/educational*games/physics/microscopes/tem/ 94...

d. Go to the

,rit ,i!

CASE FILE

4

or three wee]$ in spring 2001, nine cases of pneumonia occurred in elderly residents (median age of 86 years) living at a long-term care facility in New Jersey. Seven of the nine patients had Streptococcus pneumonloe isolated from blood cultures, with capsular serotyping revealing that all isolates were the same strain-type'1 4. Seven of the nine patients also lived in the same wing of the nursing home. Even the two patients with negative blood cultures had gram-positive diplococci in their sputum and chest X rays consistent with pneumonia. Epidemiological studies of the patients and uninfected controls revealed that all who developed pneumonia had no documented record of vaccination with the pneumococcal polysaccharide vaccine (PPV). In contrast about 50olo of infection-free patients had been vaccinated with PPV. Even though other risk factors were assessed, the lack of vaccination with PPV was the only one strongly associated with illness. Despite treatment, four of the nine patients with pneumonia died. Once the outbreak was recognized, PPV was offered to those 55 residents who had not yet been vaccinated. Thirty-seven of these were vaccinated, whereas the other 18 were either ineligible or refused the vaccine. As a control measure, the facility decided to admit in the future only those patients with a history of PPV vaccination.

7 ) >

What is pneumonio, and which microbes commonly cause it? Whot special advontoge does the copsule confer on the pathogen Streptococcus pneumoniaeZ Why are those who have been voccinated against Streptococcus pneumoniae more resistont to infection by this ogent? Cose File

CHAPTER OVERVIEW

ill

4 Wrop-Up oppeors on poge 96,

All organisms are composed of compact units of life called cells. Cells carry out fundamental activities such as growth, metabolism, reproduction, synthesis, and transport-clear indicators of life. Prokaryotic cells are the smallest, simplest, and most abundant cells on earth. Representative prokaryotes include bacteria and archaea, both of which lack a nucleus and organelles but are functionally complex. The structure of bacterial cells is compact and capable of adaptations to a multitude

of habitats. The cell is encased in an envelope that protects, supports, and regulates transport. Bacteria have special structures for motility and adhesion in the environment. 88

4.1

Characteristics of Cells and

Life

89

Bacterial cells contain genetic material in one or a few chromosomes, and ribosomes for synthesizi ng proteins. Bacteria have the capacity for reproduction, nutrient storage, dormancy, and resistance

to adverse conditions. = Shape, size, and arrangement of bacterial cells are extremely varied. i,, Bacterial taxonomy and classification are based on their structure, metabolism, and genetics. Archaea are prokaryotic cells that often live in extreme environments and possess unique biochemistry and genetics.

4.1 Characteristics of Cells and Life

Biologists have debated this idea for many years, but there is

The bodies of microorganisms such as bacteria and protozoa consist of only a single cell,1 while those of higher animals and plants often contain trillions of cells. Regardless of the organism, all cells share a few common characteristics. They tend to be spherical, cubical, or cylindrical, and the internal content of the cell, or cytoplasm, is surrounded by a cell membrane. Cells have chromosomes containing DNA and ribosomes for protein s;mthesis, and they are capable of performing highly complex chemical reactions. Aside from these similarities, most cell types fall into one of two fundamentally diflerent lines (discussed in chapter l): the small, seemingly simple prokaryotic cells and the larger, structurally more complicated eukaryotic cells. Eukaryotic cells are found in animals, plants, fungi, and protists. They contain a number of complex internal parts called organelles that perform useful functions for the cell. By convention, organelles are defined as cell components enclosed by membranes lhat carry out specific activities involving metabolism, nutrition, and synthesis. Organelles also partition the eukaryotic cell into smaller comparftnents. The most visible organelle is the nucleus, a roughly ball-shaped mass surrounded by a double membrane that contains the DNA of the cell. Other organelles include the Golgi apparatus, endoplasmic reticulum, vacuoles, and mitochondria (see

plest organism will have. The characteristics most inherent to

some agreement on a collection of properties that even the sim-

chapter 5).

Prokaryotic cells are found only in the bacteria and archaea. Sometimes it may seem that prokaryotes are the microbial "havenots" because, for the sake of comparison, they are described by what they lack. They have no nucleus or other organelles. This apparent simplicity is misleading, because the fine structure of prokaryotes can be complex. Overall, prokaryotic cells can engage in nearly every activity that eukaryotic cells can, and many can function in ways that eukaryotes carulot. After you have studied the cells as presented in this chapter and chapter 5, refer to table 5.2 (page 133), which summarizes the major differences between prokaryotic and eukaryotic cells.

What ls Life? We have stated that cells are the basic units of living things, but

it

may be useful to address exactly what

l.

it

means to be alive.

The word cell was originally coined from an Old English term meaning "small room" because ofthe way plant cells looked to early microscopists.

life

are:

1. heredity and reproduction;

2. growth and development; 3. metabolism, including cell synthesis and the release of energy;

4. movement and/or irritability; 5. cell support, protection, and storage mechanisms; and 6. the capacity to transport substances into and out ofthe cell. Although eukaryotic cells have specific organelles to perform these functions, prokaryotic cells must rely on a few simple, multipurpose cell components. As indicated in chapter 1, viruses are not cells, are not generally considered living things, and show certain signs of life only when they invade a host cell. Outside of their host cell, they are inert particles more similar to large molecules than to cells.

Heredity ond Reproduction The hereditary material of an organism lies in its genome (jee'nohm), a complete set of genetic information. It is composed of elongate strands ofDNA packed into discrete bodies called chromosomes. In eukaryotic cells, the chromosomes are located within a nuclear membrane.2 Prokaryotic DNA usually occurs in a special type of chromosome that is not enclosed by a membrane of any sort.

Living things devote a portion of their life cycle to making offspring through reproduction. In sexual reproduction, offspring are produced through the union of sex cells from two parents. In asexual reproduction, offspring originate through the division ofa single parent cell into two daughter cells. Sexual reproduction occurs in most eukaryotes, and eukaryotic cells also reproduce asexually by several processes. Many eukaryotic cells engage in mitosis (my-toh'-sis), an orderly division of chromosomes that usually accompanies cell division. In contrast, prokaryotic cells reproduce primarily by binary fission, a simple process of a cell splitting equally into two. They have no mitotic apparatus, nor do they reproduce by typical sexual means.

2. Eukaryotic cells mitochondria.

also carq' a different sort of chromosome within their

90

Chapter

4

A Survey of Prokaryotic Cells and Microorganisms

Metobolism: Chemicol and Physicol Life Processes Cells synthesize proteins using hundreds of tiny particles called ribosomes. In eukaryotes, ribosomes are dispersed throughout the cell or inserted into membranous sacs known as the endoplasmic

versatile and adaptable. The general cellular organization prokaryotic cell can be represented with this flowchart:

Piti

Fimbriae Glycocalyx Capsule, slime layer

Pr.kary.,c"

.

ueil enverope

purpose. Photosynthetic microorganisms (algae and some bacteria) trap solar energy by means of pigments and convert it to chemical energy. Photosynthetic eukaryotes contain compact, membranous units called chloroplastso which contain the pigment and perform

I

--'lI

Internal

the photosynthetic reactions. Photosynthetic reactions and pigments of prokaryotes occur not in chloroplasts but in specialized areas of the cell membrane. These structures are described in more detail in this chapter and in chapter 5.

Movement ond lrritability Although not present in all cells, true motility, or self-propulsion, is a notable sign of life. Eukaryotic cells move by locomotor organelles such as cilia, flagella, or pseudopods. Motile prokaryotes move by means of unusual flagella unique to bacteria or by special fibrils that produce a gliding form of motiliry They have no cilia or pseudopods. All cells have the capacity to respond to chemical, mechanical, or light stimuli. This quality, called irritability, helps cells adapt to a changing environment and obtain nutrients.

Protection ond Tronsport Many cells are supported and protected by rigid cell walls, which prevent them from rupturing while also providing support and shape. Among eukaryotes, cell walls occur in plants, microscopic algae, and fungi but not in animals or protozoa. The majority of prokaryotes have cell walls, but they differ in composition from the eukaryotic varieties. Cell survival depends on drawing nutrients from the external environment and expelling waste and other metabolic products from the internal environment. This two-directional transport is accomplished in both eukaryotes and prokaryotes by the cell membrane. This membrane (shown in figure 4.16) has a very similar structure in both eukaryotic and prokaryotic cells. Eukaryotes have an additional organelle, the Golgi apparatus, that assists in sorting and packaging molecules for transport and removal from the cell.

a

Appendages Flagella

reticulum. Prokaryotes have smaller ribosomes scattered throughout the cytoplasm, because they lack an endoplasmic reticulum. Eukaryotes generate energy by chemical reactions in the mitochondria, whereas prokaryotes use their cell membrane for this

of

Cell wall cett membrane Cytoplasmic matrix Ribosomes Inclusions Nucleoid/chromosome Actin cytoskeleton Endospore

Structures that are essential to the functions of all prokaryotic cells are a cell membrane, cytoplasm, ribosomes, and one (or a few) chromosome(s). The majority also have a cell wall and some form ofsurface coating or glycocalyx. Specific structures that are found in some, but not all, bacteria are flagella, pili, fimbriae, capsules, slime layers, inclusions, an actin cytoskeleton, and endospores.

The Structure of a Generalized Bacterial Cell Prokaryotic cells appear featureless and two-dimensional when viewed with an ordinary microscope, but this is only because of their small size. Higher magnification provides increased insight into their intricate and often complex structure (see figures 4.28 and 4.30). The descriptions ofprokaryotic structure, except where otherwise noted, refer to the bacteria, a category of prokaryotes with peptidoglycan in their cell walls. Figure 4.1 shows a three-dimensional illustration of a generalized (rod-shaped) bacterial cell with most of the structures fromthe flowchart. As we survey the principal anatomical features of this cell, we begin with the outer cell structures and proceed to the internal contents. Archaea-the other major group of prokaryotes-are discussed in section 4.8.

4.3 External Structures Appendages: Cell Extensions Bacteria often bear accessory appendages sprouting from their surfaces. Appendages can be divided into two major groups: those that provide motility (flagella and axial filaments) and those that provide attachments or channels (fimbriae and pili). FI a g e I I

o-

Bo cte ri o I P ro pel le rs

The prokaryotic flagellum (flah-jel'-em) is an appendage of truly

4.2 Prokaryotic Profiles: The Bacteria and Archaea The evolutionary history ofprokaryotic cells extends back over 3.5 billion years. It is now generally thought that the very first cells to appear on the earth were similar to archaea that live on sulfur componnds in geothermal ocean vents (see figure 4.33). The fact that these organisms have endured for so long in such avariety of habi-

tats indicates a cellular structure and function that are anazingly

amazing construction and is certainly uniquo in the biological world. Flagella provide the power of motility or self-propulsion. This allows a cell to swim freely through an aqueous habitat. The bacterial flagellum when viewed under high magnification displays three distinct parts: the filament, the hook (sheath), and the basal body (figure 4.2\.The filament is a helical structure composed of a protein called flagellin. It is approximately 20 nm in diameter and varies from I to 70 pm in length. It is inserted into a curved tubular hook. The hook is anchored to the cell by the basal body, a stack of rings firmly anchored through the cell wall to the cell membrane.

4.3 External Structures Fimbriae

Ribosomes

Gell

wall

91

Cell membrane

Gapsule

Slime layer

Cytoplasmic matrix Mesosome

Figure 4.1 Structure of a typical bacterialcell. Cutaway view of a rod-shaped bacterium,

showing major structural features. Note that not all components are found in all cells.

Flagellum

Figure 4.2 Details of the flagellar basal body and its position in the cellwall. The hook, rings, and rod function together as a tiny device that rotates the filament 360'. (a) Structure in gram-negative cells (b) Structure in grampositive cells.

(a)

l-

22 nm -------..1

The hook and its filament are free to rotate 360"-like a tiny propeller. This is in contrast to the flagella of eukaryotic cells, which undulate back and forth. One can generalize that all spirilla, about half of the bacilli, and a small number of cocci are flagellated (these bacterial shapes

in figure 4.23). Flagella vary both in number and arrangement according to two general patterns: (1) In a polar arrangement, the flagella are attached at one or both ends of the cell. Three subtypes of this pattern are: monotrichous* with a single flagellum; Iophotrichous* with small bunches or tufts of flagella are shown

* monotrichous (mah"-noh-trik'-us) Ctr- mono, ofte, rr[d tricho, hax. a lophotrichous (lo"-foh-trik'-us) Gr. lopho, tttft or idge.

emerging from the same site; and amphitrichous* with flagella at both poles of the cell. (2) In a peritrichous* arrangement, flagella are dispersed randomly over the surface of the cell (figure 4.3). The presence of motility is one piece of information used in the laboratory identification of various groups of bacteria. Special stains or electron microscope preparations must be used to see arrangement, because flagella are too minute to be seen in live preparations with a light microscope. Often it is sufficient to know simply whether a bacterial species is motile. One way to detect motility is to stab a tiny mass of cells into a soft (semisolid) medium in a test tube (see * amphitrichous (am"-fee-trik'-us).Gl amphi, on both sides. * peritrichous (pe1'-ee-trik'-us) Gr. peri. arottnd.

92

ffi (a)

Chapter

Figure

4.3

4

A Survey of Prokaryotic Cells and Microorganisms

Electron micrographs depicting types of flagellar arrangements.

Monotrichous flagellum on the predatory bacterium Bdellovibrio. (b) Lophotrichous flagella on Vibrio fischeri, a common marine bacterium (23,000x). (c) Unusual flagella on Aquaspirillum are amphitrichous (and lophotrichous) in arrangement and coil up into tight loops. (d) An unidentified bacterium discovered inside Porameciurn cells exhibits peritrichous flagella.

figure 3.5). Growth spreading rapidly through the entire medium is indicative of motility. Alternatively, cells can be observed microscopically with a hanging drop slide. A truly motile cell will flit, dart, or wobble around the field, making some progress, whereas one that is nonmotile jiggles about in one place but makes no progress.

Flagellar Responses Flagellated bacteria can perform some rather sophisticated feats. They can detect and move in response to chemical signals-a type of behavior called chemotaxis.* Positive chemotaxis is movement of a cell in the direction of a favorable chemical stimulus (usually a nutrient); negative chemotaxis is movement away from a repellent (potentially harmful) compound. The flagellum can guide bacteria in a certain direction because the system for detecting chemicals is linked to the mechanisms that drive the flagellum. Located in the cell membrane are clusters of receptorss that bind specific molecules coming from the immediate environment. The atlachment of sufficient numbers of these molecules transmits signals to the flagellum and sets it into rotary motion. If several flagella are present, they become aligned and rotate as a group (figure 4.4). As * chemotaxis (ke"-moh-tak'-sis)Ctr.chemo, arrangement.

chemicais, tndtaxis, anorderingor

3. Cell surface molecules that bind soecificallv with other molecules.

(b)

4.4 The operation of flagella and the mode of locomotion in bacteria with polar and peritrichous flagella. Flgure

(a) In general, when a polar flagellum rotates in a counterclockwise direction, the cell swims forward in runs. When the flagellum reverses direction and rotates clockwise, the cell stops and tumbles. (b) In peritrichous forms, all flagella sweep toward one end of the cell and rotate as a single group. (c) During tumbles, the flagella rotate in the opposite direction and cause the cells to lose coordination.

4.3 External Structures

93

Key

)ffi-# )t# Tumble

(T)

(a) No attractant

repellent

Run

or

(R)

Tumble (T)

(b) cradient o,

",*-*-l**entration

Flgure 4.5 Chemotaxis in bacteria. ff (a) A cell moves via a random series short runs and tumbles

(b)

of when there is no attractant or repellent. (b) The cell spends more time on runs as it gets closer to an attractant.

flagellum rotates counterclockwise, the cell itself swims in a smooth linear direction toward the stimulus; this action is called a run. Runs are intemrpted at various intervals by tumbles caused by the flagellum reversing its direction. This makes the cell stop and change its course. It is believed thal at1r:aclarfi molecules inhibit tumbles. increase runs. and permit progress toward the stimulus (figure 4.5). Repellents cause numerous tumbles, allowing the bacterium to redircct itself away from the stimulus. Some photosynthetic bacteria erJtibit phototaxis, a type of movement in response to light rather than chemicals. a

Periplosmic Flogello Corkscrew-shaped bacteria called spirochetes* show an unusualo miggly mode of locomotion caused by two or more long, coiled threads, the periplasmic flagella or axial Jilaments. A periplasmic flagellum is a type ofinternal flagellum that is enclosed in the space between the outer sheath and the cell wall peptidoglycan (figure 4.6). The filaments curl closely around the spirochete coils yet are free to contract and impart a twisting or flexing motion to the cell. This form of locomotion must be seen in live cells such as the spirochete ofsyphilis to be truly appreciated.

Other Appendages: Fimbrioe ond Pili The structures termed fimbria* and pilus* both refer to bacterial surface appendages thatare involved in interactions with other cells but do not provide locomotion. Fimbriae are small, bristlelike fibers emerging from the surface of many bacterial cells (figure 4.7). Their exact composition varies, but most of them contain protein. Fimbriae have an inherent

a

spiroc hete (spy'-roh-keet) Gr. speira, corl, and chaite, hair

*

fimbria (fim'-bree-ah) pl. fimbriae; L., a fringe.

* piftrs (py'-lus) pt. pili; L., hair.

tlagella (PF)

Peptidoglycan

Cell membrane (c)

Flgure

4.6

The orientation of periplasmic flagella on the

spirochete cell. (a) Electron micrograph of the spirochete Borrelia burgdorferi, the agent of Lyme disease. (b) Longitudinal section. (c) Cross section. Contraction of the filaments imparts a spinning and undulating pattern of locomotion. tendency to stick to each other and to surfaces. They may be responsible for the mutual clinging of cells that leads to biofilms and other thick aggregates ofcells on the surface ofliquids and for the microbial colonization of inanimate solids such as rocks and glass (Insight 4.1). Some pathogens can colonize and infect host tissues because of a tight adhesion between their fimbriae and epithelial cells (figure 4.7b).For example, the gonococcus (agent ofgonorrhea) colonizes the genitourinary tract, and Escherichia coli colonizes the intestine by this means. Mutant forms of these pathogens that lack fimbriae are unable to cause infections. A pilus (also called a sex pilus) is an elongate, rigid tubular structure made of a special protein, pilin. So far, true pili have been found only on gram-negative bacteria, where they ge utilized in a o'mating" process between cells called conjugationr* which involves 4. Although the term mating is sometimes used for this process, it is not a form sexual reproduction.

of

94

Chapter

4

A Survey of Prokaryotic Cells and Microorganisms

E. coli cells

Intestinal microvilli

Figure

4.7

@ Figure 4.8

Clearly evident are the sex pili forming mutual conjugation bridges between a donor (upper cell) and two recipients (two lower cells). Fimbriae can also be seen on the donor cell.

Form and function of bacterialfimbriae.

(a) Several cells of pathogenic Escherichia coli covered with numerous stiff fibers called fimbriae (30,000x). Note also the dark blue masses, which are chromosomes. (b) A row of E coli cells tightly adheres by their fimbriae to the surface of intestinal cells (12,000x). This is how the bacterium clings and gains access to the inside of cells during an infection. (6

:

Three bacteria in the process

of conjugating.

Slime layer

glycocalyx)

partial transfer of DNA from one cell to another (figure 4.8). A pilus from the donor cell unites with a recipient cell, thereby providing a cytoplasmic connection for making the transfer. Production of pili is controlled genetically, and conjugation takes place

(a)

only between compatible gram-negative cells. Conjugation in grampositive bacteria does occur but involves aggregation proteins rather than sex pili. The roles of pili and conjugation are further explored in chapter 9.

The Bacteriol Surfoce Cooting, or Glycocolyx The bacterial cell surface is frequently exposed to severe environmental conditions. The glycocalyx develops as a coating of macromolecules to protect the cell and" in some cases, help it

Capsule

(b)

adhere to its environment. Glycocalyces differ among bacteria in thickness, organization, and chemical composition. Some bacteria are covered with a loose shield called a slime layer that evi-

Figure

dently protects them from dehydration and loss of nutrients (figure 4.9a). Other bacteria produce capsules composed of

(a) The slime layer is a loose structure that is easily washed off. (b) The capsule is a thick, structured layer that is not readily removed.

4.9

Bacterial cells sectioned to show the types

of glycocalyces.

4.3

ExternalStructures

95

Biofilms-The Glue of Life Microbes rarely live a solitary existence. More

%

?

l----7

first colonisrs

often they cling together in complex masses called biofilms. The formation of these living layers is actually a universal phenomenon that all ofus have Organic surface coating observed. Consider the scum that builds up in toilet bowls and shower stalls in a short time if they \Surface are not cleaned; or the algae that collect on the walls of swimming pools; and more intimatelythe constant deposition ofplaque on teeth. Cells stick to coating. Fossils from ancient deposits tell us that microbes have been making biofilms for billions ofyears. It is through this process that they have Glycocalyx colonized most habitats on earth and created staAs cells divide, they ble communities that provide access to nutrients form a dense mat and other essential factors. Biofilms are often bound together by cooperative associations among several microbial sticky extracellular groups (bacteria, fungi, algae, and protozoa) as deposits. well as plants and animals. Substrates favorable to biofilm development have a moist. thin layer of organic material such as polysaccharides or glycoproteins deposited on Additional microbes their exposed surface. Deposition gives rise to a are attracted to slightly sticky texture that attracts primary colodeveloping film and create a mature nists, usually bacteria. These ear$ cells attach and community with begin to multiply on the surface. As they secrete complex function. their glycocalyx (receptors, fimbriae, slime layers, capsules). the cells bind to the substrate and thicken the biofilm. As the biofilm evolves, it undergoes specific Right: Experimental biofilm on a slide suspended in culture. Cells are stained red, and adaptations to the habitat in which it forms. In extracellular deposit is orange. Numbers show the progress from 3 hours to 11 hours. many cases, the earliest colonists contribute nutrients and create microhabitats that serve as a matrix for other microbes to microbes signal each other as well as human cells in ways that shape the attach and grow into the film, forming complete communities. The biofilm conditions there. varies in thickness and complexity, depending upon where it occurs and Biofilms also have serious medical implications. By one estimate how long it keeps developing. Complexity ranges from single cell layers 60% ofinfections involve the effects ofbiofilm interactions. They acto thick microbial mats with dozens of dynamic interactive layers. cumulate on damaged tissues (such as rheumatic heart valves), hard Biofilrns are a profoundly important force in the development of tertissues (teeth). and foreign materials (catheters. IUDs, artificial hip joints). Microbes in a biofilm are extremely difficult to eradicate with restrial and aquatic environments. They dwell permanently in bedrock and play roles in the earth's sediments, where they essential recycling elements, antimicrobials. New evidence indicates that bacteria in biofilms turn on leaching minerals. and forming soil. Biofilms associated with plant roots different genes when they are in a biofilm than when they are "free-floathg." promote the mufual exchange of nutrients between the microbes and roots. This altered gene expression gives the bacteria a different set ofcharacInvasive biofilms can wreak havoc with human-made structures such as teristics, often making them impervious to antibiotics and disinfectants. cooling towers, storage tanks, air conditioners, and even stone buildings. For additional discussions of biofitms. see chapters 7 and 12. New evidence now points to biofilm formation by our owrr "normal Describe some possible benefits of having biofilm flora in or on the flora"-microbes that live naturally on the body. These associations are human body. Answer available at http://www.mhhe.com/talaroT common on the skin, the oral cavity, and large intestine. In these locations,

d

t

repeating polysaccharide units, ofprotein, or ofboth. A capsule is bound more tightly to the cell than a slime layer is, and it has a

thicker, gummy consistency that gives a prominently sticky (mucoid) character to the colonies of most encapsulated bacteria

(figure 4.10u).

Specialized Functions of the Glycocalyx Capsules are formed by many pathogenic bacteria, such as Streptococcus pneumoniae (a cause of pneumonia, an infection of the lung), Haemophilus inJluenzae (one cause of meningitis), and Bacillus anthracis (the cause ofanthrax). Encapsulated bacterial cells eenerallv have

96

Chapter

4

A Survey of Prokaryotic Cells and Microorganisms

Colony without a capsule

CASE FILE

4

Wrap-Up

The outbreak of pneumococcal pneumonia described at the beginning of the chapter points out that certain bacterial structures, such as a capsule, enhance virulence. Studies have shown that the capsule allows the bacterium to avoid being engulfed by white blood cells called phagocytes. lt is an accepted fact that strains of Streptococcus pneumonioe with capsules are virulent, whereas those lacking a capsule are not. This knowledge has been used to make a vaccine using 23 types of polysaccharide capsular antigens. When this vaccine is administered, it will elicit specific antibodies that provide protection from the most common strains causing pneumococcal pneumonia. The serum antibodies that arise after vaccination specifically coat the bacterial capsule and allow for uptake of the bacteria by the host phagocytes. This disease is significant, as the Centers for Disease Control and Prevention (CDC) estimate that about a half million cases occur each year, resulting in about 40,000 deaths in the United States.

As was the case with this outbreak, the highest mortality rate (3oo/o-40olo) occurs in the elderly or in those with underlyCell body Capsule

ing medical conditions. The CDC estimates that about half of these deaths could be prevented through use of the pneumococcal vaccine. See: CDC. 2001. Outbreok of pneumococcal pneumonio among unvaccinated residents of a nursing home-New Jersey, April 2001. MMWR 50:707-710.

Figure 4.1O Appearance of encapsulated bacteria. (a) Close-up view of colonies of Bacillus species with and without capsules. Even at the macroscopic level, the moist slimy character of the capsule is evident. (b) Special staining reveals the microscopic appearance of a large, well-developed capsule (the clear "halo" around the cells) of Klebsiello.

greater pathogenicity because capsules protect the bacteria against white blood cells called phagocytes. Phagocytes are a natural body

defense that can engulf and deshoy foreign cells, which helps to prevent infection. A capsular coating blocks the mechanisms that phagocytes use to attach to and engulfbacteria. By escaping phagocytosis, the bacteria are free to multiply and infect body tissues. Encapsulated bacteria that mutate to nonencapsulated forms usually lose their pathogeniciry Other types of glycocalyces can be important in formation of biofilms. The thick, white plaque that forms on teeth comes in part from the surface slimes produced by certain streptococci in the oral cavity. This slime protects them from being dislodged from the teeth and provides a niche for other oral bacteria that, in time, can lead to dental disease. The glycocallx of some bacteria is so highly adherent that it is responsible for persistent colonization ofnonliving materials such as plastic catheters, intrauterine devices, and metal pacemakers that are in common medical use (figure 4.11).

Glycocalyx slime

Catheter surface

'p

Flgure

4.11

Biofilm.

Scanning electron micrograph of Stophylococcus oureus cells attached to a catheter bv a slime secretion.

4.4

The Cell Envelope: The Boundary Layer of Bacteria

97

The Gram Stain: A Grand Stain In 1884, Hans Christian Gram discovered a staining technique that could be used to make bacteria in infectious specimens more visible. His technique consisted of timed, sequential applications of crystal violet (the primary dye), Gram's iodine (IKI, the

Microscopic Appearance of Gell

Chemical Reaction in.Cell Wall (very magnified view)

Step

mordant), an alcohol rinse (decolorizer), and a con1 Crystal trasting counterstain. The initial counterstain used violet was yellow or brown and was later replaced by the (pnmary red dye, safranin. Since that substitution, bacteria dye) that stained purple are called gram-positive, and 2 Gram's those that stained red are called gram-negative. iodine Although these staining reactions involve an (mordant) attraction ofthe cell to a charged dye (see chapter 3), it is important to note that the terms gram-positive and gram-negative arc used to indicate not the elec3 Alcohol trical charge ofcells or dyes but whether or not a cell (decolorizer) retains the primary dye-iodine complex after decolorization. There is nothing specific in the reaction of gram-positive cells to the primary dye or in the reac4 Safranin tion of gram-negative cells to the counterstain. The (red dye different results in the Gram stain are due to differcounterstain ences in the structure of the cell wall and how it reacts to the series ofreagents applied to the cells. In the first step, crystal violet is added to the cells in a smear and stains them all the same purple color. The second and key differentiating step is the addition of the mordant-Gram's iodine. The mordant is a stabilizer that causes the dye to form large crystals that get trapped by the peptidoglycan meshwork of the cell wall. Because the peptidoglycan layer in grampositive cells is thicker, the entrapment of the dye is far more extensive in them than in gram-negative cells. Application of alcohol in the third step dissolves lipids in the outer membrane and removes the dye from the gram-negative cells. By contrast, the crystals of dye tightly embedded in the gram-positive bacteria are relatively inaccessible and resistant to removal. Because gram-negative bacteria are colorless after decolorization, their presence is demonstrated by applying the counterstain safranin in the final step. This staining method remains an important basis for bacterial clas-

sification and identification. It permits differentiation of four major

4.4 The Cell Envelope: The Boundary Layer of Bacteria The majority of bacteria have a chemically complex external covering, termed the cell envelope, that lies outside of the cytoplasm. It is composed of two main layers: the cell wall and the cell membrane. These layers are stacked together and often tightly bound into a unit like the outer husk and casings of a coconut. Although each envelope layer performs a distinct function, together they act as a sinsle unit that maintains cell intesritv.

Gram

(+)

Gram

(-)

Gram (+)

Gram

(-)

ffi ffi l'*,w

Both cell walls stain with the dve.

K

::=:::E E@ .".""."-:m No effect of iodine

Dye crystals trapped in cell

w.

f r' .. 444 -t-t-tffi ffi

Crystals remain

Outer membrane

in cell.

weakened; cell loses dye.

::.:w F{eo oye nas no etfect.

====

M

Red dye stains the colorless cell.

categories based upon color reaction and shape: gram-positive rods, gram-positive cocci, gram-negative rods, and gram-negative cocci (see table 4.4). The Gram stain can also be a practical aid in diagnosing infection and in guiding drug treatment. For example. Gram staining a fresh urine or throat specimen can help pinpoint the possible cause of infection, and in some cases it is possible to begin drug therapy on the basis ofthis stain. Even in this day ofelaborate and expensive medical technology, the Gram stain remains an important and unbeatable first tool in diagnosis.

What would be the primary concerns in selecting a counterstain dye for the Gram stain? Answer available at hup://www.mhhe.com/ talaroT

Differences in Cell Envelope Structure More than

a

hundred years ago, long before the detailed anatomy

of

bacteria was even remotely known, a Danish physician named Hans Christian Gram developed a staining technique, the Gram stainr5 that delineates two generally different groups of bacteria (Insight 4.2).The two major groups shown by this technique are

5. This text follows the American Society of Microbiology style, which calls for capitalization ofthe terms Gram stain and Gram staining and lowercase treatmenl

ofgram-negative and gram-positive, except in headings.

98

Chapter

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A Survey of Prokaryotic Cells and Microorganisms

the gram-positive bacteria and the gram-negative

bacteria. Because the Gram stain does not actually reveal the nature of these physical differences, we must turn to the electron micro-

Cell membrane

scope and to biochemical analysis. Peptidoglycan

The extent of the differences between grampositive and gram-negative bacteria is evident in the physical appearance of their cell envelopes (figure 4.12'1.1n gram-positive (see footnote 5) cells, a microscopic section resembles an openfaced sandwich with two layers: the thick cell wall, composed primarily of peptidoglycan (defined in the next section), and the cell membrane. A similar section of a gram-negative cell envelope shows a complete sandwich with three layers: an outer membrane, a thin peptidoglycan layer, and the cell membrane. Table 4.1 provides a summary of the major similarities and differences between the wall types.

Structure of Cell Walls

(a)

The cell wall accounts for a number of important bacterial characteristics. In general, it helps de-

4.f 2 @ Figure gram-negative

termine the shape of a bacterium, and it also provides the kind of strong structural support necessary to keep a bacterium from bursting or

(a) A section through a gram-positive cell wall/membrane with an interpretation of the main layers visible (85,000x). (b) A section through a gram-negative cell wall/ membrane with an interpretation of its three sandwich-style layers (90,000x).

collapsing because of changes in osmotic pressure. The cell walls of most bacteria gain their relative strength and stability from a unique macromolecule called peptidoglycan (PG). This compound is composed of a repeating framework of long glycan* chains crossJinked by

short peptide fragments (figure 4.13).

The amount and exact composition of peptidoglycan vary among the major bacterial groups. Because many bacteria live in aqueous habitats with a low solute concentration, they are constantly absorbing excess water by osmosis. Were it not for the structural support ofthe peptidoglycan in the cell wall, they would rupture from internal presswe. Several types ofdrugs used to treat infection (penicillin, cephalosporins) are effective because they target the peptide cross-links in the

(b)

and

ffi

Comparative views of the envelopes of gram-positive cells.

Comparison of Gram-Positive and Gram-Negative Cell Walls

Characteris,tic

Gram-PosJtive

Gram'Negative

Number of major layers

One

Two

Chemical composition

1

Lipopolysaccharide (LPS)

l'PTd:ttt".": lelcnotc ztclo

Lipoprotein

Lipoteichoic acid Mycolic acids and

Peptidoglycan Porin proteins

polysaccharides* Overall thickness Orr{er

rnembrane ..-.:

Periplasmic

space

Permeability to molecules

Thicker (20-80 nm)

Thinner (8-11 nm)

No

Y€$.,;r

Narrow

Extensive

More penetrable

Less penetrable

.,,.

*In

some cells. peptidoglycan, thereby disrupting its integrity. With their cell walls incomplete or missing, such cells have very little protection from lysis.* Lysozyme, afi enrpe contained in tears and saliva, provides a natural defense against certain bacteria by hydrolyzing the bonds in the g$can chains and causing the wall to break down. (Chapters I I and 12 discuss the actions of antimicrobial

chemical agents and dtogs.)

The Gram-Positive Cell Wall The bulk of the gram-positive cell wall is a thick, homogeneous sheath ofpeptidoglycan ranging from 20 to 80 nm in thickness. It also contains tightly bound a-cidic polysaccharides, including * glycan (gly' -kan) Gr., sugar. These are large polymers of simple sugan.

*/1sn (ly'-sis)Gr.,toloosen.Aprocessofcelldestruction,asoccursinbursting.

teichoic acid directly attached to the peptidoglycan and lipoteichoic acid (figure 4.14). Wall teichoic acid is a polymer of ribitol or glycerol and phosphate embedded in the peptidoglycan sheath. Lipoteichoic acid is similar in structure but is atlached to the lipids in the plasma membrane. These molecules appear to function in cell wall maintenance and enlargement during cell division. They also move cations into and out of the cell and stimulate a specific immune response (antigenicity). The cell wall of gram-positive bacteria is often pressed tightly against the cell membrane with very little space between them, but in some cells, a thin periplasmic* space is evident between the cell membrane and cell wall. * periplasmic (pe1'-ih-plaz'-mik) Grsubstances

ofa cell.

peri,

arcLrnd, and plastos, the

flfid

4.4

The Cell Envelope: The Boundary Layer of Bacteria

99

(a) The peptidoglycan of a cell wall forms a pattern similar to a chainlink fence. lt is actually one giant molecule that surrounds and supports the cell.

(b) This shows the molecular pattern ol peptidoglycan. lt has alternating glycans (G and M) bound together in long strands.The G stands for N-acetyl glucosamine, and the M stands for N-acetyl muramic acid. Adjacent muramic acid molecules on parallel chains are bound by a crosslinkage of peptides (yellow).

(c) An enlarged view of the links between the muramic acids. Tetrapeptide chains branching off the muramic acids connect by amino acid interbridges. The amino acids in the interbridge can vary or may be lacking entirely. lt is this linkage that provides rigid yet flexible support to the cell.

chains

ffi'o

--GE-

?o

HsC

-C-H c

NH

C=O I

CHs I

L--alanine o)

I

.E

o o.

L-alanine

E

D-glutamate

,o

|glutamate I

L-lysine

I

-- -

L-lVsine

T

I

\|uJn" - llydne \-gtycine

D-alanine

.- falanine

-

-{ycine

-glVclne

Interbridge

@ Figure 4.13

Structure of peptidoglycan in the cetl watl.

The Grom-Negotive Cell Wall The gram-negative cell wall is more complex in morphology because it is composed of an outer membrane (OM) and a thinner shell of peptidoglyoan (see figwes 4.12 and 4.14). The outer membrane is somewhat similar in construction to the cell membrane, except that it contains specialized types of lipopolysaccharides and lipoproteins. Lipopolysaccharides are composed of lipid molecules bound to polysaccharides. The lipids form the matrix of the top layer of the OM and the polysaccharide strands project from the lipid surface. The lipid portion may become toxic when it is released during infections. Its role as an endotoxin is described in chapter 13. The polysaccharides give rise to the

somatic (O) antigen in gram-negative pathogens and can be used

in identification. They may also function as receptors and in blocking host defenses. Two types of proteins are located in the OM. The porins are inserted in the upper layer of the outer membrane. They have some

regulatory control over molecules entering and leaving the cell. Many qualities ofthe selective permeability of gram-negative bacteria to bile, disinfectants, and drugs are due to the porins. Some structural proteins are also embedded in the upper layer of the OM. The bottom layer of the outer membrane is similar to the cell membrane in its overall structure and is composed of phospholipids and lipoproteins.

100

Chapter

4

A Survey of Prokaryotic Cells and Microorganisms

Gram-Positive

Key

It

Pentdodvcan

I

Teichoic acid

t

Phosphotipid

lt$:[ii3*

fl @

to,'n t-ipoprotein

Lipopotysaccharide

{ Flgure

4.14

A comparison of the detailed structure of gram-positive and gram-negative cetl envelopes and walls.

The bottom layer of the gram-negative wall is a single, thin nm) sheet of peptidoglycan. Although it acts as a somewhat protective rigid structure as previously describe4 its thinness gives gram-negative bacteria a relatively greater flexibility and sensitivity to lysis. There is a well-developed periplasmic space above and below the peptidoglycan. This space is an important reaction site for a large and varied pool ofsubstances that enter and leave the cell.

(l-3

Procticol Considerotions of Differences

in Cell Woll Structure Variations in cell wall anatomy contribute to several differences between the two cell types besides staining reactions. The outer membrane contributes an exta barrier in gram-negative bacteria that makes them more impervious to some antimicrobic chemicals such as dyes and disinfectants, so they are generally more difficult to inhibit or kill than are gram-positive bacteria. One exception is for alcohol-based compounds, which can dissolve the lipids in the outer membrane and disturb its integrify. Treating infections caused by gram-negative bacteria often requires different drugs from gram-positive infections, especially drugs that can cross the outer membrane. The cell wall or its parts can interact with human tissues and contribute to disease. The lipids have been referred to as endotoxins because they stimulate fever and shock reactions in gram-negative infections such as meningitis and typhoid fever. Proteins attached to the outer portion of the cell wall of several gram-positive species,

including Corynebacterium diphtheriae (the agent of diphtheria) and Streptococcus pyogenes (the cause of sfiep throat), also have toxic properties. The lipids in the cell walls of certain Mycobaeterium species are harmful to human cells as well. Because most macro-

molecules in the cell walls are foreign to humans, they stimulate antibody production by the immune system (see chapter 15).

Nontypicol Cell Wolls Several bacterial groups lack the cell wall structure of gram-positive

or gram-negative bacteria, and some bacteria have no cell wall at all. Although these exceptional forms can stain positive or nega-

tive in the Gram stain, examination of their fine structure and chemistry shows that they do not fit the descriptions for typical gram-negative or -positive cells. For example, the cells of Mycobqcterium and Nocardia contain peptidoglycan and stain grampositive, but the bulk of their cell wall is composed of unique types of lipids. One ofthese is avery-long-chain fatty acidcalledmycolic acid, or cord factor, that contributes to the pathogenicity of this group (see chapter l9). The thick, waxy nature imparted to the cell wall by these lipids is also responsible for a high degree of resistance to certain chemicals and dyes. Such resistance is the basis for the acid-fast stain used to diagnose tuberculosis and leprosy. In this stain, hot carbol fuchsin dye becomes tenaciously attached (is held fast) to these cells so that an acid-alcohol solution will not remove the dye. Because they are from a more ancient and primitive line of prokaryotes, the archaea exhibit unusual and chemically distinct cell walls. In some, the walls axe composed almost entirely of polysaccharides, and in others, the walls are pure protein; but as a group, they all lack the true peptidoglycan structure described previously. Because a few archaea and all mycoplasmas (next section) lack a cell wall entirely, their cell membrane must serve the dual functions of support as well as transport.

4.4

Flgure

4.15

The Cell Envelope: The Boundary Layer of Bacteria

101

Scanning electron micrograph of

Mycoplasmo pneumonioe (62,000x). Cells like these that naturally lack a cell wall exhibit extreme variation in shape.

Mycoplasmas and Other Cel l-Wal l- Deficient Bacteria Mycoplasmas are bacteria that naturally lack a cell wall. Although other bacteria require an intact cell wall to prevent the bursting of the cell, the mycoplasma cell membrane contains sterols that make it resistant to lysis. These extremely tiny bacteiannge from 0.1 to 0.5 pm in size. They range in shape from filamentous to coccus or doughnut-shaped. This property of extreme variations in shape is a type of pleomorphism.* They can be grown on artificial media,

although added sterols are required for the cell membranes of some species. Mycoplasmas are found in many habitats, including plants, soil, and animals. The most important medical species is Mycoplasma pneumoniae (figure 4.15), which adheres to the epithelial cells in the lung and causes an atypical form of pneumonia in humans. Some bacteria that ordinarily have a cell wall can lose it during part of their life cycle. These wall-deficient forms are referred to as L forms or L-phase variants (for the Lister Institute, where they were discovered). L forms arise naturally from a mutation in the wallforming genes, or they can be induced artificially by teatnent with a chemical such as lysozyme or penicillin that disrupts the cell wall. When a gram-positive cell is exposed to either of these two chemicals, it will lose the cell wall completely and become a protoplast* a frag-

ile cellbounded onlyby amembrane that is highly susceptible to $sis. A gram-negative cell exposed to these same substances loses its peptidoglycan but retains its outer membrane, leaving a less fragile but nevertheless weakened spheroplast * Evidence points to a role for L forms in certain chronic infections.

Cell Membrane Structure Appearing just beneath the cell wall is the cell, or cytoplasmico membrane, a very thin (5-10 nm), flexible sheet molded completely around the cytoplasm. In general composition, it is a lipid bilayer with proteins embedded to varying degrees (figure 4.16). a pleomorphism pleon, more, and morph, form or shape. Qt lee"-oh-mor'-fizn) Gr. The tendency for cells ofthe same species to vary to some extent in shape and size. * protoplast Gttoh'-toh-plast) Gr. p/oto, fitst, and plasfos, formed. a spheroplast (sfet'-oh-plast) Ctr. sphaira, spherc.

Flgure

4.16

Cell membrane structure.

A generalized version of the fluid mosaic model of a cell membrane indicates a bilayer of lipids with globular proteins embedded to some degree in the lipid matrix. This structure explains many characteristics of membranes, including flexibility, solubility,

permeability, and transport.

The structure, first proposed by S. J. Singer and G. L. Nicolson,

is called the fluid mosaic model. The model describes a membrane as a continuous bilayer formed by lipids with the polar heads oriented toward the outside and the nonpolar heads toward the center of the membrane. Embedded at numerous sites in this bilayer are various-size globular proteins. Some proteins are situ'

ated only at the surface; others extend fully through the entire membrane. The configuration of the inner and outer sides of the membrane can be quite different because of the variations in protein shape and position. Membranes are dynamic and constantly changing because the lipid phase is in motion and many proteins can migrate freely about. This fluidity is essential to such activities as engulfrnent of food and discharge or secretion by cells. The structuxe ofthe lipid phase provides an impenetrable barrier to many substances. This property accounts for the selective permeability and capacity to regulate transport of molecules. Bacterial cell membranes have this typical structure, containing primarily phospholipids (making up about 30o/a40yo of the membrane mass) and proteins (contributing 60%70%). Major exceptions to this description are the membranes of mycoplasmas, which contain high amounts of sterols-rigid lipids that stabilize and reinforce the membrane-and the membranes of axchaea, which contain unique branched hydrocarbons rather than

fatty acids. In some locations, the cell membrane forms internal folds in the cytoplasm called mesosomes* (see figure 4.1). These are prominent in gram-positive bacteria but are harder to see in gramnegative bacteria because of their relatively small size. Mesosomes presumably increase the internal surface area available for membrane activities. It has been proposed that mesosomes participate in cell wall synthesis and guiding the duplicated bacterial chromosomes into the two daughter cells during cell division (see figure 7.14). Some scientists arq not convinced that mesosomes exist in live cells and believe they are artifacts that appear in * mesosome (mes'-oh-sohm) Gr, zesoq middle, and soma,body,

102

Chapter

4

A Survey of prokaryotic Cells and Microorganisms

bacteria fixed for electron microscopy. Support for the existence of functioning internal membranes comes from specialized prokaryotes such as cyanobacteria and even mitochondria, considered as a type of prokaryotic cell. Photosynthetic prokaryotes such as cyanobacteria contain dense stacks of internal membranes that cany the photosynthetic pigments (see figure 4.28a).

Functions of the Cell Membrone Because bactsria have none of the eukaryotic organelles, the cell membrane provides a site for energy reactions, nutrient processing, and slmthesis. A major action of the cell mernbrane is to regulate transport, that is, the passage of nutrients into the cell and the discharge of wastes. Although water and small uncharged molecules can diffirse across the membrane unaided, the membrane is a selectively permeable structure with special carrier mechanisms for passage of most molecules (see chapter 7). The glycocalyx and cell wall can bar the passage of large molecules, but they are not the primary transport apparatus. The cell membrane is also involved in secretion, or the release of a metabolic product into the extracellular environment. The membranes of bacteria are an important site for a number of metabolic activities. Most en4rrnes of respiration and AIp synthesis reside in the cell membrane because prokaryotes lack mitochondria (see chapter 8). En4rme structures located in the cell membrane also help synthesize structural macromolecules to be incorporated into the cell envelope and appendages. Other products (enzymes and toxins) are secreted by the membrane into the extracellular environment.

s =

s €

Cells demonstrate a number of characteristics essential to life. parts of cells and macromolecules do not show these characteristics. Cells can be divided into two basic types: prokaryotes and eukaryotes. Prokaryotic cells include the bacteria and archaea. Prokaryotes are the oldest form of cellular life. They are also the most widely dispersed, occupying every conceivable microclimate on the planet.

The external structures ofbacteria include appendages (flagella, fimbriae, and pili) and the glycocalyx.

ffi Flagella vary in number and arrangement

as

4.5 Bacterial Internal Structure Contents of the Cell Cytoplasm The cell membrane surrounds a complex solution referred to as cytoplasm, or cytoplasmic mafrix. This chemical ..pool" is a prominent site for many of the cell's biochemical and synthetic activities. Its major component is water (70o/o-80o/o), which serves as a solvent for a complex mixture of nutrients including sugars, amino acids, and other organic molecules and salts. The components of this pool serve as building blocks for cell synthesis or as sources of energy. The cytoplasm also holds larger, discrete bodies such as the chromosome, ribosomes, granules, and actin strands.

Bacteriol Chromosomes ond Plosmids: The Sources of Genetic Informotion The hereditary material of most bacteria exists in the form of a single circular strand of DNA designated as the bacterial chromosome, although a few bacteria have multiple or linear chromosomes. By definition, bacteria do not have a frue nucleus. Their DNA is not enclosed by a nuclear membrane but instead is aggregated in a central area of the cell called the nucleoid. The chromosome is actually an extremely long molecule of DNA that is tightly coiled to fit inside the cell compartment. Arranged along its length are genetic units (genes) that carry information required for bacterial maintenance and growth. When exposed to special stains or observed with an electron microscope, chromosomes have a granular or fibrous appearance (figure 4.17). Although the chromosome is the minimal genetic requirement for bacterial survival, many bacteria contain otheq nonessential pieces of DNA called plasmids. These tiny strands exist apart from the chromosome, although at times they can become integrated into it. During bacterial reproduction, they are duplicated and passed on to offspring. They are not essential to bacterial growth and metabo-

lism, but they often confer protective traits such as resisting drugs and producing toxins and enzrymes (see chapter 9). Because they can be readily manipulated in the laboratory and transferred from

well as in the type and

rate of motion they produce.

s

The cell envelope is the complex boundary structure surrounding bacterial cell. It consists of the cell wall and membrane. In gramnegative bacteia, the cell envelope has three layers-the outer membrane, peptidoglycan, and cell membrane. In gram-positive bacteria, there are two-a thick layer of peptidoglycan and the cell a

membrane.

€ F

s s

In a Gram stain, gram-positive bacteria retain the crystal violet and stain purple. Gram-negative bacteria lose the crystal violet aad stain red from the safranin counterstain. Gram-positive bacteria have thick cell walls of peptidoglycan and acidic polysacchaiides such as teichoic acid, and they have a thin periplasmic space. The cell walls of gram-negative bacteria are thinner and have a wide periplasmic space. The outer membrane of gram-negative cells contains lipopolysaccharide (LPS). LPS is toxic to mammalian hosts. The bacterial cell membrane is typically composed ofphospholipids and proteins, and it performs many metabolic functions as well as transport activities.

Figure

4.17

Chromosome structure.

Fluorescent staining highlights the chromosomes of the bacterial pathogen Salmonella enteriditis. The cytoplasm is orangg and the chromosome fluoresces bright yellow (1,5OOx).

4.5

Bacterial lnternal Structure

103

Fibosome (70S)

Large subunit

Small subunit

(50s)

(30s)

4.18 A modelof a prokaryotic ribosome, showing the small (30S) and targe (5OS) subunits, both separate and ioined. Flgure

one bacterial cell to anotheq plasmids are an important agent in modern genetic engineering techniques'

Ribosomes: Sites of Protein Synthesis A bacterial cell contains thousands of ribosomes, which are made of RNA and protein. When viewed even by very high magnification, ribosomes show up as fine, spherical specks dispersed throughout the cytoplasm that often occur in chains (polysomes). Many are also attached to the cell membrane. Chemically, a ribosome is a

combination of a special type of RNA called ribosomal RNA, or rRNA (about 600/o), andprotein (40%). One method of characteizing ribosomes is by S, or Svedberg,o units, which rate the molecular sizes of various cell parts that have been spun down and separated by molecular weight and shape in a centrifuge. Heavieq more compact structures sediment faster and are assigned a higher S rating. Combining this method of analysis with high-resolution electron micrography has revealed that the prokaryotic ribosome, which has an overall rating of 70S, is actually composed of two smaller subunits (figure 4.18). They fit together to form a miniature "factoty" where protein synthesis occurs. We examine the more detailed functions of ribosomes in chapter 9.

lnclusions, or Gronules: Storoge Bodies Mostbacteria are exposedto severe shifts in the availabil$ of food. During periods of nutrient abundance, some can compensate by storing nutrients as inclusion bodies, or inclusions, of varying size, number, and content. As the environmental source of these nutrients becomes depleted, the bacterial cell can mobilize its own storehouse as required. Some inclusion bodies contain condensed, energy-rich organic substances, such as glycogen and poly p-hydroxybutyrate

(P[IB), within special single-layered membranes (figure 4.L9a). A unique type of inclusion found in some aquatic bacteria is gas vesicles that provide buoyancy and flotation. Other inclusions, also 6. Named in honor ofT. Svedberg, the Swedish chemist who developed the ultracentrifuge in 1926.

Flgure

4.19

Bacterial inclusion bodies.

(a) Large particles (pink) of polyhydroxybutyrate are deposited in an insoluble, concentrated form that provides an ample, long-term supply of that nutrient (32,500x). (b) A section through Aquospirillum reveals a chain of tiny iron magnets (magnetosomes = MP). These unusual bacteria use these inclusions to orient within their habitat ('123,000x).

called granules, contain crystals of inorganic compounds and are not enclosedby membranes. Sulfur granules of photosynthetic bacteria and po$phosphate granules of Corynebacterium and Mycobacterium are of this type. The latter represent an important source ofbuilding blocks for nucleic acid and ATP synthesis. They have been termed metachromatic granules because they stain a contrasting color (red, purple) in the presence of methylene blue dye. Perhaps the most remarkable cell granule is involved not in nutrition but rather in navigation. Magnetotactic bacteria contain crystalline particles of iron oxide (magnetosomes) that have magnetic properties (figure 4.19b). These granules occur in a variety of bacteria living in oceans and swamps. Their primary function is to orient the cells in the earth's magnetic fiel4 somewhat like a compass. It is thought that magnetosomes direct these bacteria into locations with favorable oxygen levels or nutrient-rich sediments.

The Bacterisl Cytoskeleton Until very recently, bacteriologists thought bacteria lacked any real form of cytoskeleton.T The cell wall was considered to be the sole

7. An inbacellular fiamework of fibers and tubules that bind and support eukaryotic cells.

r04

Chapter

4

A Survey of Prokaryotic Cells and Microorganisms

Actin filaments

Figure

4.2O

Bacterial cytoskeleton

of

Bacillus.

Fluorescent stain of actin fibers appears as fine helical ribbons wound inside the cell.

framework involved in support and shape. Research into the fine structure of certain rod- and spiral-shaped bacteria has provided several new insights. It seems that many of them possess an internal network of protein polymers that is closely associated with the wall (figure 4.20). The proteins are chemically similar to the actin filaments universal in the cytoskeleton of eukaryotic cells. presently this bacterial actin is thought to help stabilize shape. It may also influence cell wall formation by providing sites for synthesis when the wall is being repaired or enlarged.

Bacterial !1_dospores: An Extremely Resistant Life Form Ample evidence indicates that the anatomy of bacteria helps them adjust rather well to adverse habitats. But of all microbial structures, nothing can compare to the bacterial endospore (or simply spore) for withstanding hostile conditions and facilitating survival. Endospores are dormant bodies produced by the bacteria Bacillus, Clostridium, and Sporosarcina. These bacteria have a two-phase

life cycle that shifts between a vegetative cell and an endospore (figure 4.21). The vegetative cell is the metabolically active and growing phase. When exposed to certain environmental signals, it forms an endospore by a process termed sporulation. The spore exists in an inert, resting condition that is capable ofhigh resistance and very long-term survival.

Endospore Formation ond Resistance The major stimulus for endospore formation is the depletion of nutrients, especially amino acids. Once this stimulus has been received by the vegetative cell, it converts to a committed sporulating cell called a sporangium. Complete transformation of a vegetative cell into a sporangium and then into an endospore requires 6 to 12 hours in most spore-forming species. Figure 4.22 illustrates some major physical and chemical events in this process.

Figure

4.21 Development of endospores.

These biological "safety pins,, are actually stages in endospore formation of Bocillus subtilis, stained with fluorescent proteins. The large red and blue cell is a vegetative cell in the early stages of sporulating. The developing spores are shown in green and orange.

Bacterial endospores are the hardiest of all life forms, capable of withstanding extremes in heat, drying, freezing, radiation, and chemicals that would readily kill ordinary cells. Their survival under such harsh conditions is due to several factors. The heat resistance ofspores has been linked to their high content ofcalcium and dipicolinic acid, although the exact role of these chemicals is not yet clear. We know, for instance, that heat destroys cells by inactivating proteins and DNA and that this process requires a certain amount of water in the protoplasm. Because the deposition of cal_ cium dipicolinate in the endospore removes water and leaves the endospore very dehydrated, it is less vulnerable to the effects of heat. It is also metabolically inactive and highly resistant to damage from further drying. The thick, impervious cortex and spore coats also protect against radiation and chemicals. The longevity of bacterial spores verges on immortality! One record describes the isolation ofviable endospores from a fossilized bee that was 25 million years old. More recently, microbiologists unearthed a viable endospore from a 250-million-year-old salt crystal. Initial analysis of this ancient microbe indicates it is a species of Bacillus that is genetically different from known species.

A.NOTE ABOUT TERMINOLOGY word spore can have more than one usage in microbiology. lt is a generic term that refers to any tiny compact cells that are produced by vegetative or reproductive structures of microorganisms. Spores can be quite variable in origin, form, and function. The bacterial type discussed here is called an endospore, because it is produced inside a cell. lt functions in sirvival, not in reproduction, because no increase in cell numbers is involved in its formation. In contrast, the fungi produce many different typ-es of spores for both survival and reproduction (see chapter 5). The

4.5

Bacterial Internal Structure

105

Chromosome Chromosome

Core of spore

Sporulation Cycle Exosporium Spore coat Cortex Core

Foresoore Sporangium

e.ro13ss Flgure 4.22 @ germrnauon.

A typical sporulation cycle

in

Bocillusspecies from the active vegetative cell

to release and

(1o,ooox) cross section of a single spore showing the dense This process takes, on average, about l0 hours. lnset is a high magnification protective layers that surround the core with its chromosome'

The Germinotion of EndosPores After lying in a state of inactivity, endospores can be revitalized when favorable conditions arise. The breaking of dormancy, or germination, happens in the presence of water and a specific germination agent. Once initiated, it proceeds to completion quite rapidly (1% hours). Although the specific germination agent varies among species, it is generally a small organic molecule such as an amino

acid or an inorganic salt. This agent stimulates the formation of

hydrolytic (digestive) enz5imes by the endospore membranes' These enzymes digest the cortex and expose the core to water' As the core rehydrates and takes up nutrients, it begins to grow out ofthe endospore coats. In time, it reverts to a fully active vegetative cell, resuming the vegetative cycle (figxe 4.22).

Medicol Significonce of Bocterial Spores Although the majority of spore-forming bacteria are relatively

harmless, several bacterial pathogens are sporeformers' In fact, some aspects of the diseases they cause are related to the persistence and resistance oftheir spores' Bacillus qnthracis, the agent of anthrax, has been a frequent candidate for bioterrorism. The genus Clostridium includes even more pathogens, including C. tetqni, the cause of tetanus (lockjaw), and C. perfringens, the cause of gas gangrene. When the spores of these species are embedded in a wound that contains dead tissue, they can germinate, grow, and release potent toxins. Another toxin-forming species, C, botulinum, is the agent of botulism, a deadly form of food poisoning. These diseases are discussed further in chapter 19.

106

Chapter

4

A Survey of prokaryotic Cells and Microorganisms

Because they inhabit the soil and dust, encospores are a constant intruder where sterility and cleanliness are important. They resist ordinary cleaning methods that use boiling water, soaps, and disinfectants, and they frequently contaminate cultures and media. Hospitals and clinics must take precautions to guard against the potential harmful effects of endospores in wounds. Endospore destruction is a particular concern ofthe food-canning industry. Sev_ eral endospore-forming species cause food spoilage or poisoning. Ordinary boiling (100"C) will usually not destroy such spores, so canning is carried out in pressurized steam at l2C,C for 20 to 30 minutes. Such rigorous conditioris will ensure that the food is sterile and free from viable bacteria.

mentioned earlier in conjunction with periplasmic flagella is the spirochete, a more flexible form that resembles a spring. Refer to table 4,2 for a comparison of other features of the two helical bacterial forms. Because bacterial cells look two-dimensional and flat with traditional staining and microscope techniques, they are seen to best advantage using a scanning electron microscope to empha_ size their striking three-dimensional forms (figtrre 4.23). It is common for cells of a single species to show pleomorphism (figure 4.24\.Tttts is due to individual variations in cell wall struc_ ture caused by nutritional or slight hereditary differences. For exam_ ple, although the cells of Corynebacterium diphtheriae aregenerally considered rod-shaped, in culture they display club-shaped swollen, curved, filamentous, and coccoid variations. pleomorphism reaches an extreme in the mycoplasmas, which entirely lack cell walls and thus display extreme variations in shape (see figure 4.15).

s x

The cytoplasm of bacterial cells serves as a solvent for materials used in all cell functions.

The genetic material of bacteria is DNA. Genes are arranged on large, circular chromosomes. Additional genes are carried on plasmids. E Bacterial ribosomes are dispersed in the cytoplasm in chains (polysomes) and are also embedded in the cell membrane. E Bacteria may store nutrients in their cytoplasm in structures called inclusions. Inclusions vary in structure and the materials that are

*

stored. Some bacteria manufacture long actin filaments that help stabilize

their collular shape. F A few families ofbacteria produce dormant bodies called endo_ spores, which are the hardiest of all life forms, surviving for thou_ sands and even millions of years.

s

Examples of sporeformers arethe generuBacillus andClostridium. both ofwhich contain deadly pathogens.

4.6 Bacterial

Shapes, Arrangements,

and Sizes For the most part, bacteia function as independent single-celled, or unicellular, organisms. Although it is fue that an individual bacterial cell can live attached to others in colonies or other such groupings, each one is fully capable of carrying out all necessary life activities, such as reproduction, metabolism, and nutrient processing (unlike the more specialized cells of a multicellular organism;. Bacteria exhibit considerable variety in shape, size, and colonial arrangement. It is convenient to describe most bacteria by one of three general shapes as dictated by the configuration ofthe cell wall (figure 4.23). Ifthe cell is spherical or ball-shaped, the bacterium is described as a coccus (kok'-us). Cocci can be perfect spheres, but they also can exist as oval, bean-shaped, or even pointed variants. A cell that is cylindrical (longer than wide) is termed a rod, or bacillus (bah-sil'-lus). There is also a genus named Baciilus. Asmight be expected, rods are also quite varied in their actual form. Depending on the bacterial species, they can be blocky, spindle-shape{ round_ ended, long and threadlike (filamentous), or even clubbed or drum_ stick-shaped. when arod is shortandplump, it is calleda coccobacillusl

ifit

is gent$ curved, it is a

vibrio (vib,-ree-oh). A bacterium having the shape ofa curviform or spiral-shaped cylinder is called a spirillum (spy-ril,-em), a rigid helix, twisled twice or more along its axis (like a corkscrew). Another spiral cell

The cells ofbacteria can also be categoized according to ar_ rangement, or style of grouping. The main factors influencing the arrangement of a particular cell type are its pattern of division and how the cells remain attached afterward. The greatest variety in ar_ rangement occurs in cocci (figure 4.25).They may exist as singles, in pairs (diplococci*), in tetrads (groups offour), in irregular clusters (both staphylococci* and micrococci*), or in chains ofa few to hundreds of cells (streptococci). An even more complex group_ ing is a cubical packet of eight, sixteen, or more cells called a sarcina (sar'-sih-nah). These different coccal groupings are the re_ sult of the division of a coccus in a single plane, in two perpendicu_ lar planes, or in several intersecting planes; after division, the resultant daughter cells remain attached. Bacilli are less varied in arrangement because they divide only in the transverse plane (perpendicular to the axis). They oc_ cur either as single cells, as a pair of cells with their ends attached (diplobacilli), or as a chain of several cells (streptobacilli). A palisades (pal'-ih-saydz) arrungement, typical of the corynebac_ teria, is formed when the cells of a chain remain partially attached by a small hinge region at the ends. The cells tend to fold (snap) back upon each other, forming a row of cells oriented side by side (see figure 4.24).The reaction can be compared to the behavior of boxcars on a jackknifed train, and the result looks superficially like an irregular picket fence. Spirilla are occasionally found in short chains, but spirochetes rarely remain attached after division. Comparative sizes of typical cells are presented in figure 4.26.

* e

a

Most bacteia have one ofthree general shapes: coccus (sphere), bacillus (rod), or spiral, based on the configuration ofthe cell wall. Two types of spiral cells are spirochetes and spirilla. Shape and arrangement ofcells are key means ofdescribing bacteria. Arrangements of cells are based on the number of planes in which a given cell type divides.

Cocci can divide in many planes to form pairs (diplococci), chains (sheptococci), packets (sarcinae), or clusters (micrococci or staphy_ lococci). Bacilli divide only in the transverse plane. If they remain attached, they form pairs, chains, or palisades arrangements.

a

diplot:otc'i Ctr.diplo, double. staphylot:occi (staf'-ih-1oh-kok,-seye) * mitrococci Gt. mihos, small. +

Gr. staphyle, a bunch

ofgrapes.

.o{Wn

Key to MicrograPhs (d) Aquaspirillum (7,500x) (a) Deinococcus (2,OO0x) (b) Lactobacillus bulgaricus (5,000x) (c) Vibrio cholerae (13,000x) (e) Spirochetes on a lilter (14,000x) (t) Streptomyces (1 ,500x)

Flgure

4.23

Common bacterial shapes.

and branching filaments' Below each shape is Drawings show examples of shape variations for cocci, rods, vibrios, spirilla, spirochetes, micrograph of a representative example.

ffi Spirilla

Comparison of the Two Spiral-Shaped Bacteria

of

Overall Appearance

Mode of Locornotion

Number of

Gram Reaction (Cell willType)

Examples

Helical Turns

Rigid helix

Polar flagella; cells swim by rotating around like corkscrews; do not

Varies from

Grarn'negative

Most are harmless;

{#

SPirilla

Spirochetes

a

db#

one species,

1to20

Spirillum minori causes rat bite fever.

flex; have one to several flaeella: can be in tufts

Periplasmic fl agella within sheath; cells flex; can swim by rotation or by creeping on surfaces: have 2 to 100 periplasmic fl agella Curved or spiral forms: Flexible helix

lmportant Types

Varies from

3to70

Gram-negative

Treponema pallidum, cause

of syphilis;

ia

and Leptospira, important pathogens

B orrel

Spirillum/Spirochete

t07

108

Chapter

4

A Survey of prokaryotic Cells and Microorganisms

(a) Division in one plane

Diplococcus (two cells)

Streptococcus (variable number of cocci in chains)

e(D-ee9gqg

C fl ,E)

*-Et (b) Division in two perpendicular planes

Flgure 4.-24 Pleomorphism and other morphological features of Corynebacterium. Cells are irregular in shape and size (gOOx). This genus typically exhibits a palisades arrangement, with cells in parallel ariay (inseQ. Close examination will also reveal darkly stained granules called metachromatic granules.

4.7 Classification

in the Prokaryotae

lutionary origins. Since classification was staxted uto*d 200 years ago, several thousand species of bacteria and axchaea have been identified, named, and cataloged. Tracing the origins of and evolutionary relationships among bacteria has not been an easy task. As a rule, tiny, relatively soft organisms do not form fossils very readily. Several times since the 1960s, however, scientists have discovered microscopic fossils of prokaryotes that look very much like modern bacteria. some of the rocks that contain these fossils have been dated back billions of years (see figure 4.28c). One ofthe questions that has plagued taxonomists is, What char_

acteristics are the most indicative of closeness in ancestry? Early bacteriologists found it convenient to classify bacteria according to shape, variations in arrangement, growth characteristics, and habitat. However, as more species were discovered and as techniques for studying their biochemistry were developed, it soon became clear that similarities in cell shape, arrangement, and staining reactions do not automatically indicate relatedness. Even though the gram_ negative rods look alike, there are hundreds of different species, with highly significant differences in biochemistry and genetics. If we at_ tempted to classiff them on the basis of Gram stain and shape alone, we could not assign them to a more specific level than class. Increasingly, classification schemes are turning to genetic and molecular traits that cannot be visualized under a microscope or in culture. The methods that a microbiologist uses to identify bacteria to the level of genus and species fall into the main categories of morphology (microscopic and macroscopic), bacterial physiology

Tetrad (cocci in packets of four)

{\ Sarcina (packet of cells)

fb

Systems

classification systems serve both practical and academic purposes. They aid in differentiating and identifring unknown species in medical and applied microbiology. They are also useful in organiz_ ing bacteria and as a means of studying their rerationships and evo-

C

(c) Division in several planes

&S4

ffi

lrregular cluslers (number of cells varies)

Staphylococcus and Micrococcus

ofl

Flgure 4.25 Arrangement of cocci resulting from different planes of cell division. (a) Division in one plane produces diplococci and streptococci.

(b) Division in two planes at right angles produces tetrads and

packets. (c) Division in several planes produces irregular clusters.

or biochemistry, serological analysis, and genetic techniques (chap_ ter 17 and appendix table D.8). Data from a cross section of such tests can produce a rurique profile of each bacterium. Final differentiation of any unknown species is accomplished by comparing its profile with the characteristics ofknovrm bacteria in tables, charts, and keys (see figure 17.5). Many of the identification systems are automated and computerized to process data and provide a "best fit" identification. However, not all methods are used on all bacteria. A few bacteria can be identified by placing them in a machine that analyzes only the kind of fatty acids they contain; in contrast, some are identifiable by a Gram stain and a few physiological tests; others may require a di_ verse specfrum of morphological, biochemical, and genetic tests.

4.7

Classification Systems in the Prokaryotae

109

Human hair

Ragweed pollen

Lymphocyte

Yeast cell

Ragweed pollen

E. coli

2wm

Flgure 4.27 A universal phylogenetic tree

as proposed

by Norman Pace. Staphylococcus

1pm

Rhinovirus 0.03 pm (30 nm)

Flgure 4.26 The dimensions of bacteria. The sizes of bacteria range from those iust barely visible with light microscopy (0.2 Um) to those measuring a thousand times that size. Cocci measure anywhere from 0.5 to 3.0 pm in diameter; bacilli range from O.2to 2.0 pm in diameter and from 0.5 to 20 pm in length. Note the range of sizes as compared with eukaryotic cells and viruses. Comparisons are given as average sizes.

BacterialTaxonomy Based on Bergey's Monuol There is no single official system for classi$,ing the prokaryotes. Indee4 most plans are in a state of flux as new information and methods of analysis become available. One widely used reference has been Bergey's Mqnual of Systematic Bacteriology, a major resource that covers all of the world's known prokaryotes, In the past, the classification scheme in this guide has been based mostly on characteristics such as Gram stain and metabolic reactions. This is called a phenetic, or phenotypic, method of classification. The second edition of this large collection has introduced a significant new organization.It is now based more on recent genetic

This oattern is based on ribosomal RNA sequences' Balloons compare the two prokaryotic domains (Bacteria and Archaea) with the Domain Eukarya. Branches indicate the origins of malor taxonomic groups and their positions show degrees of relatedness.

information that clarifies the phylogenetic (evolutionary) history and relationships of the thousands of known species (figure 4.27). This change has somewhat complicated the presentation of their classification, because prokaryotes are now placed in five major subgroups and 25 different phyla instead of two domains split into four divisions. The Domains Archaea and Bacteria are based on genetic characteristics (see chapter 1) and have been retained. But the bacteria of clinical importance are no longer as closely aligned, and the 250 or so species that cause disease in humans are found within seven or eight ofthe revised phyla. The grouping by Gram reaction remains significant but primarily at the lower taxonomic levels.

The second edition of Bergey s Manual is presented in five volumes as summarized in table 4.3 and illustrated in figvre 4.27 . A more complete version of the major genera in this organization can be found in appendix table D.9. The following section is an overview of some features of the new system. Photographs that represent various phyla and classes are found throughout the chapter.

o

Volume 1 This volume includes all of the Domain Archaea and the most ancient members of Domain Bacteria.

1A TheArchaea are unusual primitive prokaryotes

adapted

to extreme habitats and modes of nutrition. There are two phyla

110

chapter

4

#re.The

A survey of prokaryotic Ceils and Microorganisms

Generalclaisification scherne ot Bergey's Maiaor(2nd Ed.)

Taxonomic Rank

Volume l. The Archaea and the Deeply Branching and Phototrophlc Bacteria IA. DomainArchaea

1B. Domain Bacteria

Volume 3. The Low G

Phylum Crenarchaeota Phylum Euryarchaeota Class I. Methanobacteria Class II. Methanococci Class III. Halobacteria Class IV Thermoplasmata Class V Thermococci Class VI. Archaeoglobi Class VII. Methanopyri

PhylumAquificae

Phylum Firmicutes Class I. Clostridia Class II. Mollicutes

Phylum Thermotogae Phylum Thermodesulfobacteria Phylum "Deinococcus-Thermus" Phylum Chrysiogenetes Phylum Chloroflexi Phylum Thermomicrobia Phylum Nitrospira Phylum Deferribacteres Phylum Cyanobacteria Phvlum Chlorobi

Class

*

C Gram-positive Bacteria

III. Bacilli

Volume 4. The High G * C Gram-positlve Bacteria Phylum Actinobacteria Class Actinobacteria

Volume 5. The Planctomycetes, Splrochaetes, Fibrobacteres, Bacteriodetes, and Fusobacteria Phylum Planctomycetes Phylum Chlamydiae Phylum Spirochaetes Phylum Fibrobacteres Phylum Acidobacteria Phylum Bacteroidetes Phylum Fusobacteria Phylum Verrucomicrobia Phylum Dictyoglomus

Domain Bacteria Volume 2. The Proteobacteria Phylum Proteobacteria Class L Alphaproteobacteria Class II. Betaproteobacteria Class Il l. Gammaproteobacteria Class lV Deltaproteobacteria Class V Epsilonproteobacteria

DomainArchaea

I

a significant poltion of species. Gram-positive cocci such as Staphylococcus and Streptococcu.r (see figure 4.25) are included as well. Significantly, the mycoplasmas are genetically allied with this group even though they have lost their cell wall at some point in time (see figure 4.15).

and seven classes in this group. More information on these prokaryotes is covered in section 4.8.

lB Domain Bacteria. The section of deeply branching and phototropic bacteria contains l l phyla. These are among the earliest inhabitants of earth. It contains members with a wide variety of adaptations. Many of them are photosynthetic (see figures 4.28 and 4.29 depicting cyanobacteria and green sulfur bacteria), others are inhabitants of extreme environments such as thermal vertts (Aquifex), and one genus is highly radiation resistant (Deinococcus) (see figure 4.23a). Volume 2 This includes representatives of the Phylum proteobacteria, a group with five classes and an extremely complex and diverse cross section ofover 2,000 species ofbacteria. One characteristic they all share is a gram-negative cell wall. This group includes several medically significant members, including the obligate intracellular parasites called rickettsias (see figure 4.3 l), gram-negative enteric rods such as -Es cherichia and Salmonella (see figure 4.17), and some spiral pathogens (Helicobacter and Campylobacter). Other examples are photosynthetic bacteria with brilliant purple pigments or unusual gliding bacteria with complex fruiting bodies (see figure 4.30). Volume 3 This collection represents the Phylum Firmicutes,

whose members are characterized as being mostly grampositive and having low G + C content (less than 50%). The three classes in the phylum contain a wide diversity of members, many of which are medically important. The endospore formers Clostridium and Bacillus (see figure 4.2 1) account for

DomainBacteria

o Volume 4 This includes the Phylum Actinobacteria,

o

the

taxonomic category that includes the high G + C (over 50%) gram-positive bacteria. The single class represents bacteria of many different shapes and life cycles. prominent members are the branching filamentous actinomycetes, the spore-producing streptomycetes (a source of antibiotics), and the common genera Corynebacterium (see figure 4.24), Mycobacterium, and Micrococcus. Volume 5 The final volume contains a loose assemblage of nine phyla that may or may not be related, but all of them are gramnegative. Subgroups include the Planctomyces, Spirochaetes, Fibrobacteres, Bacteriodetes, and Fusobacteria. Once again, there is extreme variefy in the members included, and many ofthem are medically important. Chlamydia are tiny obligate parasites that live inside their host cells like rickettsias. Other significant members are spirochetes such as Treponema, the cause ofsyphilis, and

Bonelia

(see

figure 4.6),the cause of Lyme disease.

Diognostic Scheme for Medicol

IJse

Many medical microbiologists prefer an informal working system that outlines the major families and genera (table 4,4). This scheme uses the phenotypic qualities of bacteria in identification. It is

4.7

ililililil;ffiH$ff#j$

111

Classification Systems in the Prokaryotae

Diseases* Medicauy tmportant Famities and cenera of Bacteria. with Notes on Some

l. Bacteria with gram-positive cell wall structure (Firmicutes) Cocci in clusters or packets that are aerobic or facultative Family Micrococcaceae: Staphylococcus (members cause boils' skin infections)

€B

4n

Cocci in pairs and chains that are facultative Family Streptococcaceae: Streptococcus (species cause strep throat, dental caries) Anaerobic cocci in pairs, tetrads, irregular clusters Family Peptococcaceae: Peptococcus, Peptostreptococczs (involved in wound infections) Spore-forming rods Family Bacillaceae'. Bacillus (anthrax), Clostridium (tetanus, gas gangrene, botulism)

.T#tilt;:".tffiil:::"",

Lactobaciilus, Listeria(milk-bome disease), Erysipetorhrix(erysipeloid) Family Propionibacteriaceae : Propionibacterium (involved in acne) Family Corynebacteriaceae: Corynebacterian (diphtheria) Family Mycobacteriaceae: Mycobacterium (tuberculosis, leprosy) Family Nocard iaceae: Nocardia (lung abscesses) Family Actinomycetaceae: Actinomyces (1umpy jaw)' Bifi

do

b acterium

Family Streptomycetaceae: streptomyces (important source of antibiotics)

_l

ll. Bacteria with gram-negative cell wall structure (Gracilicutes) Aerobic cocci Neis s eria (gonorrhea, meningitis),

Branhamella

Aerobic coccobacilli

M oraxell a, A c in e t o b a cter

_.: ...

'I

(td

1

"

'"***

l

Anaerobic cocci Family Veillonellaceae Veillonell a (dental disease) Miscellaneous rods Brucella (undulant fever), Bordetel/a (whooping cough), Francisella (tularemia)

e;

: Ps eudomonas (pneumonia, burn infections) Miscellaneous'. Legionella (Legionnaires' disease)

^T:H;"*:"domonadaceae

Facultative or anaerobic rods and vibrios Shigella (dysentery), Family Enterobacteriaceae: Escherichia, Edwardsiella, Citrobacter Salmonella (typhoid fever) , plague) (one causes species Yersinia Proteus, Serratia, Klebsiella, EnterobacteL

€6e

e

Family Mbron aceae: Vibrio (cholera, food infection), Campylobacter, Aeromonas Miscellaneous genefa: chromobacterium, Flavobacterium, Haemopftilus (meningitis), Pasteurella, Cardiobacterium, Streptobacillus Anaerobic rods

q?fr""

Family Bacteroidaceae: Bacteroides, Fusobacterium (anaerobic wound and dental infections)

Helical and curviform bacteria Family Spirochaetaceae: Treponema (ryphilis), Borrelia (Lyme disease), Leptospira (kidney infection) Obligate intracellular bacteria Family Rickettsiaceae: Rickettsia (Roclcy Mountain spotted fever), Coxiella (Q fever) Family Bartonellaceae: Bartonella (trench fever, cat scratch disease) Family Chlamydiaceae: Chlamydia (sexually transmitted infection)

ofl

lll. Bacteria with no cell walls (Tenericutes) Family Mycoplasmataceae: Mycoplasma (pneumonia), [Jreaplasma (urinary infection) *Details ofpathogens and diseases in later chapters.

__J

s_j

fffl

I

.

I

ll2

chapter

4

A survey of prokaryotic ceils and Microorganisms

restricted to bacterial disease agents and depends less on nomenclature. It also divides the bacteria into gram-positive, gram_negative, and those without cell walls and then subgroups them accordlg to cell shape, arrangement, and certain physiological traits such u, o^ygen usage: Aerobic bacteria use oxygen in metabolism: anaerobic bacteria do not use oxygen in metabolism; and facultgtive bacteria may or may not use oxygen. Further tests not listed on the table would be required to separate closely related genera and species. Many of these are included in later chapters on specific bacterial groups.

Species

ond

Subspecies in Bacteria

Among most organisms, the species level is a distinct, readily de_ fined" and natural taxonomic category. In animals, for instance, a species is a distinct type of organism that can produce viable offspring only when it mates with others of its ornm kind. This definition does not work for bacteria primarily because they do not exhibit atypical mode of sexual reproduction. They can accept ge_ netic information from unrelated forms, and they can also aier their genetic makeup by a variety of mechanisms. Thus, it is neces_ sary to hedge a bit when we define a bacterial species. Theoretically, it is a collection of bacterial cells, all of which share an ou"rutll similar pattern of traits, in contrast to other groups whose pattern differs significantly. Although the boundaries that separate two closely related species in a genus are in some cases very arbitrary, this definition still serves as a method to separate the bacteria inio various kinds that can be cultured and studied. As additional information on bacterial genomes is discovered, it may be possible to define species according to specific combinations ofgenetic codes found only in a particular isolate. Because the individual members of given species can show variations, we must also define levels within species (subspecies) called strains and types. A strain or variety ofbacteria is a culture derived from a single parent that differs in structure or metabolism from other cultures ofthat species (also called biovars or morpho_ vars). For example, there are pigmented and nonpigmented stiains of serratia marcescens and flagellated and nonflagellated strains of Pseudomonas Jluorescens. A type is a subspecies that can show dif_

ferences in antigenic makeup (serotype or serovar), in susceptibility to bacterial viruses (phage type), and in pathogenicity (pathotypej.

m Key traits that are used to identify a bacterial species include

(l)

cell morphology, (2) Gram and other staining characteristics,

(3) presence ofspecialized structures, (4) macroscopic appearance, (5) biochemical reactions, and (6) nucleotide composition ofboth DNA and rRNA.

ffi Bacteria

s

*

are formally classified by phylogenetic relationships and phenotypic characteri stics.

Medical identification of pathogens uses a more informal system of classification based on Gram stain, morphology, biochemical reactions, and metabolic requirements. A bacterial species is loosely defined as a collection ofbacterial cells tlat shares an overall similar pattern of traits diferent from other groups of bacteria.

ffi Variant forms within a species (subspecies) include strains and rypes.

Stlr.ve_y of Prokaryotic Groups with Unusual Characteristids The bacterial world is so diverse that we cannot do complete justice to it in this introductory chapter. This variety extends into all areas of bacterial biology, including nutrition, mode of life, and behavior. certain fypes ofbacteria exhibit such unusual qualities that they deserve special mention. In this minisurvey, we consider some medically important groups and some more remarkable representatives of bacteria living free in the environment that are ecologically important. Many of the bacteria mentioned here do not have the morphology typical of bacteria discussed previously, and in a few cases, they are vividly different (Insight 4.3).

Free-Living Nonpathogenic Bacteria P h otosy ntheti c B a cteri a The nutrition of most bacteria is heterotrophic, meaning that they derive their nutrients from other organisms. photosynthetic bacteria, howeveq are independent cells that contain special lighttrapping pigments and can use the energy of sunlight to synthesize ali re_ quired nutrients from simple inorganic compounds. The two general types ofphotosynthetic bacteria are those that produce oxygin dur_ ing photosynthesis and those that produce some other substance, such as sulfur granules or sulfates.

Cyonobacteria : Blue-Green Bocteria The cyanobacteria were called blue-green algae for many years and were grouped with the eukaryotic algae. However, further study verified that they are indeed bacteria with a gram-negative cell wail and general prokaryotic strucfure. These bacteria range in size from I pm to 10 pm, and they can be unicellular or can occur in colonial or filamentous groupings (figure 4.25b').

Cyanobacteria are among the oldest types of bacteria on earth. Fossil forms have been isolated in rocks that are over 3

billion years old. Interesting remnants of these microbes exist in stromatolites-fossil biofilms in oceanic deposits. When magni_ fied, they reveal ancient cells that look remarkably like modern ones (figure 4.28c).A specialized adaptation ofcyanobacteria is extensive internal membranes called thylakoids, which contain granules of chlorophyll a and other photosynthetic pigments (fig_ ure 4.28a'). They also have gas inclusions, which permit them to

float on the water surface and increase their light exposure, and cysts that convert gaseous nitrogen (N) into a form usable by plants. This group is sometimes called the blue-green bacteria in

reference to their content of phycocyanin pigment that tints some members a shade of blue, although other members are colored yellow and orange. Some representatives glide or sway gently in

the water from the action of filaments in the cell envel,ope ihat cause wavelike contractions.

Cyanobacteria are very widely distributed in nature. They grow profusely in freshwater and seawater and are thought to be responsible for periodic blooms that kill offfish. Some members are so pollution-resistant that they serve as biological indicators of polluted water. Cyanobacteria inhabit and flourish in hot springs and have even exploited a niche in dry desert soils and rock surfaces.

4.7

Classification Systems in the Prokaryotae

113

Flgure 4.29 Photosyntheticbacteria. Purple colored masses in a fall pond contain a concentrated bloom of purple sulfur bacteria (inset 1,500x). The pond also harbors a mixed population of algae (green clumps).

Green ond Purple Sulfur Bscteria The green and purple bacteria are also photosynthetic and contain pigments. They differ from the cyanobacteria in having a different type of chlorophyll called bacteriochlorophyll andby not giving off oxygen as a product of photosynthesis. They live in sulfur springs, freshwater lakes, and swamps that are deep enough for the anaerobic conditions they require yet where their pigment can still absorb wavelengths of light (figure 4.29). These bacteria are named for their predominant colors, but they can also develop brown, pink, purple, blue, and orange coloration. Both groups utilize sulfur compounds (II2S, S) in their metabolism.

Gliding, Fruiting Bocteris The gliding bacteria are a mixed collection of gram-negative bacte-

iathat live in water

and soil. The name is derived from the ten-

(a) Electron micrograph of a cyanobacterial cell (80,000x) reveals folded stacks of membranes that contain the photosynthetic pigments and increase surface area for photosynthesis. (b) Two striking cyanobacteria t Anoboeno (left) and Arthrospira (right) display their striking color and shapes (750x). At the bottom is a stromatolite sectioned to show layers laid down over a billion years by cyanobacteria. (c) A microfossil of a cyanobacterial filament from

dency of members to glide over moist surfaces. The gliding property evidently involves rotation of filaments or fibers just under the outer membrane of the cell wall. They do not have flagella. Several morphological forms exist, including slender rods; long filaments; cocci; and some miniature, tree-shaped fruiting bodies. Probably the most intriguing and exceptional mernbers of this group are the slime bacteria, or myxobacteria. What sets the myxobacteria apart from other bacteria are the complexity and advancement of their life cycle. During this cycle, the vegetative cells respond to chemotactic signals by swarming together and differentiating into a manycelle4 colored structure called the fruiting body (figure 4.30). The fruiting body is a survival structure that makes spores by a method very similar to that of certain fungi. These fruiting structures are often large enough to be seen with the unaided eye on tree bark and

Siberia.

plant debris.

Figure 4.28 Structure and examples of cyanobacteria.

tl4

Chapter

4

A Survey of Prokaryotic Cells and Microorganisms

Redefining Bacterial Size Many microbiologists believe we are still far from having a complete assessment of the bacterial world, mostly because the world is so large and bacteria are so small. Every few months we see reports of exceptional bacteria discovered in places like the deep ocean volcanoes orAntarctic ice. Among the most remarkable are giant and dwarf bacteria.

Big Bacteria Break Records In

1985, biologists discovered a new bacterium living in the intestine of surgeonfish that at the time was a candidate for the Guinness Book ofWorld Records. The large cells, narned, Epulopiscium fishelsoni ("guest at a banquet of fish";, measure around 100 pm in lengh, although some specimens were as T hiomo rga rito lo no nomtbtrytant mibia-giant cocci. cocci. large as 300 pm. This record was broken when a marine microSection throuqh sandstone shows tiny blobs thai some scientists biologist discovered an even larger species ofbacteria living in ocean sediments neartheAfrican country ofNamibia. These gigantic cocci are arranged in strands that look like pearls and contain hundreds ofgolden rhese minute cells have been given o, nanobes sulfur granules, inspiring their name, Thiomargarita namibia (,,sulfur pearl (Gr. nano s, one-billionth). of Namibia") (see photo). The size of the individual cells ranges from 100 Nanobacterialike forms were first isolated from blood and serum up to 750 pm (314 mm), and many are large enough to see with the naked samples. The tiny cells appear to grow in culture, have cell walls, and eye. By way of comparison. if the average bacterium were the size of a contain protein and nucleic acids, but their size range is only from 0.05 mouse, Thiomargarita wottld,be as large as a blue whale! to 0.2 pm. Similar nanobes have been extracted by minerologists studying Closer study revealed that they are indeed prokaryotic and have sandstone rock deposits in the ocean at temperatures of l00oC to 170"C bacterial ribosomes and DNA but that they also have some unusual and deeply embedded in billion-year-old minerals. The minute filaments adaptations to their life cycle. They live an attached existence embedwere able to grow and are capable of depositing minerals in a test tube. ded in sulfide sediments (HrS) that are free ofgaseous oxygen. They Many geologists are convinced that these nanobes are real. that they are obtain energy through oxidizing these sulfides using dissolved nitrates probably similar to the first microbes on earth, and that they play a stra(NO3). These bacteria are found in such large numbers in the sediments tegic role in the evolution ofthe earth's crust. that it is thought that they are essential to the ecological cycling of Microbiologists tend to be more skeptical. It has been postulated that H2S and other substances in this region, converting them to less toxic the minimum cell size to contain a functioning genome and reproductive substances. and synthetic machinery is approximately 0. l4 pm. They believe that the nanobes are reallyjust artifacts or bits oflarger cells that have broken free. Miniature Microbes-The Smallest of the Small It is partly for this reason that most bacteriologists rejected the idea that At the other extreme, microbiologists are being asked to reevaluate the small objects found in a Martian meteor were microbes but were more lower limits of bacterial size. Up until now it has been generally accepted likely caused by chemical reactions (see chapter I ). Additional studies are that the smallest cells on the planet are some form of mycoplasma with needed to test this curious question ofnanobes and possibly to answer some dimensions of 0.2 to 0.3 pm, which is right at the limit of resolution with questions about the origins oflife on earth and even on other planets. light microscopes. A new controversy is brewing over the discovery of List additional qualities a nanobe would require to be considered a tiny cells that look like dwarf bacteria but are 10 times smaller than living cell. Answer available at http://www.mhhe.com/talaroT mycoplasmas and a hundred times smaller than the average bacterial cell.

rJH:;Tff,..iu

Unusual Forms of Medically Significant Bacteria Most bacteria are free-living or parasitic forms that can metabolize and reproduce by independent means. Two groups of bacteria-the rickettsias and chlamydias-have adapted to life inside their host cells, where they are considered obtigate intracellular parasites.

RickettsiasB Rickeffsias are distinctive, very tiny, gram-negative bacteria (figure 4.31). Although they are somewhat typical in morphology, they 8. Named for Howard Ricketts, a physician who first worked with these organisms and later lost his life to tlphus.

are atypical in their life cycle and other adaptations. Most are pathogens that alternate between a mammalian host and bloodsucking arthropods,e such as fleas, lice, or ticks. Rickettsias cannot survive or multiply outside a host cell and cannot carry out metabolism completely on their own, so they are closely attached to their hosts. Several important human diseases are caused by rickettsias. Among these are Rocky Mountain spotted fever, caused by Rickettsia rickettsii (transmitted by ticks), and endemic typhus, caused by Rickettsia typhi (Iransmitted by lice).

9. An arthropod is an invertebrate with jointed legs, such

as an insect,

tick, or spider

4.8

Archaea: The Other ProkarYotes

115

Rickettsial cells

Nucleus

Figure4.3O

Myxobocterium.

A photograph of the mature fruiting body and its cluster of myxospores held by a stalk. Vacuole

Chlomydios Bacteria of the genera Chlamydia

4.31 Transmission electron micrograph of the rickettsia Coxietlo burnetii (20,000x), the cause of Q fever.

Figure and Chlamydophila, termed

chlamydias, are similar to the rickettsias in that they require host cells for growth and metabolism; but they are not closely related to them and are not transmitted by arthropods. Because of their tiny size and obligately parasitic lifestyle, they were at one time considered a type of virus. Species lhat carry the greatest medical impact Chlamydia trachomatis, the cause ofboth a severe eye infection (trachoma) that can lead to blindness and one of the most common sexually transmitted diseases, and Chlamydophila pneumoniae, an agent in lung infections. Diseases caused by rickettsias and chlamydias are described in more detail in the infectious disease chapters. are

4.8 Archaea: The Other Prokaryotes The discovery and characletization ofnovel prokaryotic cells that have unusual anatomy, physiology, and genetics changed our views of microbial taxonomy and classification (see chapter I and table 4.3). These single-celled, simple organisms, called archaea' or archaeons are considered a third cell type in a separate superkingdom

Its mass growth inside a host cell has filled a vacuole and displaced the nucleus to one side.

(the Domain Archaea). We include them in this chapter because they are prokaryotic in general structure and they do share many bacterial characteristics. But evidence is accumulating that they are actually more closely related to Domain Eukarya than to bacteria. For example, archaea and eukaryotes share a number of ribosomal RNA sequences that are not found in bacteria, and their protein synthesis and ribosomal subunit structures are similar. Table 4.5 outlines selected points of comparison of the three domains. Among the ways that the archaea differ significantly from other cell tlpes are that certain genetic sequences are found only in their ribosomal RNA and that they have unique membrane lipids and cell wall construction. It is clear that the archaea are the most primitive of all life forms and have retained characteristics of the first cells that originated on the earth nearly 4 billion years ago. The early earth is thought to have contained a hot, anaerobic "soup" with sulfuric gases and salts in abundance. The modern archaea still live in the remaining habitats on the earth that have some of the

Comparison of Three Cellular Domains

Characteristic

Bacteria

Archaea

Eukarya

Cell tlpe

Prokaryotic

Prokaryotic

Eukaryotic

Chromosomes

Single, or few, circular

Single, circular

Several, linear

Types of ribosomes

70s

70S but structure is similar to 80S

80s

Contains unique ribosomal RNA signature sequences

+

+

+

Number of sequences shared with Eukarya

One

Three

(A1D

+

Protein synthesis similar to Eukarya Presence ofpeptidoglycan in cell wall

+

Cell membrane lipids

Fatty acids with ester linkages

Sterols in membrane

-

(some exceptions)

Long-chain, branched hydrocarbons with ether linkages

Fatfy acids with ester linkages

+

116

Chapter

4

A Survey of prokaryotic Cells and Microorganisms

same ancient conditions-the most extreme habitats in nature. It is for this reason that they are consid ered extremophiles , meaning that they "love" extreme conditions in the environment.

Metabolically, the archaea exhibit nearly incredible adaptations to what would be deadly conditions for other organisms. These hardy microbes have adapted to multiple combinations of heat, salt, acid, pH, pressure, and atmosphere. Included in this group are methane producers, hyperthermophiles, extreme halophiles, and sulfur reducers. Members of the group called methanogens can convert CO2 and H, into methane gas (CHa) through unusual and complex pathways. These archaea are common inhabitants of anaerobic mud and the bottom sediments of lakes and oceans. The gas they produce collects in swamps and may become a source of fuel. Methane may also contribute to the "greenhouse effect," which maintains the earth's temperature and can contribute to global warming. Not all methanogens live in extreme environments. Some are commonly found in the oral cavity and large intestine of humans. Other types of archaea-the extreme halophiles-require salt to grow and may have such a high salt tolerance that they can multiply in sodium chloride solutions (36% NaCl) that would desrroy most cells. They exist in the saltiest places on the earth-inland seas, salt lakes, and salt mines. They are not particularly common in the ocean because the salt content is not high enough to support them. Many of the "halobacteit'use a red pigment to synthesize ATP in the presence of light. These pigments are responsible for "red herrings," the color ofthe Red Sea, and the red color of salt ponds (figure 4.32). Archaea adapted to growth Nvery low temperatures are called psychrophilic floving cold temperatures); those growing at very high temperatures are ltyperthermophilic (loving high temperatures). Hyperthermophiles flourish at temperatures between 80.C and 121.C (boiling temperature) and cannot grow at 50oC. They live in volcanic waters and soils and submarine vents and are also often salt- and acidtolerant as well (figure 4.33). Researchers sampling sulfur vents in the deep ocean discovered thermophilic archaea flourishing at temperatures up to 250"C-150o above the temperature of boiling water! Not only were these archaea growing prolifically at this high temperature,

Figure 4.32 Halophiles around the world. (a) A solar evaporation pond in Owens Lake, California, is extremely high in salt and mineral content. The archaea that dominate in this warm, saline habitat produce brilliant red pigments that absorb light to drive cell synthesis. (b) A sample taken from a saltern in Australia viewed by fluorescent microscopy (1,000x). Note the range of cell shapes (cocci, rods, and squares) found in this community.

but they were also living at 265 atonospheres of pressure. (On the earth's surface, pressure is about one atmosphere.) For additional discussion ofthe unusual adaptations ofarchaea, see chapter 7.

I

Bacteria exist in a tremendous variety of structure and lifestyles. Most ofthem are free-living ratherthanparasitic. Notable examples are the photosynthetic bacteria and gliding bacteria, which have unique adaptations in physiology and development. f, The rickettsias are a group of intracellular parasitic bacteria dependent on their eukaryote host for energy and nutrients. Most are pathogens that altemate between artbropods and mammalian hosts. w The chlamydias are also small, intracellular parasites that infect humans, marnmals, and birds. They do not require arthropod vectors. Archaea are another type of prokaryotic cell that constitute ttre third domain of life. They exhibit unusual biochemistry and genetics that make them different from bacteria. Many members are adapted to extreme habitats with low or high temperature, salt, pressure, or acjd.

r

Flgure 4.33 An electron micrograph of the "hottest,, microbe on earth. This tiny archaea (bar is 1 pm) thrives deep in hydrothermal vents that regularly reach 121'C-the working temperature of an autoclave. (S = slime layer, CM cell membrane.)

:

Chapter Summary with

KeY Terms

tt7

Chapter Summary with KeY Terms Gram-negative bacteria have a thinner twolayer cell wall with an outer membrane, thin layer of peptidoglycan, and a well-developed periplasmic space. 3. Wall structure gives rise to differences in staining, toxicity, and effects of drugs and disinfectants.

2.

4.1 Characteristics of Cells and Life

A.

living things are composed of cells, which are complex collections of macromolecules that carry out living processes. All cells must have the minimum structure of an outer cell membrane, cytoplasm, a chromosome, and ribosomes. Cells can be divided into two basic types: prokaryotes and

A11

B.

4,5 Bacterial

1. Prokaryotic cells are the basic structural unit ofbacteria and archaea. They lack a nucleus or organelles. They are

highly successful and adaptable single-cell life forms.

2.

C.

Eukaryotic cells contain a membrane-surrounded nucleus and a number of organelles that function in specific ways. A wide variety of organisms, from single-celled protozoans to humans, are composed of eukaryotic cells. 3. Viruses are not generally considered living or cells, and rely on host cells to rePlicate' Cells show the basic essential characteristics of life. Parts of cells and macromolecules do not show these characteristics

4.6

Bacterial Shapes, Arrangements' and Sizes

A.

1. Growth: Living entities are able to grow and increase in

2.

information to their offspring, whether asexually (with one parent) or sexually (with two parents). 3. Metabolism: This refers to the chemical reactions in the cell, including the synthesis of proteins on ribosomes and the capture and release of energy using AIP. 4. Movement: Motility originates from special locomotor structures such as flagella and cilia; irritability is responsiveness to external stimuli'

5. 6.

Transport: Nutrients must be brought into the cell through the membrane and wastes expelled from the cell. Cell protection, supporto and storage: Cells are protected by walls, membranes, and outer shells of various types.

B.

C.

B.

4.3 ExternalStructures

A.

Appendages: Cell Extensions: Some bacteria have projections that extend from the cell.

l.

Flagella (and internal axial filaments found in spirochetes) are used for motility. 2. Fimbriae function in adhering to the environment; pili provide a means for genetic exchange. 3. The glycocalyx may be a slime layer or a capsule. Envelope: The Boundary Layer of Bacteria Most prokaryotes are surrounded by a protective envelope that consists of the cell wall and the cell membrane. The wall is relatively rigid due to peptidoglycan. Structural differences give rise to gram-positive and gramnegative cells, as differentiated by the Gram stain. 1. Gram-positive bacteria contain a thick wall composed ofpeptidoglycan and teichoic acid in a single layer.

4.4 The Cell

A. B. C.

unicellulax and are found in a great variety

of

and are pleomorphic. Other variations include coccobacilli, vibrios. and filamentous forms. Prokaryotes divide by binary fission and do not utilize mitosis. Various arrangements result from cell division and are termed diplococci, streptococci, staphylococci, tetrads, and sarcina for cocci; bacilli may form pairs, chains, or palisades. Variant members of bacterial species are called strains and t1pes.

Bacteria

Domain Bacteria Volume Volume Volume Volume

4.2 Prokaryotic Profiles: The Bacteria andArchaea Prokaryotes consist oftwo major groups, the bacteria and the archaea. Life on earth would not be possible without them. Prokaryotic cells lack the membrane-surrounded organelles and nuclear comparfinent ofeukaryotic cells but are still complex in their structure and function. All prokaryotes have a cell membrane, cytoplasm, ribosomes, and a chromosome.

axe

4.7 Classification Systems in the Prokaryotae A. An important taxonomic system is standardized by Bergqt's Manual of Determinative Bacteriologt, which presents the prokaryotes in five major volumes. Volume l DomainArchaea Domain Bacteria: Deeply Branching and Phototropic

They store nutrients to survive periods of starvation.

A.

Most bacteria

shapes, arrangements, and sizes. General shapes include coccit bacilli, and helical forms such as spirilla and spirochetes. Some show great variation within the species in shape and size

independently. size, often renewing and rebuilding themselves over time. Reproduction and heredity: Cells must pass on genetic

Internal Structure

The cell cytoplasm is a watery substance that holds some or all of the following internal structures in bacteria: the chromosome(s) condensed in the nucleoidl ribosomes, which serve as the sites ofprotein synthesis and are 70S in size; extra genetic information in the form ofplasmids; storage structures known as inclusionsl a cytoskeleton ofbacterial actin, which helps give the bacterium its shape; and in some bacteria an endospore, which is a highly resistant structure for survival, not reproduction.

eukaryotes.

2 Proteobacteria (gram-negative cell walls)

G

f

C gram-positive Bacteria C gram-positive Bacteria 5 Planctomyces, Spirochaetes, Fibrobacteres, Bacteriodetes, and Fusobacteria (gram-negative

3 Low

4 High G

*

cell walls)

B.

Several groups of bacteria have unusual adaptations and life cycles. Medically important bacteria: Rickettsias and chlamydias are within the gram-negative group but are small obligate intracellular parasites that replicate within cells of the hosts they invade.

l.

2.

Nonpathogenic bacterial groups: The majority of bacterial species are free-living and not involved in disease. Unusual groups include photosynthetic bacteria such as cyanobacteria, which provide oxygen to the environment, and the green and pwple bacteria.

4.8 Archaea: The Other Prokaryotes Archaea share many characteristics with bacteria but vary in certain genetic aspects and structures such as the cell wall and ribosomes. Many are adapted to extreme environments similar to the earliest of earth's inhabitants. They are not considered medically important, but are of ecological and potential

economic importance.

Chapter

118

Mu

$F

4

A Survey of Prokaryotic Cells and Microorganisms

ltiple-choice Questions

Select the correct answer from the answers provided. For questions with blanks, choose the combination of answers that most accurately completes the statement. 1

. Which

structure is not a component of all cells? c. genetic material d. ribosomes

9. Metachromatic granules are concentrated

a. cell wall b. cell membrane

a.

b, dipicolinic

2. Viruses are not considered living things because a. they are not cells

11

3. Which of the following is not found in all bacterial cells?

4.

c. ribosomes d. actin cytoskeleton

c. fimbriae d. cilia

5. Pili are tubular shafts in bacteria that serve a. gram-positive, genetic exchange b. gram-positive, attachment

c. gram-negative, genetic

as a means of

_.

exchange

d. gram-negative, protection 6. An example of a glycocalyx is a. a capsule b. pili

c.

outer membrane

d.

a cell

wall

7. Which of the following is a primary bacterial cell wall function?

c. support d. adhesion

a. transport b. motility

Bacillus

8. Which of the following is present in both gram-positive and gramnegative cell walls? a. an outer membrane

c. teichoic acid

b. peptidoglycan

d. lipopolysaccharides

. An arrangement in

_

found in

_.

c. sulfur, Thiobacillus d. PO4, Corynebacterium c. protein synthesis d. storage

packets of eight cells is described as a

a. mlcrococcus

c. tetrad

b. diplococcus

d.

_.

sarcina

12. The major difference between a spirochete and a spirillum is a. presence offlagella c. the nature of motility b. the presence of twists d. size

The major locomotor structures in bacteria are

a. flagella b. pili

acid,

10. Bacterial endospores function in a. reproduction b. survival

b. they cannot reproduce by themselves c. they lack metabolism d. all ofthese are correct a. cell membrane b. anucleoid

fat. Mvcobacterium

13. Which phylum contains bacteria with a gram-positive cell wall? a. Proteobacteria c. Firmicutes

b. Chlorobi

d.

Spirochetes

14. To which taxonomic group do cyanobacteria belong? a. Domain Archaea c. Domain Bacteria b. PhylumActinobacteria d. Phylum Fusobacteria 15. Which stain is used to distinguish differences between the cell walls of medically important bacteria? a. simple stain c. Gram stain b. acridine orange stain d. negative stain 16. The first living cell on earth was most similar to a. acyanoDacrcnum c. a gram-positive cell

b. an endosoore former

d. an archaea

writingtoLearn

W

These questions are suggested as a writinglo-learn expeience. For each question, compose a one- or two-paragraph answer that includes the factual information needed to completely address the question. General page references for these topics are given in parenlheses.

l.

a. Name several general characteristics that could be used to define the prokaryotes. (89, 90) b. Do any other microbial groups besides bacteria have prokaryotic

cells?

(90, I 15)

c. What does it mean to

say that bacteria are ubiquitous? In what habitats are they found? Give some general means by which bacteria derive nutrients. (1, I 10-1 14) d. Label the parts on the bacterial cell featured here and write a brief description of its function. (91,ll'7)

tt9

Critical Thinking Questions

2. a. Describe the structure of a flagellum b.

c. 3. a. b.

c.

and how it operates. What

are the four main types of flagellar arrangement? (90' 9l ' 92) How does the flagellum dictate the behavior of a motile bacterium? Differentiate between flagella and periplasmic flagella. (92,93) List some direct and indirect ways that one can determine bacterial motility. (91,92) Explain the position of the glycocalyx. (91, 94) What are the fi.rnctions of slime layers and capsules? (94' 95,96) How is the presence ofa slime layer evident even atthe level ofa colony? (96)

4. Differentiate between

pili

and

10.

b. 11.

12.

serve? (98' 99) of its structure. (98' 99)

causes some to stain (97, 98) red? to stain purple and others c. How does the precise structure of the cell walls differ in grampositive and gram-negative bacteria? (98' 100) d. What other properties besides staining are different in grampositive and gram-negative bacteria? (100) e. What is the periplasmic space, and how does it firnction? (98' 100) f. What characteristics does the outer membrane confer on gram-

negativebacteria? (99) 7. a. b. c. d.

and the name

8. a. Compare the composition of the bacterial chromosome (nucleoid)

plasmids.

(102) b. What are the functions of

each? (102, 103)

9. a. What is unique about the structure of bacterial ribosomes? (103) b. What is their function and where are they located? (103)

efu4ff

(106) Staphylococcus and

13. a. Rankthe size ranges inbacteria accordingto shape. (109) b. Rank the bacteria in relationship to viruses and eukaryotic cell size. (109) c. Use the size bars to measure the cells in figure 4.32 and

Insight4.3. (116,114) d. Describe the arrangements of cells in figure 4.23a,b,andc. (107) 14. a. Whatcharacteristics are usedto classifu bacteria? (108' 109, 110' Appendix D) b. What are the most useful characteristics for categorizing bacteria into families? (111)

(l12)

b. Name at least three ways bacteria are classified below the species

level.

(112)

16. Name several ways in which bacteria are medically and ecologically

important. (109, ll0, ll2, 113,114) I

7. a. Explain the characteristics of archaea that indicate that they constitute a unique domain of living things that is neither

bacterialnoreukaryotic. (115, ll6)

b. What leads microbiologists to believe the archaea are more closely related to eukaryotes than to bacteria? (l 15) c. What is meant by the term extremophile? Describe some archaeal adaotations to extreme habitats. (l l6)

concePt MaPPins

Appendix E provides guidance for working with concept maps' 1. Construct your own concept map using the following words as the concepts. Supply the linking words between each pair of concepts.

@ff

Bacillus?

staphylococcus? (107)

15. a. Howisthe specieslevelinbacteriadefined?

Describe the structure of the cell membrane. (l0l) Why is it considered selectively permeable? (101) What are mesosomes and some proposed roles they play? (101) List five essential functions that the cell membrane performs in bacteria. (102) and

a. Draw the most common bacterial shapes and arrangements. (106, 107, 108) b. Howare spirochetes and spirilla different? (107) c. What is a vibrio? A coccobacillus? (106) d. What is pleomorphism? (106) e. What is the difference between the use of the shape bacillus

d. What happens to

6. a. What is the Gram stain? (97,98) b. What is there in the structure of bacteria that

an endospore is not considered a reproductive

(104)

d. Why are endospores so difficult to destroy? (104)

fimbriae. (93,94)

a cell that has its peptidoglycan disrupted or removed? (98) e. What functions does the LPS layer serve? (99)

(103)

a. Describe the formation ofbacterial endospores. (104' 105) b. Describe the structure of an endospore' and explain its

body.

does peptidoglycan

a simple description

contain?

tunction. (104, 105)

bacteria. (9'7,98) c. Give

and function ofinclusions

and granules. (103) What are metachromatic granules, and what do they

c. Explain why

5. a. Compare the cell envelopes of gram-positive and gram-negative b. What function

a. Compare and contrast the structure

genus serotlpe Borrelia Spirochaetes

specles

domain

burgdorferi phylum

critical rhinking Questions

Critical thinking is the ability to reason and solve problems using facts and concepts. These questions can be approached from a number ofangles, and in most cases, they do not have a single correct answer.

t.

What would happen if one stained a gram-positive cell only with safranin? A gram-negative cell only with crystal violet? What would happen to the two types if the mordant were omitted?

2. What is required to kill endospores? How do you suppose archaeologists were able to date some spores as being thousands (or millions) of years old?

120

Chapter

4

A Survey of Prokaryotic Cells and Microorganisms

3. Using clay, demonstrate how cocci can divide in several planes and show the outcome of this division. Show how the arransements bacilli occur, including palisades.

4. Using

a corkscrew and a spring to compare the

9. a. Name two main groups of obligate intracellular parasitic bacteria. b. Why can't these groups live independently?

of I

flexibility and

locomotion of spirilla and spirochetes, explain which cell type is represented by each object.

5. Under the microscope, you

see a rod-shaped cell that is swimming rapidly forward. a. What do you automatically know about that bacterium's skucture? b. How would a bacterium use its flagellum for phototaxis? c. Propose another function of flagella besides locomotion.

6. a. Name a bacterium that has no cell walls. b. How is it protected from osmotic destruction?

7. a. Name a bacterium that is aerobic, gram-positive,

0. a. Name a bacterium that uses chlorophyll to photosynthesize. b. Describe the two major groups of photosynthetic bacteria.

c. How are they similar? d. How are they different? I

L a. What are some possible adaptations

I

2. Propose

that the giant bacterium Thiomargarita has had to make because of its large size? b. If a regular bacterium were the size of an elephant, estimate the size of a nanobe at that scale. a

hypothesis to explain how bacteria and archaea could

have, togetheq given rise to eukaryotes. and spore-

forming.

b. What habitat would you expect this species to occupy? 8. a. Name an acid-fast bacterium. b. What characteristics make this bacterium different from other gram-positive bacteria?

13. Explain or illustrate exactly what will happen to the cell wall if the synthesis of the interbridge is blocked by penicillin. What if the glycan is hydrolyzed by lysozyme? 14. Ask your lab instructor to help you make a biofilm and examine it under the microscope. One possible technique is to suspend a glass slide in an aquarium for a few weeks, then carefully air-dry, fix, and Gram stain it. Observe the diversity ofcell types.

Virual Understinding 1. From chapter 3, figure 3.10. Do you believe that the bacteria that are spelled "Klebsiella" or the bacteria that are spelled "5. aureus"

2. Using figure 4,22 as a guide, label the

stages in the endospore cycle shown in the figure, and explain the events depicted.

possess the larger capsule? Defend your answer.

'@ @ @ @

ffi @

ffi €*

@ff

Internet search Topics

1. Explore these websites for information on nanobacteria. Give convincing evidence that indicates they are alive, using the characteristics from the first section ofthe chapter.

a. http ://www.sciencecases.org/nanobacteria/nanobacteria.asp

b. http://serc.carleton.edu/microtrelife/topics/nanobes/ 2.

Go to: http://www.mhhe.com/talaro7, and click on chapter 4. Open the URLs listed under Internet Search Topics, and research the

following:

a. Go to the Cells Alive website as listed. Click on "Microbiology" and go to the "Dividing Bacteria" and "Bacterial Motility" options to observe short clips on these topics.

b. Click on this website to fl

see an animated version of a functioning agellum: http ://www.life.uiuc.edu/crofts/bioph354/

flag_motor_ani.gif

c. Go to this website to

see excellent coverage ofbacterial staining techniques and results: http:i/www.spjc.edu/hec/vettech/vtde/

ATE2639LGs/gramstain.htm 3. Research

biofilms and find examples in which bacteria are involved.

4. Go to this governmental website and search for the taxonomic status

of bacteria from the chapter: http://www.itis.gov/info.html

A Survey of Eukaryotic and Micro

CASE FILE

J-

isms

uring June of 2000, several children in Delaware, Ohio, were hospitalized after experiencing watery diarrhea, abdominal cramps, vomiting, and loss of appetite. Dr. McDermott a new gastroenterologist at the hospital, who also had a strong interest in infectious diseases, was asked to examine the children. Their illness lasted from 1 to 44 days and nearly half of them complained of intermittent bouts of diarrhea. By July 20, over 150 individuals-mainly children and young adults between the ages of 20 and 4O+xperienced similar signs or symptoms. Dr. McDermott suspected that their illness was due to a microbial infection and contacted the local County Health Department to request further investigation of this mysterious outbreak. Dr. McDermott helped the Health Department team survey individuals hospitalized for intermittent diarrhea. They questioned individuals about recent travel, their sources of drinking water, visits to pools and lakes, swimming behaviors, contactwith sick persons or animals, and day-care attendance. The investigation indicated that the outbreaks were linked to a swimming pool located at a private club in central Ohio. The swimming pool was closed on July 28, after at least five fecal accidents had been observed. A total of 700 clinical cases among residents of Delaware County and three neighboring counties were eventually identified during the outbreak that began late in June and continued through September. Outbreaks of gastrointestinal distress associated with recreational water activities have increased in recent years, with most being caused by the organism in this case.

) )

Suggest what microorganism might be the cause of the outbreak. How can o single fecol accident contominate on entire pool and cause so many clinical coses of go stroi ntesti n a I di stress?

)

What possible characteristics of this pathogen contributed to its survival in a chlorinated

swimming pool? Cose File

CHAPTER OVERVIEW

5 Wrop-Up oppears on page 149.

Eukaryotic cells are large complex cells divided into separate compartments by membrane-bound components called organelles. Each major organelle-the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Colgi apparatus, and locomotor appendages-serves an essential function to the cell, such as heredity, generating energy, synthesis, transport, and movement. Eukaryotic cells are found in fungi, protozoa, algae (protists), plants, and animals, and they exhibit single-celled, colonial, and multicellular body plans.

tzl

t22

Chapter

5

A Survey of Eukaryotic Cells and Microorganisms

Fungi are eukaryotes that feed on organic substrates, have cell walls, reproduce asexually and sexually by spores, and exist in macroscopic or microscopic forms. Most fungi are free-living decomposers that are beneficial to biological communities; some may cause infections in animals and plants. Microscopic fungi are represented by spherical budding cells called yeasts and elongate filamentous cells called molds. Algae are aquatic photosynthetic protists with cell walls and chloroplasts containing

chlorophyll and other pigments. Algae are classified into several groups based on their type of pigments, cell wall, stored food materials, and body plan. Protozoa are protists that feed on other cells, lack a cell wall, usually have some type of locomotor organelle, and may form dormant cysts. Subgroups of protozoa differ in their organelles of motility (flagella, cilia, pseudopods) or lack thereof.

Most protozoa are free-living aquatic cells that engulf other microbes, and a few parasitize animals. The infective helminths are flatworms and roundworms that have greatly modified body organs so as to favor their parasitic lifestyle.

5.1 The History of Eukaryotes Evidence from paleontology indicates that the first eukaryotic cells appeared on the earth approximately 2 billion years ago. Some fos-

silized cells that look remarkably like modern-day algae or protozoa appear in shale sediments from China, Russia, and Australia that date from 850 million to 950 million years ago (figure 5.1). Biologists have discovered convincing evidence to suggest that the eukaryotic cell evolved from prokaryotic organisms by a process of intracellular symbiosis* (Insight 5.1). It now seems clear that

* syrrblosn (sim-beye-oh'-sis) Gr. syn, togethet and bios, to live. A close association between two organisms.

Flgure 5.t Ancient eukaryotic protists caught up in fossilized

some of the organelles that distinguish eukaryotic cells originated from prokaryotic cells that became trapped inside them. The structure of these first eukaryotic cells was so versatile that eukaryotic microorganisms soon spread out into available habitats and adopted great$ diverse styles of living. The first primitive eukaryotes were probably single-celled and independent, but, over time, some forms began to cluster in

permanent groupings called colonies. With further evolution, some of the cells within colonies became specialized, or adapted to perform a particlalar function advantageous to the whole colony, such as locomotion, feeding, or reproduction. Complex multicellular organisms evolved as individual cells in the organism lost the ability to survive apart from the intact colony. Although a multicellular organism is composed of many cells, it is more than

rocks.

(a) A cell preserved in Siberian shale deposits dates from 850 million to 950 million years ago. (b) A disclike cell was recovered from a Chinese rock dating 590 million to 610 million years ago. Both cells are relatively simple, with (a) showing chloroplastlike bodies like an algae. Example (b) has a cell waf f with spines, very similar to the modern algae, Pediostrum.

The Extraordinary Evolution of Eukaryotic Cells For years, biologists have grappled with the problern ofhow a cell as complex

mutualistic existence would maintain both cell types and create the

eukaryotic cell originated. One ofthe most fascinating explanations is that of endosymbiosis. This theory proposes that eukaryotic cells arose when a much larger prokaryotic cell engulfed smaller bacterial cells that began to

eukaryotic cell and its organelles. This scenario also has the advantage of explaining the relationships among the major Domains. So well accepted is the theory that bacteriologists have placed both mitochondria and chloroplasts on the family tree ofbacteria (see figure 4.27).

as the

live and reproduce inside the prokaryotic cell rather than being destroyed. As the smaller cells took up permanent residence, they came to perform specialized functions for the larger

cell, such as food synthesis and oxygen utilization, that enhanced the cell's versatility and survival. In time, the cells evolved into a single functioning entity, and the relationship

A smaller prokaryotic cell similar to purple bacteria that can use oxygen

A larger prokaryotic cell such as an archaea has a,flexible outer envelope and mesosomelike internal membranes to enclose the nucleoid.

became obligatory.

At first, the idea of endosymbiosis was greeted with

some contro-

Nuclear enverope

The larger cell engulfs the smaller one; smaller one survives and remains surrounded by the vacuolar memDrane.

versy-however, we now know that associations of this sort ate rathet common in the microbial world. Hundreds of protozoa have been discovered harboring living microbes internally. In some cases, this is a temporary symbiosis and not obligatory but certain members of Euplotes contain algae and bacteria that they need to stay alive. The biologist most responsible for validation ofthe theory ofendosymbiosis is Dr. Lym Margulls. Using modern molecular techniques, she has accumulated convincing evidence ofthe relationships between the organelles of modern eukaryotic cells and the structure of bacteria. In many ways, the mitochondrion of eukaryotic cells behaves as a tiny cell within a cell. It is capable of independent division, contains a circular chromosome with bacterial DNA sequences, and has ribosomes that are clearly prokaryotic. Mitochondria also have bacterial membranes and can be inhibited by drugs that affect only bacteria. One possible origin ofchloroplasts could have been endosymbiotic cyanobacteria that provided their host cells with a built-in feeding mechanism. Evidence is seen in a modern flagellated protist that harbors specialized chloroplasts with cyanobacterial chlorophyll and thylakoids. Dr. Margulis also has convincing evidence that eukaryotic cilia and flagella have arisen from endosymbiosis between spiral bacteria and the cell membrane of early eukaryotic cells. Most notably is a myxotrich found in termites that has spirochetes fi.rnctioning as flagella. The accompanying figure shows a possible sequence ofevents. A large archaeal cell with its flexible envelope could engulf a smaller bacterial cell, probably similar to a modern purple bacterium. The archaea would contribute its cytoplasmic ribosomes and some unique aspects of protein synthesis, as predicted by known characteristics (see table 4.5). Folds in the cell membrane could wrap around the chromosome to form a nuclear envelope. For its part, the bacterial cell would forge a metabolic relationship with the archaea, making use of archaeal molecules, and contributing energy through aerobic respiration. Evolution of a stable

Early nucteus

Smaller bacterium becomes a permanent resident of its host's cytoplasm; it multiplies and is passed on during cell division..lt utilizes aerobic metabolism and increases energy availability for the host. Early mitochondria

Early endoplasmic reticulum

Ancestral eukaryotic cell develops additional membrane pouches that become the . endoplasmic reticulum and Golgi apparatus.

Photosynthetic bacteria (similar to cyanobacteria) are also engulfed; they develop into chloroplasts: Ancestral cell

Many protozoa, animals

Algae, higher plants

Explain some of the ways the early mitochondria and chloroplasts could have assisted the nutrition ofthe larger cell. Answer available Dr. Lynn Margulis

at http : //www. mhhe. c om/talaro 7

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124

5

A Survey of Eukaryotic Cells and Microorganisms

Eukaryotic Organisms Studied

in Microbiology Unicellular

May Be or

Protozoa

Fungi

Always

Unicellular

Multicellular

Algae

Multicellular except Reproductive Stages Helminths (animals

with unicellular egg or larval forms)

In general, eukaryotic microbial cells have a cytoplasmic membrane, nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, cytoskeleton, and glycocalyx. A cell wall, locomotor appendages, and chloroplasts are found only in some groups. In the following sections, we cover the microscopic structure and functions of the eukaryotic cell. As with the prokaryotes, we begin on the outside and proceed inward through the cell.

Locomotor Appendages: Cilia and Flagella just

a disorganized assemblage of cells like a colony. Rather,

it is

composed of distinct groups of cells that cannot exist independently of the rest of the body. The cell groupings of multicellular organisms that have a specific function are termed tissues, and groups of tissues make up organs, Looking at modern eukaryotic organisms, we find examples of many levels of cellular complexity (table 5.1). All protozoa, as well as numerous algae and firngi, are unicellular. Truly multicellular organisms are found only among plants and animals and some of the fungi (mushrooms) and algae (seaweeds). Only certain eukaryotes are taditionally studied by microbiologists-primarily the protozoa, the microscopic algae and fungi, and animal parasites, or helminths.

5.2 Form and Function of the Eukaryotic Cell: External Structures The cells of eukaryotic organisms are so varied that no one member can serve as a typical example. The composite structure of a eukaryotic cell is depicted in figure 5.2. Due to the organelles, it has much greater complexity and compartmentalization than a prokary-

otic cell. No single type of microbial cell would contain all structures represented. Differences among fungi, protozoa, algae, and animal cells are introduced in later sections. The follo*ing flowchart maps the organization of a eukaryotic cell. Compare this flowchart to the one found on page 90 in chapter 4.

Motility allows a microorganism to locate life-sustaining nutrients and to migrate toward positive stimuli such as sunlight; it also permits avoidance of harmful substances and stimuli. Locomotion by means of flagella is common in protozoa, algae, and a few fungal and animal cells. Cilia are found only in protozoa and

animal cells.

Although they share the same name, eukaryotic flagella are much different from those ofprokaryotes. The eukaryotic flagellum is thicker (by a factor of 10), structurally more complex, and covered by an extension of the cell membrane. A single flagellum is a long, sheathed cylinder containing regularly spaced hollow tubules-microtubules-that extend along its entire length (figure 5.30). A cross section reveals nine pairs of closely attached microtubules surrounding a single central pair. This scheme, called the 9 + 2 arrangement, is a universal pattern of flagella and cilia (figure 5.3a). The nine pairs are linked together and anchored to the pair in the center. This architecture permits the microtubules to "walk" by sliding past each other, whipping the flagellum back and forth. Although details of this process are too complex to discuss here, it involves expenditure ofenergy and a coordinating mechanism in the cell membrane. Flagella can move the cell by pushing it forward like a fishtail or by pulling it by a

lashing or twirling motion (figure 5.3c). The placement and number of flagella can be useful in identifying flagellated protozoa and, certain algae. Cilia are very similar in overall architecture to flagella, but they are shorter and more numerous (some cells have several thousand). They are found only in certain protozoa and animal cells. In

l-Rppenoages

Ftageila I Cilia Exrernat-l ^. I urycocaryx I Capsules L Stimes

Eucary"c"l Boundary of ceil --JI

cell wall Cytoplasmic membrane

Cytoplasmic matrix

-'-j

Nucleus

l-Nuclear envelope Nucleolus

-----------.1 l_Chromosomes Endoplasmic reticulum Golgi complex Mitochondria Chloroolasts

Organelles

Ribosomes cytosr<ereton

--- l-

M

i:;}li:Hff ,"

-

-

Ribosomes Lysosomes

5.2

125

Form and Function of the Eukaryotic Cell: External Structures

Cell wall*

Mitochondrion

Cell membrane

Golgi apparatus

Rough endoplasmic reticulum with ribosomes

Microfilaments

Flagellum'

Nuclear membrane wlth pores Nucleus Lysosome

Nucleolus

Microtubules

Chloroplast* Centrioles*

*Structure not present in all cell types

Flgure

5.2

Overview of composite eukaryotic cell.

This drawing represents all structures associated with eukaryotic cells, but no microbial cell possesses all of them. See figures 5.'16, 5.26, and 5.28for examples of individual cell types.

the ciliated protozoa, the cilia occur in rows over the cell surface, where they beat back and forth in regular oarlike strokes (figure 5.4) and provide rapid motility. The fastest ciliated protozoan can swim up to 2,500 pm/s-a meter and a half per minute! On some cells, cilia also function as feeding and filtering structures.

The Glycocalyx Most eukaryotic cells have a glycocdyx, an outermost boundary that comes into direct contact with the environment. This structure is usually composed ofpolysaccharides and appears as a network offibers,

a slime layer, or a capsule much like the glycocalyx of prokaryotes. From its position as the exposed cell layeq the g$cocalyx serves a variety of functions. Most prominently, it promotes adherence to environmental surfaces and the development of biofilms and mats. It also serves.important receptor and communication functions and offers some protection against environmental changes. The nature of the layer beneath the glycocalyx varies among the several eukaryotic groups. Fungi and most algae have a thick, rigid cell wall surrounding a cell membrane. Protozoa, a few algae, and all animal cells lack a cell wall and are encased primarily by a cell membrane.

Chapter

126

5

A Survey of Eukaryotic Cells and Microorganisms

Oral groove with gullet

Micronucleus

(a)

Contractile vacuole

,ffi

Microtubules

/

(b) Figure

Power stroke

5.4

,-**-* 1 ,

Recovery stroke

Structure and locomotion in ciliates.

(a) The structure of a simple representative , Holophryo, with a regular pattern of cilia in rows. (b) Cilia beat in coordinated waves, driving the cell forward and backward. View of a single cilium shows that it has a pattern of movement like a swimmer, with a power forward stroke and a repositioning stroke.

Form and Function of the Eukaryotic Cell: Boundary Structures The Cell Woll The cell walls of the fungi and algae are rigid and provide structural support and shape, but they are different in chemical composition from prokaryotic cell walls. Fungal cell walls have a thick, inner layer of polysaccharide fibers composed of chitin or cellulose and a thin outer layer of mixed glycans. The cell walls of algae are quite

in chemical composition. Substances commonly found among various algal groups are cellulose, pectin,l mannans,2 and minerals such as silicon dioxide and calcium carbonate. varied

The Cytoplosmic M embrane

(c) Whips back and forth and pushes in snakelike pattern

Flgure

5.3

I V

Lashes, grabs the substrate, and pulls

The cytoplasmic (cell) membrane of eukaryotic cells is a typical bilayer of phospholipids in which protein molecules are embedded (see figure 4.16). In addition to phospholipids, eukaryotic membranes also contain sterols of various kinds. Sterols are different from phospholipids in both structure and behavior, as you may recall from chapter 2. Their relative rigidity confers stability on eukaryotic membranes. This strengthening feature is extremely important in cells that lack a cell wall. Cytoplasmic

The structures of microtubules.

(a) A cross section that reveals the typical 9 * 2 arrangement found in both flagella and cilia. (b) Longitudinal section through a flagellum, showing orientation of microtubules. (c) Locomotor patterns seen in flagellates.

l.

A polysaccharide composed ofgalach:ronic acid subunits.

2. A polymer of the sugm knom

as mannose-

5.3

Form and Function of the Eukaryotic Cell: Internal Structures

eukaryotes are functionally similar to those of prokaryotes, serving as selectively permeable barriers in transport. Unlike prokaryotes, eukaryotes have extensive membranemembranes

of

bound organelles that can account for

600/o

127

Chromatin

to 80% of their

volume.

I

Eukaryotes have cells with a nucleus and organelles compartrnentalized by membranes. Evidence shows that they originated from prokaryote ancestors abovt 2 billiol years ago. Eukaryotic cell structure enabled eukaryotes to diversifu from single cells into a huge variety of complex multicellular forms. u The cell structures common to most eukaryotes are the cell membrane, nucleus, vacuoles, mitochondria' endoplasmic reticulum, Golgi apparatus, and a cytoskeleton. Cell walls, chloroplasts, and locomotor organs are present in some eukaryote groups. Microscopic eukaryotes use locomotor organs such as flagella or cilia for moving themselves or their food. The glycocalyx is the outermost boundary of most eukaryotic cells. Its functions are adherence, protection, and reception ofchemical signals from the environment or from other organisms. The glycocalyx is supported by either a cell wall or a cell membrane. w The cytoplasmic (cell) membrane of eukaryotes is similar in function to that ofprokaryotes, but it differs in composition, possessing sterols as additional stabilizing agents.

r

Nuclear envelope

Nuclear pore

Nucleolus

(a)

Nuclear pore

r

5.3 Form and Function of the Eukaryotic Cell : Internal Structures The Nucleus: The Control Center The nucleus is a compact sphere that is the most prominent organelle of eukaryotic cells. It is separated from the cell cytoplasm by an external boundary called a nuclear envelope. The envelope has a unique architecture. It is composed of two parallel membranes separated by a narrow space, and it is perforated with small, regular$ spaced openings, or pores, at sites where the two membranes unite (figure 5.5). The pores are structured to serve as selective passageways for molecules to migrate between the nucleus and cytoplasm. The main body of the nucleus consists of a matrix called the nucleoplasm and a granular mass, the nucleolus. The nucleolus is the site for ribosomal RNA synthesis and a collection area for ribosomal subunits. The subunits are transported through the nuclear pores into the cytoplasm for final assembly into ribosomes. A prominent feature of the nucleoplasm in stainedpreparations is a network of dark fibers known as chromatin because of its attraction for dyes. Analysis has shown that chromatin actually comprises the eukaryotic chromosomeso large units of genetic information in the cell. The chromosomes in the nucleus of nondividing cells are not readily visible because they are long, linear DNA molecules bound in varying degrees to histone proteins, and they are far too fine to be resolved as distinct structures without extremely high magnification. During mitosis, however, when the

Flgure

5.5

The nucleus. (a) Electron micrograph section of an interphase nucleus, showing its most prominent features. (b) Cutaway three-dimensional view of the relationships of the nuclear envelope and pores.

duplicated chromosomes are separated equally into daughter cells, the chromosomes themselves become readily visible as discrete bodies (figure 5.6). This appeamnce arises when the DNA becomes highly condensed by forming coils and supercoils around the histones to prevent the chromosomes from tangling as they are separated into new cells. This process is described in more detail in chapter 9.

Although we correctly view the nucleus as the primary genetic center, it does not function in isolation. As we show in the next three sections, it is closely tied to cytoplasmic organelles that perform elaborate cell functions.

r28

Chapter

5

A Survey of Eukaryotic Cells and Microorganisms

Centrioles

Interphase (resting state prior

Chromatin

to cell division)

Cell membrane Nuclear envelope

Prophase

Nucleolus Cytoplasm Daughter cells

Spindle fibers

Cleavage furrow

Chromosome

Centromere Chromosome

Telophase

Early metaphase

Metaphase

anaphase

ffi

Early anaphase

@ Process Flgure 5.6 Changes in the cell and nucleus that accompany mitosis in a eukaryotic cell. P (1) Before mitosis (at interphase),

chromosomes are visible only as chromatin. (2) As mitosis proceeds (early prophase), chromosomes take on a fine, threadlike appearance as they condense, and the nuclear membrane and nucleolus are temporarily disrupted. (3)-(4) By metaphase, the chromosomes are fully visible as X-shaped structures. The shape is due to duplicated chromosomes attached at a central point, the centromere. (5)-(6) Spindle fibers attach to these and facilitate the separation of individual chromosomes durinq anaphase. (7)-(8) Telophase completes chromosomal separation and division of the cell proper into daughter cells.

Endoplasmic Reticulum: A Passageway in the Cell The endoplasmic reticulum (ER) is a microscopic series of tunnels used in transport and storage. Two kinds of endoplasmic reticulum are the rough endoplasmic reticulum (RER) (figure 5.7) and

the smooth endoplasmic reticulum (SER). Electron micrographs show that the RER originates from the outer membrane of the nu-

clear envelope and. extends in a continuous network through the cytoplasm, even out to the cell membrane. This architecture permits the spaces in the RER, or cisternae, to transport materials from the nucleus to the cytoplasm and ultimately to the cell's exterior. The RER appears rough because of large numbers of ribosomes partly attached to its membrane surface. Proteins syrthesized on the ribosomes are shunted into the cavity of the reticulum and held

there for later packaging and transport. In contrast to the RER, the SER is a closed tubular network without ribosomes that functions in nutrient processing and in synthesis and storage of nonprotein

macromolecules such as lipids.

Golgi Apparatus: A Packaging Machine Golgf apparatus, also called the Gotgi complex or body, is the site in the cell in which proteins are modified" stored, and packaged for transport to their final destinations. It is a discrete organelle consisting of a stack of flattened, disc-shaped sacs called cisternae The

3. (gol'-jee) Named for C Golgi, an Italian histologist who first described the apparatus

in

1898.

5.3

Form and Function of the Eukaryotic Cell: lnternal

Structures

129

Polyribosomes Cistern

Small subunit mRNA Ribosome

Large subunit

RER membrane Cistern

Protein being synthesized

Figure 5.7 The origin and detailed structure of the rough endoplasmic reticulum

(RER).

(b) Three-dimensional projection of the RER. lafschematic view of the origin of the RER from the outer membrane of the nuclear envelope. the RER cisternae and distributed through its within collected (c) Detail of the orientation of a ribosome on the RER membrane. Proteins are network to other destinations.

(think of pita bread!). These sacs have outer limiting membranes and cavities like those of the endoplasmic reticulum, but they do not form a continuous network (figure 5.8). This organelle is always closely associated with the endoplasmic reticulum both in its location and function. At.a site where it borders on the Golgi apparatus, the endoplasmic reticulum buds off tiny membranebound packets of protein called transitional vesicles lhat are picked up by the forming face of the Golgi apparatus' Once in the complex itself, the proteins are often modified by the addition of polysaccharides and lipids. The final action ofthis apparatus is to pinch off finished condensing vesicles that will be conveyed to organelles such as lysosomes or transported outside the cell as secretory vesicles (figure 5.9).

Nucleus, Endoplosmic Reticulum, ond Golgi Apporotus: Noture's AssemblY Line As the keeper of the eukaryotic genetic code, the nucleus ultimately governs and regulates all cell activities. But, because the nucleus remains fixed in a specific cellular site, it must direct these activi-

ties through a structural and chemical network (figure 5.9). This network includes ribosomes, which originate in the nucleus, and the rough endoplasmic reticulum, which is continuously connected

with the nuclear envelope.

Flgure

5.8

Detail of the Golgi apparatus.

The flattened layers are cisternae. Vesicles enter the upper surface and leave the lower surface.

130

Chapter

5

A Survey of Eukaryotic Cells and Microorganisms

5:ii","

G

Engulfment of food

Food vacuole

Formation of food vacuole

Lysosome

Figure

5.9

The transport process.

The cooperation of organelles in protein synthesis and transport: nucleus -+ RER -+ Golgi apparatus --+ vesicles -+ secretion.

Initially, a segment of the genetic code of DNA containing the instructions for producing a protein is copied into RNA and passed out through the nuclear pores directly to the ribosomes on the endoplasmic reticulum. Here, specific proteins are synthesized from the RNA code and deposited in the lumen (space) of the endoplasmic reticulum. Details of this process are covered in chapter 9. After being transported to the Golgi apparatus, the protein products are chemically modified and packaged into vesicles that can be used by the cell in a variety of ways. Some of the vesicles contain enzymes to digest food inside the cell; other vesicles are secreted to digest materials outside the cell, and yet others are important in the enlargement and repair of the cell wall and membrane.

A lysosome is one type of vesicle originating from the Golgi apparatus that contains a vaiety of enzymes. Lysosomes are involved in intracellular digestion offood particles and in protection against invading microorganisms. They also participate in digestion and removal of cell debris in damaged tissue.

Other types of vesicles include vacuoles (vak'-yoo-ohl), which are membrane-bound sacs containing fluids or solid particles to be digested excreted, or stored. They are formed in phagocytic cells (certain white blood cells and protozoa) in response to food and other substances that have been engulfed. The contents ofa food vacuole are digested through the merger ofthe vacuole with a lysosome. This merged structure is called a phagosome (figure 5.10). Other types ofvacuoles are used in storing reserve

Merger of Iysosome and vacuole Phagosome

Digestion Digestive vacuole

@ Figure 5.lO

The origin and action of lysosomes in

phagocytosis.

food such as fats and glycogen. Protozoa living in freshwater habitats regulate osmotic presswe by means of contractile vacuoles, which regularly expel excess water that has diffused into the cell (described later).

Mitochondria: Energy Generators of the Cell None of the cellular activities of the genetic assembly line could proceed without a constant supply of energy, the bulk of which is

5.3

Form and Function of the Eukaryotic Cell: lnternal

Structures

131

circular strands of DNA, and have prokaryote-sized 70S ribosomes. These findings have given support to the endosymbiotic theory oftheir evolutionary origins discussed in Insight 5.1.

Chloroplasts: Photosynthesis Machines Chloroplasts are remarkable organelles found in algae and plant cells that are capable of converting the energy of sunlight into

Gristae (darker lines)

Matrix (lighter spaces)

chemical energy through photosynthesis. The photosynthetic role of chloroplasts makes them the primary producers of organic nutrients upon which all other organisms (except certain bacteria) ultimately depend. Another important photosynthetic product of chloroplasts is oxygen gas. Although chloroplasts resemble mitochondria, chloroplasts axe larger, contain special pigments, and are much more varied in shaPe. There are differences among various algal chloroplasts, but most are generally composed of two membranes, one enclosing the other. The smooth, outer membrane completely covers an inner membrane folded into small, disclike sacs called thylakoids that are stacked upon one another into grana. These structures carry the green pigment chlorophyll and sometimes additional pigments as well. Surrounding the thylakoids is a ground substance called the stroma (figure 5.12). The role of the photosynthetic pigments is to absorb and transform solar energy into chemical energy, which is then used during reactions in the stroma to synthesize carbohydrates. We further explore some important aspects of photosynthesis in chapters 7 and 8.

Ribosomes: Protein SYnthesizers Figure 5.11

General structure of a mitochondrion.

(a) A three-dimensional projection. (b) An electron micrograph. ln most cells, mitochondria are elliptical or spherical, although in certain fungi, algae, and protozoa, they are long and filamentlike.

generated

in most

eukaryotes by mitochondria (my'-toh-kon'-

In an electron micrograph of a eukaryotic cell, ribosomes are nu-

*dotted" appearance to the cytomerous, tiny particles that give a plasm (see figue 5.31b). Ribosomes are distributed in two ways: Some are scattered freely in the cytoplasm and cytoskeleton; others are intimately associated with the rough endoplasmic reticulum as

Chloroplast enveloPe

dree-uh). When viewed with light microscopy, mitochondria appear as round or elongated particles scattered throughout the cytoplasm.

Higher magnification reveals that a mitochondrion consists of a smooth, continuous outer membrane that forms the external contour. and an inneq folded membrane nestled neatly within the outer membrane (figure 5.114). The folds on the inner membrane, called

cristae (kris'-te), vary in exact structure among eukaryotic cell types. Plant, animal, and fungal mitochondria have lamellar cristae folded into shelflike layers. Those ofalgae and protozoa are tubular, fingerlike projections or flattened discs. The cristae membranes hold the enzymes and electron carri-

of aerobic respiration. This is an oxygen-using process that extracts chemical energy contained in nutrient molecules and stores it in the form of high-energy molecules, or AIP. More detailed functions of mitochondria are covered in chapter 8. The spaces around the cristae are filled with a chemically complex fluid called the matrix, which holds ribosomes, DNA, and the ers

pool of en-4mes and other compounds involved in the metabolic cycle. Mitochondria (along with chloroplasts) are unique among organelles in that they divide independently of the cell, contair

Flgure

5.12

Detail of an algal

t32

Chapter

5

A Survey of Eukaryotic Cells and Microorganisms

Rough endoplasmic reticulum memorane Microfilaments

Flgure 5.13

A model of the cytoskeleton.

(a) Depicted is the relationship between microtubules, microfilaments, and organelles. (Not to scale.) (b) The pattern of microtubules highlighted by a fluorescent dye.

previously described. Multiple ribosomes axe often found arranged in short chains called polyribosomes (polysomes). The basic structure of eukaryotic ribosomes is similar to that of prokaryotic ribosomes, described in chapter 4. Both are composed of large and small subunits of ribonucleoprotein (see figure 5.7). By contrast, however, the eukaryotic ribosome (except in the mitochondrion) is the larger 80S variety that is a combination of 605 and 40S subunits. As in the prokaryotes, eukaryotic ribosomes are the staging areas for protein synthesis.

is

spindle fibers that play an essential role in mitosis are actually mi-

crotubules that attach to chromosomes and separate them into daughter cells. As indicated earlier, microtubules are also responsible for the movement of cilia and flagella. At this point, you have been introduced to the major features of eukaryotic cells. Table 5.2 provides a general surnmary of these characteristics. It also presents an opportunity to compare and contrast eukaryotic with prokaryotic cells (chapter 4) on the basis of significant anatomical and physiological traits. Also included for comparison purposes are viruses described in chapter 6.

The Cytoskeleton: A Support Network All cells share a generalized region encased by the cell membrane called the cytoplasm or, in eukaryotic cells, the cytoplasmic matrix. This complex fluid

houses the organelles and sustains ma-

jor metabolic and synthetic activities. It also provides the framework of support in cells lacking walls. The mahix is criss-crossed by a flexible framework of molecules called the cytoskeleton (figure 5.13). This framework appears to have several functions, such as anchoring organelles, providing support, and permitting shape changes and movement in some cells. The two main types of cytoskeletal elements arc microJilaments and microtubules. Microfilaments are thin strands composed of the protein actinthat attach to the cell membrane and form a network through the cytoplasm. Some microfilaments are responsible for movements of the cytoplasm, often made evident by the streaming of organelles around the cell in a cyclic pattern. Other microfilaments are active in amoeboid motion, atype of movement typical of cells such as amoebas and phagocytes that produces extensions of the cell membrane (pseudopods) into which the cytoplasm flows. Microtubules are long, hollow tubes that maintain the shape of eukaryotic cells such as protozoa that lack cell walls. They may also serve as an alternative transport system for molecules. The

e

The genome of eukaryotes is located in the nucleus, a spherical skucture surrounded by a double membrane. The nucleus contains the nucleolus, the site of ribosome synthesis.

DNA is organized into

chromosomes in the nucleus. m The endoplasmic reticulum (ER) is an internal network of membranous passageways extending throughout the cell. x The Golgi apparatus is a packaging center that receives materials from the ER and then forms vesicles around them for storage or for transport to the cell membrane for secretion. F The mitochondria generate energy in the form ofATp to be used in numerous cellular activities. ffi Chloroplasts, membranous packets found in plants and algae, are used in photosynthesis. Ribosomes are the sites for protein synthesis present in both eukaryotes and prokaryotes. € The cytoskeleton, consisting of microfilaments and microtubules, maintains the shape of ceils and produces movement of cytoplasm within the cell, movement of chromosomes at cell division, and in some groups, movement of the cell as a unit.

*

5.3

,,

Form and Function of the Eukaryotic Cell: Internal Structures

133

A General Comparison of Prokaryotic and Eukaryotic Cells and Viruses

Function or Structure

Characteristic*

Prokaryotic Cells

Eukaryotic Cells

Viruses*n

Genetics

Nucleic acids

+

+

+

Chromosomes

+ **x

+

Reproduction

True nucieus

+

Nuclear envelope

+

Mitosis

T

Production of sex cells

fission Independent Golgi apparatus

Binary Biosynthesis

+l-

+

+

+

+

+

-

+

+ +

Endoplasmic reticulum Ribosomes

Enzymes

Respiration

+

+

+

Mitochondria Pigments

Photosynthesis

T/-

Chloroplasts

Motility/locomotor

structures

Flagella

Tt-

+t-

Cilia Shape/protection

+l+l+l-

Cell membrane

+

+

+t-

Cell wall

+ {.*t

doubles with each new cell product of reproduction goes on to divide by binary fission, the population (b) Plotting the logarithm numbers' simple or by exponent) (2 raised to-an division or generation. This process can be represented by togiritnms gives a curved slope' arithmetically numbers cell the plotting whereas of the cells produces a straight line indicative of exponeniial trowth,

(a) starting with

a single cell, if each

208

Chapter

Z

Elements of Microbial Nutrition, Ecology, and Growth

number 2 with an exponent

(2t,2r,2t,20); (2)

the exponent in_

creases by one in each generation; and (3) the number

oith.

nent is also the number of the generation. This growth pattern ""pois termed exponential. Because these populations often u..u "orrtuin large numbers of cells, it is useful to express them by means of ex_ ponents or logarithms (see appendix A). The data from growing a bacterial population are graphed by plotting the number of cells as a function of time. The cell number can be represented rogarithmi-

cally or arithmetically. plotting the logarithm number over time provides a straight line indicative of exponential growth. plouing the data arithmetically gives a constantly curved sLpe. In g"n"ruf logarithmic graphs are preferred because an accurate cell number is easier to read, especially during early growth phases.

Predicting the number of cells that will arise during a long growth period (yielding millions of cells) is based on a relativelv simple concept. One could use the method of addition 2 + 2 : i; 4 + 4 : 8; 8 + 8 : 16; 16 * 16 : 32, andso on, oramethodof multiplication (for exampl e, 2s : 2 X 2 X 2 X 2 x 2), bntit is easy to see that for 20 or 30 generations, this calculation could be very tedious. An easier way to calculate the size of a population over time is to use an equation such as:

Nt: (Nt)" In this equation' NTis the totar number of cells in the population at some point in the grouth phase, ,Ay', is the starting number, the exponent n denotes the generation numbet, and2" represents the nurnber of cells in that generation. If we know any two of the values, the other values can be calculated. Let us use the example of staphylococan aureus to calculate how many cells (N) will be present in an egg salad sandwich after it sits in a wafin car for 4 hours. we will assume that N, is l0 (number of cells deposited in the sandwich while it was being pre_ pared). To derive n, we need to divide 4 hours (240 minutesl tv tfr. generation time (we will use 20 minutes). This calculation comes out to 12,so2" is equal to 212. Using acalculator,6 we findthat 212 is4,096. Finar number

(t'

: i3,;

t'll^!r",^rceus in the sandwich

This same equation, with modifications, is used to determine the generation time, a more complex calculation that requires know_ ing the number of cells at the beginning and end of a growth period.

Such data are obtained through actual testing uy a methoo ais-

cussed in the following section.

The Population Growth Curve In reality, a population of bacteria does not maintain its potential grouth rate aod does not double endlessly, because in mosisystems numerous factors prevent the cells from continuously dividing at their maximum rate. Quantitative laboratory studies indicate that a population typically displays a predictable pattern, or growth curve,-over time. The method traditionally used to observe the population growth pattern is a viable count technique, in which the total number of live cells is counted over a given time period. In brief, this method entails 1.

)

6.

placing a few cells into a sterile liquid medium, incubating this culture over a period ofseveral hours, See appendix

A for a table with powers of2.

3. sampling the broth at regular intervals during incubation, 4. plating each sample onto solid media, and 5. counting the number of colonies present after incubation. Insight 7.4 gives the details ofthis process.

Stages in the Normal Growth Curve The system of batch culturing described in Insight 7.4 is crosed, meaning that nutrients and space are finite and there is no mechanism for the removal of waste products. Data from an entire growth

period of 3 to 4 days typically produce a curye with a series of phases termed the lag phase, the exponential growth (log) phase, the stationary phase, and the death phase (figure 7.16). The lag phase is an early.,flat,, period on the graph when the population appears not to be growing or is growing at less than the exponential rate. Growth lags primarily because

1. the newly inoculated cells require a period of adjustment, en_ largement, and synthesis of DNA, enzymes, and ribosomes; 2. the cells are not yet multiplying at their maximum rale; and 3. the population of cells is so sparse or dilute that the sampling misses them. The length ofthe lag period varies from one population to another, depending on the condition of the microbes and medium. It is important to note that even though the population ofcells is not increasing (growing), individual cells are metabolically active as they increase their contents and prepare to divide. The cells reach the maximum rate of cell division durins the

exponential growth (logarithmic or log) phase, a period aidng which the curve increases geometrically. This phase will continui

as long as cells have adequate nutrients and the environment is favorable. During this phase, the population fulfilts its potential gen_ eration time, and growth is balanced and genetically coordinated.

At the stationary grorrth phase, the population enters a survival mode in which cells stop growing or grorv slowly. The curve levels off because the rate of cell inhibition or death balances out the rate ofmultiplication. The number of viable cells has reached maximum and remains constant during this period. The decline in the growth rate is caused by several factors. A common reason is the depletion of nutri_ ents and oxygen. Another is that the increased cell density often causes an accumulation of organic acids and other toxic biochemicals. As the limiting factors intensify, cells begin to die at an exponen_ tial rate (literally perishing in their own wastes), and most are unable to multiply. The curve now dips downward as the death phase be_ gins. The speed with which death occurs depends on the relative resistance of the species and how toxic the conditions are. but it is usually slower than the exponential growth phase. viable cells often remain many weeks and months after this phase has begun. In the laboratory, refrigeration is used to slow the progression of the death phase so that cultures will remain viable as long as possible.

Procticol Importance of the Growth Curve The tendency for populations to exhibit phases ofrapid growth, slow growth, and death has important implications in microbial control, infection, food microbiology, and culture technology. Antimicrobial agents such as heat and disinfectants rapidly accelerate the death phase in all populations, but microbes in the exponential growth phase

7.3

The Studv of Microbial Crowth

209

steps in a Viable Plate count-Batch culture Method A growing population is established by inoculating a flask containing

such bacteria, the CFU is the smallest unit of colony formation and dispersal.

a

Multiolication ofthe number of colonies in a single sample by the container's volume gives a fair estimate of the total population size (number of cells) at any given point. The grouth curve is determined by graphing the number for each sample in sequence for the whole incubation period (see figure 7'16)' Because of the scarcity of cells in the early stages of growth, some samples can give a zero reading even if there are viable cells in the culture. The sampling itself can remove enough viable cells to alter the tabulations, but since the purpose is to compare relative trends in growth, these factors do not significantly change the overall pattern'

known quantity of sterile liquid medium with few cells of a pure culture' The flask is incubated at that bacterium's optimum temperature and timed' The population size at any point in the growth cycle is quantified by removing a tiny measured sample of the culture from the growth chamber and plating it out on a solid medium to develop isolated colonies' This procedure is repeated at evenly spaced intervals (i'e., every hour for 24 hours)' Evaluating the samples involves a common and important principle in microbiology: One colony on the plate represents one cell or colony-forming unit (CFU) from the original sample. Because the CFU of some bacteria is actually composed of several cells (consider the clustered arrangement of staphylococcus, for instance), using a colony count can underestimate the a

exact population size to an extent. This is not a serious problem because,

Predict the outcome with this technique if it were carried out for an additional 5 hours. Answer available at http ://www'mhhe'com/talaroT

in

Flask inoc ulated +

i5 500 ml

Samples t rken at equi llly spaced ntervals (0.1 ml) 60 min

Plates are incubated, colonies are counted Number of colonies (CFU) per 0.1 ml Total estimated cell population in flask

180 min

I

I

,**T..t-"

360 min

420 min

480 min

I

i

I

|

540 min

600 min

I

r

0.1

ml

I Sample is diluted in liquid agar medium and poured or spread over surface of solidified meotum

120 min

x

i

I

i:::

ry

n ,i

il

,ry

ffi

L#

ffi

fl$

hffi

ffiffi

I

I

I

I

I

I

n

*-l

,on: Po+{

@

8.2O The reactions of a single turn of the Krebs cycle. will produce two spins of this pathway. Note that this is an enlarged, more detailed view of the middle

Process Flgure

Each glucose

in figure 8.17o (inset). lt occurs in the cytoplasm of prokaryotes and the mitochondrial matrix of eukaryotes.

phase depicted

8.3 Pathways of Bioenergetics

23s

biological systems for extracting larger amounts of energy from the remaining acetyl fragment. As we take a single spin around the Krebs cycle, it will be help-

metabolic schemes, it is essential for generating small organic molecules that microbes require for synthesis (see figure 8.26).

ful to keep track of

The Respiratory Chain: Electron Transport and Oxidative PhosPhorYlation

1. the numbers ofcarbons (#C) ofeach substrate andproduct,

2. reactions where CO2 is generated 3. the involvement of the electron carriers NAD+ and EAD, and 4. the site ofATP synthesis. Be aware that the terms used for organic acids can be shown as either the acid form (oxaloacetic acid) or its salt (oxaloacetate). We use the acid form in this text for the most part. The eight reactions in the Krebs cycle are: reacts with the acetyl group (2C) on acetyl CoA, thereby forming citric acid (citrate; 6C) and releasing coenzyme A so it can join with another acetyl group'

1. Oxaloacetic acid (oxaloacetate; 4C)

Citrrc acid is converted to its isomer, isocitric acid (isocitrate; 6C), to prepare this substrate for the decarboxylation and redox reaction ofthe next steP. 3. Isocitric acid is acted upon by an enzyme complex including NAD+ or NADP (depending on the organism), in a reaction that generates NADH or NADPH, splits off a carbon dioxide, and leaves a-ketoglutaric acid (cr-ketoglutarate; 5C). 4. Alpha-ketoglutaric acid serves as a substrate for the final decarboxylation reaction and yet another redox reaction, involving coenzyme A and yielding NADH. The product is the high-energy compound succinyl CoA ( C).

2.

At this point, the cycle has completed the formation of 3 CO2 molecules that balance out the original 3-carbon pynrvic acid released by glycolysis. The remaining steps are needed not only to regenerate the oxaloacetic acid to start the cycle again but also to extract more energy from the intermediate compounds leading to oxaloacetic acid.

5. Succinyl CoA is the source ofthe one substrate level phosphorylation in the Krebs cycle. In most bacteria, it proceeds with the formation of ATP, although eukaryotes produce guanosine triphosphate (GTP), a similar source of energy' The other product at this step is succinic acid (succinate; 4C). 6. Succinic acid undergoes a redox reaction, but in this case, the electron and H* acceptor is flavin adenine dinucleotide (FAD). The enzyme that catalyzes this reaction, succinyl dehydrogenase, is found in the bacterial cell membrane and mitochondrial crista of eukaryotic cells. The FADH2 generated directly enters the electron transport system. Fumaric acid (fumarate; 4C) is the product of this reaction. ?. The addition of water to fumaric acid results in malic acid (malate; 4C). This is one of the few reactions in respiration that directly incorporates water.

8. Malic acid is dehydrogenated (with formation of a final NADH), and oxaloacetic acid is formed. This step brings the cycle back to its original starting position, where oxaloacetic acid can react with acetyl coenzyme A. An important feature of this pathway is that acetyl groups from the breakdown of certain fats can enter the pathway and be used as an energy source (discussed in section 8.4).

The Krebs cycle is not present in all cells and it may not function under all metabolic conditions. But even in cells with alternate

We now come to the energy chain, which is the final "processing mill" for electrons and hydrogen and the major generator of AIP. Overall, the electron transport system @TS) consists of a chain of special redox carriers that receive electrons from reduced carriers (NADH, FADH2) generated by glycolysis and the Krebs cycle. The ETS shuttles the electrons in a sequential and orderly fashion (see figure 8.1 7a). The flow ofelecffons down this chain is highly energetic and gives offATP at various points. The step that finalizes the transport process is the acceptance ofelectrons and hydrogen by oxygen, producing water. Some variability exists from one organism to another, but the

principal compounds that carry out these complex reactions are NADH dehydrogenase, fl avoproteins, coenzyme Q (ubiquinone)'* and cytochromes.* The cytochromes contain a tightly bound metal atom at their center that is actively involved in accepting electrons and donating them to the next carrier in the series. The highly compartmenlalized structure of the respiratory chain is an important factor in its function. Note in figure 8.21 that the electron transport carriers and enzymes are embedded in the inner mitochondrial membranes in eukaryotes. The cell membrane is the equivalent structure for housing them in bacteria.

Elements

of Electron Transport: The Energy Cascade

The principal questions about the electron transport system are: How are the electrons passed from one carrier to another in the series? How is this progression coupled to AIP synthesis? and, Where and

how is oxygen utilized? Although the biochemical details of this process are rather complicated, the basic reactions consist of a number ofredox reactions now familiar to us. In general, the seven carrier compounds and their enzymes are arranged in linear sequence and are reduced and oxidized in turn (figure 8.21, lower view).

The sequence of electron carriers in the respiratory chain most aerobic organisms is

of

1. NADH dehydrogenase, which is closely associated in a complex with the adjacent carrier, which is

2. flavin mononucleotide (FMN) ; 3. coenzyme Q; 4. cytochrome b; 5. cytochrome c1; 6. cytochrome c; and 7. cytochromes a and 43, which

are complexed together.

The NADHs formed during glycolysis and the Krebs cycle are conveyed to the first carrier in the ETS. This sets in motion the remaining six steps. With each redox exchange, the energy level of the reactants is lessened. The released energy is captured and used by

* ttbiquinone (yoo-bik'-wih-nohn) L. ubique, everywhere. A type of chemical, similar to vitamin K, that is very common in cells. * ctrochr"ome (sy'+oh-krohm) Gl cyto, cell, and kroma, color. Cyochromes are pigmente{ iron-containing molecules similar to hemoglobin.

236

Chapter

8

An Introduction to Microbial Metabolism

F[ure 8.21 The etectron fltransport system and

Outer mitochondrial membrane

oxidative phosphorylation on the mitochondrial crista.

Starting at NADH dehydrogenase, electrons brought in from the Krebs cycle by NADH are passed along the chain of electron transport carriers. Each adjacent pair of transport molecules undergoes a redox reaction. Coupled to the transport of electrons is the simultaneous active

transport of H+ into the outer compartment by specific carriers. These processes set the scene for ATP synthesis and final H+ and e-

dehydrogenase

I

Puru

I

ICoenzymeQ f

f

Cytochrome

c, I

cytochrome a

acceptance by oxygen.

Cytochromec ICytochromea.

Cytochrome b

"r*{

From Krebs

ee 1/20,2- oe-

and glycolysis

f--l !

{De-

Outer compartment (intermembrane space) Inner compartment (mitochondrial matrix)

29

i"o tK {Ye

ATP synthase complexes, stationed along the cristae in close

The Formation of ATP and Chemiosmosis What biochemi-

association with the ETS carriers. Each NADH that enters electron transport can potentially give rise to 3 AIPs. This coupling ofAlp

qmthesis to elechon transport is termed oxidative phosphorylation. Because EADII, from the Krebs cycle enters the cycle after the NAD+ and FMN complex reactions, it has less energy to re-

cal processes are involved in coupling electron transport to the production of AfP? We first look at the system in eukaryotes, which have the components of electron fiansport embedded in a precise sequence on mitochondrial membranes. They are stationed between the inner mitochondrial matrix and the outer intermembrane space

ledse, and 2 ATPs result from its processing

(figure 8.21). According to a widely accepted concept called

8.3 Pathways of Bioenergetics

237

lntermembrane space

f,

+ Charoed

ffi -cnaroeo (a) As the carriers in the mitochondrial cristae transport electrons, they also actively pump H+ ions (protons) to the intermembrane space, producing a chemical and charge gradient between the outer and inner mitochondrial compartments.

(b) The distribution of electric potential and the concenhation gradient of protons across the membrane drive the synthesis of Ate Uy Rfe synthase. The rotation of this enzyme couples - . _ _ diffusion of H+ to the inner compartment with the bonding of ADP and Pi.The final event of electron transport is the reaction of the electrons with the H+ and 02 to form metabolic H2O. This step is catalyzed by cytochrome oxidase (cytochrome aa3).

* Cytoplasm ATP synthase

(c) Enlarged view of bacterial cell envelope to show the

ADP

*

ffi

Flgure

8.22

e e *

Chemiosmosis-the force behind ATP synthesis.

relationship of electron transport and ATP synthesis. Bacteria have the ETS and ATP synthase stationed in the cell membrane. ETS carriers transport H+ and electrons from the cytoplasm to the exterior of the membrane. Here, it is collected to create a gradient iust as it occurs in mitochondria.

238

Chapter

8

An Introduction to Microbial Metabolism

chemiosmosis, as the electron hansport carriers shuttle electrons, they actively pump hydrogen ions (protons) into the outer compaxt_ ment of the mitochondrion. This process sets up a concentration gradient of hydrogen ions called the proton motive force (pMF). The PMF consists of a difference in charge between the outer mem_ brane compartment (+) and the inner membrane compartment (-) (figure 8.22a). Separating the charge has the effect of a baltery, which can temporarily store potential energy. This charge will be maintained by the impermeability of the inner cristae membranes to H+. The only site where H+ can diffirse into the inner compartment is at the AIP synthase complex, which sets the stage for the final processing of H* leading to ATP synthesis.

AIP

synthase is a complex er.zqe composed oftwo large units, Fe and F1 (figure 8.22b).It is embedded in the membrane but paxt of it rotates like a motor and traps chemical energy. As the H+ ions flow through the Fo center of the enzyme by diffirsion, the F, compart-

ments pull in ADP and P,. Rotation causes a three-dimensional change in the en4me that bonds these two molecules, thereby releas-

ing ATP into the inner compartment (figure 8.22b). The enzyme is then rotated back to the start position and will continue the process. Bacterial AIP synthesis occurs by means of this same overall process. HoweveE bacteria have the ETS stationed in the cell membrane, and the direction of the proton movement is from the cytoplasm to the periplasmic space between the membrane and cell wall. Bacteria may also lack one or more of the electron carriers (figure 8.22c'5. These differences will affect the amount of AIp produced (discussed in the next section). In both cell types, the chemiosmotic theory has been supported by tests showing that oxidative phosphorylation is blocked if the mitochondrial or bacterial cell membranes are disrupted.

Potential Yield of ATPs from Oxidative phosphorylation The total of five NADHs (four from the Krebs cycle and one from glycolysis) can be used to synthesize; 15 ATPs

(5 X

15

x

3 per electron pair) during ETS:

2 per glucose

=

30

AIps

The single FADH produced during the Krebs cycle results in: 2 ATPs

2

x

per electron pair

2per glucose = 4AIPs

Thus, electron transport yields a potential tolal of 34 AIps from a single glucose.

others have several alternative electron transport schemes. Because many bacteria lack cytochrome c oxidase, this variation can be used to differentiate among certain genera of bacteria. An oxidase detection test can be used to help identifz members of the genera Ners_

seria and Pseudomonas and some species of Bacillus. Another variation in the cytochrome system is evident in certain bacteria

(Klebsiella, Enterobacter) that can grow even in the presence of cyanide because they lack cytochrome oxidase. Cyanide will cause rapid death in humans and other eukaryotes because it blocks cyto_ chrome oxidase, thereby completely terminating aerobic respira_ tion, but it is harmless to these bacteria. A potential side reaction ofthe respiratory chain in aerobic or_ ganisms is the incomplete reduction ofoxygen to superoxide ion (O2-) and hydrogen peroxide (H2O2). As mentioned in chapter 7 ,these toxic oxygen products can be very damaging to cells. Aerobes have neutral-

izing enrymes to deal with these products, including superoxide dis_ mutase andcatqlase. One exception is the genus Streptococcus, which can grow well in oxygen yet lacks both cytochromes and catalase. The tolerance of these organisms to oxygen can be explained by the neu-

Iralizing effects of a special peroxidase. The lack of cytochromes, catalase, and peroxidases in anaerobes as a rule limits their ability to process free oxygen and contributes to its toxic effects on them.

Summary of Aerobic Respiration Originally, we presented a surnmary equation for respiration on page 231. We are now in a position to tabulate the input and output

ofthis equation at various points in the pathways and sum up the final ATPs.

Figure 8.23 summarizes the total of AIp and other products for the entire aerobic pathway. These totals are the potential yields possible but may not be fulfilled by many organisms.

1. The total possible yield ofATP is 40: 4 from glycolysis, 2 from the Krebs cycle, and 34 from electron transport. However, because 2 AIPs were expended in early glycolysis, this leaves a maximum of 38ATPs. The actual totals may be lower in certain eukaryotic cells because energy is expended in transporting the NADH produced during glycolysis across the mitochondrial membrane. Certain aerobic bacteria come closest to achieving the full total of 38 because they lack mitochondria and thus do not have to use ATp in transport of NADH across the outer mitochondrial membrane.

2. Six carbon dioxide molecules

are generated by reactions just

prior to and during the Krebs cycle.

The Terminol Step of Electron Tronsport The terminal step, during which oxygen accepts the electrons, is catalyzed by cytochroma aa3, a,lso called cytochrome oxidase. This large enzyme complex is specifically adapted to receive electrons from cltochrome c, pick up hydrogens from the solution, and react with oxygen to form a molecule of water (figure 8.22b).This reaction, though in actuality more complex, is summarized as follows:

2H* + 2e- *

yzo2--->H2O

Most eukaryotic aerobes have a fully frurctioning cytochrome system, but bacteria exhibit wide-ranging variations in this part of the system. Some species lack one or more of the redox steps;

3. Six oxygen molecules are consumed during electron transport. 4. Six water molecules are produced in electron transport and 2 in glycolysis, but because 2 are used in the Krebs cvcle. this leaves a net number of 6.

Alte r n ate Coto bo I i c P othwoys Certain bacteria follow a different pathway in carbohydrate catabolism.

The phosphogluconate pathway (also called the hexose monophosphate shunt) provides ways to anaerobically oxidize glucose and other hexoses, to release AIP, to produce large amounts of NADpH, and to process pentoses (5-carbon sugars). This pathway, common in hetero-

lactic fermentative bacteria, yields various end products, including

8.3 Pathways of Bioenergetics

239

Some species of Pseudomonas and Bacillus possess enzymes that can further reduce nitrite to nitric oxide Q.{o), nitrous oxide

(NzO),andevennitrogengas(N).Thisprocess,calleddenitrification, is a very important step in recycling nitrogen in the biosphere.

other oxygen-containing nutrients reduced anaerobically by vari-

2

NADHs--a---------+ uxrdat|ve

Substrate-level

6

ATPs

2

ATPs

phosphorylation

2 NADHs

in ETS

--:---..----> UXIOAIIVE

ous bacteria are carbonates and sulfates. None of the anaerobic pathways produce as much ATP as aerobic respiration' Most obligate anaerobes use the H* they generate during glycolysis and the Krebs cycle to reduce some compound other than o, but still use the oxidative phosphorylation pathway. Methanogens (described in chapter 7) reduce CO2 or CO3- (carbonate) to CHo (methane gas), and certain sulfate bacteria reduce SOo'- to S sulfide or hydrogen sulfide gas (H2S). Some use metals or organic compounds as the final H+ acceptor. These microbes live in subterranean habitats such as swamps and deep oceanic vents that are devoid of 02 gas'

6 ATPs

phosphorylation

CASE FILE

8

Wrop-Up

==-;.:#=::' 6

in ETS

NADHs--# uxrdaflve

18 ATPs

permanently colonized by three unusual microbes from the

phosPhorylation in ETS

2 FADHTs 2 Krebs cycles

---:---..-+

uxrdaltve phosphorylation

4 ATP"

2 ATPs"

Substrate-level

phosphorylation Total aerobic

yield

36-38 ATP

*This amount can vary among mrcrobes.

Figure

8.23

The chapter opening case was about a man whose skin became

Theoretic ATP yield from aerobic respiration.

To attain the theoretic maximum yield of ATP, one must assume a ratio of 3 for the oxidation of NADH and 2for FADH2. The actual yield is generally lower and varies between eukaryotes and

prokaryotes and among prokaryotic species.

lactic acid ethanol, and carbon dioxide. Furthermore, it is a significant intermediate source ofpentoses for nucleic acid rymthesis.

same bacterial genus (Ctostridium). These unusual species altered the composition of the normal flora-microbes that reside in the external surfaces of the body. Laboratory techniques determined that the man's odor was caused by compounds produced by the clostridial bacteria during their metabolism' In simplified terms, the energy comes from an electron donated by an atom at the beginning of the process and accepted by another atom at the end of the process' One of the odor-causing chemical compounds produced was N-butyric acid (or normalbutyric acid). N-butyric acid is a fatty acid commonly found in rancid butter that has an unpleasant odor. In this case, the Nbutyric acid was produced by the Clostridium species through fermentation of carbohydrates in the absence of oxygen' This process often gives off organic acids and other volatile and strong smelling Products. N-butyric acids have been shown to inhibit the growth of other bacteria. This may help to explain how the three new Clostridium species were able to establish themselves and outgrow the normal flora. See:

C. M. Mitls, M. B. Llewetyn, D. R. Kelly, and P. Holt, "A Mon Who putria for Years. Case Report," Lancet 348

prirnii iit Fiiiier and smeila

i

(1996):1 282.

Anaerobic Respiration

r.ea

Some bacteria have evolved an anaerobic respiratory system that functions like the aerobic cytochrome system except that it utilizes

oxygen-containing ions rather than free oxygen as the final electron acceptor. Of these, the nitrate (NO:-) and nitrite (NOr-) reduction systems are best known. The reaction in species suchas Escherichia

coli is represented

as:

: r

Nitrate reductase

NO: + NADH *

J H+

-+

NO2- + H2O + NAD-

The enzyme nitrate reductase catalyzes the removal of oxygen from nitrate, leaving nitrite and water as products. This reaction is the basis for a physiological test used in identifying certain bacteria'

Bioenergetics describes metabolism in terms ofrelease, utilization, and transfer of energy by cells. Catabolic pathways release energy through three pathways: glycolysis, the Krebs cycle, and the respiratory electron transport system. Cellular respiration is described by the nature ofthe final electron acceptor. Aerobic respiration implies thatC2 is the final electron acceptor. Anaerobic respiration implies that some other molecule is the final electron acceptor. In some instances such as fermentation, the acceptor is an organic molecule.

240

Chapter

8

An Introduction to Microbial Metabolism

Carbohydrates are often used as cellular energy sources because they are superior hydrogen (electron) donors. ffi Glycolysis is the catabolic process by which glucose is oxidized and converted into two molecules of pynrvic acid, with anet gatn of 2 AIPs. The formation ofAIP is via substrate-level phosphorylation. € The Krcbs cycle processes the 3-carbon pynrvic acid and generates three CO2 molecules. The electrons it releases are transferred to redox carriers for energy harvesting. It also generates 2 AIps. ffifi The electron transport chain generates free energy through sequential redox reactions collectively called oxidative phosphorylation. This energy is used to generate 28 to 34 AIps for each glucose molecule catabolized.

The lmportance of Fermentation Of all the results of pyruvate metabolism, probably the most varied is fermentation. Technically speaking, fermentation* is the incomplete oxidation ofglucose or other carbohydrates in the absence of oxygen. This process uses organic compounds as the terminal electron acceptors and yields a small ilmount ofATp Over time, the term fermentation has acquired several looser meanings. Originally, Pasteur called the microbial action of yeast during wine productionferments, and to this day, biochemists use the term in reference to the production of ethyl alcohol by yeasts acting on glucose and other carbohydrates. Fermentation is also what bacteriologists call the formation of acid" gas, and other products by the action of various bacteria on pyruvic acid. The process is a common metabolic strategy among bacteria. Industrial processes that produce chemicals on a massive scale through the actions

of microbes are also called fermentations. Each of these usages is acceptable for one application or another. It may seem that fermentation would yield only meager amounts of energy (2 ATPs maximum per glucose) and that would slow down growth. What actually happens, however, is that many bacteria can grow as fast as they would in the presence of oxygen. This rapid growth is made possible by an increase in the rate of glycolysis. From another standpoint, fermentation permits survival and growth in the absence of molecular oxygen and allows colonization of anaerobic environments. It also enables microorganisms with a versatile metabolism to adapt to variations in the availability ofoxygen. For these facultative microbes, fermentation provides a means to function even when oxygen levels are too low for aerobic respiration. Bacteria that digest cellulose in the rumens of cattle are largely fermentative. After initially hydro$zing cellulose to glucose, they ferment the glucose to organic acids, which are then absorbed as the bovine's principal energy source. Even human muscle cells can undergo a form of fermentation that permits short periods of activity after the oxygen supply in the muscle has been exhausted. Muscle cells convert pynlic acid into lactic acid, which allows anaerobic production of AIP to proceed for a time. But this cannot go on indefinitely, and after a few minutes, the accumulated lactic acid causes muscle fatigue.

Products of Fermentotion in Microorgonisms Alcoholic beverages (wine, beer, whiskey) are perhaps the most prominent among fermentation products; others are solvents *

.fermentation (fi.u-men-tay'-shun) L.fenere,

nboll, otfermentafiim,leaverr oryeast.

System: Yeasts

Glucose

System: Homolactic bacteria Human muscle

]",,".,,.,, I

t

F@:

Pyruvic

ar

-Hoo

5-l

*

*,\>H-C-C-C' TP tl

&

/,O

'ox

Lactic acid

Figure 8.24 The fermentation systems that produce acid and alcohol. ln both cases, the final electron acceptor is an organic compound. In yeasts, pyruvic acid is decarboxylated to acetaldehyde, and the NADH given off in the glycolytic pathway reduces acetaldehyde to ethyl alcohol. In homolactic fermentative bacteria, pyruvic acid is reduced by NADH to lactic acid. Both systems regenerate NAD+ to feed back into glycolysis or other cycles.

(acetone, butanol), organic acids (lactic, acetic), dairy products, and many other foods. Derivatives of proteins, nucleic acids, and other organic compounds are fermented to produce vitamins, antibiotics, and even hormones such as hydrocortisone. Fermentation products can be grouped into two general catego-

ries: alcoholic fermentation products and acidic fermentation products (figure 8.24). Alcoholic fermentation occurs in yeast or bacterial species that have metabolic pathways for converting pymvic acid to ethanol. This process involves a decarboxylation of pyruvic acid to acetaldehyde, followed by a reduction ofthe acetaldehyde to ethanol. In oxidizing the NADH formed during glycolysis, NAD+ is regenerated, thereby allowing the glycolytic pathway to continue. These processes are crucial in the production ofbeer and wine, though the actual techniques for arriving at the desired amount of ethanol and the prevention of unwanted side reactions are important tricks of the brewerb trade Qnsight 8.4). Note that the products of alcoholic fermentation include both ethanol and COr, a gas that accounts for the bubbles in champagne and beer (and the rising ofbread dough). Alcohols other than ethanol can be produced during bacterial fermentation pathways. Certain clostridia produce butanol and isopropanol through a complex series of reactions. Although this process was once an important source of alcohols for industrial use, it has been largely replaced by a nonmicrobial petroleum process.

8.4

Biosynthesis and the Crossing Pathways of Metabolism

241

Pasteur and the Wine-to-Vinegar Connection The microbiology of alcoholic fermentation was greatly clarified by Louis Pasteur after French winemakers hired him to uncover the causes of periodic spoilage in wines. Especially troublesome was the conversion

of wine to vinegar and the resultant sour flavor. Up to that time, wine formation had been considered strict$ a chemical process. After extensively studying beer making and wine grapes' Pasteur concluded that wine, both fine and not so fine, was the result of microbial action on the juices of the grape and that wine "disease" was caused by contaminating organisms that produced undesirable products such as acid. Although he did not know it at the time, the bacterial contaminants responsible for the acidity of the spoiled wines were likely to be Acetobacter ot Gluconobacter introdvced by the grapes, air, or wine-making apparatus. These common gram-negative genera further oxidized ethanol to acetic acid and are presently used in commercial vinegar production. The following

formula shows how this is accomplisbed:

HH ll H_C_C-OH ll HH Ethanol

02 -

H

lt/ H-9-C. + 2H2O | 'oH H Acetic acid

Pasteur's far-reaching solution to the problem is still with us today-mild heating, or pasteurizatioin, ofthe grapejuice to destroy the contaminants, followed by inoculation of the juice with a pure yeast culture. The topic

of pasteurization is explored further in chapter

1

1.

An analysis of wine shows that it contains thousands of different organic compounds (see http://www.vinterus.com./pdffilesAiline.pdf.). What does this tell you about the metabolism of yeasts during alcoholic fermentation? Answer available at http : //www.mhhe.com/talaroT

The pathways of acidic fermentation are extremely varied. Lactic acid bacteria ferment pyruvate in the same way that humans do-by reducing it to lactic acid. If the product of this fermentation is mainly lactic acid" as in certain species of Streptococcus and Lactobacillus, it is termed homolactic. The souring of milk is due largely to the production of this acid by bacteria. When glucose is fermented to a mixture of lactic aci4 acetic acid, and carbon dioxide, as is the case wilh Leuconostoc and other species of Lactobac il lus, lhe process is termed h e t erol a ctic ferm e nt ati o n. Many members of the family Enterobacteriaceae (Escherichia, Shigella, and Salmonel/a) possess enzyme systems for converting pyruvic acid to several acids simultaneously (figure 8.25). Mixed acid fermentation produces a combination of acetic, lactic, succinic, and formic acids, and it lowers the pH of a medium to about 4.0. Propionibacterium produces primarily propionic aci{ which gives the characteristic flavor to Swiss cheese while fermentation gas (CO2) produces the holes. Some members also further decompose formic acid completely to carbon dioxide and hydrogen gases. Because enteric bacteria commonly occupy the intestine, this fermentative activity accounts for the accumulation of some types of gas-primarily CO2 and l{2-in the intestine. Some bacteria

reduce the organic acids and produce the neutral end product 2,3-butanediol. Several ofthese end products are the basis ofbiochemical tests discussed in chapters 17 and20. We have provided only a brief survey of fermentation products, but it is worth noting that microbes can be harnessed to synthesize a variety of other substances by varying the raw materials provided them. In fact, so broad is the meaning of the wordprmetttationthat the large-scale industrial syntheses by microorganisms often utilize entirely different mechanisms from those described here, and they even occur aerobically, particularly in antibiotic, hormone, vitamin, and amino acid production (see chaptet 27).

8.4 Biosynthesis and the Crossing Pathways of Metabolism Our discussion now turns from catabolism and energy extraction to anabolic functions and biosynthesis. In this section we present aspects of intermediary metabolism, including amphibolic pathways, the synthesis of simple molecules, and the synthesis of macromolecules.

242

Chapter

8

An Introduction to Microbial Metabolism

Streptococc us, Laetobaci II us

€l@ ffi*ffi

-FrO

\_ffi

ffi

Escherichia, Shigeila

Acetobacterium

ffit Propionibacterium

Flgure

8.25

Enterobacter

Miscellaneous products of pyruvate fermentation and some of the bacteria involved in their production.

The Frugality of the CellWaste Not Want Not It must be obvious by now that cells have mechanisms for careful management of carbon compounds. Rather than being dead ends, most catabolic pathways contain shategic molecular intermediates (metabolites) that can be diverted

Cell structure E .9

Ito(g

Macromolecule

tr

Building block

into anabolic pathways. In this way, a given molecule can serve multiple pulposes, and the maximum benefit can be derived from all nutrients and metabolites of the cell pool. The property ofa system to integrate catabolic and anabolic pathways to improve cell efficiency is

Metabolic pathways

termed amphibolism.*

At this point in the chapter, you can appreciate a more complex view of metabolism than that presented at the beginning in figure 8.1. Figure 8.26 demonstrates the amphibolic nature of intermediary metabolism. The pathways of glucose catabolism are an especially rich "metabolic marketplace." The principal sites of amphibolic interaction occur during glycolysis (glyceraldehyde-3-phosphate and pynrvic acid) and the Krebs cycle (acetyl coenzyme A and various organic acids).

Amphibolic Sources of Cellulor Building Blocks

E .9

o

lt

G (g

f^,"4 cycle

o

\t-t""\ / t\

&&

Figure 8.26 An amphibolic view of metabolism.

Intermediate compounds such as pyruvic acid and acetyl coenzyme A serve multiple functions. with comparatively small modifications, these compounds can be converted into other compounds and enter a different pathway. Note that catabolism of glucose (center) furnishes numerous intermediates for anabolic pathways that synthesize amino acids, fats, nucleic acids, and carbohydrates. These building blocks can serve in further synthesis of larger molecules to construct various cell components.

Glyceraldehyde-3-phosphate can be diverted away from glycolysis and converted into precursors for amino acid, carbohydrate, and triglyceride (fat) synthesis. (A precursor molecule is a compound that is the source of another compound') Earlieq we noted the numerous directions that pynrvic acid catabolism can take. In terms of synthesis, pyruvate also plays a pivotal role in providing intermediates for amino acids. In the event of an inadequate glucose supply, pyruvate serves as the starting point in * amphibolisn (am-fee-bol'-izm)

Simple products

Cn.amphi,two-sided, and bo le,

athrow.

glucose slmthesis from various metabolic intermediates, a process called gluconeogenesis.* The acetyl group that starts the Krebs cycle is another extremely versatile metabolite that can be fed into a number of synthetic * gluconeogenesis (gloo"-koh-nee"-oh-gen'-uh-sis) ..new,'glucose.

Literatly, the formation

of

243

8.4 Biosynthesis and the Crossing Pathways of Metabolism

M

ffi

l-^

"-;-_l l"'\-3-l-"1

l'/

l,

".ti:L; l"/ ll

NADH

+

+

f

I

I

-

Pyruvic acid

p-alanine

(a) Amination

FJ:i-ll I I '"'l l"'f

\

+

OH

Aspartic acid (4C) (b)

W

ffi

W

OHH

lt tl

-c -ctl-c HH

t2

t-l

-

H

-ctl-c

OH

OH

c-ketoglutaric acid (5C)

NH,

\

HH

ffi

! -/: t\

ioH

+

[--;--:l I \-3-l-"/ l"'i I a'l I

Oxaloacetic acid (4C)

Glutamic acid (5C)

Transamination

W

@ \ OH

NH,H

H

\-! \ r/\^., | | | HHHvn Glutamic acid

(c) Deamination

oo ilil

NAD*

,C

oH Hzo

-

H H ^ ll d -:C.

-C -C -C

Hi

oH

+

a-ketoglutaric acid

Figure 8.27

Reactions that produce and convert amino acids. (the addition of an ammonium molecule ntt of ttre reactions require energy as ATp or NADH and specialized enzymes. (a) Through amination transamination (transfer of an amino group from an amino (b) Through acid. an amino to converted ian Ue carbohydrate group]), a [amino (removal of icid to i carbohydrate), metabolic intermediates can be converted to amino acids that are in low supply. (c) Through deamination used to derive proteins are is how This catabolism. carbohydrate of an amino group), an amino acid can be converted to a useful intermediate in blue. acids are and amino brown in are structures Note that carbohydrate product. energy. Ammonium is one waste

pathways. This 2-carbon fragment can be converted as a single unit into one of several amino acids, or a number of these fragments can be condensed into hydrocarbon chains that are important building blocks for fatty acid and lipid synthesis. Note that the reverse is also true-fats can be degraded to acetyl and thereby enter the Krebs cycle via acetyl coen-4rrne A. This aerobic process, called beta oxidation, can provide a large amount of energy. Oxidation of a 6-carbon fatty acid yields 50 AIPs, compared with 38 for a 6-carbon sugar. Two metabolites of carbohydrate catabolism that the Krebs cycle produces, oxaloacetic acid and a-ketoglutaric acid" are essential intermediates in the synthesis of certain amino acids. This occurs through amination, the addition of an amino group to a carbon skeleton (figure 8.27u\. Acertain core group of amino acids can then be used to synthesize others. Amino acids and carbohydrates can be interchanged through transamination (figure 8.27 b)-

that synthesize the nitrogen bases

(purines, in RNA, originate pyrimidines), which are components of DNA and from the intermediates amino acids and so can be dependent on Krebs cycle as well. Because the coenzymes NAD+, NADP, EAD, and others contain purines and pyrimidines similar to the nucleic acids, their synthetic pathways are also dependent on amino acids. During times of carbohydrate deprivation, organisms can likewise

Pathways

convert amino acids to intermediates of the Krebs cycle by deamination (removal of an amino group) and thereby derive energy from proteins. Deamination results in the formation of nitrogen waste products such as ammonium ions or urea (figure 8-27c)-

Formation of Mocromolecules Monosaccharides, amino acids, fatty acids, nitrogen bases, and vitamins-the building blocks that make up the various macromolecules and organelles of the cell----come from two possible sources. They can enter the cell from the outside as nutrients, or they can be syrthesized through various cellular pathways. The degree to which an organism can synthesize its own building blocks (simple molecules) is determined by its genetic makeup, a factor that varies tremendously from group to group. In chapter 7, you learned that autotrophs require only CO2 as a carbon source, a few minerals to synthesize all cell substances, and no organic nutrients. Some heterotrophic organisms (E coli, yeasts) are so efficient that they can synthesize all cellular substances from minerals and one organic carbon source such as glucose. Compare this with a strict parasite that has few synthetic abilities of its own and obtains all of its nutrient molecules from the host. Whatever their source, once these building blocks are added to the metabolic pool, they are available for synthesis of polymers by the cell. The details of synthesis vary among the types of macromolecules, but all of them involve the formation of bonds by specialized enzymes and the expenditure ofAIP.

Ca rbohyd

rote Biosynthesis

The role of glucose in bioenergetics is so crucial that its biosynthesis is ensured by several alternative pathways' Cerlatn structures in the cell depend on an adequate supply of glucose as well' It is the major

244

Chapter

8

An Introduction to Microbial Metabolism

component of the cellulose cell walls of some eukaryotes and of certain storage granules (starch, glycogen). One ofthe intermediaries in glycolysis, glucose-6-P, is used to form glycogen. Monosaccharides other than glucose are important in the synthesis of bacterial cell walls. Peptidoglycan contains a linked polyner of muramic acid and glucosamine. Fructose-6-P from glycolysis is used to form these two sugars. Carbohydrates (deoxyribose, ribose) are also essential building blocks in nucleic acids. Polysaccharides are the predominant components ofcell surface structures such as capsules and the glycocalyx, and they are commonly found in slime layers (dextran). Remember that most polymenzationreactions occw via loss of a water molecule

indirectly dependent on photosyrthesis. A few chemoautotrophs derive their energy and nutrients solely from inorganic substrates. The other major products of photosynthesis are organic carbon compounds, which are produced from carbon dioxide through a process called carbon fixation. On land, green plants are the primary photosynthesizers; and in aquatic ecosystems, where 80% to 90% of all photosynthesis occurs, algae, green and purple bacteria, and cyanobacteria fill this

role. Otherphotosynthetic prokaryotes are grcen sulfir, purple sulfur, and purple nonsulfur bacteria.

The summary equation for the main reactants and products

(see figure 2.16) andthe input of energy (see figure 8.8) 6CO2 + 6H2O

Assembly of the Cell The component parts ofa bacterial cell are being synthesized on a continuous basis, and catabolism is also taking place, as long as nutrients are present and the cell is in a nondormant state. When anabolism produces enough macromolecules to serve two cells, and when DNA replicationproduces duplicate copies of the cell,s genetic material, the cell undergoes binary fission, which results in two cells from one parent cell. The two cells will need twice as many ribosomes, twice as many en-4/mes, and So on. The cell has created these during the initial anabolic phases we have described. Before cell division, the membrane(s) and the cell wall will have increased in size to create a cell that is almost twice as big as a "newborn" cell. Once synthe-

of

photosynthesis is

ightgg-gcryE c6H12o6 + 60rl Glucose

The anatomy of photosynthetic cells is adapted to trapping sunlight, and their physiology effectively uses this solar energy to produce high-energy glucose from low-energy CO2and water. photoslmthetic organisms achieve this remarkable feat through a series of reactions involving light, pigment,Co2,and water, which is used for electrons. Photosynthesis proceeds in two phases: the light-dependent reactions, which proceed only in the presence ofsunlight, and the as a source

light-independent reactions, which proceed regardless of the lighting conditions (light or dark) (figure 8.28).

sized, the phospholipid bilayer components of the membranes assemble themselves spontaneously with no energy input. But proteins and other components must be added to the membranes. Growth of the cell wall, accomplished by the addition and coupling of sugars and peptides, requires energy input. The catabolic processes provide all the energy for these complex building reactions.

€

$

"Intermediary metabolism" refers to the metabolic pathways that use intermediate compounds and connect anabolic and catabolic reactions.

e Amphibolic compounds

are the "crossroads compounds" of metabolism. They not only participate in catabolic pathways but also are precursor molecules to biosynthetic pathways. w Biosynthetic pathways utilize building-block molecules from two sources: the environment and the cell's own catabolic pathways. Microorganisms construct macromolecules from these monomers using ATP and specialized enzymes.

w

Carbohydrates are crucial as energy sources, cell wall constituents, and components of nucleotides.

€ Proteins

*

are essential macromolecules in all cells because they function as structural constituents, enzymes, and cell appendages.

Final assembly ofa new cell occurs via binary fission and requires an input ofenergy.

8.5 Photosynthesis: The Earth's Lifeline As mentioned earlieq the ultimate source of all the chemical energy in cells comes from the sun. Because this source is directly available only to the cells ofphotosynthesizers, most organisms are either directly or

Flgure

8.28

Overview of photosynthesis.

The general reactions of photosynthesis, divided into two phases called light-dependent reactions and light-independent reactions. The dependent reactions require light to activate chlorophyll pigment

and use the energy given off during activation to split an HrO molecule into oxygen and hydrogen, producing ATp and NADpH. The independent reactions, which occur either with or without light, utilize ATP and NADPH produced during the light reactions to fix CO2 into organic compounds such as glucose.

8.5

Photosvnthesis: The Earth's Lifeline

245

(a) cefl of the motile alga Chlamydomonas, with a single ' ' Alarge chloroplast (magnifled cutaway view)'The chloroplast contains membranous compartments called grana where chlorophyll molecules and the photosystems lor the light reactions are located,

Flagellum

Chloroplast

(b) A chlorophyll molecule, with a central magnesium atom held by a porPhYrin ring.

(c) The main events of the light reactions shown as an exploded vlew in

one granum.

O .9

o

() c E

o

Wnen light activates photosystem ll, it sets up a chain reaction, in which electrons are released from chlorophyll. fnese electrons are transported along a chain of carriers to @ photosystem l. CO fne empty position in photosystem ll is replenished by photolysis of HzO.Other products of photolysis are 02 and H-.

-

@ Pumping of H+ into the interior

-

of the granum produces conditions for

ATP to be synthesized.

final electron @ fne photosystem

and H+ acceptor is NADB which receives these trom

l.

@ Thylakoid membrane

ffi

Process Flgure

Interior of

eotn NADPH and ATP are fed into the stroma forthe Calvin cycle.

granut

@

8.29

The reactions of photosynthesis.

Solar energy is delivered in discrete energy packets called photons (also called quanta) that travel as waves. The wavelengths of light operating in photosynthesis occur in the visible spectrum between 400 (violet) and 700 nanometers (red). As this

light strikes photosynthetic pigments, some wavelengths are absorbed, some pass through, and some are reflected. The activity that has the greatest impact on photosynthesis is the absorbance

of light by photosynthetic pigments. These include the chlorophylls, which are green; carotenoids, which are yellow, orange, or ied; and phycobilins, which are red or blue-green.4 By far the most important of these pigments are the bacterial chlorophylls, which contain a photocenter that consists of a magnesium atom held in the center of a complex ringed molecule called a porphy' rin (figure 5.29b). As we will see, the chlorophyll molecule harvests the energy ofphotons and converts it to electron (chemical) energy. Accessory photosynthetic pigments such as carotenes trap light energy and shuttle it to chlorophyll, thereby functioning like antennae. These light-dependent reactions are catabolic (energy-

producing) reactions, which pave the way for the next set

of

reactions, the light-independent reactions, which use the extracted energy for synthesis. During this phase, carbon atoms from CO2 are fixed to the carbon backbones oforganic molecules. The detailed biochemistry of photosynthesis is beyond the scope of this tex! but we will provide an overview of the general process as it occurs in green plants, algae, and cyanobacteria (see figure 8.28). Many of the basic activities (electon transport and phosphorylation) are biochemically similar to certain pathways of respiration.

Light-Dependent Reactions The same systems that carry the photosynthetic pigments are also the sites for the light reactions. They occur in the thylakoid membranes of compartments called grana Gingula., granum) in chloroplasts (figure 8.29a) and in specialized parts of the cell membranes in prokaryotes (see figure 4.28a). These systems exist as two separate complexes called photosystem I (P700) and photosysternll(P680)' (figure 8.29c). Both systems contain chlorophyll and they are simultaneously activated by light, but the reactions in photosystem II help drive photosystem I.

5. The mrmbers refer to the wavelength of light to which each system is most

4. The color ofthe pigrnent corresponds to

the wavelength

oflight it reflects.

sensitive.

246

Chapter

8

An Introduction to Microbial Metabolism

Together the systems are activated by light, transport electrons, pump hydrogen ions, and formATP andNADPH.

When photons enter the photocenter of the p680 system (pS II), the magnesium atom in chlorophyll becomes excited and releases 2 electrons. The loss of electrons from the photocenter has two major effects:

1.

It

creates

a vaaancy in the chlorophyll molecule forceful

enough to split an HrO molecule into hydrogens (H+) (elec-

trons and hydrogen ions) and oxygen (O). This splitting of water, termed photolysis, is the ultimate source of the 02 gas that is an important product ofphotosynthesis. The electrons released from the lysed water regenerate photosystem II for its next reaction with light. 2. Electrons generated by the first photoevent are immediately boosted through a series of carriers (cytochromes) to the p700 system. At this same time, hydrogen ions accumulate in the internal space of the thylakoid complex, thereby producing an

electrochemical gradient.

CO,

"Rn*;6r,s'ini*

,,re*6'Jtf*"'" \ riib,ros"-r,s-bi.pnoipr,"t"

*'- )r

^rr\l

-\r.Phos'hos'|Ycer.

F*& aArpxz carvincyc,e ;lt ilS^",

\

7-carbon \ intermediates \

Series of and S-carbon

FH

nn ,;€ i.;?. *-.t/

1,3-bisphosphoglyceric

;-,\*'

6- tf^5 1,,.-

The P700 system (PS I) has been activated by light so that it is ready to accept electrons generated by the pS II. The electrons it receives are passed along a second transport chain to a complex that

NADGlyceraldehyde-3phosphate

uses electrons and hydrogen ions to reduce NADp to NADpH. (Recall that reduction in this sense entails the addition ofelectrons and hydrogens to a substrate.)

A second energy reaction involves synthesis of ATp by a chemiosmotic mechanism similar to that shown in figure 8.22. Channels in the thylakoids of the gramrm actively pump H+ into the inner chamber, producing a charge gradient. ATp synthase located in this same thylakoid uses the energy from H+ transport to phosphorylate ADP to AIP. Because it occurs in light, this process is termed photophosphorylation. Both NADPH and ATp are released into the stroma of the chloroplast, where they drive the reactions of the Calvin cycle.

Light-l ndependent Reactions The subsequent photosynthetic reactions that do not require light occur in the chloroplast stroma or the cytoplasm of cyanob acteia. These reactions use energy produced by the light phase to synthesize glucose by means of the Calvin cycle (figure 8.30). The cycle begins at the point where CO2 is combined with a doubly phosphorylated 5-carbon acceptor molecule called

ribulose-1,5-bisphosphate (RuBP). This process, called carbon fixation, generates a 6-carbon intermediate compound that immediately splits into two 3-carbon molecules of 3-phosphoglyceric acid (PGA). The subsequent steps use the AIp and NADPH generated by the photosystems to form high-energy intermediates. First, AIP adds a second phosphate to 3-PGA and produces 1,3-

bisphosphoglyceric acid (BPG). Then, during the same step, NADPH contributes its hydrogen to BPG, and one high-energy

@@@@@@

-->

Grucose

Fructose intermediates

@ Figure 8.3O

The Catvin cycte.

The main events of the reactions in photosynthesis that do not require light. lt is during this cycle that carbon is fixed into organic form using the energy (ATP and NADPH) released by the light reactions. The end product, glucose, can be stored as complex carbohydrates, or it can be used in various amphibolic pathways to

produce other carbohydrate intermediates or amino acids.

Other Mechanisms of Photosynthesis The oxygenic, or oxygen-releasing, photosynthesis that occurs in plants, algae, and cyanobacteria is the dominant type on the earth. Other photosynthesizers such as green and purple bacteria possess bacteriochlorophyll, which is more versatile in capturing light. They have only a cyclic photosystem I, which routes the electrons from the photocenter to the electron carriers and back to the photosystem again. This pathway generates a relatively small amount ofATp, and

it may not produce NADPH. As photolithotrophs, these bacteria use Hz, I{zS, or elemental sulfur rather than I{2O as a source of electrons

and reducing power. As a consequence, they are anoxygenic (nonoxygen-producing), and many are skict anaerobes.

w The sun

is the primary energy source for most swface ecosystems. Photosynthesis captures this energy and utilizes it for carbon fixation by producer populations.

phosphate is removed. These events give rise to glyceraldehyde-3-

phosphate (PGAL). This molecule and its isomer dihydroxyacetone phosphate (DHAP) are key molecules in hexose synthesis leading to fructose and glucose. You may notice that this pathway is very similar to glycolysis, except that it runs in reverse (see figure 8. I 8). Bringing the cycle back to regenerate RuBp requires PGAL and several steps not depicted in figure 8.30.

NADpHx2

M Producers include plants. algae, cyanobacteria, and certain bacterial species.

ffi

Photosynthesis proceeds in two stages: (1) photophosphorylation, in which light is trapped, energy is extracted to make ATB and oxy-

gen is evolved; (2) Calvin cycle, in which ATp is used to

-*J:::-,::9#::.::m:*::,*::_"::*::__

fix

CO2

Chapter Summary with

Chapter Summary 8.1 The Metabolism

A.

B.

with&Y

C.

3.

of Microbes

A.

process that gives rise to the enzymes.

1. A substance that resembles the normal substrate and can occupy the same active site is said to exert

competitive inhibition.

2. Noncompetitive inhibition

occurs when the regulator molecule does not bind to the same site as the substrate.

The mechanisms of enzyme repression and enryme induction exert control at the genetic level by controlling the slmthesis of key enzymes'

Pursuit and Utilization of Energy

8.2 The

Cell Energetics Energy is the capacity

ofa system to perform work- It is consumed in endergonic reactions and is released in exergonic reactions.

B. A Closer Look at Biological

Oxidation and Reduction energy requires a series ofelectron carriers arcayed in a downhill redox chain between electron donors and electron acceptors. NAD+ is the primary carrier of electrons from one

1. Extracting

2.

pathway to another.

3. In oxidative phosphorylation,

energy is transferred to

high-energy compounds such as AIP. 8.3 Pathways of Bioenergetics A. Principal Pathways in Oxidation of Glucose: Carbohydrates' such as glucose, are energy-rich because they can yield a large number of electrons per molecule' Glucose rs

dismantled in stages. 1. Glycolysis is a pathway that degrades glucose to pyruvic acid without requiring oxygen. 2. Pyuvic acid is an important key molecule in several

B.

substrate-sPecific. Cofactors: Metallic cofactors impart greater reactivity

to the enzyme-substrate complex. Coenzymes such as NAD+ (nicotinamide adenine dinucleotide) are transfer agents that pass functional groups from one substrate to another. Coenzymes usually contain vitamins. 5. Enzyme Classification: Enzyme names often consist of a prefix derived from the tlpe of reaction or the substrate and the ending -ase' 6. Microbial Enzymes and Disease: Many pathogens secrete en4lmes or toxins, which are referred to as virulence factors, that enable them to avoid host defenses. 7 . Types of Enzyme Function: The release ofwater that comes with formation of new covalentbonds is a condensation reaction. Hydrolysis reactions involve addition ofwater to break bonds. Functional groups may be added, remove{ or traded in many reactions. Coupled redox reactions transfer electrons and protons (H+) from one substrate to another. 8. Enzyme Sensitivity: Enzymes are labile (unstable) and function only within narrow operating ranges of temperature, osmotic pressure, and pH; extreme conditions can cause denaturation. Enzymes are frequently the targets for physical and chemical agents used in control. Regulation of Enzymatic Activity Regulatory controls can act on enzymes directly or on the

247

.:r.

Terms

Metabolism 1. Metabolism is the sum of cellular chemical and physical activities; it involves chemical changes to reactants and the release ofproducts using well-established pathways' 2. Metabolism is a complementary process consisting of anabolism, synthetic reactions that convert small molecules into large molecules, and catabolism, in which large molecules are degraded. Together' they generate thousands of intermediate molecular states, called metabolites, which are regulated at many levels. Enzymes: Metabolic CatalYsts 1. Metabolism is made possible by organic catalysts, or enzymes' that speed up reactions by lowering the energy of activation. Enzymes are not consumed and can be reused. Each enzyme acts specifically upon its matching molecule or substrate. 2. En4,rne Sttucture: Depending upon its composition, an enz;We is either conjugated or simple. A conjugated enzyme consists of a protein component called the apoenzyme and one or more activators called cofactors. 3. Enzyme Specificity: Substrate attachment occurs in the special pocket called the active, or catalytic' site. In order to fit, a substrate must conform to the active site of the enzyme. This three-dimensional state is determined by the amino acid content, sequence, and folding of the apoenzyme. Thus, enzymes are usually

4.

KeY Terms

pathways. Fate of Pyruvic Acid in Krebs Cycle and Electron TTansport Pynrvic acid is processed in aerobic respiration via the Krebs cycle and its associated electron transport chain. 2. Acetyl coenzyme A is the product ofpyruvic acid processing that undergoes further oxidation and decarboxylation in the Krebs cycle, which generates

l.

ATR CO2, and H2O. The respiratory chain completes energy extraction' 4. The chemiosmotic theory is a conceptual model that explains the origin and maintenance of electropotential gradients across a membrane that leads to ATP sy,nthesis, by ATP synthase. 5. The final electron acceptor in aerobic respiration is oxygen. In anaerobic respiration, compounds such as sulfate. nitrate. or nitrite serve this function. Bacteria serve as important agents in the nitrogen cycle (denitrification). Fermentation is anaerobic respiration in which both the electron

3.

C.

donor and final electron acceptors are organic compounds. Fermentation enables anaerobic and facultative

l.

2.

microbes to survive in environments devoid of oxygen. Production of alcohol, vinegar, and certain industrial solvents relies upon fermentation. The phosphogluconate pathway is an alternative anaerobic pathway.

D.

Versatility of Glycolysis and Krebs Cycle: Glycolysis and the Krebs cycle are central pathways that link catabolic and anabolic pathways, allowing cells to break down different classes of molecules in order to synthesize compounds required by the cell. l. Metabolites of these pathways double as building blocks and sources ofenergy. Intermediates such as pynrvic acid are convertible into amino acids through amination. Amino acids can be deaminated and used as a source of glucose and other carbohydrates (gluconeogenesis).

248

Chapter

2.

8

An Introduction to Microbial Metabolism

TWo-carbon acetyl molecules from pyruvate decarboxylation can be used in fatty acid synthesis.

A. During the lighfdependent reactions, photons ofsolar energy are absorbed by chlorophyll, carotenoid, and phycobilin pigments in thylakoid membranes. Light energy captured by two photosystems splits water by photolysis and releases oxygen gas; it provides electrons to drive photophosphorylation; released light energy is used to sl.nthesize ATP and NADPH.

8.4 Biosynthesis and the Crossing Pathways of Metabolism

A. B.

Amphibolism The ability ofa cell or system to integrate catabolic and anabolic pathways to improve efficiency is called amphibolism. Anabolism: furmation of Macromolecules Macromolecules, such as proteins, carbohydrates, and nucleic acids, are made of building blocks from two possible sources: from outside the cell (preformed) or via synthesis in one ofthe anabolic pathways.

8.5 Photosynthesis: The

B.

The Calvin cycle (light-independent) uses AIP to fix carbon dioxide to a carrier molecule, ribulose-1,5-bisphosphate, and convert it to glucose in a multistep process. The t)?e of photosyrrthesis commonly found in plants, algae, and cyanobacteria is called oxygenic because

it liberates oxygen. Anoxygenic photosynthesis occws in photolithotrophs, which do not produce oxygen.

Earth's Lifeline

Photosynthesis takes place in two stages-Iight-dependent and

light-independent reactions.

euestions

e#Muttipte-choice

Select the correct answer from the answers provided. For questions with blanks, choose the combination ofanswers that most accuratelv comoletes the statement. 1.

is another term for biosynthesis. c. metabolism -b. anabolism d. catalyst

11. A reduced compound is

a. catabolism

2. Catabolism is

a

converted into a. large, small b. small, large

3. An

enzrr'me

a. increases

b. converts -

form of metabolism in which

a.

b. molecules

are

d.

b. addition of electrons and hydrogens

c. addition ofoxygen d. removal ofoxygen lJ.

catalyzes

4. Anenzyme a. becomes part of the final products

c. is consumed by the reaction

a,4 b.2

d. is heat andpH labile 5. An apoenzyme is where the is located. a. cofactor c. redox reaction b. coenzyme d. active site 6. Many coenzymes are a. metals c. proteins b. vitamins d. substrates a. endoenzyme

c.

b. exoenzyme

d.

I

I

a. electrical

c. radiant

b. chemical

d. mechanical

of-

t'7

a. 40

c.

b.6

d.2

38

The FADH, formed during the Krebs cycle enters the electron transport system at which site?

a. NADH dehydrogenase b. cytochrome

c. coenzyme Q d. ATP synthase 18.

AIP

synthase complexes can generate

NADH that enters electron transoort.

a. ADP

a.7

b. high-energyAlP bonds

b.2

coenzymes

d. inorganic phosphate 10. Exergonic reactions a. release potential energy b. consume energy bonds

d. occur only outside the cell

a net

AIPs.

6. The compound that enters the Krebs cycle is a. citric acid c. pyruvic acid b. oxaloacetic acid d. acetyl coenzyme A

9. Energy is carried from catabolic to anabolic reactions in the form of

c. form

40

d.0

output

a fungus produces a./an

catalase polyrnerase

c.

has the potential to produce a

5. Complete oxidation of glucose in aerobic respiration can yield

8. Energy in biological systems is primarily

c.

A product or products of glycolysis is/are a. AIP c. CO2 b. pyruvic acid d. both a and b

t4 Fermentation of a glucose molecule net number ofAIPs.

b. is nonspecific for substrate

7. To digest cellulose in its environment,

c. NADH d. ADP

12. Most oxidation reactions in microbial bioenergetics involve the a. removal of electrons and hydrogens

molecules. c. amino acid" proteind. food, storage the activation energy required for a chemical reaction. c. lowers

NAD+ FAD

I

c.3 d.4

for each

-ATP/AIPs

9. Photosynthetic organisms convert the energy of energy. a. electrons

b. protons

c. photons d. hydrogen atoms

into chemical

-

Writing to

H*

20. The Calvin cycle operates during which part ofphotosynthesis? c. in both light and dark a. only in the light b. only in the dark d. only during photosystem I 21..

Multiple Matching. Match the process

a, b, or c

249

e- are delivered to 02 as the final acceptor.

_ Pyruvic acid is formed. -_ GTP is formed. _ H:O is produced. _COzis formed.

with the metabolic

events in the list.

Fructose diphosphate is split into two 3-carbon fragments.

a. glycolysis b. Krebs cycle

_ -_

c. electron transporVoxidative phosphorylation

g#

and

Learn

NADH is oxidized. ATP slnthase is active.

writins to Learn

These questions are suggested as awriting-to-learn experience. For each question, compose a one- or two-paragraph answer that includes the factual information needed to completely address the question. General page references for these topics are given in parentheses.

1. a. Describe the chemistry of enzl'rnes and explain how the apoenzyme forms. (218, 219, 220) b. Show diagrammatically the interaction of holoenzyme and its substrate and general products that can be formed from a reaction. (21.9,220,221)

2. Differentiate among the chemical composition and functions of various cofactors. Provide examples ofeach

9. Name the major ways that substrate-level phosphorylation is different from oxidative phosphorylation. (229, 232, 236) I

1

type. (220)

3. a. Two steps in glycolysis are catalyzedby allosteric enzymes. These are : ( I ) step 2, catabolized by phosphoglucoisomerase, and (2) step 9, catabolized by pyruvate kinase. Suggest what (224,232) metabolic products might regulate these enzymes

.

b. How might one place these regulators in figure

8.

18? (232)

4. a. Explain how oxidation of a substrate proceeds without oxygen. b. Refer to the redox equation on page 227 for glucose, and name which substance is reduced, which is oxidized" the oxidizing agent, and the reducing agent. 5. In the following redox pairs, which compound is reduced and which is oxidized? (228, 234, 239, 240)

a. NAD+ andNADH b. FADH2 and FAD

c. lactic acid and pyruvic acid d. NO3- and NOre. Ethanol and acetaldehyde

6. a. Describe the roles played by AIP and NAD- in metabolism. (228,229) b. What particular features of their structure lend them to these

tunctions? (228) 7. Discuss the relationship of a. anabolism to catabolism (217\ b. ATP to ADP (229) c. glycolysis to fermentation (230) d. electron transport to oxidative phosphorylation (235) 8. a. What is meant by the concept of the "final electron acceptor"? (227,228) b. What are the final electron acceptors in aerobic, anaerobic, and fermentative metabolism? (230, 231) c. Describe the reaction between H- and oxygen at the final step of electrontransport. (236,238)

0.

Compare and contrast the location of glycolysis, Krebs cycle, and electron transport in prokaryotic and eukaryotic cells. (232,234,237)

1. a. Outline the basic steps in glycolysis, indicating where ATP is used and given off (232) b. Where does NADH originate, and what is its fate in an aerobe? (232) c. What is the fate of NADH in a fermentative organism? (233,242)

12. a. What is the source of ATP in the Krebs cycle? (234,235) b. How many AIPs could be formed from the original glucose molecule carried through aerobic respiration? (239) c. How does the total ofAIPs generated differ between bacteria and many eukaryotes? What causes this difference? (238,239) 13. Name the sources of oxygen in bacteria that use anaerobic

respiration. (239) 14. a. Summarizethe chemiosmotic theory ofAIP formation. (236,237 , 238) b. What is unique about the actions of ATP synthase? (236,237) 1

5

. How

are aerobic and anaerobic respiration

different? (230, 231 , 239)

16. Compare the general equation for aerobic metabolism with figure 8.23 and verify that all figures balance. (231,239) 17. Give the general name of the enzyme that a. synthesizes ATP; digests RNA

b. phosphorylates glucose c. reduces pyruvic acid to lactic acid d. reduces nitrate to nitrite (222,229,233,239) 18. a. Tell whether each of the following is produced during the lightindependent or light-dependent reactions of photosynthesis: 02, ATR NADPH, and glucose. (244,245,246) b. When are water and CO2 consumed? (244,245,246) c. What is the function of chlorophyll and the

photosystems? (245,246)

d. What is the fate of the AIP and NADH? (246) e. Compare oxygenic with nonoxygenic photosynthesis. (246)

250

Chapter

8

An lntroduction to Microbial Metabolism

Concept Mapping Appendix E provides guidance for working with concept maps.

l.

Supply your owrr linking words or phrases in this concept map, and provide the missing concepts in the empty box.

2. Construct your own concept map using the following words

as the

concepts. Supply the linking words between each pair of concepts. anabolism

nucleotides

catabolism precursor molecules

DNA ATP

bacterial cell

Critical Thinking Questions Critical thinking is the ability to reason and solve problems using facts and concepts. These questions can be approached from a number of angles, and in most cases. thev do not have a sinele correct answer. 1. Using the following sirnplified chart, fill in a summary of the major starting compounds required and the products given offby each phase of metabolism. Use arrows to pinpoint approximately where the reactions take olace on the oathwavs.

2. Use the following graph to diagram the energetics of

a chemical reaction, with and without an enzyme. Be sure to position reactants and products at appropriate points and to indicate the stages in the reaction and the energy levels.

'F (5

o

E.

.c

o

=

;s, q)

IJJ

Progress of Reaction

--------------->

Internet Search Topics

J. Describe the relationship between photosl'nthesis and respiration with regard to CO2 and 02.

9. Observe

251

figure 8.17. Outline the major differences between the three

metabolic pathways.

ofAIP slmthase in three dimensions, showing how works. Where in the mitochondrion does the ATP supply collect?

it

A

Explain how it is possible for certain microbes to survive and grow in the presence of cyanide, which would kill many other organisms.

10. Draw a model

5.

Suggest the advantages ofhaving metabolic pathways staged in specific membrane or organelle locations rather than being free in

11. Microorganisms are being developed to control human-made pollutants

the cytoplasm.

6. What adaptive advantages does

a fermentative metabolism confer on

a microbe?

7. Describe some of the special adaptations of the enzymes found in

extremophiles.

and oil spills that are metabolic poisons to animal cells. A promising approach has been to genetically engineer bacteria to degrade these chemicals. What is actually being manipulated in these microbes?

12. Explain the relationship between enzyme sensitivity and the adaptations microbes make to their environments.

ofreactions that are exergonic and some that are endergonic, using figures 8.18, 8.26, and 8.30.

13. Find examples

an early period ofrapid aerobic metabolism ofglucose by yeast. Given that anaerobic conditions are necessary to produce alcohol, can you explain why this step is necessary?

8. Beer production requires

Visual Understanding 1. From chapter 7, figure 7.11. Describe the likely types ofmetabolism that are happening in tubes I through 4. Specify the pathways, find electron acceptor, and end products.

Internet Search Topics I

. Look up fermentation

on a search engine, and outline some of the

products made by this process.

2. Find websites that feature three-dimensional views of enzymes. Make simple models of three different enzlmes indicating enzyme structure, active site, and substrate.

3.

Research anaerobic infections online. Outline the major microorganisms

involved, their source, the types of diseases they cause, and how they are treated.

and click on chapter 8. Access the URLs listed under Internet Search Topics. Log on to the websites listed to view animations and tutorials of the biochemical pathways and enzymes.

4. Go to: http://www.mhhe.com/talaro7,

CASE FILE

9

ancomycin-resistant Staphylococcus oureus (VRSA) was isolated from the exit site of a dialysis catheter in a 40-year-old diabetic with a history of peripheral vascular disease, renal failure, and foot ulcers. A few months earlier, the patient's gangrenous toe had been amputated. Following that surgery the patient developed a bacterial infection of the blood caused by methicillin-resistant S. aureus (MRSA) following a hemodialysis graft. Treatment with vancomycin, rifampin, and graft removal had successfully cured this first infection. A few months later, when the catheter exit site infection appeared, the area was cultured and the catheter removed. A week later, one of the patient's foot ulcers developed an infection. This time, vancomycin-resistant Enterococcus foecolis (VRE) and Klebsielto oxytocd were cultured from the ulcer. The patient finally recovered after stringent wound care and systemic treatment with trimethoprim/su lfamethoxazole. Anafysis of the VRSA isolate revealed that it contained the vanA gene for vancomycin resistance and the mecA gene for oxacillin resistance.

I

How do you think the Staphylococcus aureus VRSA strain ended up with the gene for va ncomycin resisto nce?

)

What ore some likely mechanisms for genetic transfer of antibiotic resistance from one organism to another?

)

Why would this porticulor patient be at increased risk for infection with VRSA? Case File

CHAPTER OVERVIEW

Cenetics is the study of the expression of biological information and its transfer between organisms. The molecules most important to this endeavor are DNA and RNA, which carry chemical codes, and proteins, which are involved in most cellular functions. DNA is a very long molecule composed of small subunits called nucleotides. The sequence of the nucleotides contains information needed to direct the synthesis of all proteins in the cell. The DNA molecule must be copied so that the genetic material can be transferred to

=

offspring. DNA's chemical structure makes its replication possible.

ts The codes

on DNA are transferred to various RNA molecules, which carry out numerous

functions, including protein synthesis and genetic regulation. 252

9 Wrop-Up oppears on poge 282.

9.1

Introduction to Cenetics and Cenes: Unlocking the Secrets of Heredity

2s3

Viruses contain various forms of DNA and RNA that are translated by the genetic machinery of their host cells to form functioning viral particles. The genetic activities of bacterial cells are highly regulated by operons, groups of genes that interact as a unit to control the use or synthesis of metabolic substances. Permanent changes in the sequence of the DNA, called mutations, can occur. Because mutations may alter the function or expression of genes, they serve as a force in the

evolution of organisms. Bacteria undergo genetic recombination through the transfer of small pieces of DNA to other bacteria, as well as through the uptake of DNA from the environment.

Introduction to Genetics and Genes: Unlocking the Secrets of Heredity

9."1

Genetics is the science that studies the inheritance of biological characteristics by living things. This subject, also known as heredity, is a wide-ranging science that examines

1. the transmission of biological properties (traits) from parent to offspring,

2. the expression and variation ofthose traits, 3. the structure and function of the genetic material, and 4. how this material changes.

The Nature of the Genetic Material For a species to survive, it must give rise to offspring through some form ofreproduction. In bacteria, reproduction involves division of the cell by means of binary fission or budding. But this involves a more significant activity than just a random splitting of one cell into two daughter cells. Because the genetic material is responsible for inheritance, it must be accurately duplicated and separated into each daughter cell to ensure normal function. The genetic material itself is a long molecule of DNA that can be studied on several levels. Before we look at how DNA is copied, let us explore the organization of this genetic material, proceeding from the general to the specific.

The study of genetics takes place on several levels (figure 9.1). Organismal genetics observes the transmission and expression of genetic factors in the whole organism or cell; chromosomal genetics examines the characteristics and actions of chromosomes; and molecular genetics deals with the biochemistry of gene function. All of these levels have value in understanding the expressions of microbial structure, physiology, mutations, and pathogenicity. But in order to gain full knowledge of these processes, we must begin with a study of genes at the cellular and molecular levels. The study of microbial genetics will provide a greater understanding of human genetics and an increased appreciation for the astounding advances in genetic engineering we are currently witnessing (see chapter 10).

Organism level

Cell level

The Levels of Structure and Function of the Genome The genome is the sum total of genetic material of a cell. Although most of the genome exists in the form of chromosomes, genetic mateial can appear in nonchromosomal sites as well (figure 9.2). For example, bacteia and some fungi contain tiny exha pieces of

DNA (plasmids), and certain organelles or eukaryotes (the mitochondria and chloroplasts) are equipped with their own genetic programs. Genomes of cells are composed exclusively of DNA, but viruses contain either DNA or RNA as the principal genetic material. Although the specific genome of an individual organism is

Chromosome level

Molecular level

ffi3 Figure

9.1

Levels of genetic study.

The operations of genetics can be observed at the levels of organism, cell, chromosome, and DNA sequence (molecular level). Levels shown here are for a eukaryotic organism.

Chapter

254

9

Microbial Genetics

Chromosomes Nucleus

Plasmids Mitochondrion

Plasmid (in some fungi and protozoa)

Chloroplast

Figure

9.2

The general location and forms of the genome in two cell types and selected viruses (not to scale).

unique, the general pattern ofnucleic acid structure and function is similar among all organisms. In general, a chromosome is a discrete cellular structure composed of a neatly packaged DNA molecule. The chromosomes of eukaryotes and bacterial cells differ in several respects. The structure of eukaryotic chromosomes consists of a DNA molecule tightly wound around histone proteins, whereas a bacterial chromosome is condensed and secured into a packet by means ofhistonelike pro-

teins. Eukaryotic chromosomes are located in the nucleus; they vary in number from a few to hundreds; they can occur in pairs (diploid) or singles (haploid); and they are linear in format. In contrast, most bacteria have a single, circular (double-stranded) chromosome, although many bacteria have multiple circular chromosomes and some have linear chromosomes.

All chromosomes contain a series of basic informational "packets" called genes. A gene can be defined from more than one perspective. In classical genetics, the term refers to the fundamental unit of heredity responsible for a given trait in an organism. In the molecular and biochemical sense, it is a site on the chromosome that provides information for a given cell function. More specifically still, it is a certain segment of DNA that contains the necessary code to make a protein or RNA molecule. This last definition of a gene is emphasized in this chapter. Genes fall into three basic categories: structural genes that code for proteins, genes that code for RNA, and regulatory genes Ihat control gene expression. The sum ofall ofthese types ofgenes constitutes an organism's distinctive genetic makeup, or genotype (jee'-nohEp).The expression of the genotype creates traits (certain structures or functions) referred to as the phenotype (fee'-noh-typ). Just as a

The Size and Pockaging of Genomes Genomes vary greatly in size. Viruses have anywhere from three to several hundred genes; the bacterium Escherichia colihas a single chromosome containing 4,288 genes, and a human cell packs

about five times that many into 46 chromosomes. The chromosome of E. coli would measure about 1 mm if unwound and stretched out linearly, and yet this fits within a cell that measures just over I pm across, making the stretched-out DNA 1,000 times longer than the cell (figure 9.3). Still, the bacterial chromosome takes up only about one-third to one-half of the cell's volume. Likewise, if the sum of all DNA contained in the 46 human chromosomes were unraveled and laid end to end it would measure about 6 feet. How can such elongated genomes fit into the minuscule volume of a cell, and in the case of eukaryotes, into an even smaller compafirnent, the nucleus? The answer lies in the complex coiling of the DNA chain (Insight 9.1).

person inherits a combination of genes (genotype) that gives a certiain eye color or height (phenotype), a bacterium inherits genes that direct

the formation of a flagellum, or the ability to metabolize a ceriain substrate, and a virus contains genes for its capsid structure. All organisms contain more genes in their genotypes than are being seen as a

phenotype at any given time. In other words, the phenotype can change depending on which genes are "turned on" or expressed.

Figure 9.3 A disrupted Escherichia coli cell has spewed out its single, uncoiled DNA strand.

9.'l

Introduction to Cenetics and Cenes: Unlocking the Secrets of Heredity

255

The Packaging of DNA: Winding, Twisting, and Coiling Packing the mass of DNA into the cell involves several levels of DNA structure called supercoils or superhelices. In the simpler system ofprokaryotes, the circular chromosome is packaged by the action ofa special enzyme called a topoisomerase (specifically, DNA gyrase). This enzyme coils the chromosome into a tight bundle by introducing a reversible series of twists into the DNA molecule. The system in eukaryotes is more complex. with three or more levels of coiling. First. the DNA molecule of a chromosome. which is linear, is wound twice around the histone proteins, creating a chain of nucleosomes. The nucleosomes fold in a spiral formation upon one another. An even greater supercoiling occurs when this spiral arrangement further twists on its radius into a giant spiral with loops radiating from the outside. This extreme degree of compactness is what makes the eukaryotic chromosome visible during mitosis.

The expression of many traits, especially in eukaryotes, also seems to be related to the coiling of DNA. While the condensation of DNA and protein into chromatin was once thought to be a very regular process whose primary function was to provide

a

compact molecule, new research

twisting of DNA often serves to make certain segments ol the genetic program either more or less available to the cell. This previously unknown regulatory function creales an entirely new set of

has shown that the

information to guide the eventual production of the cell's phenotype. The reahzalion that the phenotype of a cell is due to more than just the order of nucleotides in the DNA has led to a rethinking of some of the most basic aspects ofgenetics {see Insight 9.3 ). Because genetic codes cannot be

properly accessed when DNA is

supercoiled, what other system would a cell need to manage DNA? Answer available at http ://www.mhhe.com/talaroT

DNA double helix

Condensed metaphase chromosomes

Chemical tags attached to histone proteins may increase the expression of nearby genes.

DNA wrapped around histones with linker DNA between them

DNA

I

Histone_f

Nucleosome

Supercoiled condensed chromatin Condensed nucleosomes Loosely condensed chromosome

Uncondensed chromatin fiber

The packaging of DNA. Eukaryotic DNA is intimately associated with a variety of proteins and small molecules. This relationship modulates the expression of many genes while at the same time allowing for the ordered condensation of DNA into a compact molecule.

The Structure of DNA: A Double Helix with lts Own Language Examining the function of DNA at the molecular level requires an even closer look at its structure. To do this, we will imagine being able to magnif, a small piece of a gene about 5 million times to disclose one of the great marvels of biology. James Watson and Francis Crick put the pieces of the molecularpuzzle together in 1953 (Insight 9.2). They discovered that DNA is a giant molecule, a type of nucleic acid with two shands forming a double helix. The general structure of DNA is universal, except in some viruses that contain single-stranded DNA. The basic unit of DNA structure is a nucleotide, composed of phosphate, deoxyribose sugar, and a nitrogen base, as shown in this simplified model (see figure 2.23):

Backbone Deoxyribose

t'Tt

-e-o

Y Phosphate

O€

N base t,tPl

>o)_€r ^rf lB\ v{

-rf rI))

lffi>

DNA

--T-Hydrogen bonds I

€hapter

256

9

Microbial Genetics

Deciphering the Structure of DNA The search for the primary molecules of heredity was a serious focus throughout the first half of the 20th cenhry. At first, many biologists thought that protein was the genetic material. An important milestone occurred in 1944 when Oswald Avery Colin Macleod, and Maclyn McCarty purified DNA and demonstrated at last that it was indeed the blueprint for life. This was followed by an avalanche ofresearch, which continues today.

"We Have Discovered the Secret of Life.' One area of extreme interest concerned the molecular structure of DNA.

In

American biologist James Watson and English physicist Francis Crick collaborated on solving the DNA puzzle. Although they did little of the original researchn they were intrigued by several findings from other scientists. It had been determined by Erwin Chargaffthat any model of DNA structure would have to contain deoxyribose, phosphate, purines, and pyrimidines arranged in a way that would provide variation and a simple way of copying itself. Watson and Crick spent long hours constructing models with cardboard cutouts and kept alert for any and every bit of information that might give them an edge. Two English biophysicists, Maurice Wilkins and Rosalind Franklin, had been painstakingly collecting data on X-ray crystallographs of DNA for several yearc. With this technique, molecules of DNA bombarded by X rays produce a photographic image that can predict the three195

1,

The molecule appeared to be a double helix. Gradually, the pieces of the

ptzzLe fell into place, and a final model was assembled-a model that explained all of the qualities of DNA, including how it is copied. A1though Watson and Crick were rightly hailed for the clarity of their solution, it must be emphasized that their success was due to the considerable efforts of a number of English and American scientists. This historic discovery showed that the tools of physics and chemistq, have useful applications in biological systems, and it also spawned ingenious research in all areas ofmolecular genetics. Since the discovery ofthe double helix in 1953, an extensive body of biochemical, microscopic. and crystallographic analysis has left little doubt that the model first proposed by Watson and Crick is correct. Newer techniques using scanning tunneling microscopy produce threedimensional images of DNA magnified 2 million times. These images veri8r the helical shape and twists of DNA represented by models.

What particular requirements didWatson and Crick's model of DNA have to fit to be accurate? Answer available at http://www. mhhe.com/talaroT

dimensional structure of the molecule. After being allowed to view certain X-ray data, Watson and Crick noticed an unmistakable pattern:

The first direct glimpse at DN,{s structure. This false-color scanning tunneling micrograph of calf thymus gland DNA (2,000,000x) brings out the well-defined folds in the helix.

The men who cracked the code of life. Dr. lames Watson (left) and Dr. Francis Crick (right) stand next to their model that finally explained the structure of DNA in 1953.

9.'l

Introduction to Genetics and Cenes: Unlocking the Secrets of Heredity

257

Base pairs

&

Phosphate

5)

4',n

Deoryribose

3'1321', with carbon number 2',

o o

o o

Cytosine Guanine

Thymine Adenine Hydrogen bond Covalent bond (b)

9.4 Three views of DNA structure. fl(a) AF[ure schematic nonhelical model shows the basic layout of the two strands. Note that the order

(a)

of phosphate and sugar bonds goes in the opposite direction for the two strands, going from the 5' carbon to the 3' carbon on one strand, and from the 3' carbon to the 5' carbon on the other strand. lesets show details of the nitrogen base pairs. (b) Simplified model that highlights the antiparallel arrangement and the major and minor grooves. (c) Space-filling model that more accurately depicts the three-dimensional structure of DNA.

Each deoxyribose sugar bonds covalently in a repeating pattern with two phosphates. One of the bonds is to the number 5' (read "five prime") carbon on deoxyribose, and the other is to the 3' carbon, which specifies the order and direction ofeach strand (figure 9.4). This formation results in an elongate strand supported by a sugar-phosphate backbone. The nitrogen bases, purines and pyrimidines, altach by covalent bonds at the 1' position ofthe sugar (figure 9.4c). They span

the center of the molecule and pair with appropriate complementary bases from the other strand, thereby forming a doublestranded helix. The paired bases are so aligned as to bejoined by hydrogen bonds. Such weak bonds are easily broken, allowing the molecule to be "unzipped" into its complementary strands. This feature is of great importance in gaining access to the information encoded in the nitrogen base sequence. Pairing of purines and

pyrimidines is not random;

it is dictated by the formation of

258

Chapter

9

Microbial Cenetics

hydrogen bonds between certain bases. Thus, in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T), and the purine guanine (G) pairs with the pyrimidine cytosine (C). Note that adenine forms two hydrogen bonds with thymine, and cytosine forms three hydrogen bonds with guanine. This difference will influence a number of DNA functions. New research also indicates that the bases are attracted to each other in this pattern because each has a complementary three-dimensional shape that matches its pair. Although the base-pairing partners generally do not vary, the sequence of base pairs along the DNA molecule can assume any order, resulting in an infinite number of possible nucleotide sequences. Other important considerations of DNA structure concern the nature of the double helix itself. The two strands are not oriented in the same direction. One side of the helix runs in the op-

posite direction of the other, in what is called an antiparallel arrangement (figure 9.4b). The order of the bond between the carbon on deoxyribose and the phosphates is used to keep track of the direction of the two sides of the helix. Thus, one helix runs from the 5' to 3' direction, and the other runs from the 3' to 5' direction. This characteristic is a significant factor in DNA synthesis and translation. As apparently perfect and regular as the DNA molecule may seem, it is not exactly symmetrical. The torsion in the helix and the stepwise stacking of the nitrogen bases produce two different-size surface features, the major and minor grooves (figure 9.4c). One of the significant findings that has emerged from sequencing the DNA of chromosomes is knowing how long it can be. An average bacterial chromosome consists of 5 to 6 million nucleotides; the 46 chromosomes of humans amount to about 3 billion

4

w

Template strands

E€(a)

fl

Figure

(b)

9.5

Simplified steps to show the

semiconservative replication of DNA. (a, b) The two strands of the double helix are unwound and separated by a helicase, which disrupts the hydrogen bonds and exposes the nitrogen base codes of DNA. Each single strand formed will serve as a template to synthesize a new strand of DNA. (c) A DNA polymerases (D) proceed along the DNA molecule, attaching the correct nucleotides according to the pattern of the template. An A on the template will pair with a T on the new molecule, and a C will pair with a G. (d) The resultant new DNA molecules contain one strand of the newly synthesized DNA and the original template strand. The integrity of the code is kept intact because the linear arrangement of the bases is maintained during this process. Note that the fine details of the process are presented in figure 9.6.

nucleotides.

The Significance of DNA Structure The arrangement of nitrogen bases in DNA has two essential effects.

1. Maintenance of the code during reproduction. The constancy of base-pairing guarantees that the code will be retained during cell growth and division. When the two strands are separate{ each one provides a template (pattern or model) for the replication (exact copying) of a new molecule (figure 9.5). Because the sequence of one strand automatically gives the sequence ofits partner, the code can be duplicated with fidelity. 2. Providing variety. The order ofbases along the length ofthe DNA strand provides the information needed to produce RNA and protein molecules, which in turn are responsible for the phenotype of the cell. As we will see, changing the identity of the bases or their order in the DNA molecule can have a dramatic effect on the phenotype of the organism.

It is tempting to consider how such a seemingly simple code can account for the extreme differences among forms as diverse as

bacterial gene may contain 1,000 nucleotides-so while DNA contains fewer "letters," they are arranged into much longer "words." Mathematically speaking, 1,000 nucleotides can be arranged in 41'000 different sequences, a number so large (1.5 x t0602;1hat it provides nearly endless degrees ofvariation.

DNA Replication: Preserving the Code and Passing lt On It is the sequence of bases along the length of DNA that constitutes its "language." For this language to be preserved for hundreds of generations, it will be necessary for its codes to be duplicated and passed on to each offspring. This process of duplication is called DNA replication. In the following example, we will show replication in bacteria, but with some exceptions it also applies to the process as it works in eukaryotes and some viruses. Recall from chapter 7 that early in binary fission, the metabolic machinery of a bacterium responds to a message and initiates the duplication of the chromosome. Despite its complexity DNA replication can be very rapid-it must be completed in a single generation time (around 20 minutes n E. coli).

a virus, E. coli, and a human. The English language, based on 26 letters, can create an infinite variety ofwords, but how can an

The Overoll Replication Process

apparently complex genetic language such as DNA be based on just four nitrogen base "letters"? The answer lies in the fact that the four bases of DNA are arranged into much longer sequences. An average

What features allow the DNA molecule to be exactly duplicated, and how is its integrity retained? DNA replication requires a careful orchestration of the actions of 30 different en-4/mes (partial list in

9.'l

Introduction to Cenetics and Cenes: Unlocking the Secrets of Heredity

Enzyme

Function

Helicase

Unzipping the DNA helix Synthesizing an RNA primer Adding bases to the new DNA chain;

Primase

DNA polymerase III

proofreading the chain for mistakes

DNA polymerase I

Removing primer. closing gaps. repairing mismatches

Ligase

Final binding of nicks in DNA during syrthesis and repair

Gyrase

Supercoiling

table 9.1), which separate the strands ofthe existing DNA molecule, copy its template, and produce two complete daughter molecules. A simplified version of replication is shown in figure 9.5 and includes the following:

1. uncoiling the parent DNA molecule, beginning at a predetermined point of origin: 2. unzipping the hydrogen bonds between the base pairs, thus separating the two strands and exposing the nucleotide sequence of each strand (which is normally buried in the center of the helix) to serve as templates; and

3. synthesizing two new strands by attachment of the correct complementary nucleotides to each single-stranded template.

A critical feature of DNA replication is that each daughter molecule will be identical to the parent in composition, but neither one is completely new; the shand that serves as a template is an original parental DNA strand. The preservation of the parent molecule in this way, termed semiconservative replication, helps explain the reliability and fidelity of replication.

Details of Replicotion The process of synthesizing a new daughter strand of DNA using the parental strand as a template is carried out by the enzyme DNA polymerase III. The entire process of replication involves several other enzymes working in concert with DNA polymerase III. This enzyme can decipher and duplicate DNA codes, but it has two major restrictions: DNA polymerase III is unable to begin synthesizing a chain ofnucleotides but can only continue to add nucleotides to an already existing chain; and it can only add nucleotides in one direction, so a new strand is always synthesized 5' to 3' . All chromosomes have a specific origin of replication site that serves as the place where replication will be initiated. It is recognized by a short sequence rich in adenine and thymine that, you will recall, are held together by only two hydrogen bonds rather than three. Because the origin ofreplication is AT-rich, less energy is required to separate the two strands than would be required if the origin were rich in guanine and cyosine. Prior to the start of replication, enzymes called helicases (unzipping enzymes) bind to the DNA at the origin. These enzymes untwist the helix and break the hydrogen bonds holding the two strands together, resulting in two

259

separate strands, each of which will be used as a template for the synthesis of a new strand. Replication begins when an P.NA primer is synthesized at the origin of replication (figure 9.6, step 1). DNA polymerase III must have this short strand of RNA to serve as a starting point for adding nucleotides. Because the bacterial DNA molecule is circular, opening of the circle forms two replication forks, each containing its own set of replication enzymes. DNA polymerase III is actually a huge enzl'rne complex containing two active DNA polymerases along with several other proteins whose main functions are to stabilize the polymerase and provide a means of removing improperly incorporated nucleotides. Because two copies of most of the major enzymes are present in the complex, both strands of DNA can be copied at (nearly) the same time. The enzyme complex encircles the DNA near the replication fork and synthesizes new DNA using the parent strand as a template. As synthesis proceeds, the forks continually open up to expose the template for replication (figure 9.6' steps 2 and3). Because DNA polymerase III can only synthesize new DNA in the 5' to 3' direction,just one strand, called the leading strand, can be synthesized continuously. One enzyme does this as it moves

along the template strand toward the replication fork. The other strand which is oriented 3' to 5' with respect to the polymerase, cannot be synthesized continuously and is termed the lagging strand (figure 9.6, steps 4 and 5). Because it cannot be synthesized continuously, the polymerase adds nucleotides a few at a time in the direction away from the fork (5' to 3'). As the fork opens up a bit, the next segment is synthesized backward near the point of the previous segment, a process repeated at both forks

until synthesis is complete. In this way, the two new strands can be synthesized simultaneously. This manner of synthesis produces short fragments of DNA (100 to 1,000 bases long) called Okazaki fragments. The lagging strand is completed when an enzyme, DNA ligase, fills in the spaces between Okazaki fragments with the correct nucleotides.

Elongation and Termination of the Daughter Molecules The addition of nucleotides proceeds at an astonishing pace, estimated in some bacteria to be 750 bases per second at each fork! As replication proceeds, one new double strand loops down (figure 9.7a).The DNA polymerase I removes the RNA primers used to initiate DNA synthesis and replaces them with DNA. When the forks come fulI circle and meet, ligases move along the lagging

strand to begin the initial linking of the fragments and to complete

synthesis and separation of the two circular daughter molecules (figure 9.76). Like any language, DNA is occasionally "misspelled" when an incorrect base is added to the growing chain. Studies have shown that such mistakes are made once in approximately 108 to l0e bases, but most ofthese are corrected.

Ifnot corrected

they

are referred to as mutations and can lead to serious cell dysfunc-

tion. Because continued cellular integrity is very dependent on accurate replication, cells have evolved their own proofreading function for DNA. DNA polymerase III, the enzyme that elongates the molecule, can also detect incorrect, unmatching bases;

excise them; and replace them with the correct base. DNA I is involved in proofreading the molecule and repairing damaged DNA. polymerase

Chapter

260

9

Microbial Cenetics

Origin of replication

(p

nt tne replication origin, the DNA template strands (blue) are separated. At the point of separation, short RNA primers (black) are synthesized and 2 replication forks are formed.

@

Since replication proceeds at both forks and for both template strands, 2 DNA polymerases will be needed on each side of the origin. The polymerase encircles a single strand in preparation for replication. Arrows here show the direction in which the polymerases will be moving.

Q

/_.-..-\\ Primer molecules',

,'

earfy stage in replication showing the newly synthesized strands in red. Note that the numbers in red refer to the direction of the synthesis of this new strand, i.e., 5'to 3'.

@

Cbse-up view of one replication fork. The lagging strand is synthesized when the polymerase loops the DNA template backward for a short distance (not shown in illustration). This presents the correct orientation for forming a short piece or Okazaki fragment that is in the 5'to 3' direction. These fragments are later linked together to form the finished double DNA strand.

DNA polymerase acts only in the 5'to 3'direction, it forms a continuous leading strand lrom that orientation (-). The lagging strand must be made backward in short sections, 5'to which are later linked together.

QSince

3'(**-),

@ Pto""ts Flgure 9.6

The bacteriat replicon: a modelfor DNA synthesis.

(1) Circular DNA has a special origin site where replication originates. (2) When strands are separated, two replication forks form, and a DNA polymerase lll complex enters at each fork. (3) Starting at the primer sequence, each polymerase moves along the template strands (blue), synthesizing the new strands (red) at each fork. (4) DNA polymerase works only in the 5' to 3' direction, necessitating a different pattern of replication at each fork. Because the leading strand orients in the 5' to 3' direction, it will be synthesized continuously. The lagging strand, which orients in the opposite direction, can only be synthesized in short sections, 5' to 3', which are later linked together. (5) Inset presents details of process at one replication fork and shows the Okazaki fragments and the relationship of the template, leading, and lagging strands.

9.2

Applications of the DNA Code: Transcription and Translation

26r

9.2 Applications of the DNA Code: Transcription and Translation We have explored how the genetic language of the DNA molecule is conserved through replication. Next, we consider exactly what DNA does in the cell. Given that the sequence of bases in DNA is a genetic code, just what is the nature of this code and how is it utilized? Although the genome is full of critical information, the DNA molecule itself cannot perform cell processes directly. In-

(a)

\


/.1\

\"/

Factors that influence the rate at which microbes are killed by antimicrobial agents. population, even a pure culture, do (a) Length of exposure to the agent. During exposure to a chemical or physical agent, all cells of a microbial giving a straight-line logarithmically, decreases population the in remaining viable organisms of not die simultaneously. over time, the number (b) Effect of the microbial sterilization. is considered small infinitesimally is of survivors number the which point at graph. The relationship on a or microbistatic. load. (c) Relative resistance of spores versus vegetative forms. (d) Action of the agent, whether microbicidal

Figure

11.2

instantaneous but begins when a certain threshold of the microbicidal agent (some combination of time and concentration) is met' Death continues in a logarithmic manner as the time of exposure or concentration ofthe agent is increased (figure 11.2). Because many microbicidal agents target the cell's metabolic processes, active cells (younger, rapidly dividing) tend to die more quickly than those

that are less metabolically active (older, inactive). Eventually, a point is reached at which survival of any cells is highly unlikely; this point is equivalent to sterilization. The effectiveness of a particular agent is governed by several factors besides time. These additional factors influence the action of antimicrobial agents:

1. The number of microorganisms (figure ll.2b). A higher load of contaminants requires more time to destroy. 2. The nature of the microorganisms in the population (figure ll.2c\.In most actual circumstances of disinfection and sterilization,the target population is not a single species of microbe but a mixture of bacteria, fungi, spores, and viruses, presenting a broad spectrum of microbial resistance. 3. The temperature and pH of the environment. 4. The concentration (dosage, intensity) of the agent. For example, UV radiation is most effective at260 nm and most disinfectants are more active at higher concentrations. 5. The mode of action of the agent (figure ll.zd). How does it kill or inhibit the microorganism?

6. The presence of solvents, interfering organic matter, and inhibitors. Saliva, bloo4 and feces can inhibit the actions of disinfectants and even ofheat.

Practicql Concerns in Microbiol Control Numerous considerations govern the selection of a workable method of microbial control. These are among the most pressing concerns:

1. Does the application require sterilization, or is disinfection adequate? In other words, must spores be destroyed, or is it necessary to destroy only vegetative pathogens?

2. Is the item to be reused or permanently discarded? If it will

be

discarde4 then the quickest and least expensive method should be chosen.

3. If it will be reused can the item withstand heat, pressure' radiation, or chemicals?

4. Is the control method suitable for a given application? (For example, ultraviolet radiation is a good sporicidal agent, but it will not penetrate solid materials.) Oa in the case of a chemical, will it leave an undesirable residue? 5. Will the agent penetrate to the necessary extent? 6. Is the method cost- and labor-efficient, and is it safe? A remarkable variety of substances can require sterilization. They range from durable solids made of glass or rubber to sensitive liquids such as serum. Even entire buildings or large pieces of equipment

322

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Physical and Chemical Agents for Microbial Control

such as space probes and satellites are sterilized. Hundreds ofsituations requiring sterilization confront the network of persons involved in health care, whether technician, nurse, doctor, or manufacturer, and no universal method works well in every case. Health care workers especially must practice very high standards of infection prevention. Considerations such as cost, effectiveness, and method of disposal are all important. For example, the disposable plastic items such as catheters and syringes that are used in invasive medical procedures have the potential for infecting the tissues. These must be sterilized during manufacture by a nonheating method (gas or radiation), be-

Surfactant molecules

Membrane lipids

cause heat can damage most plastics. After these items have been use{ it is often necessary to destroy or decontaminate them before they are discarded because ofthe potential risk to the handler (from needle-

sticks). Steam sterilization, which is quick and sure, is a sensible choice it does not matter if the plastic is destroyed.

at this point, because

How Antimicrobial Agents Work: Their Modes of Action An antimicrobial agent's adverse effect on cells is known as its mode (or mechanism) of action.Agents affect one or more cellular targets, inflicting damage progressively until the cell is no longer able to survive. Antimicrobials have a range of cellular targets, with the agents that are least selective in their targeting tending to be effective against the widest range of microbes (examples include heat and radiation). More selective agents (drugs, for example) tend to target only a single cellular component and are much more restricted as to the microbes they are effective against. The cellular targets of physical and chemical agents fall into four general categories :

cell wall, ) the cell membrane, 3. cellular synthetic processes (DNA, RNA), and 4. proteins. 1. the

The Effects of Agents on the Cell Woll The cell wall maintains the structural integrity of bacterial and fungal cells. Several types of chemical agents damage the cell wall by blocking its synthesis, digesting it, or breaking down its surface. A cell deprived of a functioning cell wall becomes fragile and is lysed very easily. Examples of this mode of action include some antimicrobial drugs (penicillins) that interfere with the synthesis of the cell wall in bacteria (described in chapter l2). Detergents and alcohol can also disrupt cell walls, especially in gram-negative bacteria.

How Agents Affect the Cell Membrone All microorganisms have a cell membrane composed of lipids and proteins, and even some viruses have an outer membranous envelope. As we learned in previous chapters, a cell's membrane provides a two-way system of transport. If this membrane is disrupted" a cell loses its selective permeability and can neither prevent the loss of vital molecules nor bar the entry of damaging chemicals. Loss of those functions usually leads to cell death. Detergents called surfactants* work as microbicidal agents because they lower * sufiactant (si-fak'-tunt) A word derived from surface-acting agent.

Flgure 11.3 Mode of action of surfactants on the cell membrane. Surfactants inserting in the lipid bilayer disrupt it and create abnormal channels that alter permeability and cause leakage both into and out of the cell.

the surface tension of cell membranes. Surfactants are polar molecules with hydrophilic and hydrophobic regions that can physically bind to the lipid layer and penetrate the internal hydrophobic region of membranes. In effect, this process "opens up" the once tight interface, leaving leaky spots that allow injurious chemicals to seep into the cell and important ions to seep out (figure 11.3).

Agents That Affect Protein and Nucleic Acid Synthesis Microbial life depends upon an orderly and continuous supply of proteins to function as enzymes and structural molecules. As we saw in chapter 9, these proteins are synthesized via the ribosomes through a complex process called translation. For example, the antibiotic chloramphenicol binds to the ribosomes of bacteria in a way that stops peptide bonds from forming. In its presence, many bacterial cells are inhibited from forming proteins required in growth and metabolism and are thus inhibited from multiplying. Many drugs used in antimicrobial therapy are simply chemicals that block protein synthesis in microbes without adversely affecting human cells. These drugs are discussed in greater detail in chapter 12.

The nucleic acids are likewise necessary for the continued functioning of microbes. DNA must be regularly replicated and transcribed in growing cells, and any agent that either impedes these processes or changes the genetic code is potentially antimicrobial. Some agents bind irreversibly to DNA, preventing both transcription and translation; others are mutagenic agents. Gamma, ultraviolet, or X radiation causes mutations that result in permanent inactivation of DNA. Chemicals such as formaldehyde and ethylene oxide also interfere with DNA and RNA function.

Agents That Alter Protein Function A microbial cell contains large quantities of proteins that function properly only if they remain in a normal three-dimensional

11.2 Methods of Physical Control (a) Native State Enzyme Substrate

Substrate

(b) Complete

Denaturation

o

o) a6

l(d

rlo Ya

(d) Blocked Active Site

Active site can no longer accept the substrate, and the enzyme is inactive.

ff

Flgure

11.4

323

The population ofmicrobes that cause spoilage or infection varies widely in species composition, resistanceo and harmfulness, so

microbial control methods must be adjusted to

fit

individual

situations. The type ofmicrobial control is indicated by the terminology used. Sterilization ageats destroy all viable organisms, including viruses. Antisepsis, disinfection, and s anitization agents reduce the numbers of viable microbes to a specified level. Antimicrobial agents are described according to their ability to destroy or inhibit microbial growth. Microbicidal agents cause microbial death. They are described by what they are - cidal fot: sporicides' bactericides, fi.rngicides, virucides. An antiseptic agent is applied to living tissue to destroy or inhibit microbial growthA disinfectant agent is used on inanimate objects to destroy vegetative pathogens but not bacterial endospores. Sanitization reduces microbial numbers on inanimate objects to safe levels by physical or chemical means. Degermation refers to the process of mechanically removing microbes from the skin. Microbial death is defined as the permanent loss of reproductive capability in microorganisms. Antimicrobial agents attack specific cell sites to cause microbial death or damage. Any given antimicrobial agent attacks one offour major cell targets: the cell wall, the cell membrane, biosynthesis pathways for DNA or RNA, or protein (enzyme) function.

Modes of action affecting protein

frinction. (a) The native (functional) state is maintained by bonds that create active sites to fit the substrate. Some agents denature the protein by breaking all or some secondary and tertiary bonds. Results are

(b) complete unfolding or (c) random bonding and incorrectfolding. (d) Some agents react with functional groups on the active site and interfere with bonding. configuration called the native stqte.The antimicrobial properties of some agents arise from their capacity to disrupt the structure of, or denature, proteins. In general, denaturation occurs when the bonds that maintain the secondary and tertiary structure of the protein are broken. Breaking these bonds will cause the protein to unfold or create random, irregular loops and coils (fig-

ure 11.4). One way that proteins can be denatured is through coagulation by moist heat (the same reaction seen in the irrevers-

ible solidification of the white of an egg when boiled). Chemicals such as strong organic solvents (alcohols, acids) and phenolics also coagulate proteins. Other antimicrobial agents, such as metallic ions, attach to the active site of the protein and prevent it from interacting with its correct substrate. Regardless of the exact mechanism, such losses in normal protein function can promptly arrest metabolism. Most antimicrobials of this type are nonselective as to the microbes they affect.

methods involve the use ofphysical and chemical agents to eliminate or reduce the numbers of microorganisms from a specific environment so as to prevent the spread of infectious agents, retard spoilage, and keep commercial products safe.

w Microbial control

','a.2 Methods of Physical Control Many microorganisms have adapted to a tremendous diversity of habitats on earth, even severe conditions of temperature, moisture, pressure, and light. For microbes that normally withstand such ex-

treme physical conditions, our usual attempts at control would probably have little effect. Fortunately for us' we are most interested in controlling microbes that flourish in the same environment in which humans live. The vast majority of these microbes are readily controlled by abrupt changes in their environment. Most prominent among antimicrobial physical agents is heat' Other less widely used agents include radiation and filtration. The following sections examine these and other methods and explore their practical applications in medicine, commerce, and the home.

Heat as an Agent of Microbial Control a microbe's temperature of adaptation is detrimental effect on it. As a rule, elevated temperatures (exceeding the maximum growth temperature) are microbicidal, whereas lower temperatures (below the minimum growth temperature) are microbistatic. The two physical states of heat used in microbial control are moist and dry. Moist heat occurs in the form of hot water, boiling water, or steam (vaporized water). In practice, the temperature of moist heat usually ranges from 60"C to 135'C. As we shall see, the temperature of steam can be regulated by adjusting its pressure in a closed container. The expression dry heat denotes air with a low moisture content that has been heated by a flame or electric heating coil. In practice, the temperature of dry heat ranges from 160"C to several thousand degrees Celsius.

A sudden departure from likely to have

a

324

Chapter

11

Physical and Chemical Agents for Microbial Control

Comparison of Times and Temperatures to Achieve Sterilization with Moist and Dry Heat Temperature

Moist Heat

Time to Sterilize

121"C

15 min

125"C

10 min

134'C

l2l'c

Dry Heat

3

min

180

160'C

120 min

170'C

60 min

Temperature

Time (Min)

Non-spore-forming bacteria

58'C

28

Non-spore-forming bacteria

6l"c

l8

58'C

l9

ofspore-

forming bacteria

min

spores Yeasts Fungal

76"C 59'C

22 19

Viruses

Mode of Action qnd Relqtive Effectiveness of Heot

Nonenveloped Enveloped

Moist heat and dry heat differ in their modes of action

as

well

as

in

their efficiency. Moist heat operates at lower temperatures and shorter exposure times to achieve the same effectiveness as dry heat (table f 1.3). Although many cellular structures are damaged by

moist heat, its most microbicidal effects are the coagulation and denaturation of proteins, which quickly and permanently halt cellular metabolism. Dry heat of a moderate temperature dehydrates the cell, removing the water necessary for metabolic reactions, and it also alters protein strucfure. However, the lack of water actually increases the stability of some protein conformations, necessitating the use of higher temperatures when dry heat is employed as a method of microbial control. At very high temperatures, dry heat oxidizes cells, burning them to ashes. This method is the one used in the laboratory when a loop is flamed or in industry when medical waste is incinerated.

Heot Resistonce qnd Thermol Deoth of Spores and Vegetqtive Cells Bacterial endospores exhibit the greatest resistance, and vegetative states ofbacteria and fungi are the least resistant to both moist and dry heat. Destruction of spores usually requires temperatures above boiling (table 11.4), although resistance varies widely.

Thermal Death Times of Various Endospores Time of Exposure

Organism

MicrobialType

Vegetative stage

600 min

140'C

Average Thermal Death Times of Vegetative Stages of Microorganisms

Temperature to Kill Spores

Moist Heat Bacillus subtilis

121"C

I min

B. stearothermophilis

127"C

12 min

Clostridium botulinum

120"c

10

C. tetani

105"C

10

min min

Bacillus subtilis

t2t"c

120

min

B. stearothermophilis

140'C

Dry Heat

Clostridium botulinum

120"c

min 120 min

C. tetani

100"c

60 min

5

Protozoan trophozoites Protozoan cysts

Worm eggs Worm larvae

57"C 54"C 46"C 60'c 54"C 60'c

29 22 16

6 3

10

Vegetative cells also vary in their sensitivity to heat, though not to the same extent as spores (table 11.5). Among bacteria, the death times with moist heat range from 50oC for 3 minutes (Neisseria gonorrhoeae) to 60"C for 60 minute s (Staphylococcus aureus). lt ts worth noting that vegetative cells of sporeformers are just as susceptible as vegetative cells ofnon-sporeformers, and that pathogens

are neither more nor less susceptible than nonpathogens. Other microbes, including fungi, protozoa, and worms, are rather similar in their sensitivity to heat. Viruses can be surprisingly resistant to heat, with a tolerance range extending from 55"C for 2 to 5 minutes (adenoviruses) to 60'C for 600 minutes (hepatitis A virus). For practical purposes, all non-heat-resistant forms of bacteria, yeasts, molds, protozoa, worms, and viruses are destroyed by exposure to 80'C for 20 minutes.

Procticql Concerns in the Use of Heot: Thermol Deqth Meosurements Adequate sterilization requires that both temperature and length of exposure be considered. As a general rule, higher temperatures allow shorter exposure times, and lower temperatures require longer exposure times. A combination of these two variables constitutes the thermal death time, or TD! defined as the shortest length of time required to kill all test microbes at a specified temperature. The TDT has been experimentally determined for the microbial species that are common or important contaminants in various heat-treated materials. Another way to compare the susceptibility of microbes to heat is the thermal death point (TDP), defined as the lowest temperature required to kill all microbes in a sample in 10 minutes.

Many perishable substances are processed with moist heat. Some of these products are intended to remain on the shelf at room temperature for several months or even years. The chosen heat treatment must render the product free of agents of spoilage or

11.2 Methods of PhYsical Control

disease.

At the same time, the quality of the product and the

speed

and iost of processing must be considered. For example, in the commercial preparation of canned green beans, one of the cannery3 greatest concerns is to prevent growth of the agent of botulism. From several possible TDTs (that is, combinations of time and temperature) for Clostridium botulinum spores' the cannery must choose one that kills all spores but does not turn the beans to mush' These many considerations produce an optimal TDT for a given processing method. Commercial canneries heat low'acid foods at 121'C for 30 minutes, a treafinent that sterilizes these foods. Because ofsuch strict controls in carmeries, cases ofbotulism from commercially canned foods are rare.

Common Methods of Moist Heot Control The four ways that moist heat is employed to contol microbes are

1.

steam under pressure; nonpressurized steam;

2. 3. boiling water; and 4. pasteurization.

Sterilization with Steam Under Pressure At

sea level,

normal atmospheric pressue is 15 pounds per square inch (psi), or 1 atmosphere. At this pressure, water will boil (change from a

325

liquid to a gas) at 100oC, and the resultant steam will remain at exactly that temperature, which is unfortunately too low to relia-

bly kill all microbes. In order to raise the temperature of steam, the pressure at which it is generated must be increased. As the pressrue is increase4 the temperature at which water boils and the temperature of the steam produced both rise' For example, at a pressure of 20 psi (5 psi above normal), the temperature of steam is 109"C. As the temperature is increased to 10 psi above normal, the steam's temperature rises to 115oC,. and at 15 psi above normal (a total of 2 atmospheres), it will be l2loC.It is not the pressure by itself that is killing microbes but the increased temperature it produces. Such pressure-temperature combinations can be achieved only with a special device that can subject pure steam to pressures greaterthan 1 atnosphere. Health and commercial industries use an autoclave for this purpose, and a comparable home appliance is the pressure cooker. Autoclaves have a frrndamentally similar plan: a cylindrical metal chamber with an airtight door on one end and racks to hold materials (figure 11.5). Its construction includes a complex network of valves, pressure and temperature gauges, and ducts for regulating and measuring pressure and conducting the steam into the chamber. Sterilization is achieved when the steam condenses against the objects in the chamber and gradually raises their temperature.

Pressure regulator Exhaust to atmosphere Steam from jacket to chamber or exhaust from chamber

Steam jacket Steam supply valve

Steam supply

Temperaturesensing bulb

(b)

Flgure ll.5

Sterilization using steam under pressure.

(a) A small, benchtop autoclave used in many small laboratories. (b) Cutaway section, showing autoclave components and the flow of steam itriougtr the autoclave. From tohn J. perkens, Principlu ond Methods of Steritizotion in Heolth Science,2nd ed., 1969. Courtesy of Charles C. Thomas, Publisher, Springfield, lllinois'

326

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Physical and Chemical Agents for Microbial Control

Experience has shown that the most efficient pressuretemperature combination for achieving sterilization is l5 psi, which yields 121"C. It is possible to use higher pressure to reach higher temperatures (for instance, increasing the pressure to 30 psi raises the temperature to 132'C), but doing so will not significantly reduce the exposure time and can harm the items being sterilized. The duration ofthe process is adjusted according to the bulkiness of the items in the load (thick bundles of material or large flasks of liquid) and how fullthe chamber is. The range of holding times varies from l0 minutes for light loads to 40 minutes for heavy or bulky ones; the average time is 20 minutes. The autoclave is a superior choice to sterilize heat-resistant materials such as glassware, cloth (surgical dressings), rubber (gloves), metallic instruments, liquids, paper, some media, and some heat-resistaat plastics.

Petri dishes) but

If the items are heat-sensitive (plastic

will be discarded the autoclave is still a good

choice. However, the autoclave is ineffective for sterilizing substances that repel moisture (oils, waxes, powders).

Nonpressurized Steam Selected substances that cannot withstand the high temperature of the autoclave can be subjected to lntermittent sterilization, also called tyndallization.' This technique requires a chamber to hold the materials and a reservoir for boiling water. Items in the chamber are exposed to free-flowing steam for 30 to 60 minutes. This temperature is not sufficient to reliably kill spores, so a single exposure will not suffice. On the assumption that surviving spores will germinate into less resistant vegetative cells, the items are incubated at appropriate temperatures for 23 Io 24 hours, and then again subjected to steam treatment. This cycle is repeated for 3 days in a row. Because the temperature never gets above 100'C, highly resistant spores that do not germinate may survive even after 3 days of this treatment. Intermittent sterilization is used most often to process heatsensitive culture media, such as those containing sera, egg, or carbohydrates (which can break down at higher temperatures) and some canned foods. It is probably not effective in sterilizing items such as instruments and dressings that provide no environment for spore germination, but it certainly can disinfect them.

Boiling Water: Disinfection A simple boiling water bath or chamber can quickly decontaminate items in the clinic and home. Because a single processing at 100'C will not kill all resistant cells, this method can be relied on only for disinfection and not for sterilization. Exposing materials to boiling water for 30 minutes will kill most non-spore-forming pathogens, including resistant species such as the tubercle bacillus and staphylococci. Probably the greatest disadvantage with this method is that the items can be easily recontaminated when removed from the water. Boiling is also a recommended method of disinfecting unsafe drinking water. In the home, boiling water is a fairly reliable way to sanitize aad disinfect materials for babies, food utensils, bedding, and clothing from the sickroom.

Pasteurization: Disinfection of Beverages Freshbeverages such as milk, fruit juices, beer, and wine are easily contaminated during collection and processing. Because microbes have the 1. Named for the British physicist John Tyndall, who did early experiments with sterilizing procedures.

potential for spoiling these foods or causing illness, heat is frequently used to reduce the microbial load and destroy pathogens. Pasteurization is a technique in which heat is applied to liquids to kill potential agents of infection and spoilage, while at the same time retaining the liquidb flavor and food value. Ordinary pasteurization techniques require special heat exchangers that expose the liquid to 71.6'C for 15 seconds (flash method) or to 63'C to 66oC for 30 minutes (batch method). The first method is preferable because it is less likely to change flavor and nutrient content, and it is more effective against certain resistant pathogens such as Coxiella and Mycobacterium. Although these treatments inactivate most viruses and destroy the vegetative stages of 97o/o to 99%o ofbacteria and fungi, they do not kill endospores or thermoduric microbes (mostly nonpathogenic lactobacilli, micrococci, and yeasts). Milk is not sterile after regular pasteurization. In

fact, it can contain 20,000 microbes per milliliter or more, which explains why even an unopened carton of milk will eventually spoil. Newer techniques can also produce sterile milk that has a storage life of 3 months. This milk is processed with ultrahigh temperature (UHT)-134'C for I to 2 seconds. One important aim in pasteurization is to prevent the transmission of milk-borne diseases from infected cows or milk handlers. The primary targets ofpasteurization are non-spore-forming pathogens: Salmonella species (a common cause of food infection), Campylobacter j ejuni (acute intestinal infection), Listeria monocytogenes (listeriosis), Brucella species (undulant fever), Coxiella burnetii (Q fever), Mycobacterium bovis, M. tuberculo.vs, and several enteric viruses. Pasteurization also has the advantage of extending milk storage time, and it can also be used by some wineries and breweries to stop fermentation and destroy contaminants.

Dry Heat: Hot Air ond lncinerotion Dry heat is not

as versatile or as widely used as moist heat, but it has several important sterilization applications. The temperatures and times employed in dry heat vary according to the particular method, but in general, they are greater than with moist heat. Incineration in a flame or electric heating coil is perhaps the most rigorous of all heat treatments. The flame of a Bunsen burner reaches l,870oC at its hottest point, and furnaces/incinerators operate at temperatures of 800"C to 6,500oC. Direct exposure to such intense heat ignites and reduces microbes and other substances to ashes and gas. Incineration of microbial samples on inoculating loops and needles using a Bunsen burner is a very common practice in the microbiology laboratory. This method is fast and effective, but it is also limited to metals and heat-resistant glass materials. Tabletop infrared incinerators (figure 11.6), an alternative to Bunsen burners, are safer and prevent splatter of the inoculum. Large incinera-

tors are regularly employed in hospitals and research labs for complete destruction and disposal of infectious materials such as syringes, needles, cultural materials, dressings, bandages, bedding, animal carcasses, and pathology samples. The hot-air oven provides another means of dry-heat sterilization. The so-calleddry oven is usually electric (occasionally gas) and has coils that radiate heat within an enclosed compartment. Heated, circulated air transfers its heat to the materials in the oven. Depending on the type of oven and the material being decontaminated, a

'i.1.2 Methods of

327

PhYsical Control

others are not killed and some are even preserved. Endospores of Bacillus and Clostridium are viable for millions of years under ex-

tremely arid conditions. Staphylococci and streptococci in dried

secretions, and the tubercle bacillus surrounded by sputum, can remain viable in air and dust for lengthy periods. Many viruses (especially nonenveloped) and fungal spores can also withstand long periods of desiccation. Desiccation can be a valuable way to preserve foods because it greatly reduces the amount of water available

to support microbial growth. It is interesting to note that a combination

of freezing and preserving micromethod of common drying-lyophilization*-is years. Pure culmany for viable state in a organisms and other cells rapidly that vacuum to a exposed and tures are frozen instantaneously removes the water. This method avoids the formation of ice crystals that would damage the cells. Although not all cells swvive this process, enough ofthem do to permit futrue reconstitution of that culture. As a general rule, chilling,freezing,and desiccation should not a

be construed as methods of disinfection or sterilization

Flgure

11.6

Dry heat incineration.

Infrared incinerator with shield to prevent spattering of microbial samples during flaming. cycle takes 12 minutes to 4 hours to complete and involves temperatures of 150'C to 180"C. The dry oven is used in laboratories and clinics for heat-resistant items that do not sterilize well with moist heat. Substances appropriate for dry ovens are glassware, metallic instruments, powders, and oils that steam does not penetrate well. This method is not suitable for plastics, cotton, and paper, which may burn at the high temperatures,

or for liquids, which will evaporate. Another limitation is the time required for it to work.

The Effects of Cold and Desiccation The principal benefit of cold treatment is to slow growth of cultures and microbes in food during processing and storage. It must be emphasized that cold merely retards the activities of most microbes. Although it is tnre that some microbes are killed by cold temperatures, most are not adversely affected by gradual cooling, long-term refrigeration, or deep-freezing. In fact, fteezingtemperatures, ranging from -70'C to - 135"C, provide an environment that can preserve cultures of bacteria, viruses, and fungi for long periods. Some psychrophiles grow very slowly even at freezing temperatures and can continue to secrete toxic products. Pathogens able to survive

several months in the refrigerator are Staphylococcus aureus; Clostridium species (sporeformers); Streptococczs species; and several types of yeasts, molds, and viruses. Outbreaks of Sqlmonella food infection traced backed to refrigerated foods such as ice cream and eggs are testimony to the inability of cold temperatures to reliably kill pathogens. Vegetative cells directly exposed to normal room air gradually become dehydrate{ or desiccated.* Delicate pathogens such as Streptococcus pneumoniae, the spirochete of syphilis, and Neisseria gonorrhoeae can die after a few hours of air-drying, but many

a desiccate (des'-ih-kayt) To dry at normal environmental temperatues.

because

their antimicrobial effects are erratic and uncertain, and one cannot be sure that pathogens subjected to them have been killed.

Radiation as a Microbial Control Agent Another way in which energy can serve as an antimicrobial agent is through the use of radiation. Radiation is defined as energy emitted from atomic activities and dispersed at high velocity through matter or space. Although radiation exists in many states and can be described and characterized in various ways, we will consider only those types suitable for microbial control: gamma rays, X rays, and ultraviolet radiation.

Modes of Action of lonizing versus Nonionizing Rodiotion The actual physical effects ofradiation on microbes can be understood by visualizing the process of irradiation, or bombardment with radiation, at the cellular level (figure 11.7). When a cell is bombarded by certain waves or particles, its molecules absorb some of the available energy, leading to one of two consequences: (l) If the radiation ejects orbital electrons from an atom, it causes ions to form; this type of radiation is termed ionizing radiation. One of the most sensitive targets for ionizing radiation is DNA, which will undergo mutations on a broad scale. Secondary lethal effects appear to be chemical changes in organelles and the produc-

tion of toxic substances. Gamma rays, X rays, and high-speed electrons are all ionizing in their effects. (2) Nonionizing radiation' best exemplified by U! excites atoms by raising them to a higher energy state, but it does not ionize them. This atomic excitation, in turn, leads to the formation of abnormal bonds within molecules such as DNA and is thus a source of mutations.

lonizing Rodiation: Gomma ond Cathode Roys

Roys,

X Roys,

Over the past several years, ionizing radiation has become safer and more economical to use, and its applications have mushroomed. It

+ lt;ophilization (ly-off'-il-ih-za'-shun) Gr. /yelz, to dissolve, andphilein,tolove

328

Chapter

11

Physical and Chemical Agents for Microbial Control

lonizing Radiation

machines and irradiated for a short time with a carefully chosen dosage. The dosage of radiation is measured in grays (which has replaced the older term, rads). Depending on the application, exposure ranges from 5 to 50 kilograys (kGy; a kilogray is equal to 1,000 grays). Although all ionizing radiations can penetrate liquids and most solid materials, gamma rays are most penetrating, X rays are intermediate, and cathode rays least penetrating.

Applications of lonizing Rodiation Radiation source

Barrier

DNA (breakages)

(a)

Nonionlzing Radiation

Foods have been subject to irradiation in limited circumstances for more than 50 years. From flour to pork and ground beef to fruits and vegetables, radiation is used to kill not only bacterial pathogens but also insects and worms and even to inhibit the sprouting of white potatoes. As soon as radiation is involved, however, consumer concern arises that food may be made less nutritious, unpalatable, or even unsafe by subjecting it to ionizing radiation. But irradiated food has been extensively studied, and each ofthese concerns has been addressed.

Irradiation may lead to a small decrease in the amount of thia-

mine (vitamin B,) in food" but this change is small enough to be inconsequential. The irradiation process does produce shortlived free radical oxidants, which disappear almost immediately (this same type of chemical intermediate is produced through cooking as DNA (abnormal bonds)

UV does not penetrate No effect on cell

well). Certain foods do not irradiate well and are not good candidates for this type of antimicrobial control. The white of eggs becomes milky and alfalfa seeds do not germinate properly. It should be emphasized that food is not made radioactive by the irradiation process, and many studies, in both animals and humans, have concluded that there are no ill effects from eating irradiated food. In fact, NASA relies on irradiated meat for its astronauts. One of the potential benefits of irradiated foods has to do with

Radiation source

infection conkol. It has been estimated that irradiation of 50% of the meat and poultry in the United States would result in 900,000 fewer cases of infection, 8,500 fewer hospitalizations, and 350 fewer deaths each year. Radiation is currently approved in the United States for the reduction of bacterial pathogens such as E. coli and Salmonella inbeef and chicken, reduction of Trichinella worms in pork, and the reduction of insects on fruits and vegetables. An additional benefit of irradiation is that microbes responsible for food spoilage are killed along with pathogens, leading to an increased shelflife. In any event, no irradiated food can be sold to consumers without clear labeling that this method has been used (figure 11.8). See chapter 27 for further discussion about irradiation of food. Sterilizing medical products with ionizing radiation is a rap-

Barrier

(c)

Flgure 11.7

Cellular effects of irradiation.

(a) lonizing radiation can penetrate a solid barrier, bombard a cell, enter it, and dislodge electrons from molecules. Breakage of DNA creates massive mutations. (b) Nonionizing radiation enters a cell, strikes molecules, and excites them. The effect on DNA is mutation by formation of abnormal bonds. (c) A solid barrier cannot be

penetrated by nonionizing radiation.

is a highly effective alternative for sterilizing materials that are sensitive to heat or chemicals. Because it sterilizes in the absence

of

heat,^irradiation is a type of cold (or low-temperature) sterilization.'Devices that emit ionizing rays include gamrna-ray machines containing radioactive cobalt, X-ray machines similar to those used in medical diagnosis, and cathode-ray machines that operate like the vacuum tube in a television set. Items are placed in these

idly expanding field. Drugs, vaccines, medical instruments

(espe-

cially plastics), syringes, surgical gloves, tissues such as bone and skin, and heart valves for grafting all lend themselves to this mode of sterilization. Its main advantages include speed, high penetrating power (it can sterilize materials through outer packages and wrappings), and the absence ofheat. Its main disadvantages are potential dangers from factory exposure to radiation and possible damage to some materials.

2. This

is possibly a confusing use ofthe word "cold." In this context, it only means the absence ofheat. Beer manufacturers have sometimes used this terminology as

well. When they say that their product is "cold-filtered,,' they mean that it has been freed ofcontaminants via filtration, i.e., in the absence ofheat.

Nonionizing Rodiation : Ultroviolet Roys Ultraviolet (UV) radiation ranges in wavelength from approximately 100 nm to 400 nm. It is most lethal from 240 nmto 280 nm

'11.2 Methods of

Thymine dimer

PhYsical Control

329

lru

Y

EI Details of bonding

Figure

11.8

Sterilization with ionizing radiation.

Many foods can be effectively sterilized by utilizing the penetrating power of ionizing radiation. The symbols at the upper corners of the photo signify that the food has been subiect to irradiation and are required to be displayed on all food so treated.

(with

a peak at 260

nm). In everyday practice, the source of UV ra-

diation is the germicidal lamp, which generates radiation at254 nm. Owing to its lower energy state, UV radiation is not as penetating as

ionizing radiation. Because W radiation passes readily through air, slightly through liquids, and only poorly through solids, the object to be disinfected must be directly exposed to it for full effect. As UV radiation passes through a cell, it is initially absorbed Specific molecular damage occurs on the pyrimidine DNA. by (thymine and cytosine), which form abnormal linkages with bases each other called pyrimidine dimers (figure 11.9). These bonds occur between adjacent bases on the same DNA strand and interfere with normal DNA replication and transcription. The results are inhibition of growth and cellular death. In addition to altering DNA directly, UV radiation also disrupts cells by generating toxic photochemical products called free radicals. These highly reactive mole-

lt.9 Formation of pyrimidine dimers by the action of ultraviolet (UV) radiation. Flgure

This shows what occurs when two adiacent thymine bases on one strand of DNA are induced by UV rays to bond laterally with each

other. The result is a thymine dimer shown in greater detail. Dimers can also occur between adiacent cytosines and thymine and cytosine bases. lf they are not repaired, dimers can prevent that segment of DNA from being correctly replicated or transcribed. Massive dimerization is lethal to cells.

and plasma from contaminants. The surfaces of solid' nonporous materials such as walls and floors, as well as meat' nuts, tissues for grafting, and drugs, have been successfirlly disinfected with UV One major disadvantage of UV is its poor powers of penetration through solid materials such as glass, metal, cloth, plastic, and even paper. Another drawback to UV is the damaging effect of overexposure on human tissues, including sunburn, retinal damage, cancer, and skin wrinkling.

cules interfere with essential cell processes by binding to DNA, RNA, and proteins. Ultraviolet rays are a powerful tool for destroying fungal cells and spores, bacterial vegetative cells, protozoa, and viruses. Bacterial spores are about 10 times more resistant to radiation than are vegetative cells, but they can be killed by increasing the time of exposure.

Applications of UV Radiation Ultraviolet radiation is usually directed at disinfection rather than sterilization. Germicidal lamps can cut down on the concentration of airborne microbes as much as 99%.They are used in hospital rooms, operating rooms' schools, food preparation areas, and dental offices. Ultraviolet disinfection ofair has proved effective in reducing postoperative infections, preventing the transmission of infections by resPiratory droplets, and curtailing the growth of microbes in food-processing plants and slaughterhouses. Ultraviolet irradiation of liquids requires special equipment to spread the liquid into a thin, flowing film that is exposed directly to a lamp. This method can be used to treat drinking water (figure 11.10) and to purify other liquids (milk and fruit juices) as an alternative to heat. Ultraviolet treatment has proved effective in freeing vaccines

Flgure lt.lO An ultraviolet (UV) treatment system for disinfection of water. Water flows through tunnels at a water treatment plant, past racks of UV lamps. This system has a capacity of several million gallons per day and can be used as an alternative to chlorination. Home systems that fit under the sink are also available.

330

Chapter

ll

Physical and ChemicalAgents for Microbial Control

Sterilization by Filtration: Techniques for Removing Microbes Filhation is an effective method to remove microbes from air and liquids. In practice, a fluid is strained through a filter with openings large enough for the fluid to pass through but too small for microorganisms to pass through (figure 11.11).

Most modern microbiological filters are thin membranes of cellulose acetate, polycarbonate, and a variety of plastic materials (Teflon, nylon) whose pore size can be carefully controlled and standardized. Ordinary substances such as charcoal, diatomaceous earth, or unglazed porcelain are also used in some applications. Viewed microscopically, most filters are perforated by very precise, uniform pores (figure 11.110). The pore diameters vary from coarse (8 trm) to ultrafine (0.02 pm), permitting selecrion ofthe minimum particle size to be trapped. Those with even smaller pore diameters permit true sterilization by removing viruses, and some will even remove large proteins. A sterile liquid filtrate is typically produced by suctioning the liquid through a sterile filter into a presterilized container. These filters are also used to separate mixtures of microorganisms and to enumerate bacteria in water analvsis.

Ap p I icotion s of F i ltratio

n

can cause severe reactions in the body. It has the disadvantage ofnot removing soluble molecules (toxins) that can cause disease. Filtration is also an efficient means of removing airborne contaminants that are a cofirmon source of infection and spoilage.

High-efficiency particulate air (HEPA) filters are widely used to provide a flow of sterile air to hospital rooms and sterile rooms.

r r r r r

SteriI izati on

Filtration sterilization is used to prepare liquids that cannot withstand heat, including senrm and other blood products, vaccines, drugs, IV fluids, en4{mes, and media. Filtration has been employed as an alternative method for sterilizing milk and beer without altering their flavor. It is also an important step in water purification. Its use extends to filtering out particulate impurities (crystals, fibers, and so on) that

r I r r r r

Physical methods ofmicrobial control include heat, cold, radiation,

drying, and filtration. Heat is the most widely used method of microbial control. It is used in combination with water (moist heat) or as dry heat (oven, flames). The thermal death time (TDT) is the shortest length oftime required to kill all microbes at a specific temperature. The TDT is longest for spore-forming bacteria and certain viruses. The thermal death point (TDP) is the lowest temperature at which all microbes are killed in a specified length of time (10 minutes). Autoclaving, or steam sterilization, is the process by which steam is heated under pressure to sterilize a wide range of materials in a comparatively short time (minutes to hours). It is effective for most materials exceptwater-resistant substances such as oils, waxes, and powders.

Boiling water and pasteurization ofbeverages disinfect but do not sterilZe materials. Dry heat is microbicidal under specified times and temperatures. Flame heat, or incineration, is microbicidal. It is used when total destruction of microbes and materials is desired.

Chilling, freezing, and desiccation are microbistatic but not microbicidal. They are not considered true methods ofdisinfection because they are not consistent in their effectivenessIonizing radiation (cold sterilization) by garnma rays and X rays is used to sterilize medical products, meatso and spices. It damages DNA and cell organelles by producing disruptive ions. Ulffaviolet light, or nonionizing radiation, has limited peneuating ability. It is therefore restricted to disinfecting air and certain liquids.

Sterilization by filtration removes microbes from heat-sensitive liquids and circulating air. The pore size of the filter determines what kinds of microbes are removed.

11.3 Chemical Agents in Microbial Control

Vacuum

(a)

Flgure 11.11 Membrane filtration. (a) Vacuum assembly for achieving filtration of liquids through suction. lnset shows filter as seen in cross section, with tiny passageways (pores) too small for the microbial cells to enter but large enough for liquid to pass through. (b) Scanning electron micrograph of filter, showing relative size of pores and bacteria trapped on its surface (5,9OOx).

Chemical control of microbes probably emerged as a serious science in the early 1800s, when physicians used chloride of lime and iodine solutions to treat wounds and to wash their hands before surgery. At the present time, approximately 10,000 different antimicrobial chemical agents are manufactured; probably 1,000 of them are used routinely in the health care af,ena and the home. A genuine need exists to avoid infection and spoilage, but the abundance of

products available to o'clean

"kill

germs," "disinfect," ..antisepticize,,,

and sanitize," "deodorize," "fight plaque," and ..puriSr the air" indicates a preoccupation with eliminating microbes from the environment that, at times, seems excessive (Insight ll.2). Antimicrobial chemicals occur in the liquid, gaseous, or even solid state. They serve as disinfectants, antiseptics, sterilants

11.3

Chemical Agents in Microbial Control

331

Pathogen Paranoia: "The Only Good Microbe ls a Dead Microbe" The sensational publicity over outbreaks ofinfluenza, SARS, and staphylococcal skin infections has had a dramatic impact on the public view of microorganisms. On the positive side, this glut of information has im-

proved people's awareness of the importance of microorganisms' And" certainly, such knowledge can be seen as beneficial when it leads to wellreasoned and sensible choices, such as using greater care in hand washing' food handling, and personal hygiene. But sometimes a little knowledge can be dangerous. The trend also seems to have escalated into an obsessive fear of"germs" lurking around every corner and a fixation on eliminating microbes from the environment and the human body.

As might be expected, commercial industries have found a way to capitalize on those fears. Every year, a number ofnew products are released that incorporate antibacterial or germicidal "protection." A widespread array of cleansers and commonplace materials have already had antimicrobial chemicals added. First it was hand soaps and dishwashing detergents, and

eventually the list grew to include shampoos, lat[rdry aids, hand lotions, foot pads for shoes, deodorants, sponges and scrub pads' kitty litter, acne medication, cutting boards, garbage bags, toys, and toothpaste' One chemical agent routinely added to these products is a phenolic called triclosan (Irgasan). This substance is fairly mild and nontoxic and does indeed kill most pathogenic bacteria. However, it does not reliably destroy viruses or fungi and has been linked to cases ofskin rashes due to hypersensitivity. One unfortunate result of the negative news on microbes is how it fosters the feeling that all microbes are harmful. we must not forget that most human beings manage to remain healthy despite the fact that they live in continual intimate contact with microorganisms. We really do not have to be preoccupied with microbes every minute or feel overly concerned that the things we touch, drink, or eat are sterile, as long as they are somewhat clean and free of pathogens. For most of us, resistance to infection is well maintained by our numerous host defenses' Medical experts are concerned that the widespread overuse of these antibacterial chemicals could favor the survival and growth ofresistant

(chemicals that sterilize), degermers, or preservatives (chemicals that inhibit the deterioration ofsubstances). In most cases, solid or gaseous antimicrobial chemicals are dissolved in water, alcohol, or a mixture of the two to produce a liquid solution. Solutions containing pure water as the solvent are termed aqueousr whereas

those dissolved in pure alcohol or water-alcohol mixtures are termed tinctures.

Choosing a Microbicidal Chemical The choice and appropriate use of antimicrobial chemical agents are of constant concern in medicine and dentistry' Although actual clinical practices of chemical decontamination vary widely, some desirable qualities in a germicide have been identified' including:

r o

r

rapid action in low concentrations, solubility in water or alcohol and long-term stability, broad-spectrum microbicidal action without being toxic to human and animal tissues"

The molecular structure of triclosan, also known as lrgasan and Ster-Zac, a phenol-based chemical that destroys bacteria by disrupting cell walls and membranes.

strains ofbacteria. A study reported in 2000 that many pathogens such as Mycobacterium tuberculosis and Pseudomonas afe naturally resistant to triclosan, and that E coli and Staphylococcus aureushave already demonstrated decreased sensitivity to it. The widespread use of this chemical may actually select for "super microbes" that survive ordinary disinfection' Another outcome of overuse of environmental germicides is to reduce the natural contact with microbes that is required to maintain the normal resident flora and stimulate immunities. Constant use of these agents could shift the balance in the normal flora of the body by killing off harmless or beneficial microbes. And there's one more thing' More and more studies are showing that when some bacteria become resistant to antibacterial agents, including triclosan, they simultaneously become resistant to antibiotics, such as tetracycline and erythromycin' Infectious disease specialists urge a happy medium approach' Instead of filling the home with questionable germicidal products, they encourage cleaning with traditional soaps and detergents, reserving more

potent products to reduce the spread ofinfection in clinical settings'

Can you name several side effects of rnass usage of antimicrobial chemicals in addition to those mentioned here? Answer available at http : //www. mhhe. com/talaroT

o

penetration of inanimate surfaces to sustain a cumulative or persistent action,

o resistance to becoming inactivated by organic o noncorrosive or nonstaining properties,

r

matter,

sanitizing and deodorizing properties, and and ready availability.

o affordability

As yet, no chemical can completely fulfill all of those requirements, but glutaraldehyde and hydrogen peroxide approach this ideal. At the same time, we should question the rather overinflated claims made about certain commercial agents such as mouthwashes and disinfectant air sPraYs. Germicides are evaluated in terms of their effectiveness in destroying microbes in medical and dental settings. The three levels of chemical decontamination procedures are high, intermediate, and low (table 11.6). HighJevel germicides kill endospores and, ifproperly used, are sterilants. Materials that necessitate high-level control are medical devices-for example, catheters, heart-lung equipment, and implants-that are not heat-sterilizable and are intended to enter

332

Chapter

l1

Physical and Chemical Agents for Microbial Control

Used in Health Care Agent

Target Microbes

Level of Activity

Toxicity

Chlorine

Sporicidal (slowly)

Intermediate

Gas is

Comments

highly toxic;

solution irritates skin

Inactivated by organics ; unstable in sunlight

Iodine

Sporicidal (slowly)

Intermediate

Can irritate tissue; toxic if ingested

Iodophors* are milder forms

Phenolics

Some bacteria. viruses. fungi

Low to intermediate

Can be absorbed by skin; can cause CNS damage

Poor solubility; expensive

Chlorhexidine*

Most bacteria, some vlruses, nrngl

Low to intermediate

Low toxicitv

Fast-acting,

Alcohols

Most bacteria. viruses, fungi

Intermediate

Toxic if ingested; a mild irritant; dries skin

Hydrogen peroxide,* stabilized

Sponclcat

High

Toxic to eyes; toxic

Quaternaryammonium

Somebactericidal,

compounds

Soaps

Low

Weakly microbistatic

has

Flammable, fast-acting lmproved stabiliry: works well in organic matter

Irritating to mucous

virucidal, fungicidal actlvlty

membranes; poisonous

iftaken internally

Weak solutions can support microbial growth;

easily inactivated

Very low

Nontoxic; few if any toxic effects

Used for removing soil. oils, debris

Low

Highly toxic if ingested,

Easily inactivated

species

Mercurials

if

ingested

Certain very sensitive

mil4

residual effects

inhaled, absorbed

Silver nitrate

Bactericidal

Low

Toxic, irritating

Glutaraldehyde*

Sporicidal

High

Can irritate skin; toxic absorbed

Formaldehyde

Sporicidal

Intermediate to high

Very irritating; fumes damaging, carcinogenic

Slow rate ofaction; limited applications

Ethylene oxide gas*

Sporicidal

High

Very dangerous to eyes, lungs; carcinogenic

Explosive in pure state; good penetration; materials must be aerated

Dyes

Weakly bactericidal,

Low

Low toxicity

Stains materials, skin

tungicidal

Discolors skin

if

Not inactivated by organic matter; unstable

*These chemicals approach the in organic matter. and solubility.

body tissues during medical procedures. Intermediatelevel germicides kill fungal (but not bacterial) spores, resistant pathogens such as the tubercle bacillus, and viruses. They are used to disinfect items (respiratory equipment, thermometers) that come into intimate contact with the mucous membranes but are noninvasive. Low levels

of

disinfection eliminate only vegetative bacteria, vegetative fungal cells, and some viruses. They are used to clean materials such as electrodes, straps, and fuiniture that touch the skin surfaces but not the mucous membranes.

Factors That Affect the Germicidal Activity of Chemicals Factors that control the effect of a germicide include the nature of the microorganisms being treated, the nature of the material being treated the degree of contamination, the time of exposure, and the strength and chemical action of the germicide (table ll.7). Stand_ ardized procedures for testing the effectiveness of germicides are summarized in appendix C. The modes of action of most sermicides

are to attack the cellular targets discussed earlier; proteins, nucleic acids, the cell wall, and the cell membrane.

A chemical's strength or concentration is expressed in various ways, depending upon convention and the method of preparation. The content of many chemical agents can be expressed by more than one notation. In dilutions, a small volume of the liquid chemical (solute) is diluted in a larger volume of solvent to achieve a certain ratio. For example, a common laboratory phenolic disinfectant such as Lysol is usually diluted l:200; that is, one part of chemical has been added to 200 parts of water by volume. Solutions such as chlorine that are effective in very diluted concentrations are expressed in parts per million (ppm). In percentage solutions, the solute is added to water by weight or volume to achieve a certain percentage in the solution. Alcohol, for instance, is used in percentages ranging from 50% to 95o/o. In general, solutions of low dilution or high percentage have more of the active chemical (are more concentrated) and tend to be more germicidal, but expense and potential toxicity can necessitate using the minimum strength that is effective.

11.3

Agent: Aqueous lodine

Concentration 2% 2% 2%

Staphylococcus aureus

Escherichia coli Enteric viruses

333

Germicidal Categories According

Required Concentrations and Times for Chemical Destruction of Selected Microbes

Agent/Organism

Chemical Agents in Microbial Control

to Chemical GrouP

Time

2min min

1.5

l0min

Several general groups of chemical compounds are widely used for antimicrobial purposes in medicine and commerce (see table I 1'6)' Prominent agents include halogens, phenolic compounds, alcohols, oxidizers, aldehydes, gases, detergents, and heavy metals. These groups are surveyed in the following section from the standpoint of each agent's specific forms and modes of action. Applications and methods of delivery are described in tables.

Agent Chlorine My c ob a c t er ium tub ercul o s t s Ent amo eb a cysts (Protozoa)

Hepatitis A vitus

50 ppm 0.1 ppm ? nnm 'rr-"

50 sec

min 30 min

150

Agent: Phenol dil l:75 dil 1:85

Staphylococcus aureus

Escherichia coli

10 10

min min

Agent Chlorhexidine Staphylococcus aureus

l: l0 dil

15 sec

Escherichia coli

1:10

dil

30 sec

Agent: EthylAlcohol 10% 70% 70%

Staphylococcus aureus

Escherichia coli Poliovirus

10

min

2 min 10

min

The Hologen Antimicrobial Chemicols The halogens* are fluorine, bromine, chlorine, and iodine, a group of nonmetallic elements with similar chemical properties. Although they can exist in either the ionic (halide) or nonionic state, most halogens exert their antimicrobial effect primarily in the nonionic state, not the halide state (chloride, iodide, for example). Because fluorine and bromine are difficult and dangerous to handle and no more effective than chlorine and iodine, only the latter two are used routinely in germicidal preparations. These elements are highly effective components of disinfectants and antiseptics because they are microbicidal and not just microbistatic, and they are sporicidal with longer exposure. For these reasons, halogens are the active ingredients in nearly one-third of all antimicrobial chemicals cur-

rently marketed.

Agent: Hydrogen Peroxide 12.5 sec

Staphylococcus aureus

3o/o

Neisseria gonorrhoeae Herpes simplex virus

3%

0.3 sec

Chlorine and lts Compounds Chlorine has been used for

3o/o

12.8 sec

disinfection and antisepsis for approximately 200 years. The major forms used in microbial control are liquid and gaseous chlorine (Cl2), hypochlorites (OCl), and chloramines (NH2CI). In solution, these compounds combine with water and release hypochlorous acid (HOCI):

Agent: Quaternary Ammonium ComPound 450 ppm 300 ppm

Staphylococcus aureus

Salmonella typhi

10 10

min min

Cl2 + H2O

Agent: Silver lons

i"

pglml

48h

2mglnl

48h

14mg/mI

48h

8

Staphylococcus aureus LSCnencnla cofi ui r rns (yeast) V', ii

HCIO

"t

Agent Glutaraldehyde Staphylococcus aureus Mycobacte riu m tu be rcu

2o/o I os i s

Herpes simplex virus

Agent Ethylene Oxide Strep

Ca(OCl)2 + 2H2O

t o c o c cus

fae c al is

Influenza virus

2% 2%

(l

min Dihydrofolic -----------> Tetrahydrofolic i------+ Pyrimidines

acid

{-

acid

bacteria

Only certain perform this step

""ioli6ii"

*iol

\

or,no

".,0,

A

Structural differences

N

N H- -H

Ht -H

Sulfanilamide

PABA

(b) Left: Formula of a sulfanilamide molecule compared alongside one of PABA. Note tha! despite a similarity in overall shape, sulfa drugs cannot be used to make folic acid. Right: The sulfa drug molecules can insert into the active site in the enzyme that would ordinarily fit the pABA. This will work only when the sulfa molecules are more prevalent and can outcompete with the PABA and keep the enzyme sites filled. Since little or no PABA can bind, the synthesis of folic acid will be inhibited.

Figure 12.6 Competitive inhibition

as a mode

of action.

This example shows how sulfa drugs block a metabolic pathway bacteria use to synthesize folic acid.

12.3 Survey of Major Antimicrobial Drug Groups Scores of antimicrobial drugs are marketed in the United States. Although the medical and pharmaceutical literature contains a wide anay of names for antimicrobials, most of them are variants of a

small number of drug families. About 260 different antimicrobial drugs are currently classified in around 20 drug families. Drug reference books may give the impression that there are 10 times that many because various drug companies assign different trade names to the very same generic drug. Ampicillin, for instance, is available under 50 different names. Most antibiotics are useful in controlling bacterial infections, although we shall also consider a number of antifungal, antiviral, and antiprotozoan drugs. Summaries of drug choices are located in later chapters on disease.

Antibacterial Drugs That Act on the Cell Wall The betaJactam (bey'-tuh-lak'-tam) group of antibiotics all contain a 3-carbon, l-nitrogen ring that is highly reactive (figure 12.7).

Its primary mode of action is to interfere with proteins involved in synthesis of the cell wall, leading to lysis and cell death. More than half of al1 antimicrobic drugs are beta-lactams, with the penicillins and cephalosporins being the most prominent representatives.

Penicillin ond lts Relqtives The penicillin group of antibiotics, named for the parent compound is a large, diverse group of compounds, most of which end in the suffix -cillin. Although penicillins could be completely synthesized in the laboratory from simple raw materials, it is more practical and economical to obtain natural penicillin through microbial fermentation. The natural product can then be used either in unmodified form or to make semisynthetic derivatives. Penicillium chrysogenum is the major source of the drug. All penicillins consist ofthree parts: a beta-lactam ring, athiazolidine ring, and a variable side chain that dictates its microbicidal activity (figure 12.7 and

Insight 12.2).

Subgroups and Uses of Penicillins The characteristics of certain penicillin drugs are shown in table 12.5. Penicillin G was

Chapter

356

12

Drugs, Microbes, Host-The Elements of Chemotherapy

the first antibiotic and is the parent compound for all ..-cillin', drugs. It is narrow spectrum and considered the drug of choice for infections by sensitive gram-positive bacteria (streptococci) and some gram-negative bacteria (meningococci and the syphilis spirochete). A close relative, penicillin ! has similar uses but has been modified to be stable in the stomach acid and so can be

Nucleus

(Aminopenicillanic acid) R Group

Beta-

@ @

Thiazolidine

taken orally.

\reg

Semisynthetic penicillins such as ampicillin, carbenicillin, and amoxicillin have been chemically altered with side chains that help

Nafcillin

them move across the outer membrane of gram-negative cell walls. This increases their spectrum and makes them useful in treating many types of gram-negative infections.

,cH- co

Many bacteria produce enzymes that are capable of destroying the beta-lactam ring of penicillin. The enzymes are referred to as penicillinases or beta-lactamases, and they make the bacteria that possess them resistant to many penicillins. Penicillinaseresistant penicillins such as methicillin, nafcillin, and cloxacillin are useful in treating infections caused by some penicillinaseproducing bacteria. Mezlocillin and azlocillin have such an extended spectrum that they can be substituted for combinations of antibiotics. All of the "-cillin" drugs are relatively mild and well tolerated because of their specific mode of action on cell walls (which humans lack). The primary problems in therapy include allergy, which is altogether different than toxicity, and resistant strains of pathogens. Clal.ulanic acid is a chemical that inhibits beta-lactamase enzyrnes, thereby increasing the longevity of beta-lactam antibiotics in the presence of penicillinase-producing bacteria. For this reason, cla'uulanic acid is often added to semisynthetic penicillins to augment their effectiveness. For example, clavamox is a combination of amoxicillin and clavulanate and is marketed under the trade name

fn'loo"" \./

Ticarcillin

::-=:::,:: i

o Cloxacillin

cH-co I

COONa

Carbenicillin

Figure

12.7

Chemical structure of penicillins.

The basic nucleus of penicillin drugs (blue box) consists of a thiazolidine ring (yellow) and a beta-lactam ring (red), but specific types differ in the nature of the side chain (R group), which is also responsible for differences in each compound's activity against gramneoative cells.

.

Augmentin. Zosyn, a similar combination of the beta-lactamase inhibitor tazobactum and piperacillin, is used for a wide variety of systemic infections.

Characteristics of Selected Penicillin Drugs

Name

Spectrum of

Penicillin G

Action

Uses, Advantages

Disadvantages

Narrow

Best drug ofchoice when bacteria are sensitive; low cost; low toxicity

Can be hydrolyzed by penicillinase; allergies occur: requires injection

Penicillin V

Narrow

Good absorption from intestine; otherwise. similar to penicillin G

Hvdrolvsis bv oenicillinase: allergies

Oxacillin, dicloxacillin

Narrow

Not susceptible to penicillinase; good

Allergies; expensive

absorption

Methicillin, nafcillin

Narrow

Not usually susceptible to penicillinase

Poor absorption; allergies; growing resistance

Ampicillin

Broad

Works on gram-negative bacilli

Can be hydrolyzed by penicillinase;

allergies; only fair absorption

Amoxicillin

Broad

Gram-negative infections; good absorption

Carbenicillin

Broad

Same as

Azlocillin, mezlocillin, ticarcillin

Very broad

Effective against Pseudomo n as species; low toxicity compared with

ampicillin

aminoglycosides

Hydrolysis by penicillinase; allergies Poor absorption; used only parenterally

Allergies, susceptible to many beta-lactamases

12,3

Survey of Maior Antimicrobial Drug Groups

357

A Modern Quest for Designer Drugs Once the first significant drug was developed, the world immediately witnessed a scientific scramble to find more antibiotics' This search was advanced on several fronts. Hundreds ofinvestigators began the laborious task of screening samples from soil, dust, muddy lake sediments, rivers, estuaries, oceans, plant surfaces, compost heaps' sewage, skin, and even the hair and skin of animals for antibiotic-producing bacteria and fungi. This intense effort has paid offover the past 50 years, because more than 10,000 antibiotics have been discovered (although surprisingly, only a relatively small number have actually been used clinically). Finding a new antimicrobial substance is only a first step. The complete pathway ofdrug development from discovery to therapy takes between 12 and24

ofbillions ofdollars. Antibiotics are products of fermentation pathways in many microorganisms. Among the greatest producers of antibiotics are the fungi, members of the soil-dwelling bacterial genera Streptomyces and the spore-forming bacteria. The role of antibiotics in the lives of these mi-

years at a cost

o

R

Groups

cH2- co

Aminooenicillanic acid

(a)

locHs

\

r"o ocH3

(b)

Methicillin

crobes must be important because the genes for antibiotic production are

preserved in evolution. Some experts theorize that antibiotic-releasing microorganisms can inhibit or destroy nearby competitors or predators; others propose that antibiotics play a part in spore formation. Whatever benefit the microbes derive, these compounds have been extremely profitable for humans. Every year, the pharmaceutical industry farms vast quantities ofmicroorganisms and harvests their products to treat diseases caused by other microorganisms. Researchers have facilitated the work ofnature by selecting mutant species that yield more abundant or useful products, by varying the growth medium, or by altering the procedures

for large-scale industrial production' Another approach in the drug quest is to chemically manipulate molecules by adding or removing functional groups. Drugs produced in

W'ix; (c)

Ampicillin

O"-cH2-co (d)

Penicillin V

this way are designed to have advantages over other, related drugs' Using this semisynthetic metho4 a natural product of the microorganism is joined with various preselected functional groups. The antibiotic is reduced to its basic molecular framework (called the nucleus), and to this nucleus specially selected side chains (R groups) are added. A case in point is the metamorphosis of the semisynthetic penicillins. The nucleus is an inactive peniciilin derivative called aminopenicillanic acid" which has an opening on the number 6 carbon for addition of R groups. A particular carboxylic acid (R group) added to this nucleus can "fine-tune" the penicillin, giving it special characteristics. For instance, some R groups will make the product resistant to penicillinase

(methicillin), some confer a broader activity spectrum (ampicillin)' and others make the product acid resistant (penicillin V). Cephalosporins and tetracyclines also exist in several semisynthetic versions. The potential for using bioengineering techniques to design drugs seems almost limitless, and, indee{ several drugs have already been produced by manipulating the genes ofantibiotic producers. So far virtually all of the antimicrobials used in human infections have been derived from microorganisms or artificially synthesized in the laboratory. But many scientists are investigating antimicrobial substances derived from plants and animals (called "natural products"). Plantderived antimicrobials are by no means an original idea; indigenous cultures have been using plants as medicines for centuries. In the fifth century BC, Hippocrates mentioned 300 to 400 medicinal plants. But phy.to- (plant) chemicals have been overlooked by modern infectious

A plate with several discrete colonies of soil bacteria was sprayed with a culture of Escherichio coli and incubated. Zones of inhibition (clear areas with no growth) surrounding several colonies indicate species that oroduce antibiotics.

until recently. Now modern techniques have proven the antimicrobial efficacy of a wide variety of extracts from botanical sources, and the search for new ones has intensified' disease scientists

Explain why development of effective drugs takes so many years. Answer available at hup : //www.mhhe.com/talaro7

358

Chapter

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Drugs, Microbes, Host-The Elements of Chemotherapy

have improved dosing schedules and fewer

side effects. First-generation

cepha-

losporins (cephalothin and cefazolin) are most effective against gram-positive cocci and a few gram-negative bacteria. Secondgeneration forms (cefaclor and cefonacid) are more effective than the first-generation

'cHr-o< CHs Cefotiam (second generation)

I I I

,-@""*

Y

2nd I

Nr_N

,rf''. 'N'

forms in treating infections by gram-negative

'cHr-cHr-

I

N'

tcH,

I I

Y

3fd I

I

cHg

I

I

I

I

ff1"#!3n"'"tion1

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12.8

activity against enteric bacteria that produce beta-lactamases. Fourth-generation drugs, such as cefepime, have the widest range of antimicrobial properties. They are effective with both gram-negative and gram-positive bacterial infections and are rapidly microbicidal. Additional beta-lactams are the carbroad-spectrum carbapenem, is used for infections with aerobic and anaerobic pathogens. It is active in very low concentrations and can be taken by mouth with few side effects except for allergies. Aztreonam, monobactam, is a narrow-spectrum antibi-

a

*New improved versions of drugs are referred to as new,,generations.,'

Flgure

phalosporins, such as cephalexin (Keflex) and ceftriaxone (Rocephin), are broad spectrum with especially well-developed

bapenems and monobactams. Imipenem, a

I

v 4th

bacteria such as Enterobacten Proteus, and Haemophilus. Third-generation ce-

otic for treating pneumonia, septicemia, and

The structure of cephalosporins.

Like penicillin, their main nucleus consists of a beta-lactam ring (red) and a second ring (yellow). However, unlike penicillins, they have two sites for placement of R groups (at positions 3 and 7). This makes possible several generations of molecules with greater versatility in function and complexity in structure.

urinary tract infections by gram-negative aerobic bacilli. Aztreonam is especially useful when treating persons who are allergic to penicillin, because it is chemically

different and does not cross-react with antibodies made to penicillin.

The Cepholosporin Group of Drugs The cephalosporin antibiotics currently account for one-third of all antibiotics administered. Cephalosporins are similar to penicillins; they have a beta-lactam structure that can be synthetically altered (figure 12.8) and have a similar mode of action. The generic names ofthese compounds are often recognized by the presence of the root cef, ceph, or kef intheir names.

Subgroups and Uses of Cephalosporins Thecephalosporins are versatile. They are relatively broad spectrum, resistant to most penicillinases, and cause fewer allergic reactions than penicillins. Although some cephalosporins are given orally, many are poorly absorbed from the intestine and must be administered parenterallyr* by injection into a muscle or a vein. Four generations of cephalosporins exist, with each group being more effective against gram-negative organisms than the generation before it. In addition, succeeding generations typically

* parcnteralll, Qnr-ehn'-tur-ah-lee) Gn para, beyond, arld mteron, intestine. route ofdrug administration other than the gastrointestinal tract.

A

Miscellaneous Non-Beta-Lactam Cell Wall Inhibitors Vancomycin is a narrow-spectrum antibiotic most effective in treating staphylococcal infections in cases of penicillin and methicillin resistance or in patients with an allergy to penicillins. It has also been chosen to treat Clostridium infections in children and endocarditis (infection ofthe lining ofthe heart) caused by Enterococcus faecalis. Because it is very toxic and hard to administer, vancomycin is usually restricted to the most serious, lifethreatening conditions. Bacitracin is a narrow-spectrum peptide antibiotic produced by a strain of Bacillus subtilis.Its primary effect is to block the elongation of the peptidoglycan in gram-positive bacteria. Since it was first isolated, its greatest claim to fame has been as a major ingredient in a common drugstore antibiotic ointment (Neosporin) for combating superficial skin infections by streptococci and staphylococci. For this pu{pose, it is usually combined with neomycin (an aminoglycoside) and polymyxin. Isoniazid (IllH) works by interfering with the synthesis of mycolic acid, a necessary component of the cell wall of acid-fast organisms. It is used to treat infections with Mycobacterium tuberculosis but is effective only against growing cells. Although still in

12.3 has been largely supplanted by rifampicin. Oral doses are indicated for both active tuberculosis and prophylaxis in cases ofa positive TB test. Ethambutol, a closely related compound" is effective in treating the early stages oftuberculosis. use,

it

Survey of Maior Antimicrobial Drug Groups

i'l' oH o"Vg

i'f' HxAHn-[-nn. NH2-C-NHJ.'\-NH-C-

359

6-carbon ring

Antibiotics That Damage Bacterial Cell Membranes Bacillus polymyxa is the source

of the polymyxins,

narrow-

spectrum peptide antibiotics with a unique fatty acid component that contributes to their detergent activrty (see figure 12.4). Only two polymyxins-B and E (also known as colistinFhave any routine applications, and even these are limited by their toxicity to

the kidney. Either drug can be indicated to treat drug-resistant Pseudomonas aeruginosa and severe urinary tract infections caused by other gram-negative rods. It is also one of the topical agents in ointments to prevent skin infections.

Drugs That Act on DNA or RNA Much excitement was generated by a new class of synthetic drugs chemically related to quinine called fluoroquinolones. These drugs work by binding to DNA gyrase and a related enzpq topoisomerase IV-both of which are essential for replication of the bacterial DNA. While DNA gyrase tends to be the primary target in gramnegative organisms, topoisomerase is targeted in gram-positive cells. Although the reason that the drug targets different enzymes in different cell types is unknown, this mode of action ensures that fluoroquinolones provide broad-spectrum effectiveness. In addition to being broad spectrum and highly potent, the drugs are readily absorbed from the intestine. The principal quinolones, norfloxacin and ciprofloxacin, have been successful in therapy for urinary tract infections, sexually transmitted diseases, gastrointestinal infections, osteomyelitis, respiratory infections, and soft tissue infections. Newer drugs in this category are sparfloxacin and levofloxacin. These agents are especially recommended for pneumonia, bronchitis, and sinusitis' Now that concerns have arisen regarding the overuse ofquinolone drugs, the CDC is recommending careful monitoring of theiruse to prevent the emergence of ciprofloxacin-resistant bacteria (see Insight 12.4). Side effects that can limit the use of quinolones include seizures and other brain disturbances.

Another drug that is essential to tuberculosis therapy is rlfampin. The primary action of this drug is in blocking the action of RNA polymerase, thereby preventing transcription. It is somewhat limited in speckum because the molecule cannot pass through the cell envelope of many gram-negative bacilli. It is mainly used to treat infections by selected gram-positive rods and cocci and a few gram-negative bacteria. Rifampin figures most prominently in treating mycobacterial infections, especially tuberculosis and leprosy, but it is usually given in combination with other drugs to prevent development of resistance. fufampin is also recommended for prophylaxis in Neisseria meningitidis carriers and their contacts, and it is occasionally used to treat Legionella, Brucella, and Stap hy I o c o c cus infections.

Flgure

12.9

The structure of an aminoglycoside-

streptomycin. Colored portions of the molecule are found in all numbers of this drug class.

Drugs That Interfere with Protein Synthesis The Aminoglycoside Drugs Antibiotics composed of one or more amino sugars and an aminocyclitol (6-carbon) ring are referred to as aminoglycosides (figure 12.9). These complex compounds are exclusively the products of various species of soil actinomycetes in the genera Strep tomy c es and Micromonosp ora.

Subgroups and Uses of Aminoglycosides The aminoglycosides have a relatively broad antimicrobial spectrum because they inhibit protein synthesis by binding to one of the ribosomal subunits. They are especially useful in treating infections caused by aerobic gram-negative rods and certain gram-positive bacteria. Streptomycin is among the oldest of the drugs and has graduallybeen replaced by newer forms with less mammalian toxicity. It is still the antibiotic of choice for fieating bubonic plague and tularemia and is considered an effective antituberculosis agent. Gentamicin is less toxic and is widely administered for infections caused by gram-negative rods (Escherichia, Pseudomonas, Salmonella, and Shigella). Two relatively new aminoglycosides, tobramycin and amikacin, are also used for gram-negative infections, with tobramycin especially usefi.rl for treating Pseudomonas infections in cystic fibrosis patients.

Tetra cycl i n e Anti b i otics The first antibiotic in this class was aureomycin. It was used to synthesize terramycin, tetracycline, and several semisynthetic derivatives, commonly known as the tetracyclines (figure l2.l0a). Their action ofbinding to ribosomes and blocking protein synthesis accounts for the broad-spectrum effects in the group.

360

Chapter

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Drugs, Microbes, Host-The Elements of Chemotherapy ctr3,,cH3

one type of antibiotic that is no longer derived from the natural source but is entirely synthesized through chemical processes. Although this drug is fully as broad spectrum as the tetracyclines, it is so toxic to human cells that its uses are restricted. A small number of people undergoing long-term therapy with this drug incur irreversible damage to the bone marrow that usually results in a fatal form of aplastic anemia.z Its administration is now limited to typhoid fever, brain abscesses, and rickettsial and chlamydial infections for which an alternative therapy is not available. Chloramphenicol should never be given in large doses repeatedly over a long time period, and the patient's blood must be monitored during therapy.

N

o tl

c- cH- ct2 I

HNH Noz

tl

c-c- cH2-oH

Macrolides ond Reloted Antibiotics

tl

OHH (b) Chloramphenicol

Lactone ring

cH3-cH2

(c) Erythromycin

FIgure 12.1O Structures of three broad-spectrum antibiotics. (a) Tetracyclines. These are named for their regular group of four rings. The several types vary in structure and activity by substitution at the four R groups. (b) Chloramphenicol. (c) Erythromycin, an example of a macrolide drug. lts central feature is a large lactone ring to which two hexose sugars are attached.

Erythromycin is a representative of antibiotics termed macrolides.3 Its structure consists of a large lactone ring with sugars attached (figure l2.l0c), This drug is relatively broad spectrum and of fairly low toxicity. Its mode of action is to block protein synthesis by attaching to the 50S subunit of the ribosome. It is administered orally for Mycoplasma pneumorriq legionello sis, Chlamydia infections, pertussis, and diphtheria, and as a prophylactic drug prior to intestinal surgery. It also offers a useful substitute for dealing with penicillin-resistant streptococci and gonococci and for treating syphilis and acne. Newer semisynthetic macrolides include clarithromycin and azithromycin. Both drugs are useful for middle ear, respiratory and skin infections and have also been approved for Mycobacterium (MAC) infections in AIDS patients. Clarithromycin has additional applications in controlling infection and gastric ulcers caused by Helicobacter pylori. Clindamycin is a broad-spectrum antibiotic derived from lincomycin. The tendency of clindamycin to cause adverse reactions in the gastrointestinal tract limits its applications to (l) serious infections in the large intestine and abdomen due to anaerobic bacteia (B act eroide s and Cl o s tr idium), (2) infections with penicillinresistant staphylococci, and (3) acne medications applied to the skin.

Drugs That Block Metabolic Pathways Subgroups and Uses of Tetracyclines The scope of microorganisms inhibited by tetracyclines is very broad. It includes grampositive and gram-negative rods and cocci, aerobic and anaerobic bacteria, mycoplasmas, rickettsias, and spirochetes. Tetracycline compounds such as doxycycline and minocycline are administered orally to treat several sexually hansmitted diseases, Rocky Mountain spotted fever, Lyme disease, typhus, Mycoplasma pneumonia, cholera, leptospirosis, acne, and even some protozoan infections. Although generic tetracycline is low in cost and easy to administer, its use can be limited by its side effects. In addition to gastrointestinal disruption due to changes in the normal flora and staining of the teeth, ingestion during pregnancy can interfere with fetal bone development (see table 12.8).

Chloramphenicol Chloramphenicol is a potent

broad-spectrum antibiotic

with

a

unique nitrobenzene structure (figure l2.l0b).Its primary effect on cells is to block peptide bond formation and protein synthesis. It is

Many of the drugs used to interfere with metabolism are synthetic. These antimicrobials as a group do not originate from bacterial or fungal fermentations. Some were synthesized from aniline dyes, and others were originally isolated from plants. Although they have been largely replaced by antibiotics, several types remain essential to chemotherapy. The most important group of drugs in this class is the sulfonamides, or sulfa drugs. Their name is derived from sulfanilamide, an early form of the drug (figure l2.ll). These drugs function as metabolic analogs to block the synthesis of folic acid by bacteia (see figure 12.6). Dozens ofthese drugs have been developed, most of them narrow spectrum. Because of its solubility, sulfisoxazole is an effective choice for treating shigellosis, urinary tract infections, and certain protozoan infections. Silver sulfadiazine ointment and

2. A failue ofthe blood-producing tissue that results in very low levels ofblood cells. 3. Macrolide antibiotics get their name from the type of chemical ring structure (macrolide ring) they all possess.

'1,2.3 Survey of Maior Antimicrobial Drug Croups

R Group

Nucleus

*r,@so2-NH*

361

only when no other drugs are available to slow the development of drug resistance. Another new class of drugs, the ketolides, is similar to macrolides like erythromycin but has a different ring structure. The main representative, telithromycin (Ketek), is used for respiratory tract infections caused by macrolide resistant bacteria. The oxazolidinone class of drugs operates using a unique mechanism, namely to inhibit the initiation of protein synthesis. It does this by interfering with the interaction between the mRNA and the two ribosomal subunits necessary for the initiation of translation. The sole drug in this class, linezolid (sold under the trade name Zyvox), has been used to treat infections caused by two of the most difficult clinical pathogens, methicillin-re sistant Staphy lo co ccus aureus (MRSA) and vancomycin-resi slant Enterococcas (VRE). Because this drug is synthetic and not found in nature, it is hoped that drug resistance will be slow to develop.

Agents to Treat Fungal Infections (c)

Figure 12.17

The structures of some sulfonamides.

group of the nucleus. Boxed side chains attach at the -NH (a) Sulfacetamide, (b) sulfadiazine, and (c) sulfisoxazole.

solution are prescribed for treatment ofburns and eye infections. Another drug, trimethoprim, inhibits a second enzymatic step in the synthesis of folic acid. Because of this, trimethoprim is often given in combination with sulfamethoxazole to take advantage of the synergistic effect of the two drugs (Septra, Bactrim). This combination is one of the primary treatments for Pneumocystis (carinii) jiroveci pnevmonia (PCP) in AIDS patients, urinary tract infections, and otitis media. Sulfones are compounds chemically related to the sulfonamides but lacking their broad-spectnrm effects. They still remain important as key drugs in treating Hansen's disease (leprosy). The most active form is dapsone, usually given in combination with rifampin and clofazamine (an antibacterial dye) over long periods.

Because the cells of fungi are eukaryotic, they present special problems in chemotherapy. For one, the great majority of chemotherapeutic drugs are designed to act on bacteria and are generally ineffective in combating fungal infections. For another, the similarities between fungal and human cells often mean that drugs toxic to fungal cells are also capable of harming human tissues. A few agents with special antifungal properties have been developed for treating systemic and superficial fungal infections. Five main drug groups currently in use are the macrolide polyene antibiotics, griseofulvin, synthetic azoles, flucytosine, and echinocandins (figure 12.12). See table 22.4 for a complete outline of antifungal druss,

Newly Developed Closses of Antibiotics Although most new antibiotics are formulated from the drug classes that already exist, a few classes ofdrugs with novel actions against bacteria have recently been added to drug regimens. Fosfomycin trimethamine is a phosphoric acid agent effective as alternate treatment for urinary tract infections caused by enteric bacteria. It works by inhibiting an enzpe necessary for cell wall

W

synthesis.

Synercid is an antibiotic from the streptogramin group of drugs which contains two active chemicals. It is effective against Staphylococcus and Enterococcus species that cause endocarditis and surgical infections, and against resistant strains of Streptococcrzs. It is one of the main choices when other drugs are ineffective due to resistance. Synercid works by binding to sites on the 50S ribosome, inhibiting peptide transfer and elongation. Daptomycin is a lipopeptide directed mainly against grampositive bacteia, acting to disrupt multiple aspects of membrane function. Many experts are urging physicians to use these medications

(b)

w

Flgure 12.12

TH,

NAF H

^.) (c)

Some antifungal drug structures. (a) Polyenes. The example shown is amphotericin B, a complex steroidal antibiotic that inserts into fungal cell membranes. (b) Clotrimazole, an azole that inhibits synthesis of ergesterol, a component of the fungal cell membrane. (c) Flucytosine, a structural analog of cytosine that inhibits DNA and protein synthesis.

362

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Drugs, Microbes, Host-The Elements of chemotherapy

Polyenes bind to fungal membranes and cause loss of selective

permeability. They are specific for fungal membranes because fungal membranes contain a particular sterol component called ergosterol, while human membranes do not. The toxicity of polyenes is not completely selective, however, because mammalian cell membranes contain compounds similar to ergosterol that bind polyenes to a small extent. Macrolide polyenes, represented by amphotericin B (Fungizone) (named for its acidic and basic-amphoteric-properties) and nystatin (forNewYork State, where it was discovered), have a structure that mimics the lipids in some cell membranes. Amphotericin B is by far the most versatile and effective of all antifungals. Not only does it work on most fungal infections, including skin and mucous membrane lesions caused by Candida albicans, but it is one of the

few drugs that can be injected to treat systemic fungal infections such as histoplasmosis and cryptococcus meningitis. A significant drawback to the widespread use of amphotericin centers on its serious side effects, which can include fatigue and irregular heartbeat. Nystatin (Mycostatin) is used only topically or orally to treat candidiasis of the skin and mucous membranes, but it is not useful for subcutaneous or systemic fungal infections or for ringworm. Griseofulvin is an antifungal product especially active in certain dermatophyte infections such as athlete's foot. The drug is de-

posited in the epidermis, nails, and hair, where it inhibits fungal growth. Because complete eradication requires several months and griseofulvin is relatively nephrotoxic, this therapy is given only for the most stubborn cases. The azoles are broad-spectrum antifungal agents with a complex ringed structure that also act on fungal mernbrane structure. The most effective drugs are ketoconazole (Nizoral), fluconazole @iflucan), clotrimazole (Gyne-Lotrimin), miconazole (Monistat), and itraconazole (Sporanox). Ketoconazole is used orally and topically for cutaneous mycoses, vaginal and oral candidiasis, and some systemic mycoses. Fluconazole can be used in selected patients for AlDS-related mycoses such as aspergillosis and cryptococcus meningitis. Clotrimazole and miconazole are used mainly as topical oinfinents for infections in the skin, mouth, and vagina. Itraconazole is generally used orally for fungal infections ofthe nails and systemic candidiasis.

Flucytosine is an analog of the nucelotide cytosine that has antifungal properties. It is rapidly absorbed after oral therapy, and it is readily dissolved in the blood and cerebrospinal fluid. Alone, it can be used to treat certain cutaneous mycoses. Many fungi arc tesistant to flucytosine, so it is usually combined with amphotericin B to effectively treat systemic mycoses. After many years of testing, a new antifungal drug category has been introduced. Echinocandlzs, such as caspofungin, damage the cell wall of several types of fungi, thereby making them sensitive to lysis.

Antiparasitic Chemotherapy The enormous diversity among protozoan and helminth parasites and their corresponding therapies reaches far beyond the scope of this textbook; however, a few of the more common drugs are surveyed here and described again for particular diseases in chapter 23. Presently, a small number of approved and experimental drugs are used to treat malaria,leishmaniasis, trypanosomiasis, amebic dysentery, and helminth infections, but the need for new and better drugs has spurred considerable research in this area.

Antimoloriol Drugs: Quinine and tts Relotives Quinine, extracted from the bark of the cinchona tree, was the principal treatment for malaria for hundreds of years, but it has been replaced by the synthesized quinolines, mainly chloroquine and primaquine, which have less toxicity to humans. Because there are several species of Plasmodium (the malaria parasite) and many stages in its life cycle, no single drug is universally effective for every species and stage, and each drug is restricted in application. For instance, primaquine eliminates the liver phase of infection, and chloroquine suppresses acute attacks associated with infection ofred blood cells. Chloroquine is taken alone for prophylaxis and suppression of acute forms of malaria. Primiquine is administered to patients with relapsing cases of malaria. Mefloquine is a semisynthetic analog ofquinine used to treat infections caused by chloroquine-resistant strains of Plasmodium, an increasing problem in SoutheastAsia.

Chemotheropy for Other Protozoon lnfections A widely used amoebicide, metronidazole (Flagyl), is effective in treating mild and severe intestinal infections and hepatic disease caused by Entamoeba histolytica. Given orally, it also has applications for infections by Giardia lqmblia and Trichomonas vaginalis. Other drugs with antiprotozoan activities are quinicrine (a quininebased drug), sulfonamides, and tetracyclines.

Antihelminthic Drug Therapy Treating helminthic infections has been one of the most challenging of all chemotherapeutic tasks. Flukes, tapeworms, and roundwonns are much larger parasites than other microorganisms and, being animals, have greater similarities to human physiology. Also, the usual strategy of using drugs to block their reproduction is usually not successful in eradicating the adult woftns. The most effective drugs immobilize, disintegrate, or inhibit the metabolism of all stages of the life cycle. Mebendazole and thiabendazole are broad-spectrum antiparasitic drugs used to treat several types ofroundworm infections. These drugs work locally in the intestine to inhibit the function of the microtubules of worms, eggs, and larvae, which interferes with their glucose utilization and disables them. The compounds pyrantel andpiperazine paralyze the muscles of intestinal round-

worms. Niclosamide destroys the scolex and the adjoining proglottids of tapeworms, thereby loosening the worm's holdfast. In these forms of therapy, the worms are unable to maintain their grip on the intestinal wall and are expelled along with the feces by the normal peristaltic action of the bowel. A drawback to the use of the drug is the severe abdominal cramps that often accompany treatment. Two newer antihelminthic drugs are praziquantel, a treatment for various tapeworm and fluke infections, and ivermectin, a veterinary drug now used for strongyloidiasis and oncocercosis in humans. A

ntivi ra I

C h em

oth e ro peuti c Ag ents

The chemotherapeutic treatment of viral infections presents unique problems. Throughout our discussion of infections by bacteria, fungi, protozoa, and helminths, we've emphasized differences in

'12.3 Survey of Maior Antimicrobial Drug Croups structure and metabolism that guide the choice of drug. With viruses, we are dealing with an infectious agent that relies upon the host cell for the vast majority of its metabolic functions. Disrupting viral metabolism may require that we disrupt the metabolism of the host cell to a much greater extent than is desirable. Put another way, selective toxicity with regard to viral infection is almost impossible to achieve because a single metabolic system is responsible for the well-being of both virus and host. A few viral diseases such as measles, mumps, and hepatitis are routinely prevented by effective vaccinations. Unfortunately, vaccines are unavailable for many serious viral diseases, creating a real need for antiviral medications' Over the last few years, several antiviral drugs have been developed that target specific points in the infectious cycle of viruses' But due to marked differences among the viruses' most antiviral compounds are quite limited in their spectrum. Most antiviral drugs are designed to block a step in completion of the virus cycle. Major modes of action include (table 12'6)

1. barring penetration ofthe virus into the host cell; 2. blocking replication, transcription, and/ot translation of viral

3.

genetic material; and preventing the normal maturation of viral particles.

Although antiviral drugs protect uninfected cells by keeping viruses from being synthesized and released, most are unable to destroy extracellular viruses or those in a latent state.

Drugs for Treating Influenza Amantadine and its relative, rimantidine, are restricted almost exclusively to treating infections by influenzaA virus. They frrnction by blocking the hemagglutinin receptors. In this way, they prevent fusion of the virus with cell membranes and consequently interfere with entry, uncoating, and release of the virus. Relenza and Tamiflu are slightly broaderspectrum antiviral medications because they block neuraminidase en4/mes in both influenza A and influenza B. Both tlpes of drugs may be used prophylactically and must be given rather ear$ in an infection to be most effective (table 12.6)'

Antiherpes Drugs Many antiviral

agents mimic the structure of nucleotides (i.e., act as analogs) and compete for sites on replicating DNA. Once one of these nucleotide analogs is incorporated into the growing chain of DNA, replication of that strand stops, interrupting the viral life cycle. Acyclovir (Zovitax) and the related compounds valacyclovir (Valtrex), famiciclovir (Famvir), and peniciclovir (Denavir) all work in this manner and (with small exceptions from drug to drug) can be used either orally or topically to treat common herpesvirus infections such as oral and genital herpes, chickenpox, and shingles. A related drug, ganciclovir (Cytov-

ene) is used parenterally to treat cytomegalovirus infection in patients with compromised immune systems' An interesting aspect of some of these antiviral agents (specifically valacyclovir and famciclovir) is that they are activated by an enzyme encoded by the virus itself, activating the drug only in virally infected cells. The enzyme thymidine kinase is used by the

virus to process nucleosides before incorporating them into viral RNA or DNA. When the inactive drug enters a virally infected cell, the virus'own thymidine kinase converts it to a working antiviral agent. In cells without viruses, the drug is never activated and normal DNA replication is allowed to continue.

363

Drugs for Treating HIV Infection and AIDS The global epidemic ofAIDS has been a powerful stimulus for drug discovery' Most of the newer drugs being introduced are designed to work on HIV HIV is classified as a retrovirus, meaning it carries its genetic information in the form of RNA rather than DNA (HIV andAIDS are discussed in chapter 25). Upon infection, the RNA genome is used as a template by the enzyme reverse transcriptase to produce a DNA copy of the virus'genetic information. Because this particular reaction is not seen outside of the retroviruses, it offers two ideal targets for chemotherapy. The first is interfering with the synthesis of the new DNA strand which is accomplished using nucleoside reverse transcriptase inhibitors (nucleotide analogs), while the second involves interfering with the action of the enzyme responsible for the synthesis, which is accomplished using nonnucleoside reverse transcriptase inhibitors. Azidothymidine (AZT or zidovudine) was the first drug aimed at treating AIDS. It is a thymine analog that becomes incorporated into the growing DNA chain of HIV and terminates synthesis' in a manner analogous to that seen with acyclovir. AZT is used at all

of HIV infection, including prophylactically with people accidentally exposed to blood or other body fluids. Other apstages

proved anti-HIV drugs that act as nucleotide analogs are abacaviq lamivudine (3T3), didanosine (ddl), zalcitabine (ddc), and stavudine (d4T). Nonnucleoside reverse transcriptase inhibitors accomplish the same goal (preventing reverse transcription of the HIV genome) by binding to the reverse transcriptase enzyme itself, inhibiting its ability to synthesize DNA. Drugs with this mode of action include nevirapine, efavirenz, and delaviridine. Assembly and release of mature viral particles are also targeted in HIV through the use of protease inhibitors. These drugs (indinavir, saquinavir, nelfinavir, crixivan), usually used in combination with nucleotide analogs and reverse transcriptase inhibitors, have been shown to reduce the HIV load to undetectable levels by blocking the virus multiplication cycle at several points simultaneously. This form of combined therapy also decreases the problems of drug resistance. Refer to table 12.6 for a summary of HIV drug mechanisms and see chapter 25 for further coverage of this topic.

lnterferons A sensible alternative to artificial drugs has been a human-based substance, interferon (IFN). Interferon is a glycoprotein produced primarily by fibroblasts and leukocytes in response to various immune stimuli. It has numerous biological activities, including antiviral and anticancer properties. Studies have shown that it is a versatile part of animal host defenses, having a major role in natural immunities. Its mechanism is discussed in chapter 14. The first investigations of interferon's antiviral activity were limited by the extremely minute quantities that could be extracted from human blood. Several types of interferon (Betaseron, Roferon) are currently produced by the recombinant DNA technology techniques outlined in chapter 10. Extensive clinical trials have tested interferon's effectiveness in viral infections and cancer' Some of the known therapeutic benefits of interferon include reducing the time of healing and some of the complications in certain infections (mainly of herpesviruses); 2. preventing or reducing some symptoms of cold and papillomaviruses (warts); 1..

Actions of Selected Antiviral Drugs*

xDetails of viral cycles are omitted lbr easc in observing drug effects.

364

12.3

Survey of Maior Antimicrobial Drug Croups

365

New Perspectives in Antimicrobial Therapy Researchers are constant$ "pushing the envelope"

ofantimicrobial re-

search. often the quest focuses on finding new targets in the bacterial cell and custom-designing drugs that aim for them. Interesting new strategies that could lead to a marketable drug are to target iron-scavenging

cipabilities of bacteria and to target riboswitches, a genetic control mechanism in bacteria. Recent work with Staphylococcus aureus holds a key to understanding how iron-scavengers function. Scientists have found that this bacteririm has a special pathway involving several proteins that punch holes in red blood cells and The research team

..reach

in" to bind the iron for use in cell processes. is currently investigating inhibitory substances that

block the iron-collecting pathway, which would result in inevitable bacterial death. This approach may prove effective in wiping out antibioticresistant "super bugs" like S. aureus. Riboswitches are sections of bacterial RNA that are used to control translation of mRNA. By binding various targets in the bacterial cell. they prohibit translation processes. Because riboswitches appear to be ubiquitous in bacteria. a drug that controls their action could be an effective therapy. Other novel approaches to controlling infections include the use of probiotics* and prebiotics. Probiotics are preparations oflive microorganisms that are fed to animals and humans to

modiff the intestinal flora. These

microorganisms can replace microbes lost during antimicrobial therapy or simply augment the flora that is already there. Culture-enriched dairy products are the latest offerings in the market for probiotics. several new types ofyogurt now claim specific health benefits from eating high numbers of special strains ofbacteria. According to some published research findings, the ingestion of BiJidobacterium lactis regularls can help to maintain regu-

lar bowel function, arrd Lactobacillus casei immunitas may boost the immune function of the gut. Purposely eating such high numbers of bacterial cells-*-estimated at from 1 billion to 10 billion per serving-is quite a departure from the usual view ofmicrobes as disease agents' Bon appetit! A probiotic approach may also be used in female patients with recur-

,ing urina.y traciinfections. There is evidence that vaginal inserts or perineal swabs of Lactobacillus can restore a healthy acidic environment to the region and displace disease-causing microorganisms. Probiotics also seem to inhibit the development of food allergies' *

p ro b i o t i c

Prebiotics are nutrients that encouage the growth of beneficial microbes in the intestine. For instance, certain sugars such as fructans are thought to encourage the groMh ol Bifidobacterium inthe large intestine and to discourage the growth ofpotential pathogens. Clearly, the use of these agents is a different type ofantimicrobial strategy than we are used to, but it may have its place in a future in which traditional antibiotics are more problematic.

Another category of antimicrobial is lantibiotics-short peptides produced by bacteria that inhibit the growth ofother bacteria' They are distinguished by the presence ofunusual amino acids that are not seen els$ere in the cel1. They exert their antimicrobial activity either by ouncturinq cell membranes or by inhibiting bacterial enzlmes. The most well-knorin lantibiotic is nisin. Lantibiotics have long been used in food preservation. in veterinary medicine. and more recently in personal care products such as deodorants. Because lantibiotics are apparently effective against human pathogens such as Propionibacterium acnes. methiciltinresistant s. aureus, arrd Helicobacter pylorr, they may soon be an alternate choice for some types of infections. Describe several benefits of using nondrug regimens and foods to treat or prevent infections. Answer available at http://www.mhhe.com/ talaroT

(proh' -by-ah-tik' ) Gr. pro, fot, and Dios, life'

3. slowing the progress of certain cancers' including bone cancer and cervical cancer, and certain leukemias and lymphomas; and

4. treating a rare cancer called hairy-cell leukemia, hepatitis C (a viral liver infection), genital warts, and Kaposi's sarcoma in AIDS patients. Medical science is giving close scrutiny to alternate methods ofpreventing or treating infections beyond the usual antibiotics and similar forms of therapy (Insight 12.3).

Antimicrobials are classified into around 20 major drug families, based on their chemical composition, source or origin, and their site of action. Antimicrobial drugs are classified by their range ofeffectiveness'

Broad-spectrum antimicrobials are effective against many types of microbes. Narrow-spectrum antimicrobials are effective against a limited group of microbes.

366

i'

Chapter

12

Drugs, Microbes, Host-The Elements of Chemotherapy

The majority of antimicrobials are effective against bacteria, but a limited number are effective against protozoa, helminths, fungi, and viruses.

HaemoPhilus

vancomycin, and cycloser_ ines block cell wall synthesis, pririrarily in gram_positive bacteria.

\+

. Aminoglycosides and tetracyclines block protein synthesis in prokaryotes.

serratia

'.'l Sulfonamides, trimethoprim, isoniazid, nitrofi..rantoin, and the fluoro_ quinolones are synthetic antimicrobials effective against a broad range of microorganisms. They block steps in the synthesis of nucleic acids.

,rr Most antifungal drugs are selective for cell membranes, causing lysis of the fungus, but their action on membranes makes them po_

hel_

minths or inhibit their metabolism in some manner. ..i Antiviral drugs interfere with viral replication by blocking viral entry into cells, blocking the replication process, or preventing the assembly of viral subunits into complete virions. ;: Many antiviral agents are analogs of nucleotides. They inactivate the replication process when incorporated into viral nucleic acids. HIV antivirals interfere with reverse transcriptase or proteases to prevent the maturation of viral particles. .': Interferon is effective in vivo against certain viral infections; com_ mercial interferon is mainly restricted to certain cancers and heoa_ titis B and C.

12.4 Interactions between Microbes and Drugs: The Acquisition of Drug Resistance One unfortunate outcome of the use of antimicrobials is the develop_ ment of microbial drug resistance, an adaptive response in which microorganisms begin to tolerate an amount of drug that would ordinarily

+

lzProteus

/ t\

"""ro)r'ori"

aeruginosa

Other Aer6monas pseudomonads VI nnodospiritlium

Staphylococcus

aureus

tentially toxic.

,': Antihelminthic drugs immobilize or disintegrate infesting

cori

/l \

I I I

and flucytosine, are used for systemic and superficial infections.

eral life stages, some ofwhich can be resistant to the drugs.

/U""'""'"" \\

E.

,/l\/f\ur,, Bacittus{Bacteroides/l

:,! Fungal ant' icrobials, suchasmacrolidepolyenes, griseofulvin, azoles,

There are fewer antiparasitic drugs than antibacterial drugs because parasites are eukaryotes like their human hosts and they have sev_

Other

^*^\ T*il",,,,,\T"'," f"*:*:"

, Penicillins, cephalosporins, bacitracin,

':

Salmonella

Figure 12.13

Transfer of drug resistance.

This exchange diagram traces documented evidence of known cases in which R factors have been transferred among pathogens. Such promiscuous exchange of drug resistance occurs primarily by conjugation and transduction. Most of the bacteria are gram_negative (pink section) buI Bocillus and Stophylococcus are unrelated gram-positive genera (blue). This phenomenon is responsible for the rapid spread of drug-resistant microbes.

of genes via transfer from another species. Chromosomal drug resistance usually results from spontaneous random mutations in bacterial populations. The chance that such a mutation will be advantageous is minimal, and the chance that it will confer resistance to a specific drug is lower still. Nevertheless, given the huge num_ bers of microorganisms in any population and the constant rate of mutation, such mutations do occur. The end result varies from slight changes in microbial sensitivity, which can be overcome by larger doses of the drug, to complete loss of sensitivity. Resistance occurring through intermicrobial transfer originates

from plasmids called resistance (R) factors that are transferred through conj ugation, transformation, or transduction (figure 12.13).

ited, however, to a small group of organisms and is generally not a problem with regard to antimicrobial chemotherapy. Of much greater importance is the acquisition of resistance to a drug by a microbe that was previously sensitive to the drug. In our context, the term drug re_ sistance will refer to this last tlpe of acquired resistance.

Studies have shown that plasmids encoded with drug resistance are naturally present in microorganisms before they have been exposed to the drug. Such traits are "lying in wait" for an opporfunity to be expressed and to confer adaptability on the species. Many bacteria also maintain transposable drug-resistance sequences (transposons) that are duplicated and inserted from one plasmid to another or from a plasmid to the chromosome. Chromosomal genes and plasmids containing codes for drug resistance are faithfully replicated and inherited by all subsequent progeny. This sharing ofresistance genes accounts for the rapid proliferation of drug-resistant species (figure 12.13). A growing body of evidence points to the ease and frequency ofgene transfers in nature, from totally unrelated bacte_ ria living in the body's normal flora and the environment.

How Does Drug Resistance Develop?

Specific Mechanisms of Drug Resistance

Contrary to popular belief, antibiotic resistance is not a recent phenomenon. Resistance to penicillin developed in some bacteria as early

Inside a bacterial cell, the net effect of the two events described earlier is one of the following, which actually causes the bacterium to be resistant (note that the numbers that follow correspond to the numbers seen in figure 12.14):

be inhibitory. The development of mechanisms for circumventing or inactivating antimicrobial drugs is due largely to the genetic versatility and adaptability of microbial populations. The property of drug resistance can be intrinsic as well as acquired. Intrinsic drug resistance can best be exemplified by the fact that bacteria must, of course, be resistant to any antibiotic that they produce. This type of resistance is lim_

as 1940,3 years before the drug was even approved for public use. The scope ofthe problem became apparent in the 1980s and 1990s when scientists and physicians observed treatment failures on a large scale.

Microbes become newly resistant to a drug after one of the following events occurs: (l) spontaneous mutations in critical chromosomal genes, or (2) acquisition of entire new genes or sets

l. 21

3.

Inducement of alternate enzymes can inactivate the drug (only occurs when new genes are acquired). Permeability or uptake of drug into bacterium is decreased or eliminated (can occur via mutation or acquisition of new genes).

12.4 Interactions between Microbes and Drugs: The Acquisition of Drug

CHs CHg

(]J

Penicillinase

cooH

cooH

Resistance

367

1. Inactivation of a drug like penicillin by

penicillinase, an enzyme that cleaves a portion of the molecule and renders it inactive.

Inactive Penicillin

Active Penicillin 2. Decreased Permeability

2. The receptor that transports the drug is altered, so that the drug cannot enter the cell.

Normal

//teceplol

@ Cell

surface t

of microbe

3. Activation of drug Pumps 3. Specialized membrane proteins are activated and continually pump the drug out of the cell.

lnactive drug pump

@

+ Cell surlace

ol microbe

Gell surface

of microbe 4. Change In drug binding site

4. Binding site on target (ribosome) is altered so drug has no effect.

el e\ -),

@

*rI

5. Use of alternate metabolic pathway

acts

@

A---------------= g

TDrug f----)(-->

C

------->

\., Figure 12.14

-

D

aa"

Dr'

Product

5. The drug has blocked the usual metabolic pathway (green), so the microbe circumvents it bY using an alternate, unblocked Pathway that achieves the required outcome (red).

Examples of mechanisms of acquired drug resistance.

4.

Binding sites for drug are decreased in number or affinity

5.

(can occur via mutation or acquisition of new genes)' An affectedmetabolicpathway is shut dorvnoran altemate pathway is used (occurs due to mutation of original enryme(s)).

Some bacteria can become resistant indirectly by lapsing into dormancy or, in the case of penicillin, by converting to a cell-walldeficient form (L form) that penicillin cannot affect. D

>

rug I noctivation Mechonisms

Microbes inactivate drugs by producing enzymes that permanent$ alter drug structure. One example, bacterial exoenzymes called beta-lactamases, hydrolyze the beta-lactam ring structure of some penicillins and cephalosporins rendering the drugs inactive. Tvo betalactamases-penicillinase and cephalosporinase--{isrupt the structure of certain penicillin or cephalosporin molecules so their activity is lost (figure 12.14, step 1). So many strains of Staphylococcus aureus produce penicillinase that regular penicillin is rarely a possible therapeutic choice. Now that some strains of Neisseria gonorrhoeae,

called PPNG,4 have also acquired penicillinase, alternative drugs are required to treat gonorrhea. A large number of other gram-negative species are inherently resistant to some of the penicillins and cephalosporins because of natwally occurring beta-lactamases.

Decreosed Drug Permeability or lncreosed

Drug Elimination The resistance of some bacteria can be due to a mechanism that prevents the drug from entering the cell and acting on its target' For example, the outer membrane of the cell wall of certain gram-negative bacteria is a natural blockade for some of the penicillin drugs. Resistance to the tetracyclines can arise from plasmid-encoded proteins that pump the drug out of the cell. Aminoglycoside resistance is known to develop through changes in drug permeability caused by point mutations in proteins of the transport system or outer membrane (figure 12,14, step 2). 4. Penicillinase-prodrtcir'g Neisseria gonorrhoeae

368

chapter

12

Drugs, Microbes, Host-The Erements of Chemotherapy

Many bacteria possess multidrug-resistant (MDR) pumps that actively transport drugs and other chemicals out of cells. These pumps are proteins encoded by plasmids or chromosomes. They are stationid in the cell membrane and expel molecules by a proton-motive force

similar to AIP synthesis (figure l2.l4o step 3). They confer drug re_ sistance on many gram-positive pathogens (Staphylococcus, Stripto_

coccus) and gram-negative pathogens (pseudomonas,

E.

coli).

Because they lack selectiviry one type ofpump can expel a broad ar_ ray ofantimicrobial drugs, detergents, and other toxic substances.

The action of antimetabolites can be circumvented

if

a microbe

develops an alternative metabolic pathway or enzyme (figure 12.14, step 5). Sulfonamide and trimethoprim resistance develops when microbes deviate from the usual patterns of folic acid synthesis. Fungi can acquire resistance to flucytosine by completely shutting

off certain metabolic activities.

Natural Selection and Drug Resistance

Chonge of Drug Receptors Because most drugs act on a specific target such as protein, RNA, DNA, or membrane structue, microbes can circumvent drugs by al_

tering the nature of this target. Bacteria can become resistant to aminoglycosides when point mutations in ribosomal proteins arise

(figure 12.14, step 4). Erythromycin and clindamycin resistance is associated with an alteration on the 50s ribosomal binding site. peni-

cillin resistance in streptococcus pneumoniae

Chonges in Metobolic Potterns

and

methicillin resist-

ance in Staphylococcus aureus are related to an alteration in the binding proteins in the cell wall. Several species ofenterococci have acquired resistance to vancomycin through a similar alteration of cell wall proteins. Fungi can become resistant by decreasing their synthe_ sis of ergosterol, the principal receptor for certain antifungal drugs.

A NOTE ABOUT BIOFILMS AND DRUG RESISTANCE In chapters 4 and 7, we described the development of microbial biofilms in various habitats. Recall that in these complex com-

munities, certain "pioneer" microbes anchor themselves to a substrate by secreting a sticky matrix. More than being just an inert film, this initial colonization stimulates a network of other microbes to migrate into the film. In habitats such as soil nearlv all microbes are adapted to this sort of biofilm partnership. ft is estimated that around 600/o of infections also involve biofilms containing single or multiple species of microbes. Often the infections occur on natural tissues-for example, bacterial infections of the heart valves, middle ear, and teeth. The increased use of indwelling medical devices has created yet another habitat in which biofilms may become tenaciously attached. Catheters, artificial valves and pacemakers, endotracheal tubes, and prosthetic joints can harbor persistent biofilm colonizations that are very difficult to treat and remove. Microbes in infectious biofilms have shown a tendency to be hundreds of times more drug resistant than the same free, unattached microbes. This resistance is not developed by genetic mutations-rather, it is due to the character of the biofilm itself. For one thing, microbes are protected by the impenetrable nature of the extracellular matrix-drugs may be blocked from entry. lt is increasingly clear that microbes also communicate in the regulation of resistance mechanisms such as drug pumps. These factors alone or in combination allow the persistence of the biofilm and a long-term source of infection for the patient. With knowledge of biofilms, drug companies are researching newer drug strategies that can prevent or disrupt their formation. Another concept already in use is to incorporate

antimicrobic chemicals into implants, catheters, and other metal or plastic devices so they inhibit microbial growth.

So far, we have been considering drug resistance at the cellular and molecular levels, but its fu1l impact is felt only if this resistance occurs throughout the cell population. Let us examine how this might happen and its long-term therapeutic consequences. Any large population of microbes is likely to contain a few individual cells that are already drug resistant because of prior mutations or transfer of plasmids (figure l2.lla).As long as the drug is not present in the habitat, the numbers of these resistant forms will remain low because they have no particular growth advantage (and often are disadvantaged relative to their nonmutated counterparts). But ifthe population is subsequently exposed to this drug (figure l2.l5b), sensitive individuals are inhibited or destroyed" and resistant forms survive and proliferate. During

subsequent population growth, offspring of these resistant microbes will inherit this drug resistance. In time, the replacement population will have a preponderance of the drug-resistant forms

and can eventually become completely resistant (figure l2.l5c). In ecological and evolutionary terms, the environmental factor (in this case, the drug) has put selection pressure on the population, allowing the more "fit" microbe (the drug-resistant one) to sur_ vive and grow. The end result is a population that has evolved to a condition of drug resistance.

Natural selection for drug-resistant forms is apparently

a

common phenomenon. It takes place most frequently in various natural habitats, laboratories, and medical environments, and it also can occur within the bodies of humans and animals during drug therapy. See Insight 12.4 to find out some of the medical and social implications of drug resistance. Table 12.7 summarizes a number of actions that are being considered to slow its development.

:i; Microorganisms

are termed drug resistant when they are no longer inhibited by an antimicrobial to which they were previously sensitive. i.' Drug resistance is genetic; microbes acquire genes that code for methods of inactivating or escaping the antimicrobial or acquire mutations that affect the drug's impact. Resistance is selected for in environments where antimicrobials are present in high concentrations, such as in hospitals. = Microbial drug resistance develops through the selection ofpreex_

isting random mutations and through acquisition of resistance

genes from other microorganisms. r',r

:-

Varieties of microbial drug resistance include drug inactivation, decreased drug uptake, decreased drug receptor sites, and modification of metabolic pathways formerly attacked by the drug.

Widespread indiscriminate use of antimicrobials has resulted in an explosion of microorganisms resistant to all common drugs.

12.5

369

Interactions between Drugs and Hosts

Not drug-resistant Drug-resistant mutanl

o- o o-(\/a/oooo,a

\ //o ooS^ u -o o o o

G o o"o -o o- o ^U\Jo o _ OG o uo ^^- u-ouor-lo o ^-\t-E;r0 ^ ^

a

'. ^tlo. : {.:.: oa

ooo ooo oa a o o

+

:

cells

population of microbial cells (a) Population

-----.-.-.-->

Remaining population grows over time

Exposure to clrug

(I)

(b) Sensitive cells eliminated by resistant mutants survive

drug;

o

$ m0m sqps

CI00G0

(c) All cells are now resistant reslslant

Figure 12.15

A model of natural selection for drug resistance. (here, (afpopulations of microbes can harbor some members with a prior mutation that confers drug resistance. (b) Environmental pressure population. the of members dominant the become (c) They eventually the presence of the drug) selects for survival of these mutants.

'

Strategies to Limit Drug Resistance

of Microorganisms

Drug Usage

.

Physicians have the responsibility for making an accurate diagnosis and prescribing the correct drug therapy.

.

Patients must comply with and carefully follow the physician's guidelines. It is important for the patient to take the correct dosage, by the best route, for the appropriate period. This diminishes the selection for mutants that can resist low drug levels, and ensures elimination of the pathogen.

.

Combined therapy is administration of two or more drugs together and increases the chances that at least one of the drugs will be effective and that a resistant strain ofeither drug will not be able to persist. The success depends on carefully choosing drugs that have different metabolic targets. It is unlikely that a microbe would possess resistance to multiple drugs with distinct targets simultaneously.

Drug Research

. .

. .

.

antimicrobials and the microorganisms they target. During an infection, the microbe is living in or on a host; therefore, the drug is administered to the host though its target is the microbe. Therefore, the effect of the drug on the host must always be considered. Although selective antimicrobial toxicity is the ideal constantly being sought, chemotherapy by its very nature involves contact with foreign chemicals that can harm human tissues' In fact, estimates indicate that at least 5o/o of all persons taking an antimicrobial drug experience some tlpe of serious adverse reaction to it. The major side effects of drugs fall into one of three categories: direct damage to tissues through toxicity, allergic reactions, and disruption in the balance of normal microbial flora. The damage incurred by antimicrobial drugs can be short term and reversible or permanent, and it ranges in severity from cosmetic to lethal.

Toxicity to Organs

fewer side effects.

Drugs can adversely affect the following organs: the liver (hepatotoxic), kidneys (nephrotoxic), gastrointestinal tract, cardiovascular system and blood-forming tissue (hemotoxic), nervous system

Pharmaceutical companies continue to seek new antimicrobial drugs with structures that are not readily inactivated by microbial enzymes or drugs with modes of action that are not readily circumvented. Proposals to reduce the abuse of antibiotics range from educational programs for health workers to requiring justification for prescribing certain types of antibiotics. Especially valuable antimicrobials may be restricted in their use to only one or two types of infections. The addition of antimicrobials to animal feeds must be curtailed

worldwide.

.

Until now this chapter has focused on the interactions between

Research focuses on developing shorter-term, higher-dose antimicrobials that are more effective, less expensive, and have

Long-Term Strategies

.

12.5 Interactions between Drugs and Hosts

Government programs that make effective therapy available to lowincome populations should be increased. Vaccines should be used whenever possible to provide alternative protection.

(neurotoxic), respiratory tract, skin, bones, and teeth' Because the liver is responsible for metabolizing and detoxifying foreign chemicals in the bloo{ it can be damaged by a drug or its metabolic products. Injury to liver cells can result in enzymatic abnormalities, fatty liver deposits, hepatitis, and liver failure. The kidney is involved in excreting drugs and their metabolites. Some drugs irritate the nephron tubules, creating changes that interfere with their filtration abilities. Drugs such as sulfonamides can crystallize in the kidney and form stones that can obstruct the flow ofurine' The most common complaint associated with oral antimicrobial therapy is diarrhea, which can progress to severe intestinal irritation or colitis. Although some drugs directly irritate the intestinal lining, the usual gastrointestinal complaints are caused by disruption of the intestinal microflora (discussed in a subsequent section)'

Chapter

370

l2

Drugs, Microbes, Host-The Elements of Chemotherapy

The Rise of Drug Resistance It is tempting to assume that science alone can solve the problem of drug resistance. If drug companies can discover more powerful antimicrobials, infectious diseases

be resistant to seven different antimicrobials. In the United States, a stain of fluoroquinolone-resistant

Campylobacter from chickens caused over 5,000 cases of food infection in the late 1990s. The opportunistic pathogen cal1ed VRE (vancomycin-

will

disappear. This mindset vastly underestimates the extreme versatility and adaptability of microorganisms and the complexity of the task. It is a fact of nature that if a large number of microbes are exposed to a variety of drugs, there will always be some genetically favored strains or types that survive and thrive. The AIDS virus (HIV) is so prone to drug resistance that it can become resistant during the first few weeks oftherapy in a single individual. Because HIV mutates so rapidly, in most cases, it will eventually become resistant to all drugs that have been developed so far.

? I)od* \ we* ile ouT:

I

Ironically, thousands ofpatients die every year in the United States from infections that lack effective drugs" and 600zo ofhospital infections are caused by drug-resistant microbes. For many years, con-

Worldwide Drug Resistance

gonococci. But during the past decade, the scope of the problem has escalated. It is now a common event to discover microbes that have become resistant to relatively new drugs in a very short time. In fact, many strains ofpathogens have multidrug resistance, and a few are resistant to all drugs.

The Hospital Factor The clinical setting is aprolific source ofdrug-resistant strains ofbacteria. This environment continually exposes pathogens to a variety ofdrugs. The

hospital also maintains patients with weakened defenses, making them highly susceptible to pathogens. A classic example occurredwith Staphylococcus aureus and penicillin. In the 1 950s, hospital strains began to show resistance to this drug, and because of indiscriminate use, these strains became nearly 100% resistant to penicillin in 30 years. In short order, S. aureus) strains appeared,

which can toler-

all antibiotics. To complicate this problem, strains of MRSA have been spread from the clinic into the community. Up until recently, MRSA has been sensitive to the drug vancomycin. Then, in 2002, the first ate nearly

cases

of the most tenacious of hospital-acquired infections for which there are few drug choices.

To attempt to curb this source of resistance, Europe and the United States have begun to ban the use of human drugs in animal feeds. The move seems to be working. Denmark banned all agricultural antibiotic use for growth promotion in 1998. Resistance to the drugs declined dramatically among bacteria isolated from the farm animals without significant reductions in animal size or health. Scientists are confident that this will lead to a reduction in human carriage of antibiotic-resistant bacteria.

cerned observers reported the gradual development of drug resistance in staphylococ ci, Salmonella, and,

MRSA (methicillin-resistant

resistant enterococcus) has been traced to the use of a vancomycinlike drug in cattle feed. It is now one

of complete resistance to this drug were reported (VRSA).

Drugs in Animal Feeds Another practice that has contributed significantly to growing drug resistance is the addition of antimicrobials to livestock feed, with the idea of decreasing infections and thereby improving animal health and size. This practice has had serious impact in both the United States and Europe. Enteric bacteria such as Salmonella, Escherichia coli, and enterococci that live as normal intestinal flora of these animals readily share resistance plasmids and are constantly selected and amplified by exposure to drugs. These pathogens subsequently'J.lmp" to humans and cause drugresistant infections, oftentimes at epidemic proportions. In a deadly outbreak of Sa lmonella infection in Denmark, the pathogen was found to

The drug dilemma has become a widespread prob-

lem, affecting all countries and socioeconomic groups. In general, the majority of infectious diseases, whether bacterial, fungal, protozoan, or are showing increases in drug resistance. In parts

viral, oflndia, the main drugs

used to treat cholera (furazolidone. ampicillin) have gone from being highly effective to essentially useless in I 0 years. In Southeast Asia. 98% of gonococcus infections are multidrug resistant. Malaria, tuberculosis, and typhoid fever pathogens are gaining in resistance, with few alternative drugs to control them. To add to the problem, global travel and globalization of food products mean that drug resistance can be rapidly exported. In countries with an adequate budget for antimicrobials, most infections will be treated, but at some expense. In the United States alone, the extra cost for treating the drug-resistant variety is around $ 1 0 billion per year. In many developing countries, drugs are mishandled by overuse and underuse. either of which can contribute to drug resistance. Many countries that do nol regulate the sale oflprescription drugs make them readily available to purchase over the counter. For example, the antituberculosis drug INH (isoniazid; is sometimes used as a "lLrng vitamin" to improve health, and antibiotics are taken in the wrong dose and wrong time for undiagnosed conditions. These countries serve as breeding grounds for drug resistance that can evenfually be carried to other countries. It is clear that we are in a race with microbes and we are falling behind. If the trend is not contained, the world may return to a time when there are few effective drugs left. We simply cannot develop them as rapidly as microbes can develop resistance. In this light, it is essential to fight the battle on more than one front. See table 12.7 for a summary of several critical strategies to give us an edge in controlling drug resistance.

What are some of the reasons that microbes develop drug resistance so

rapidty?

Answer available at http://www.mhhe.com/talaroT

12.5

Interactions between Drugs and Hosts

37r

Suppression and Alteration of the Micioflora by Antimicrobials Most normal, healthy body surfaces, such as the skin, large intestine, outer openings ofthe urogenitallracl, and oral cavity, provide oogarden" of microorganisms. These numerous habitats for a virtual normal colonists or residents, called the flora or microflora, consist mostly of harmless or beneficial bacteria, but a small number can potentially be pathogens. Although we defer a more detailed discussion ofthis topic to chapter 13 and later chapters, here we focus on the general effects of drugs on this population. If a broad-spectrum antimicrobial is introduced into a host to treat infection, it will destroy microbes regardless of their roles in the balance, affecting not only the targeted infectious agent but also many others in sites far removed from the original infection (figure 12.17).

When this therapy destroys beneficial resident species, resistant

Figure 72.76 Drug-induced

side effect.

An adverse effect of tetracycline given to young children is the permanent discoloration of tooth enamel.

Many drugs given for parasitic infections are toxic to the heart, causing irregular heartbeats and even catdiac arrest in extreme cases. Chloramphenicol can severely depress blood-forming cells in the bone marro\M, resulting in either a reversible or a pefinanent (fatal) anemia. Some drugs hemolyze the red blood cells, others reduce white blood cell counts, and still others damage platelets or interfere with their formation, thereby inhibiting blood clotting. Certain antimicrobials act directly on the brain and cause seizures. Others, such as aminoglycosides, damage nerves (very commonly, the Sth cranial nerve), leading to dizziness, deafrress, or motor and sensory disturbances. When drugs block the transmission of impulses to the diaphragm, respiratory failure can result. The skin is a frequent target of drug-induced side effects. The skin response canbe a symptom ofdrug allergy or a directtoxic effect. Some drugs interact with sunlight to cause photodermatitis, a skin inflammation. Tetracyclines are contraindicated (not advisable) for children

microbes that were once in small numbers begin to overgrow and cause disease. This complication is called a superinfection. Some common examples demonstrate how a disturbance in microbial flora leads to replacement flora and superinfection. A broad-spectrum cephalosporin used to treat a urinary tract infection by Escherichia coli will cure the infection, but it will also destroy the lactobacilli in the vagina that normally maintain a lnfection

Circulating drug

from birth to 8 years of age because they bind to the enamel of the teeth, creating apermanentgrayto brown discoloration (figure 12.16). Pregnant women should avoid tefacyclines because they cross the placenta and can be deposited in the developing fetal bones and teeth.

Allergic Responses to Drugs One of the most frequent drug reactions is heightened sensitivity, or allergy. This reaction occurs because the drug acts as an antigen (a foreign material capable of stimulating the immune system) and stimulates an allergic response. This response can be provoked by the intact drug molecule or by substances that develop from the bodyb metabolic alteration of the drug. In the case ofpenicillin, for instance, it is not the penicillin molecule itselfthat causes the allergic response but a product, benzylpenicilloyL Allergic reactions have been reported for every major type of antimicrobial drug, but the penicillins account for the greatest number of antimicrobial allergies, followed by the sulfonamides. People who are allergic to a drug become sensitized to it during the first contact, usually without symptoms. Once the immune system is sensitized, a second exposure to the drug can lead to a reaction such as a skin rash (hives); respiratory inflammation; and, rarely, anaphylaxis, an acuteo overwhelming allergic response that develops rapidly and can be fatal. (This topic is discussed in greater detail in chapter 16.)

Potential pathogen resistant to drug but held in check by other microbes

Figure 12.17

The role of antimicrobials in disrupting mlcrobial flora and causing superinfections. (a) A primary infection in the throat is treated with an oral antibiotic. (b) The drug is carried to the intestine and is absorbed into the circulation. (c) The primary infection is cured, but drug-resistant pathogens have survived and create an intestinal superinfection.

372

Chapter

ffffiffi1jffiffiffiffiffiii,.,W

12

Drugs, Microbes, Host-The Elements of Chemotherapy

Major Adverse roxic Reactions to common Drus Groups

Antimicrobial Drug

Primary Tissue Affected

Primary Damage or Abnormality produced

Antibacterials Penicillin G Carbenicillin

Ampicillin Cephalosporins

Tetracyclines

Skin

Rash

Platelets

Abnormal bleeding Diarrhea and enterocolitis

GI tract Platelet function White blood cells

Inhibition of prothrombin synthesis Decreased circulation

Kidney GI tract

Nephritis Diarrhea and enterocolitis Discoloration of tooth enamel; damage to fetal skeleton Reactions to sunlight fuhotosensitization)

Teeth, bones

Skin Chloramphenicol

Bone marrow

Aminoglycosides ( streptomycin. gentamicin, amikacin)

GI tract, hair cells in cochlea, vestibular cells, neuromuscular cells, kidney tubules

Tsoniazid

Liver Brain

Sulfonamides

Injury to red and white blood cell precursors Diarrhea and enterocolitis; malabsorption; loss hearing, dizziness, kidney damage Hepatltls

of

Seizures

Skin

Dermatitis

Kidney

Formation of crystals; blockage of urine flow Hemolysis Reduction in number

Red blood cells Platelets

Polyrnyxin

Kidney

Quinolones (ciprofl oxacin, norfl oxacie

Neuromuscular system Nervous system, bones, GI tract

Rifampin

Headache, dizziness, tremors, GI distress

Liver

Damage to hepatic cells

Skin

Dermatitis

Amphotericin B

Kidney

Disruption of tubular filtration

Flucytosine

White blood cells

Decreased number

Metronidazole

GI tract

Nausea, vomiting

Chloroquine

GI tract

Vomiting

Brain

Headache

Skin

Itching

GI tract GI tract Brain

lrritation

Damage to membranes of tubule cells Weakened muscular responses

Antifungals

Antiprotozoan Drugs

Antihelminthics Niclosamide Pyrantel

Nausea, abdominal pain Headache. dizziness

Antivirals Acyclovir Amantadine

Brain Skin

Seizures, confusion Rash

Brain GI tract

Nervousness, light-headedness Nausea

AZT

Bone marrow

Immunosuppression. anemia

Protease inhibitors

Liver Lipid metabolism

Hepatic damage Abnormal deposition of fats

Pancreas

Diabetes

protective acidic environment there. The drug has no effect, however, on Candida albicans, a yeast that also resides in normal vaginas. Released from the inhibitory environment provided by lactobacilli, the yeasts proliferate and cause symptoms. Candida can cause similar superinfections ofthe oropharynx (thrush) and the large intestine.

Oral therapy with tetracyclines, clindamycin, and broadspectmm penicillins and cephalosporins is associated with a serious

and potentially fatal condition known as antibiotic-associated colitis

(pseudomembranous colitis). This condition is due to the overgrowth in the bowel of Clostridium dfficile, an endospore-forming bacterium that is resistant to the antibiotic. It invades the intestinal lining and releases toxins that induce diarrhea, feveq and abdominal pain. (You'll learn more about C. dfficile in chapter 19.)

Refer to table 12.8 for a general summary of major drug groups and their side effects.

12.6

|[s 1lt . -ajor side effects

different drugs and observing the effects of the drugs on growth. The Kirby-Bauer Iechnique is an agar diffusion test that provides useful data on antimicrobial susceptibility. In this test, the surface of a plate of special medium is spread with the test bacterium, and small discs containing a premeasured amount of antimi-

"12.6 Considerations in Selecting an

Antimicrobial Drug Before actual antimicrobial therapy can begin, consideration must be given to at least three factors:

L. the nature of the microorganism causing the infection, 2. the degree of the microorganism's susceptibility (also called

3.

sensitivity) to various drugs, and the overall medical condition of the patient.

ldentifying the Agent Identification of infectious agents from body specimens should be attempted as soon as possible. It is especially important that such specimens be taken before any antimicrobial drug is given, just in case the drug eliminates the infectious agent. Direct examination ofbody fluids, sputum, or stool is a rapid initial method for detecting and perhaps even identi$'ing bacteria or fungi. A doctor often begins the therapy on the basis of such immediate findings. The choice of drug will be based on experience with drugs that are known to be effective against the microbe. For instance, if a sore throat appears to be caused by Streptococcus pyogenes, the physician may prescribe penicillin, because this species seems to be almost universally sensitive to it so far. If the infectious agent is not or cannot be isolated epidemiological statistics may be required to predict the most likely agent in a given infection. For example, Streptococcus pneumoniae accounts for the majority of cases of meningitis in children, followed by Ne i s s eria meningitidi s (discussed in detail in chapter 18).

Testing for the Drug Susceptibility of Microorganisms

crobial are dispensed onto the bacterial lawn' After incubation, the zone of inhibition surrounding the disks is measured and compared with a standard for each drug (table 12.9 and figure 12.18). The profile of antimicrobial sensitivity, or antibiogram, provides data for drug selection. The Kirby-Bauer procedure is less effective for bacteria bhat are anaerobic, highly fastidious' or slow-growing (My c ob acterium). An alternative quantitative system that provides a quantitative rating of drug effectiveness is the Etest* (figure 12.19). In addition to providing a precise numerical rating (the MIC), this method allows testing for a wide variety of drugs and microbial types, including anaerobes, mycobacteria, and fungi. More sensitive and quantitative results can also be obtained with tube dilution tests. First the antimicrobial is diluted serially in tubes of broth, and then each tube is inoculated with a small uniform sample of pure culture, incubated" and examined for growth (turbidity). The smallest concentration (highest dilution) of drug that visibly inhibits growth is called the minimum inhibitory concentration, or MIC. The MIC is useful in determining the smallest effective dosage of a drug and in providing a comparative index against other antimicrobials (figure 12.20 and tabte 12.10). In many clinical laboratories, these antimicrobial testing procedures are performed in automated machines that can test dozens of drugs

simultaneously.

Results of a Sample Kirby-Bauer Test Zone of lnhibition (mm) Required For

Susceptibility Resistance Result (mm) for (R) Stophylococcus oureus Evaluation fSl

Drug

Testing is essential in those groups ofbacteria commonly showing resistance, primarily

Bacitracin

>13

12 >15 >12 >19

enteric bacilli. However, not all infectious agents require antimicrobial sensitivity testing. When certain groups, such as group A

Neomycin

Penicillin G Polymlxin B

streptococci and all anaerobes (except Bacteroides), are known to be uniformly susceptible to penicillin G, testing may not be necessary

unless the patient

Streptomycin Vancomycin Tetracycline

is allergic to penicillin.

Testing methods are available for fungi (see

R

373

figure I2.20b), protozoa, and viruses, although testing may not be done as often for these grouPs. Selection of a proper antimicrobial agent begins by demonstrating the in vitro activity of several drugs against the infectious agent by means of standardized methods' In general, these tests involve exposing a pure culture ofthe bacterium to several

of antimicrobials are toxicity to organs' allergic reactions, and problems resulting from suppression or alteration of normal flora. ,',: Antimicrobials that destroy most but not all normal flora allow the urraffected normal flora to overgrow. causing a suPerinfectiorr,. =:;

Considerations in Selecting an Antimicrobial Drug

:

resistant,

I

:

intermediate, S

:

sensitive

20

S

t2 10

R R

ADP. riboseEF-2 (inactivated)

i

nicotinamide

Figure 13.72 A-B toxin ot Corynebocterium diphtherioe' The B chain attaches to a specific receptor on the cell membrane, then the toxin is engulfed into a vacuole. The two chains separate and the A chain enters the cytoplasm as an enzyme that inactivates a protein needed for protein synthesis. The lack of protein leads to cell

however, have incubation periods ranging between 2 and 30 days' The earliest notable symptoms of infection appear as a vague as head and muscle aches, fatigue, upset This short period ( 1-2 days) is known general malaise. stomach, and infectious agent next enters a period The prodromal stage. as the

feeling of discomfort, such

dysfunction.

'f o

tissues. Pathogens can obstruct tubular structures such as blood vessels, lymphatic channels, fallopian tubes, and bile ducts' Accumulated damage can lead to cell and tissue death, a condition called necrosis. Although viruses do not produce toxins or destructive enzymes, they deshoy cells by multiplying in and lysing them' Many of the cytopathic effects of viral infection arise from the impaired metabolism and death of cells (see chapter 6)'

o 6.

tnducing on lniurious Host Response The direct effects of virulence factors such as enzymes and toxins can cause significant harm to cells, tissues' and organs' It must be stated that many microbial diseases are actually the result of indirect damage, or the host's excessive or inappropriate response to a

E

E

t,

o

=oc

o

c

lnitial

exposure -----> to mircrobe Time

Figure 13.13

Stages in the course of infection

and disease. Dashed lines represent periods with a variable length'

400

Chapter

Localized infection (boil)

13

Microbe-Human Interactions

Systemic infection (influenza)

Focal infection

(e)

Various microbes

\q, Figure 13.14

ru,

(d)

Secondary (vaginal) infection

Mixed infection

The occurrence of infections with regard to location and sequence.

(a) A localized infection, in which the pathogen is restricted to one specific site. (b) systemic infection, in which the pathogen spreads through circulation to many sites' (c) Afocal infection occurs initially as a local infection, but circumstances cause the microbe to be carried to other sites systemically' (d) A mixed infection, in which the same site is infected with several microbes at the same time. (e) In a primary-secondary infection, an initial infection is complicated by a second one in the same or a different location and caused by a different microbe.

of invasion, during which it multiplies at high levels, exhibits its

greatest toxicity, and becomes well established in its target tissue. This period is often marked by fever and other prominent and more specific signs and symptoms, which can include cough, rashes, di_ arrhea,loss ofmuscle control, swelling, jaundice, discharge of exu_ dates, or severe pain, depending on the particular infeCtion. The length of this period is extremely variable. As the patient begins to respond to the infection, the symptoms decline-sometimes dramatically, other times slowly. During tt ,"_

" covery that follows, called the convalescent period, the patient,s strength and health gradually retum owing to the healing nature of the immure response. An infection that results in death is called terminal. The transmissibility of the microbe during these four stages must be considered on an individual basis. A few agents are ie_ leased mostly during incubation (measles, for example); many are released during the invasive period (Shigella); and others can be transmitted during all of these periods (hepatitis B). Establishment, Spreod, ond pothotogic Effects Patterns of lnfection pafterns of infection are many

and

varied. In the simplest situation, a localized infection. the microbe enters the body and remains confined to a specific tissue (figure l3.l4a). Examples of localized infections are boils, fungal skin in_ fections, and warts. - Many infectious agents do not remain localized but spread from the initial site of entry to other tissues. In fact, spreading is necessary for pathogens such as rabies and hepatitis A virus, whose target tissue is some distance from the site of entry. The rabies virus travels from a bite wound along nerve tracts to its target in the brain,

and the hepatitis A virus moves from the intestine to the liver via the circulatory system. when an infection spreads to several sites and

tissue fluids, usually in the bloodstream, it is called a systemic in_ fection (figure l3.l4b). Examples of systemic infections are viral diseases (measles, rubella, chickenpox, and AIDS); bacterial dis_ eases

(brucellosis, anthrax, typhoid fever, and syphilis); and fungal

diseases (histoplasmosis and cryptococcosis). Infectious agents can also travel to their targets by means ofnerves (as in rabiei) or cerebrospinal fluid (as in meningitis).

A focal infection is

said to exist when the infectious asent

breaks loose from a local infection and is seeded or disseminated-rnto other tissues (figure 13.14c). This pattern is exhibited by tuberculosis

or by streptococcal pharyngitis, which gives rise to scarlet fever. In the condition called toxemia,e the infection itself remains localized at the portal of entry, but the toxins produced by the pathogens are carried by the blood to the actual target tissue. In this way, the target of the bacterial cells can be different from the target of their toxin.

An infection is not always caused by a single microbe. In a mixed infection, several agents establish themselves simultaneously atihe infection site (figure l3.l4d).In some mixed or synergistic infections, the microbes cooperate in breaking down a tissui. In other mixed infections, one microbe creates an environment that enables another microbe to invade. Gas gangrene, wound infections, dental caries, and human bite infections tend to be mixed. These are sometimes called polymicrobial diseases and may be the result of biofilm formation at the site of infection. 9. Not to be confused with toxemia of pregnancy, which is and not an infection.

a metabolic disturbance

13.2

Maior Factors in the Development of an Infection

40r

A Quick Guide to the Terminology of lnfection and Disease Words in medicine have great power and economy. A single tdchnical term can often replace a whole phrase or sentence, thereby saving time and space in patient charting. The beginning student may feel overwhelmed by what seems like a mountain of new words. However, having a grasp ofa few root words and a fair amount ofanatomy can help you learn many of these words and even deduce the meaning of unfamiliar ones. Some examples of medical shorthand follow. The suffix -ifts means an inflammation and when affixed to the end of an anatomical term, indicates an inflammatory condition in that location. Thus, meningitis is an inflammation ofthe meninges surrounding the brain; encephalitis is an inflammation of the brain itself; hepatitis involves the liver;

blood." Thus, septicemia means sepsis (infection) of the blood; bacteremia, bacteria in the blood; viremia, viruses in the blood; and fungemia, fungi in the blood. It is also applicable to specific conditions such as toxemia, gonococcemia, and spirochetemia. The suffix -osis means "a disease or morbid process." It is frequently added to the names ofpathogens to indicate the disease they cause: for example, listeriosis, histoplasmosis, toxoplasmosis, shigellosis, salmonellosis, and borreliosis. A variation of this suffix is -iasis, as in trichomoniasis and candidiasis. The suffix -oma comes from the Greek word onkomas (swelling) and means tumor. Although the root is often used to describe cancers (sarcoma, melanoma), it is also applied in some infectious diseases that cause masses or swellings

vaginitis, the vagina; gastroenteritis, the intestine; and otitis media, the middle ear. Although not all inflammatory conditions are caused by infections, many infectious diseases inflame their target organs. The suffix -emia is derived from the Greek word haeima, meaning blood. When added to a word it means "associated with the

Some diseases are described according to a sequence ofrelated infections. When an initial, or primary infection is complicated by another infection caused by a different microbe, this subsequent infec-

tion is termed a secondary infection (figure l3.l4e). This pattern often occurs in a child with chickenpox (primary infection) who may scratch his pox and infect them with Staphylococcus aureus (secondary infection). The secondary infection need not be in the same site as the primary infection, and it usually indicates altered host defenses.

Infections that come on rapidly, with severe but short-lived effects, are called acute infections. Infections that progress and persist over a long period of time are chronic infections. Insight 13.4 illustrates other common terminology used to describe infectious diseases.

Signs and Symptoms: Warning Signals of Disease When an infection causes pathologic changes leading to disease, it is often accompanied by a variety of signs and symptoms. A sign is any objective evidence ofdisease as noted by an observer; a symptom is the subjective evidence ofdisease as sensed by the patient' Signs tend to be more precise than symptoms and are often measured. Both can be the result of the same underlying cause. For example, an infection of the brain may present with the sign of bacteria in the spinal fluid and symptom of headache. Or a streptococcal infection may produce a sore throat (symptom) and inflamed pharynx (sign). Disease indicators that can be sensed and observed can qualify as either a sign or a symptom depending upon how they are reported. When a disease can be identified or defined by a defined collection of signs and symptoms, it is termed a syndrome. Signs and symptoms with considerable importance in diagnosins infectious diseases are shown in table 13.9.

(tuberculoma, leproma)'

Give definitions for urethritis, endotoxemiao chlamydiosiso and

lymphoma. Answer available at http

: '

""

':

'"

:

//www. mhhe. com/talaro 7

of Infectious Diseases

Signs

Symptoms

Fever

Chills Pain, irritation

Septicemia Microbes in tissue fluids

Nausea

Chest sounds

Malaise, fatigue

Skin eruptions

Chest tightness

Leukocytosis

Itching

Leukopenia

Headache

Swollen lymph nodes

Weakness

Abscesses Tachycardia (increased heart rate)

Abdominal cramps Anorexia (lack of appetite)

Antibodies in serum

Sore throat

Signs and Symptoms of lnflommation The earliest symptoms of disease result from the activation of the body defense process called inflammation.* The inflammatory response includes cells and chemicals that respond nonspecifically to disruptions in the tissue. This subject is discussed in greater detail in chapter 14, but as noted earlier, many signs and symptoms of infection are caused by the mobilization of this system. Some common symptoms of inflammation include fever. pain. soreness. and swelling. Signs of inflammation include edemar* the accumulation of fluid in an afilicted tissue; granulomas and abscesses, walled-off collections of inflammatory cells and microbes in the tissues; and lymphadenitis, swollen lymph nodes. ' iilflLtntntulion (in-flam'-uh+o1'-eel)L. inflammtion, to set on fire. ' aclcnu (uh-dee'-muh) Gr. oidema, swelling.

402

Chapter

13

Microbe-Human Interactions

Rashes and other skin eruptions are common symptoms and signs in many diseases, and because they tend to mimic each other, it can be difficult to differentiate among diseases on this basis alone. The general term for the site of infection or disease is lesion.* Skin lesions can be restricted to the epidermis and its glands and follicles, or they can extend into the dermis and subcutaneous

regions. The lesions changes and thus

of

some infections undergo characteristic

in appearance and location during the course of fit more than one category.

disease Skin cells (open lesion)

Signs of lnfection in the Blood Changes in the number of circulating white blood cells, as determined by special counts, are considered to be signs of possible infection. Leukocytosis* is an increase in the level of white blood cells, whereas leukopenia* is a decrease. Other signs of infection revolve around the occurrence of a microbe or its products in the blood. The clinical term for blood infection, septicemia, refers to a general state in which microorganisms are multiplying in the blood and are present in large numbers. When small numbers of bacteria or viruses are found in the bloo4 the correct terminology is bacteremia or viremia, which means that these microbes are present in the blood but are not necessarily multiplying. During infection, a normal host will invariably show signs of an immune response in the form of antibodies in the serum or some type of sensitivity to the microbe. This fact is the basis for several serological tests used in diagnosing infectious diseases such as AIDS or syphilis. Such specific immune reactions indicate the body's attempt to develop specific immunities against pathogens. We concentrate on this role ofthe host defenses in chapters 14 and,15.

Infections Thot Go Unnoticed It is rather common for an infection to produce no noticeable symptoms, even though the microbe is active in the host tissue. In other words, although infected, the host does not manifest the disease. Infections of this nature are known as asymptomatic, subclinical, or inapparenl because the patient experiences no symptoms or disease and does not seek medical attention. However, it is important to note that most infections are attended by some sort of detectable sign. In section 13.3, we further address the significance of subclinical infections in the transmission of infectious asents.

The Portal of Exit: Vacating the Host Earlier, we introduced the idea that a parasite is considered unsucif iI does not have a provision for leaving its host and moving to other susceptible hosts. With few exceptions, pathogens depart by a specific avenue called the portal of exit (figure 13.ft. In most cases, the pathogen is shed or released from the body through secretion, excretion, discharge, or sloughed tissue. The usually very high number of infectious agents in these materials increases both virulence and the likelihood that the pathogen will reach other hosts. cessful

* lesion (lee'-zJlrttn)L. laesio, tohwt. * leukocfiosis (1oo"-koh'-sy{oh'-sis) From /euftocyte, awhiteblood cell, and

sufix

-osls.

the

* leukopenia (loo"-koh'-pee'-nee-tth)Fromleukocyteandpenia, alossorlackof

Figure 13.15 Major portals of exit of infectious diseases. In many cases, the portal of exit is the same as the portal of entry, but a few pathogens use a different route. As we see in the next section, the portal of exit concerns epidemiologists because it greatly influences the dissemination of infection in a population.

Respirotory ond Solivory Portals Mucus, sputum, nasal drainage, and other moist secretions are the media of escape for the pathogens that infect the lower or upper respiratory tract. The most effective means of releasing these secretions are coughing and sneezing (see figure I 3. I 8), although they can also be released during talking and laughing. Tiny particles of liquid released into the air form aerosols or droplets that can spread the infectious agent to other people. The agents of tuberculosis, influenza, measles, and chickenpox most often leave the host through airborne droplets. Droplets of saliva are the exit route for several viruses, including those of mumps, rabies, and infectious mononucleosis.

Epitheliol Cells The outer layer ofthe skin and scalp are constantly being shed into the environment. A large proportion of household dust is actually composed of skin cells. A single person can shed several billion skin cells a day, and some persons, called shedders, disseminate massive numbers of bacteria into their immediate surroundings. Skin lesions and their exudates can serve as portals ofexit in warts, fungal infections, boils, herpes simplex, smallpox, and syphilis.

13.3 Fecol Exit Feces are a very cornmon portal of exit. Some intestinal pathogens

grow in the intestinal mucosa and create an inflammation that in-

motility of the bowel. This increased motility speeds up peristalsis, resulting in diarrhea; and the more fluid stool provides a rapid exit for the pathogen. A number of helminth worrns release cysts and eggs through the feces. Feces containing pathogens are a public health problem when allowed to contaminate drinking water or when used to fertilize croPs'

Sources and Transmission of Microbes

403

can result in deafness, a strep throat can lead to rheumatic heart disease, Lyme disease can cause arthritis, and polio can produce paralysis.

creases the

Urogenital Tract A

number

of agents involved in sexually transmitted infections

leave the host in vaginal discharge or semen. This is also the source

of neonatal infections such as herpes simplex, Chlamydia, and Candida albicans, which infect the infant as it passes through the birth canal. Less commonly, certain pathogens that infect the kidney are discharged in the urine: for instance, the agents of leptospirosis, typhoid fever, tuberculosis, and schistosomiasis.

Removal

of Blood or Bleeding

Although the blood does not have a direct route to the outside, it can serve as a portal of exit when it is removed or released through a vascular puncture made by natural or artificial means. Bloodfeeding animals such as ticks and fleas are common transmitters of pathogens (see chapter 23). The AIDS and hepatitis viruses are transmitted by shared needles or through small gashes in a mucous membrane caused by sexual intercourse. Because many microbes can be transferred through blood dona-

tion, the monitoring of both donors and donated blood has to be scrupulous. In the mid-1980s, several thousand people suffering from hemophilia were infected with HIV as a result of receiving clotting factors contaminated with the virus. The current risk for transfusion-transmitted infections is about 1 case in 10 million transfusions. One factor that increases risk is the presence of emerging disease agents in the blood supply that have not yet been detected. This recently happened with West Nile virus and Chagas disease.

The Persistence of Microbes and Pathologic Conditions The apparent recovery of the host does not always mean that the microbe has been completely removed or derstroyed by the host defenses. After the initial symptoms in certain $ronic infectious diseases, the infectious agent retreats into a state/called persistence or latency. In some cases of latency, the microbe canperiodicallybecome active and produce a recurrent disease. The viral agents ofherpes simplex, herpes zoster, hepatitis B, AIDS, and Epstein-Barr can persist in the host for long periods. The agents of syphilis, typhoid fever, tuberculosis, and malaria also enter into latent stages. The person harboring a persistent infectious agent may or may not shed it during the latent stage. Ifit is shed, such persons are chronic carriers who serve as sources of infection for the rest of the population.

Some diseases leave sequelae* in the form of long-term or permanent damage to tissues or organs. For example, meningitis *

sequelae (su-kwee'Jee) L. sequi, to

follow

r r r E

!!

il

r

Exoenzymes, toxins, and the ability to induce injurious host responses are the three main types of virulence faclors pathogens utilize to combat host defenses and damage host tissue. Exotoxins and endotoxins differ in their chemical composition and tissue specificity. Characteristics or structures ofmicrobes that induce extreme host responses are a major factor in most infectious diseases. Patterns of infection vary with the pathogen or pathogens involved. They range from local and focal to systemic. A mixed infection is caused by two or more microorganisms simultaneously.

Infections can be. characterizedby their sequence as primary or secondary and by their duration as either acute or cbronic. An infectious disease is characterized by both objective signs and

subjective symptoms. Infectious diseases that are asymptomatic or subclinical nevertheless often produce clinical signs. B The portal of exit by which a pathogen leaves its host is often but not always the same as the portal of entry. s The portals of exit and entry determine how pathogens spread in a population. E Some pathogens persist in the body in a latent state; others cause

tr

long-term diseases called s equelae.

13.3 Sources and Transmission

of Microbes Reservoirs: Where Pathogens Persist For an infectious agent to continue to exist and be spread, it must have a permanent place to reside. The reservoir is the primary habitat in the natural world from which a pathogen originates. Often it is a human or animal carrier, although soil, water, and plants are also reservoirs. The reservoir can be distinguished from the infection source' which is the individual or object from which an infection is actually acquired. In diseases such as syphilis, the reservoir and the source are the same (the human body). In the case of hepatitis A, the reservoir (a human carrier) is usually different from the source of infection (contaminated food).

Living Reservoirs Many pathogens continue to exist and spread because they are harbored by members of a host population' Persons or animals with frank symptomatic infection are obvious sources of infection, but a carrier is, by definition, an individual who inconspicuously shelters a pathogen and spreads it to others without any notice. Although human carriers are occasionally detected through routine screening (blood tests, cultures) and other epidemiological devices, they are unfortunately very difficult to discover and control. As long as a pathogenic reservoir is maintained by the carrier state, the disease

404

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Microbe-Human Interactions

Asymptomatic

Incubation

Timerl! (a)

(c)

Figure 13.16 Involvement of carriers

Convalescent

Chronic

Stages of release during infection ,,

(b)

Transfer of infectious agent through contact

*

Infectious agent

in transmission of infectious agents.

(a) An asymptomatic carrier is infected without symptoms. (b) Incubation, convalescent, and chronic carriers can transmit the infection either before or after the period of symptoms. (c) A passive carrier is contaminated but not infected.

will

continue to exist in that population, and the potential for epidemics

will be a constant threat. The duration of the carrier

state can be short or long term, and the carrier may or may not have experienced disease due to the microbe.

Several situations can produce the carrier state. Asymptomatic (apparently healthy) carriers are infected, but as previously indicated, they show no symptoms (figure l3.l6a).A few asymptomatic infections (gonorrhea and genital warts, for instance) can carry out their entire course without overt manifestations. Figure l3.l6b demonstrates three types of carriers who have had or will have the disease but do not at the time they transmit the organism. Incubation carriers spread the infectious agent during the incubation period. For example, AIDS patients can harbor and spread the virus for months and years before their first symptoms appear. Re_ cuperating patients without symptoms are considere d convalescent carriers whenthey continue to shed viable microbes and convey the

infection to others. Diphtheria patients, for example, spread the microbe for up to 30 days after the disease has subsided. An individual who shelters the infectious agent for a long period after recovery is a chronic cqrrier Patients who have recovered from

tuberculosis, hepatitis, and herpes infections frequently carry the agent

chronically. About one in 20 victims

of typhoid fever continues to

harbor Salmonella typhi inthe gallbladder for several years, and sometimes for life. The most infamous of these was ..Typhoid Maryi' a cook who spread the infection to hundreds of victims in the early1900s.

The passive carrier state is ofgreat concern during patient care (see a later section on nosocomial infections). Medical and dental personnel who must constantly handle materials that are heavily con_ taminated with patient secretions and blood risk picking up patho_ gens mechanically and accidently transferring them to other patients

(figure l3.l6c). Proper hand washing, handling of contaminated materials, and aseptic techniques greatly reduce this likelihood.

Animals as Reservoirs and Sources Up to now, we have lumped animals with humans in discussing living reservoirs or carriers, but animals deserve special consideration as vectors ofinfections. The word vector is used by epidemiologists to indicate a live

animal that transmits an infectious agent from one host to another. (The term is sometimes misused to include any object that spreads disease.) The majority

of vectors are arthropods such as fleas,

13.3

Sources and Transmission of Microbes

mosquitoes, flies, and ticks; although larger animals can also spread

infection-for example, mammals (rabies), birds (ornithosis), or lower vertebrates (salmonellosis).

By tradition, vectors are placed into one of two categories, depending upon the animal's relationship with the microbe. A biological vector actively participates in a pathogen's life cycle, serving as a site in which it can multiply or complete its life cycle (see figure 23.28,page 718). A biological vector communicates the

infectious agent to the human host by biting, aerosol formation, or touch. In the case of biting vectors, the animal can inject infected saliva into the blood (the mosquito), defecate around the bite wound (the flea), or regurgitate blood into the wound (the tsetse fly). Mechanical vectors are not necessary to the life cycle of an infectious agent and merely transport it without being infected. The external body parts of these animals become contaminated when they come into physical contact with a source of pathogens. The agent is subsequently transferred to humans indirectly by an intermediate such as food or, occasionally, by direct contact (as in certain eye infections). Houseflies are noxious mechanical vectors. They feed on decaying garbage and feces, and while they are feeding, their feet and mouthparts easily become contaminated. They also regurgitatejuices onto food to soften and digest it' Flies spread more than 20 bacterial, viral, protozoan, and worm infections. Other nonbiting flies transmit tropical ulcers, yaws, and trachoma. Cockroaches, which have similar unsavory habits, play a role in the mechanical transmission of fecal pathogens, as well as contributing to allergy attacks in asthmatic children. Many vectors and animal reservoirs spread their own infections to humans. An infection indigenous to animals but naturally transmissible to humans is a zoonosis.* In these types of infections, the human is essentially a dead-end host and does not contribute to the natural persistence of the microbe. Some zoonotic infections (rabies, for instance) can have multihost involvement, and others can have very complex cycles in the wild (plague). Zoonotic spread of disease is promoted by close associations of humans with animals, and people in animal-oriented or outdoor professions are at greatest risk. At least 150 zoonoses exist worldwide; the most common ones are listed in table 13.10. Zoonoses make up a full 70% of all new emerging diseases worldwide. It is worth noting that zoonotic infections are impossible to completely eradicate without also eradicating the animal reservoirs. Attempts have been made to eradicate mosquitoes and certain rodents' In 2004, China slaughtered millions of chickens that were potential carriers of avian influenza. One technique that can provide an early warning signal for the occurrence ofcertain mosquito-borne zoonoses has been the use

of

sentinel animals. These are usually domestic animals (most often chickens or horses) that can serve as hosts for diseases such as West

Nile fever, various viral encephalitides, and malaria. Sentinel animals are placed at various sites throughout the communiry and their blood is monitored periodically for antibodies to the infectious agents that would indicate a recent infection by means of a mosquito bite. The presence of infected animals provides useful data on the potential for human exposure, and it also helps establish the epidemiological pattern of the zoonosis including where it may have spread.

* zoono.sis (zoh" -uh-noh'-sis) Gr. zoion, arrimal, and nosos, disease.

405

Common Zoonotic Infections Disease/Agent

Primarv Animal Reservoirs

Viruses Rabies

A1l mammals

Yellow fever

Wild birds, mammals, mosquitoes

Viral fevers

Wildmammals

Hantavirus

Rodents

Influenza

Chickens, swine

West Nile virus

Wild birds, mosquitoes

Bacteria Rocky Mountain spotted fever

Dogs, ticks

Psittacosis

Birds

Leptospirosis

Domestic animals

Anthrax

Domestic animals

Brucellosis

Cattle, sheep, pigs

Plague

Rodents, fleas

Salmonellosis

Variety of mammals, birds, and rodents

Tularemia

Rodents, birds, arthropods

Miscellaneous Ringworm

Domestic mammals

Toxoplasmosis

Cats, rodents, birds

Trypanosomiasis

Domestic and wild mammals

Trichinosis

Swine, bears

Tapeworm

Cattle, swine, fish

Scabies

Domestic animals

Nonliving Reservoirs Clear$, microorganisms have adapted to nearly every habitat in the biosphere. They thrive in soil and water and often find their way into the air. Although most of these microbes are saprobic and cause little harm and considerable benefit to humans, some are opportunists and a few are regular pathogens. Because human hosts are in regular contact with these environmental sornces, acquisition of pathogens from natural habitats is of diagnostical and epidemiological importance'

Soil harbors the vegetative forms of bacteria, protozoa, helminths, and fungi, as well as their resistant or developmental stages such as spores, cysts, ova, and larvae. Bacterial pathogens include the anthrax bacillus and species of Clostridium thar ate responsible for gas gangrene, botulism, and tetanus. Pathogenic fungi in the genera Coccidioides and Blastomyces are spread by spores in the soil and dust. The invasive stages ofthe hookworm Necator occur in the soil. Natural bodies of water carry fewer nutrients than soil does but still support pathogenic species such as Legionella, Crypto sp oridium, and Giardia.

The Acquisition and Transmission of Infectious Agents Infectious diseases can be categorized on the basis ofhow they are acquired. A disease is communicable when an infected host can transmit the infectious agent to another host and establish infection in that host. (Although this terminology is standard, one must

406

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realize that it is not the disease that is communicated but the microbe. Also be aware that the word infectiozs is sometimes used interchangeably with the word communicable,b\tthisis not precise usage.) The transmission of the agent can be direct or indirect, and the ease with which the disease is transmitted varies considerably from one agent to another. If the agent is highly communicable, especially through direct contact, the disease is contagious. Influenza and measles move readily from host to host and thus are contagious, whereas leprosy is only weakly communicable. Because they can be spread through the population, communicable diseases are our main focus in the following sections.

In contrast, a noncommunicable infectious disease does not arise through transmission of the infectious agent from host to host. The infection and disease are acquired through some other, special circumstance. Noncommunicable infections occur primarily when a compromised person is invaded by his or her own microflora (as with certain pneumonias, for example) or when an individual has accidental contact with a facultative parasite that exists in a nonliving reservoir such as soil. Some examples are certain mycoses, acquired through inhalation of fungal spores, and tetanus, in which Clostridium tetani Wores from a soiled object enter a cut or wound. Persons thus infected do not become a source ofdisease to others.

Potterns of Tronsmission in Communicoble Diseoses The routes or patterns of disease transmission axe many and varied. The spread of diseases is by direct or indirect contact with animate or inanimate objects and can be horizontal or vertical. The term horizontal means the disease is spread through a population from one infected individual to another; vertical signifies transmission from parent to offspring via the ovum, sperm, placenta, or milk. The extreme complexity of transmission patterns among microorganisms makes it very difficult to generalize. However, for easier organization, we divide microorganisms into two major groups, as shown in figure 13.17: transmission by direct routes or transmission by indirect routes, in which some sort of vehicle is involved.

Modes of Direct Transmission For microbes to be directly transferred, some type of contact must occur between the skin or mucous membranes of the infectedperson andthose of the infectee. It may help to think of this route as the portal of exit meeting the portal of entry without the involvement of an intermediate object or substance. Included in this category are fine droplets sprayed

t I I

I

Fecal-oral contamination can also lead to both of these types of transmission

Aerosols

Figure 13.17

Summary of how communicable infectious diseases are acquired.

13.3

Sources and Transmission of Microbes

407

directly upon a person during sneezing or coughing (as distinguished from droplet nuclei that are transmitted some distance by air). Most sexually transmitted diseases are spread directly. In addition. infections that result from kissing, or bites by biological vectors are direct. Most obligate parasites are far too sensitive to survive for long outside the host and can be transmitted only through direct contact. Diseases transmitted vertically from mother to baby

fit in this contact

category also.

of Indirect Transmission

For microbes to be indirectly transmitte{ the infectious agent must pass from an infected host to an intermediate conveyor and from there to another host. This form of communication is especially pronounced when the infected individuals contaminate inanimate objects, food' or air through their activities. The transmitter of the infectious agent can

Routes

be either openly infected or a carrier.

Indirect Spread by Vehicles: Contaminated Materials The term vehicle specifies any inanimate material commonly used by humans that can transmit infectious agents' A common vehicle is a single material that serves as the source of infection for many individuals. Some specific types of vehicles are food, water, various biological products (such as blood" serum' andtissue), and fomites. A fomite is an inanimate object that harbors and transmits pathogens. The list ofpossible fomites is as long as your imagination allows. Probably highest on the list would be objects commonly in contact with the public such as doorknobs, telephones, push buttons, and faucet handles that are readily contaminated by touching. Shared bed linens, handkerchiefs, toilet seats, toys, eating utensils, clothing, personal articles, and syringes are other examples. Although paper money is impregnated with a disinfectant to inhibit microbes, pathogens are still isolated from bills as well as coins.

Outbreaks of food poisoning often result from the role of food as a common vehicle. The source of the agent can be soil, the handler, or a mechanical vector. Because milk provides a rich growth medium for microbes, it is a significant means of transmitting pathogens from diseased animals, infected milk handlers, and environmental sources of contamination. The agents of brucellosis, tuberculosis, Q fever, salmonellosis, and listeriosis are transmitted by contaminated milk. Water that has been contaminated by feces or urine can carty Salmonella, Vibrio (cholera), viruses (hepatitis A, polio), and pathogenic protozoans (Giardia, Cryptosporidium). In the fype of transmission termed the oral-fecal route, a fecal carrier contaminates food during handling, and an unsuspecting person ingests it. Hepatitis A, amebic dysentery shigellosis, and typhoid fever are often transmitted this way. Oral-fecal transmission can also involve contaminated materials such as toys and diapers. It is really a special category of indirect transmission, which specifies the way in which the vehicle became contaminated was through contact with fecal material and that it found its way to someone's mouth.

Indirect Spread by Vehicles: Air as a Vehicle Unlike soil and water, outdoor aif cannot provide nutritional support for microbial growth and is less likely to transmit airborne pathogens.

Figure 13.18

The explosiveness of a sneeze.

Special photography dramatically captures droplet formation in an unstifled sneeze. Even the merest attempt to cover a sneeze with one's hand will reduce this effect considerably. When such droplets dry and remain suspended in air, they are droplet nuclei.

On the other hand indoor air (especially in a closed space) can serve as an important medium for the suspension and dispersal of certain respiratory pathogens via droplet nuclei and aerosols. Droplet nuclei are dried microscopic residues created when microscopic pellets of mucus and saliva are ejected from the mouth and nose. They are generated forcefully in an unstifled sneeze or cough (figure 13.18) or mildly during other vocalizations. Although the larger beads of moisture settle rapidly, smaller particles evaporate and remain suspended for longer periods. Droplet nuclei are implicated in the spread ofhardier pathogens such as the tubercle bacillus and the influenza virus. Aerosols are suspensions of fine dust or moisture particles in the air that contain live pathogens. Q fever is spread by dust from animal quarters, and psittacosis by aerosols from infected birds. Outbreaks of coccidioidomycosis (a lung infection) often occur in areas rvhere soil is disturbedby wind or digging, releasing clouds of dust bearing the spores of Coccidioides.

Nosocomial Infections: The Hospital as a Source of Disease Infectious diseases that are acquired or develop during a hospital stay are known as nosocomial* infections. This concept seems incongruous at first thoughto because a hospital is regarded as a place to get treatrnent for a disease, not a place to acquire a disease. Yet it is not uncommon for a surgical patient's incision to become infected or a burn patient to develop a case of pneumonia in the clinical setting. The rate of nosocomial infections can be as low as 0.lo/o or as high as 20oh of all admitted patients depending on the clinical setting. The average rate is about 5%. In light of the number

* nosocomial (noltz"-oh-koh'-mee-a1) of. Originating from

a hospital or

Ctr.

nosos, disease, and komeion, to take carc

infirmary.

408

Chapter

13

Microbe-Human Interactions

of admissions, this adds up to 2 to 4 million cases a year, which result in nearly 90,000 deaths. Nosocomial infections cost time and money as well as suffering. By one estimate, they amount to g million additional days ofhospitalization a year and an increased cost of 5 to

10

billion dollars.

So many factors unique to the hospital environment are tied to nosocomial infections that a certainnumber of infections are virtually unavoidable. After all, the hospital both athacts and creates compromised patients, and it serves as a collection point for pathogens. Some patients become infected when surgical procedures or lowered defenses permit resident flora to invade their bodies. Other patients acquire infections directly or indirectly from fomites, medical equipment, other patients, medical personnel, visitors, air,

and water.

The health care process itselfincreases the likelihood that inwill be transferred from one patient to another. Treatments using reusable instruments such as respirators and endoscopes constitute apossible source ofinfectious agents. Indwelling devices such as catheters, prosthetic heart valves, grafts, drainage tubes, and tracheostomy tubes form a ready portal ofentry and habitat for infectious agents. Because such high numbers of the hospital population receive antimicrobial drugs during their stay, drug-resistant microbes are selected for at a much greater rate than

fectious agents

is the case outside the hospitat.

The most common nosocomial infections involve the urinary tract, the respiratory tract, surgical incisions, and the blood (sepsis)

(figure 13.19). Gram-negative intestinal flora (Escherichia coli.

Klebsiella, Pseudomonas) are cultured in more than half of patients with nosocomial infections. Gram-positive bacteria (staphylococci and streptococci) and yeasts make up most of the remainder. True

pathogens such as Mycobacterium tuberculosis, Salmonella, hepatitis B, and influenza virus can be transmitted in the clinical settins as well.

The potential seriousness and impact of nosocomial infections have required hospitals to develop committees that monitor infectious outbreaks and develop guidelines for infection control and aseptic procedures. Medical asepsls includes practices that lowerthe microbial load in patients, caregivers, and the hospital environment. These practices include proper hand washing, disinfection, and saritization, as well as patient isolation. Table 13.11 summarizes guidelines for the major types of isolation. The goal of these procedures is to limit the spread of infectious agents from person to person. An even higher level of stringency is seen with surgical asepsis, which involves all ofthe strategies listed previously plus ensuring that all surgical procedures are conducted under sterile conditions. This includes sterilization of surgical instruments, dressings, sponges and the like, as well as clothing personnel in sterile garments and scrupulously disinfecting the room surfaces and air.

Hospitals generally employ an infection control fficer v,rho not only implements proper practices and procedures throughout the hospital but also is charged with tracking potential outbreaks, identifying breaches in asepsis, and haining other health care workers in aseptic technique. Among those most in need of this training are nurses and other caregivers whose work, by its very nature, exposes them to needlesticks, infectious secretions, blood, and physical contact with the patient. The same practices that interrupt the routes ofinfection in the patient can also protect the health care worker. It is for this reason that most hospitals have adopted universal precautions that recognize that all secretions from all persons in the clinical setting are potentially infectious and that transmission can occur in either direction.

Universal Blood and Body Fluid precautions Medical and dental settings require stringent measures to prevent the spread of nosocomial infections from patient to patient, from pa-

tient to worker, and from worker to patient. But even with precautions, the rate of such infections is rather high. Recent evidence indicates that more than one-third of nosocomial infections could be prevented by consistent and rigorous infection control methods.

Previously, control guidelines were disease-specific, and clearly identified infections were managed with particular restrictions and techniques. With this arrangement, personnel tended to handle materials labeled infectious with much greater care than those that were not so labeled. The AIDS epidemic spurred

a reexamination of that policy. Because of the potential for increased numbers of undiagnosed HlV-infected patients, the Centers for Disease Control and Prevention laid down more-shingent guide-

lines for handling patients and body substances. These guidelines have been termed universal precautions (Ups), because they are based on the assumption that all patient specimens could harbor infectious agents and so must be treated with the same degree of care. They also include body substance isolation (BSI) techniques to be used in known cases ofinfection.

It is worth mentioning that these precautions are designed to protect all individuals in the clinical setting-patients, workers, and

Flgure 13.19 Most common Relative frequency by target area.

nosocomial infections.

the public alike. In general, they include techniques designed to prevent contact with pathogens and contamination and, if prevention is not possible, to take purposeful measures to decontaminate potentially infectious materials.

13.3

Sources and Transmission of Microbes

409

Levels of lsolation Used in Clinical Settings Type of lsolation*

Protective Measures**

To Prevent Spread of

Enteric Precautions

Gowns and gloves must be worn by all persons having direct contact with patient; masks

Diarrheal diseases; Shigella, Salmonella, and Escherichia coli gastroenteritis; cholera; hepatitis A; rotavirus; and giardiasis

are not required; special precautions are taken for disposing of feces and urine.

Respiratory Precautions

Private room with closed door is necessary; gowns and gloves are not required; masks

Tuberculosis, measles, mumps, meningitis, pertussis, rubella, chickenpox

are usually indicated; items contaminated

with secretions must be disinfected.

Drainage and Secretion Precautions

Gowns and gloves are required for all persons; masks are not needed; contaminated instruments and dressings require special

Staphylococcal and streptococcal infections; gas gangrene; herpes zoster; burn infections

precautions.

Strict lsolation

Private room with closed door is required; gowns, masks, and gloves must be worn by all persons; contaminated items must be

wrapped and sent to central supply for decontamination.

Reverse lsolation (Also Called

Protective lsolation)

Same guidelines as for strict isolation are required; room may be ventilated by

unidirectional or laminar airflow filtered through a high-efficiency particulate air (HEPA) filter that removes most

Mostly highly virulent or contagious microbes; includes diphtheria, some types of pneumonia, extensive skin and burn infections, disseminated herpes simpiex and zoster Used to protect patients extremely immunocompromised by cancer therapy, surgery, genetic defects, burns, prematurity, or AIDS and therefore vulnerable to opportunistic pathogens

airborne pathogens; infected persons must be barred. *Precautions are based upon the primary portal of entry and communicability ofthe pathogen.

r*in all

cases, visitors to the patient's room must report to the nurses'station before entering the room; all visitors and personnel must wash their hands upon entering and

leaving the room.

The universal precautions recommended for all health care settings follow.

1. Barrier precautions, including masks and gloves, should be taken to prevent contact of skin and mucous membranes with patients' blood or other body fluids. Because gloves can develop small invisible tears, double gloving decreases the risk further. For protection during surgery, venipuncture, or emergency procedures, gowns, aprons, and other body coverings should be worn. Dental workers should wear eyewear and face shields to protect against splattered blood and saliva. 2. More than 10% of health care personnel are pierced each year by sharp (and usually contaminated) instruments. These accidents carry risks not only for AIDS but also for hepatitis B, hepatitis C, and other diseases. Preventing inoculation infection requires vigilant observance of proper techniques. o All disposable needles, scalpels, or sharp devices from invasive procedures must immediately be placed in punctureproofcontainers for sterilization and final discard. o Under no circumstances should a worker attempt to recap a syringe, remove a needle from a syringe, or leave unprotected used syringes where they pose a risk to others. o Reusable needles or other sharp devices must be heatsterilized in a puncture-proofholder before they are handled.

o If a needlestick or other injury occurs, immediate attention to the wound, such as thorough degermation and application ofstrong antiseptics, can prevent infection. 3. Dental handpieces should be sterilized between patients, but if this is not possible, they should be thoroughly disinfected with a high-level disinfectant (peroxide, hypochlorite).

Blood and saliva should be removed completely from all contaminated dental instruments and intraoral devices prior to sterilization. 4. Hands and other skin surfaces that have been accidently contaminated with blood or other fluids should be scrubbed immediately with a germicidal soap. Hands should likewise be washed after removing rubber gloves, masks, or other

barrier devices.

5. Because saliva can be a source of some types of infections, barriers should be used in all mouth-to-mouth resuscitations. 6. Health care workers with active, draining skin or mucous membrane lesions must refrain from handling patients or equipment that will come into contact with other patients. Pregnant health care workers risk infecting their fetuses and must pay special attention to these guidelines. Personnel should be protected by vaccination whenever possible.

Isolation procedures for known or suspected infections should

still be instifuted on a case-by-case basis.

410

Chapter

l3

Microbe-Human Interactions

of diseases seen by the medical community and reported to public health authorities. By law, certain reportable, or notifiable, The primary habitat of a pathogen is called its reservoir. A human reservoir is also called a carrier.

Animals can be either reservoirs or vectors of pathogens. An infected animal is a biological vector. Uninfected animals, especially insects, that transmit pathogens mechanically are called mechanical

vectors. Soil and water are nonliving reservoirs for pathogenic bacteria, ptotozoa, fungi, and worms.

A communicable disease can be transmitted from an infected host to others, but not all infectious diseases are communicable. The spread of infectious disease from person to person is called horizontal transmission. The spread ofinfectious disease from parent to offspring is called vertical transmission. Infectious diseases are spread by either contact or indirect routes of transmission. Vehicles of indirect transmission include soil, water, foo4 air, and fomites (inanimate objects).

Nosocomial infections are acquired in a hospital from surgical procedures, equipment, personnel, and exposure to drug-resistant microorganisms.

'13.4 Epidemiology: The Study of Disease in Populations So far, our discussion has revolved primarily around the impact of an infectious disease in a single individual. Let us now turn our at-

tention to the effects of diseases on the community-the realm of epidemiology.* By definition, this term involves the study of the frequency and distribution ofdisease and other health-related factors in defined human populations. It involves many disciplinesnot only microbiology but also anatomy, physiology, immunology, medicine, psychology, sociology, ecology, and statistics-and it considers many diseases other than infectious ones, including heart disease, caf,rcer, drug addiction, and mental illness. The epidemiologist is a medical sleuth who collects clues on the causative agent, pathology, and sources and modes oftransmission and tracks the numbers and distribution of cases of disease in the community. In fulfilling these demands, the epidemiologist asks who, when, where, how, why, and what about diseases. The outcome of these studies helps public health departments develop prevention and treatment programs and establish a basis for predictions.

whq when,

and where? Tracking in the Population

Disease

Epidemiologists are concerned with all of the factors covered earlier in this chapter: virulence, portals of entry and exit, and the course of disease. But they are also interested in surveillance-that is, collecting, analyzing, and reporting data on the rates of occurrence, mortality, morbidity, and transmission of infections. Surveillance involves keeping data for a large number

diseases must be reported to authorities; others are reported on a

voluntary basis.

A well-developed network of individuals and agencies at the local, district, state, national, and international levels keeps track of infectious diseases. Physicians and hospitals report all notifiable diseases that are brought to their attention. Case reporting can focus on a single individual or collectively on group data. Local public health agencies first receive the case data and determine how they will be handled. In most cases, health officers investigate the history and movements of patients to trace their prior contacts and to control the further spread of the infection as soon as possible through drug therapy, immunization, and education. In sexually transmitted diseases, patients are asked to name their partners so that these persons can be notified, examined" and treated. It is very important to maintain the confidentiality of the persons in these reports. The principal government agency responsible for keeping track of infectious diseases nationwide is the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, which are a part of the United States Public Health Service. The CDC publishes a weekly notice of diseases (the Morbidity and Mortality Report) that provides weekly and cumulative summaries of the case rates and deaths for about 50 notifiable and 65 reportable diseases, highlights important and unusual diseases, and presents data concerning disease occurrence in the major regions of the United States. It is available to anyone at http://www. cdc.gov/mmwr/. Ultimately, the CDC shares its statistics on disease with the World Health Organization (WHO) for worldwide tabulation and control.

Epidemiological Stotistics: Frequency of Cases The prevalence of a disease is the total number of existing cases with respect to the entire population. It is usually reported as the percentage ofthe population having a particular disease over a given period, and it provides a long-term accumulative oopicture" of a disease. Disease incidence measures the number of new cases over a certain time period, as compared with the general healthy population. This statistic, also called the case, or morbidity, rate, indicates both the rate and the risk of infection at the time of reporting. The equations used to figure these rates follow. Total number

Prevalence:

lncidence

:

of

cases in population

Total number of persons in population

Number of new cases Total number of

x 100: %

(Usually reported per 100,000 persons)

susceptible persons

As an example, let us use a classroom of 50 students exposed

to a new strain of influenza. Before exposure, the prevalence and incidence in this population are both zero (0/50). If in one *

ep ide nt io

logy (ep'Lih-dee-mee-alh'-uh-gee) Gr. epidemios, prevalent.

week, 5 out ofthe 50 people contract the disease, the prevalence is

13.4

Epidemiology: The Study of Disease in Populations

Gonorrhea 400 325

Syphilis*

300

300

18

200

200

12

Chlamydia 400 347

4tl

24

4'Z\

100

100

:.:.::::

50.8

,:::,.'.

'87 '91 '95 '99 '03

0

'06

0

'87 r91 '95 '99 ',03 '06

,:,':

'87 ',91 '95 ',99 ',03 ',06

.Rate for primary and secondary syphilis, the earliest stag,es of the infection and the most intectious periods. Source:Centers for Disease Control and Prevention.

(a) Overall rates of chlamydia, gonorrhea and syphilis have risen for the second year in a raising concerns among public health officials. Rates per 100,000 people:

Men

population) Women 1,200 600 0 Age 0 600 1,2OO 1,800 2,400 3,000 Rate (per 100,000

3,000 2,400 1,800

10-14

11.6

2,862.7

20-24

856.9

2,797.0

9.1

25-29 30-34 35-39 40-44 45-54 55-64

6.8

2.8

65+

2.2

480.8 222.2 120.8 65.1

27.8

1,141.2 415.7 174.2 69.0 25.6

517.0

Total

173.4

-

121.5

15-19

545.1

(b) Chlamydia

roq

Age- and sex-specific rates: United States, 2006

NHE

I

Indicates human disease case(s).

MAE

F--l

Avian, animal, or mosquito infections.

R!E

cr!l NJE DEE MDIE

DCT

WVf

(c) West Nile virus

Figure 13.20

-

Activity by state, 2007

Graphical representation of epidemiological data.

The Centers for Disease Control and Prevention collect epidemiological data that are analyzed with regard to (a) time frame, (b) age and sex,

and (c) geographic region.

5150

:

l0o/o, and the incidence

is I in 10 (10 is used

of 100,000 due to smaller population).

If

students contractthe flu, the prevalence becomes 5

20Yo, and the incidence becomes

instead

after 1 week, 5 more

+5:

10/50

:

2 in 10. The 20% figure for

prevalence assumes that all l0 patients were still sick in the second week. If the first 5 patients were well by the time the second group

of five became ill, the prevalence at that second time point would be 5/50, or l0%o. But the incidence for the 2-week period would still be 2 in I0 (10 cases per 50). The changes in incidence and prevalence are usually followed over a seasonal, yearly, and long-term basis and are helpful in predicting trends (figure 13.20). Statistics of concern to the

Chapter

412

13

Microbe-Human Interactions

epidemiologist are the rates of disease with regard to sex, race, or

geographic region. Also of importance is the mortality rate, which measures the total number of deaths in a population due to a certain disease. Over the past century, the overall death rate from infectious diseases has dropped, although the morbidity* rate, or the number of persons afflicted with infectious diseases, has remained relatively high. Monitoring statistics also makes it possibie to define the frequency of a disease in the population. An infectious disease that exhibits a relatively steady frequency over a long time period in a particular geographic locale is endemic (figure l3.2la). Often, the reason for endemicity is the presence of a reservoir in that location. For example, Lyme disease is endemic to certain areas of the United States where the tick vector is found. A certain number of new cases are expected in these areas every year. When a disease is sporadic, occasional cases are reported at irregular intervals in random locales (figure 13,2lb). Tetanus and diphtheria are reported sporadically in the United States (fewer than 50 cases a year).

When statistics indicate that the prevalence of an endemic or sporadic disease is increasing beyond what is expected for that population, the pattern is described as an epidemic (figure 13.2lc). An epidemic exists when an increasing trend is observed in a particular population. The time period is not

CASE FILE

13

Wrop-Up

The encephalitis victims in Queens suffered from West Nile virus infection. lt was not initially suspected because until that time,

it had never

been seen in North America. Also, most viruses

spread by arthropods are confined to specific geographic areas because their vectors are confined to those areas. West Nile virus .|996 had been seen in France in 1962 and in Romania in but never in North America before 1999. Wild birds are the primary hosts in the wild. They can harbor the virus in most any climate and season. Mosquitoes serve as vehicles to carry the virus among avian and human hosts. The virus has developed spikes that provide entry into the cells of very different animals. They probably all share a receptor for viral attachment. The virus must also multiply in both coldand warm-blooded animals. The virus has exhibited other changes besides its geographic distribution. In its "native" locale of Africa and the Middle East, West Nile virus doesn't seem to cause overt disease in birds, although it infects large percentages of them. Something has changed the effects of the virus in the United States, because it is proving lethal to birds as well as to some of the humans it infects. See: lnsight 25.2. CDC, "Outbreak of West Nile-like Virol

Encephalitis-New

York, 1999," Morbidity and Mortality Weekly Report 48 (1999): 890-92.

, (mor-bih'-dih-tee) L. morbidis, sick. A condition of being

o

diseased.

Outbreaks

(a) Endemic Occurrence (Valley fever)

(b) Sporadic Occurrence

Figure

(Measles)

t3.21

(d) Pandemic Occurrence (AIDS)

Patterns of infectious disease occurrence. (a) In endemic occurrence, cases are concentrated in one area at a relatively stable rate. (b) ln sporadic occurrence, a few cases occur randomly over a wide area. (c) An epidemic is an increased number of cases that often appear in geographic clusters. The clusters may be local, as in the case of a restaurant-related food-borne epidemic, or nationwide, as is the cale with Chlomydio, (d) Pandemic occurrence means that an epidemic ranges over more than one continent.

13.4

ffii:1f;;il'11;til,,m,ffi

.

. . . . . . .

.

Acquired immunodeficiency

Botulism Brucellosis Chancroid Chlamydia trachomatis genital infections Cholera Coccidioidomycosis Cryptosporidiosis Cyclosporiasis Diphtheria Ehrlichiosis

. . . , . .

Powassan

.

Encephalitis/meningitis, St. Louis

Giardiasis

Listeriosis

Shigellosis

Gonorrhea

Lyme disease

Haemophilus influenzae invasive disease

Malaria

Streptococcal disease, invasive, group A

Enterohemorrhagic Escherichia coli

Meningococcal disease

Hantavirus pulmonary

Mumps

Hemolytic uremic syndrome Hepatitis, viral, acute Hepatitis A, acute Hepatitis B, acute Hepatitis B virus, perinatal

infection

. .

Measles

Hansen disease (leprosy)

. . .

Encephalitis/meningitis, Encephal itisimeningitis,

disease

Nile

syndrome

. .

California serogroup viral

.

Legionellosis

Encephalitis/meningitis, West

Encephalitis/meningitis,

eastern equine

Rubella, congenital syndrome

Encephalitis/meningitis, western equine

Encephalitis/meningitis, arboviral

.

HIV infection . HIV infection, adult (>13 years) . HIV infection, pediatric (

10 Cases

Figure

25.4

United States.

Hantavirus pulmonary syndrome cases,

Cumufative data through February 2007.

hemorrhagic fever, caused by a hantavirus and transmitted by ro_ dents. Other bunyaviruses are discussed in a later section along with other arboviruses because most of them are hansmitted by insects and ticks. The most important American bunyavirus is Sin Nombre (no name) hantavirus. The first cases came to light in 1993 in the Four Corners area of New Mexico, when a cluster of unusual cases was reported to the CDC. The victims were struck with high fever, lung edema, and pulmonary failure. Most of them died within a few days. Similar cases were reported sporadically inArizona, Colorado, and other western states (figure 25.4). The disease was termed hantavirus pulmonary syndrome

(HPS). Comprehensive studies of rodent populations have

revealed that the virus is carried by deer and harvest mice. It is probably spread by dried animal wastes that have become airborne when rodent nests are disturbed. The disease occurs sporadically at a rate of 25 to 50 cases per year but with a high (33Yo) mortality rate. Because of the widespread rodent reservoir and the potential for human contact, this is considered an emers_

ing disease. Four major arenavirus diseases are Lassa feveq found in parts of Africa; Argentine hemorrhagic fever, typified by severe weakening of the vascular bed, hemorrhage, and shock; Bolivian

Bunyaviruses and Arenaviruses Between them, the bunyavirus* and arenavirus* groups contain hundreds ofviruses whose normal hosts are arthropods or rodents. Although none of the viruses are natural parasites of humans, they can be transmitted zoonotically and periodically cause epidemics in human populations. Examples of important bunyavirus diseases are California encephalitis, spread by mosquitoes; Rift Valley fever, an African disease transmitted by sand flies; and Korean

hemorrhagic fever; and lymphocytic choriomeningitis, a widely distributed infection of the brain and meninges. The viruses are closely associated with a rodent host and are continuously shed by the rodent throughout its lifetime. Transmission of the virus to humans is through aerosols and direct contact with the animal or its excreta. These diseases vary in symptomology and severity from mild fever and malaise to complications such as hemorrhage, renal failure, cardiac damage, and shock syndrome. The

lymphocytic choriomeningitis virus can cross the placenta infect the fetus. Pathologic effects include hydrocephaly,

and * bunyavirus (bun'-yah-vy''-rus) From Bunyamwera, tlpe ofvirus was first isolated.

an area of Africa where this

+ arenavirus (ah-ree'-nah-r,y''-rus) L. arena, sand. In reference to the appearance virus particles in electron micrographs.

of

blindness, deafness, and mental retardation. So dangerous are these viruses to laboratory workers that the highest level of containment procedures and sterile techniques must be used in handling them.

Life in the Hot Zone over the last several decades, many of mankind's most virulent viral few diseases have been vanquished. cases of polio are restricted to a

(biosmall areas of the globe, and smallpox has been eliminated entirely are overshadowed successes terrorism worries nofivithstanding). Yet these by an emerging viral threat'

Expertshavebeenwarningusabouttheemergenceofinfectious predicdiseases for quite some time. Arrd nature has measured up to these

in tions, delivering numerous outbreaks of diseases into a world already turmoil. Many of these diseases are viral and many are zoonotic, becoming even more virulent in a human host. Increased cases have been docu,nlnr.d for well-known viruses such as influenza. for viruses that have of experienced changes in host or distribution. and for entirely new types possibility the are viruses emerging these by viruses. The fears fostered of amplification in the population leading to rapid global transmission and the unknown. uncontrollable nature oftheir origins' Among the most dramatic of these outbreaks are

Red

Cros woikers prepare an Ebola virus victim for burial in Zaire, using

high-level containment procedures. Thousands of Africans have died from

eTheWestNilefevervirus,firstspreadfromAfricatotheMiddle East and North America. possibly by migrating birds or with hitch-

hiking mosquitoes carried by travelers' The disease is now well

o o

entrenched in America. Figure I 3.20 presents the current statistics (also see Insight 25'2.1. A worldwide epidemic of a new coronavirus (tlrc SARS virus) that first

appearedinChinain2002andquicklyspreadtoseveralcontinents. virus is no* thought io u" no* wild fruit bats' ii'.

"G^

"irne The rise of monkeypox virus, a virus of rodents related to smallpox. ItbrokeoutfirstinAfricaandthensuddenlyappearedinmidwestern America in the late spring of2003' lt appears to have been carried by imported pet store animals from Africa'

o

newly emerging influenza virus (H5N I ) that has spread Virus' page 753)' lsee A Note about the Avian Influenza

A threat from

;; ;;r".

a

Becauseofthefudeadlypotentia|,agreatdealofattentionhasbeen focused on viruses dubbed "hot agents." These viruses are so lethally infectious that they cannot be safely handled without level 4 biosafety precautions. one of these-the Ebola virus, named for ariver tnzafieis fraught with particular dangers. This virus and its German relative, the Marburg virus. belong to a category of viruses calledJ? I ov i ruses, characterized by a unique. thready appearance ( see table 25' I )' The Ebola virus '(HoI zone." has furious virulence, as described in this account from the

In this they are like HIV which also destroys the immune system' but unlike the onset of HIY the attack by Ebola is explosive' As

:' i:i

,.

The enveloped, segrnented, single-stranded RNA virus group includes the Orthomyxoviridae, the Bunyaviridae, and the Arenaviridae' Orthomyxoviruses such as the influenza A (flu) virus undergo genetic changes that can create new strains and cause failure in immunity. In antigenic drift, small mutations accumulate' In antigenic shift, two genetic variants of the flu virus infect a host and combine

to form a third genetically different strain' Like all enveloped RNA viruses, influenza viruses have two unique glycoprotein virulence factors (receptors) in the envelope, hemaggt"ti"in and neuraminidase. These assist in viral penetration and

Ebola sweeps through you, your immune system fails, and you seem to lose your ability to respond to viral attack'''' Your mouth bleeds' and you bleed around your teeth ' ' ' literally every opening in the bodv bleeds. no matter how small. Ebola does in ten days what it takes AIDS ten Years to accomPlish.

ThisdiseasehaseruptedseverallimessincelgT6.Therecentisolation of this virus in bats is strong evidence of a wild animal reservoir. It

isspreadthroughcontactwithbodyfluids'Withnoeffectivetreatments. the mortality rate is 80% to 90%. Another microbiological mystery involves an unusual agent that causes disbovine spongiform encephalopathy (BSE), or "mad cow disease." This transmissible among cows and sheep. causes overwhelming deterioration of the brain, frenzy. and death' A new variant of human creutzfeldt-Jakob disease was traced to consumption offood products con-

.ur.,

*ti.h;r

ffom taminated with the bovine agent. Nearly 200 people in Europe have died millions that epidemic a massive for concem was the great So this disease. ofcattle and sheep were ordered destroyed and incinerated. Experts in infecto tiny tious diseases anribute these and other transmissible brain infections meats' contaminated from protein particles (.prions) that may be acquired

for the increase in new and old Answer available at http://www'mhhe'com/

Suggest some reasons to account

,ooiori. viral infection

s,

talaroT

release from the host cell. Infection damages the respiratory epi-

thelium and may lead to pneumonia. Control is via drugs that shorten the duration of the disease, or yearly vaccination, which prevents infection. Bunyaviruses are grouped with the arboviruses because many ofthem are spread by insects. Specific diseases caused by bunyaviruses include hantavirus pulmonary syndrome and California encephalitis'

Arenaviruses are typically spread by rodents. They also cause fatal encephalitis and/or hemorrhagic fevers such as Lassa fever and hemorrhagic fever.

755

756

Chapter

25

The RNA Viruses That Infect Humans

Giant cell

Point of cell fusion (b)

Figure

25.5

The effects of paramyxoviruses.

(a) when they infect a host cell, paramyxoviruses induce the cell membranes of adiacent cells to fuse into large multinucleate giant cells, or syncytia (1,200x)' (b) This fusion allows direct passage of viruses from an infected cell to uninfected cells. Through this means, the virus evades antibodies.

??.2 Enveloped Nonsegmented Single-Stranded RNA Viruses Paramyxoviruses The important human paramyxoviruses are paramyxovirus (gtal;rin_ fluenza and mumps viruses), Morbilliviras (measles virus), and Pneamovirus (respiratory syncytial virus), alr of which are readily transmitted through respiratory droplets. The envelope of a para_ myxovirus possesses specialized grycoprotein spikes that initiate attachment to host cells. They also bear fusion (F) spikes that initi-

ate the cell-to-cell fusions typical of these viruses (figure 25.5). A series of multiple cell fusions produces a syncytium,* or multi_ nucleate giant cell with cytoplasmic inclusion bodies, cytopathic effect that is useful in diasnosis.

Epidemiology ond pothology of porainfluenzo One type of infection by paramlncovirus, calledparainfluenza, is influenza but usually more benign. It is spread by droplets and respiratory secretions that are inhaled or inoculated into the mucous membranes by contaminated hands. parainfluenzal respiratory disease is seen most frequently in children, most of whom have been infected by the age of 6. Newborns lacking passive as widespread as

* syncytiunt (sin-sish'-yum) Gr. syn, togethe\ arfi lrytos, cell.

antibodies are particularly susceptible, and they develop more se_ vere symptoms. The usual effects of parainfluenza are minor upper respiratory symptoms (a cold), bronchitis, bronchopneumonia, and

laryngotracheobronchitis (croup). Croup* manifests as labored and noisy breathing accompanied by a hoarse cough that is most cornmon in infants and young children.

Diagnosis, Treatment, and prevention of parainfluenza

Presenting symptoms typical of a cold are often sufficient to pre_ sume respiratory infection of viral origin. Determining the actual

viral agent, howeveq is difficult and usually unnecessary in older

children aad adults whose infection is usually self-limited and benign. Primary infection in infants can be severe enough to be life_ threatening. So far, no specific chemotherapy is available, but supportive treatment with immune serum globulin or interferon can

be ofbenefit.

Mumps: Epidemic Parotitis Another infection caused by paramyxovirus is mumps (old English for lump or bump). So distinctive are the pathologiCfeatures oithis disease that Hippocrates clearry characteized, it several hundred years BC as a self-limited, mildly epidemic illness associated with painful swelling at the angle of the jaw (figure 25.6). Also called * croup (kroop) Scot. lmtpan, to cry aloud.

25.2

Enveloped Nonsegmented Single-Stranded RNA

Viruses

757

Compfications in Mumps ln

20o/o to 30o/o of young adult the epididymis and testis, usuin localizes males, mumps infection of orchitis and epidiresultant syndrome The ally on one side only. permanent damage usually painful, no but dymitis may be rather sterilization causes readily popular that mumps belief occurs. The to the contrary. evidence medical held, despite of adult males is still that conthe tenderness by reinforced has been Perhaps this notion testis partial of one atrophy the and by infection tinues long after that occurs in about half the cases. Permanent sterility due to mumps is very rare.

Diagnosis, Treatment, and Prevention of Mumps Mumps can be tentatively diagnosed in a child with swollen parotid glands and a known exposure 2 or 3 weeks previously. Because swelling is not always present and the incubation period can range from 7 to 23 days, apractical diagnostic alternative is to test the serum by a direct fluorescent or ELISA method. The general pathology of mumps is mild enough that symptomatic treatment to relieve fever, dehydration, and pain is usually adequate. A live, attenuated mumps vaccine (paxt of the MMR vaccine) is given routinely at 12 to 15 months of age followed by at least one booster. A separate single vaccine is available for adults who require protection. Although the antibody titer is lower than that produced by wild mumps virus, protection usually lasts at least a decade.

M easles : M orbi I I ivirus I nfection

Flgure 25,6

The external appearance of swollen parotid glands in mumps (parotitis). Usually both sides are affected, though parotitis affecting one side (as shown here) occasionally develops.

epidemic parotitis, this infection typically targets the parotid salivary glands, but it is not limited to this region. The mumps virus bears morphological and antigenic characteristics similar to the parainfluenzavirus and has only a single serological type.

Epidemiology and Pathology of Mumps Humans are the exclusive natural hosts for the mumps virus. It is communicated primarily through salivary and respiratory secretions. Infection occurs worldwide, with epidemic increases in the late winter and early spring in temperate climates. High rates of infection arise among crowded populations or communities with low levels of immunity. Most cases occur in children under the age of 15, and as many as 40o/o arc subclinical. Because lasting immunity follows any form of mumps infection, no long-term carrier reservoir exists in the popu-

lation. The incidence of mumps has been reduced in the United States to around 300 cases per year.

After an average incubation period of 2 to 3 weeks, symptoms of fever, nasal discharge, muscle pain, and malaise develop. These may be followed by inflammation of the salivary glands (especially the parotids), producing the classic gopherlike swelling of the cheeks on one or both sides that can induce considerable discomfort (see figure 25.6).The virus can multiply in other organs, especially the testes, ovaries, thyroid gland, pancreas, meninges, heart, and kidney. Despite the invasion of multiple organs, the prognosis for most infections is complete, uncomplicated recovery with permanent immunity.

Measles, an acute disease causedby Morbillivirus, is also known as red measles* and rubeola.* A somewhat similar disease-rubella or German measles-is caused by an unrelated togavirus (section 25.3). Some differentiating criteria for these two forms axe summarized in table 25.3. Despite a tendency to think of red measles as a mild childhood illness, several recent outbreaks have reminded us that measles can kill babies and young children and that it is a frequent cause of death worldwide.

Epidemiology of Measles Measles is one of the most contagious infectious diseases, transmitted principally by respiratory aerosols. There is no reservoir other than humans, and a person is infectious during the periods of incubation, prodromium, and the skin rash but not usually during convalescence. Epidemic spread

is favored by crowding, low levels of herd immunity, malnutrition, and inadequate medical care. Occasional outbreaks of measles have been linked to the lack of immunization in children or the failure of a single dose of vaccine in many children. Only relatively large, dense populations ofsusceptible individuals can sustain the continuous chain necessary for transmission. In the United States, the incidence of measles is sporadic, usually less than 100 cases per year.

Infection, Disease, and Complications of Measles

The

measles virus invades the mucosal lining of the respiratory tract during an incubation period of nearly 2 weeks. The initial symptoms are sore throat, dry cough, headache, conjunctivitis, lymphadenitis,

* measles (mee' -z1z) I)fich maselen, spotted. * mbeola (roo-bee'-ohlah) L. rubex red.

Chapter

758

25

Measles German

Measles

The RNA Viruses That Infect Humans

Synonyms

Etiology

Primary Patient

Complications

Skin Rash

Koplik's Spots

Rubeola, red measles

Paramyxovirus:

child

SSPE,* pneumorua

Present

Present

Rubella, 3-day

Togavirus:

Child/fetus

Congenital

Present

Absent

Morbillivirus

measles

Rubivirus

defects**

*Subacute sclerosing panencephalitis.

**When transmitted in utero.

and fever. In a short time, unusual oral lesions called Koplik's spots appear as a prelude to the characteristic red maculopapular

exanthem* that erupts on the head and then progresses to the trunk and extremities, until most of the body is covered (figure 25.7).The rash gradually coalesces into red patches that fade to brown.

A small number of children develop laryngitis, bronchopneumonia, and bacterial secondary infections such as otitis media and sinusitis. Children afflicted with leukemia or thymic deficiency are especially predisposed to pneumonia because of their lack of the natural T:cell defense. Undernourished children may have severe diarrhea and abdominal discomfort, which add to their debilitation. The most serious complication is subacute sclerosing panencephalitis (SSPE),* a progressive neurological degeneration ofthe cerebral cortex, white matter, and brain stem. Its incidence is approximately one case in a million measles infections, and it afflicts primarily male children and adolescents. The pathogenesis of SSpE appears to involve a defective virus, one that has lost its ability to form a capsid and be released from an infected cell. Instead" it spreads unchecked through the brain by cell fusion, gradually destroys neurons and accessory cells, and breaks down myelin. The disease is known for profound intellectual and neurological impairment. The course of the disease invariably leads to coma and death in a matter of months or years. Diagnosis, Treatment, and Prevention

of Measles

The

patient's age, a history ofrecent exposure to measles, and the season of the year are all useful epidemiological clues for diagnosis. Clinical characteristics such as dry cough, sore throat, conjunctivitis, lymphadenopathy, fever, and especially the appearance ofKoplik's spots and a rash are presumptive of measles. Treatment relies on reducing fever, suppressing cough, and replacing lost fluid. Complications require additional remedies to relieve neurological and respiratory symptoms and to sustain nutrient, electrolyte, and fluid levels. Therapy includes antibiotics for bacterial complications and doses of immune globulin. Vaccination with attenuated viral vaccine administered by subcutaneous injection achieves immunity that persists for about 20 years. It must be stressed that because the virus is live, it can cause an atypical infection sometimes accompanied by a rash and fever. Measles immunization is recommended for all healthy children at the age of 12 Io 15 months (MMR vaccine, with mumps and

't'

cxunthant (eg-zan'-thum) Gr. exanthema, to bloom or flower. An eruption or rash

ofthe skin. 't .sttbttc tr/c ,vc:lerosing punertt t:1thulir,! (sub-uh-kewt' sklair-oh'-sing pan" -en-cef" Gr. skleros, hard, pan, all, and, enkephalos, brain.

-uh-ly'{is)

rubella), and a booster is given before a child enters school. A single antigen vaccine (Meruvax) is also available for older patients who require protection against measles alone. As a general rule, anyone who has had the measles is considered protected.

CASE HLE

25

wrap-t)p

..::-aaaaaa-::a:

The baby from China described at the beginning of the chapter was sick with measles. Hospital staff looked inside the baby,s

mouth for Koplik's spots. Finding these spots provides some assurance that, among all the maculopapular diseases, measles is

the likely diagnosis.

Although most cases are uncomplicated, babies may develop pneumonia, diarrhea, and secondary infections. The most serious long-term condition is a fatal brain disease called subacute sclerosing panencephalitis (SSPE).

The U.S. incidence of measles is low (usually less than 100 per year), but sporadic outbreaks do occur, even among people who were fully vaccinated as children, probably due to waning of their artificially acquired active immunity. This case highlights the need for constant vigilance and continued immunization, especially as the world "shrinks" and we come in contact with people from other parts of the world with access

less

to immunization.

See: CDC. "Measles Outbreak omong lnternationalty Adopted Chitdren Arriving in the United States, February-Morch 2001." Morbidity and Mortality Weekly Report 51 (2003): 1.115-16.

Respirotory Syncytiol Virus

: RSV I nfections

As its name indicates, respiratory syncytial virus (RSV)' also called Pneumovirus, infects the respiratory tract and produces giant multinucleate cells. Outbreaks of droplet-spread RSV disease occur regularly throughout the world, with peak incidence in the winter and early spring. Children 6 months of age or younger are especially susceptible to serious disease ofthe respiratory tract. Approximately 5 in 1,000 newborns are affbcted" making RSV the most prevalent cause of respiratory infection in this age group. An estimated 100,000 children are hospitalized with RSV infection each year in the United States. The mortality rate is highest for children who have complications such as prematurity, congenital disease, and immunodeficiency.

25.2

Enveloped Nonsegmented Single-Stranded RNA Viruses

759

include acute bouts ofcoughing, wheezing, dyspnear* and abnormal breathing sounds (rales). Adults and older children with RSV infection can experience cough and nasal congestion but are frequently asymptomatic.

Diagnosis, Treatment, and Prevention of RSV Diagnosis of RSV infection is more critical in babies than in older children or adults. The afilicted child is conspicuously ill, with signs typical of pneumonia and bronchitis. The best diagnostic procedures are those that demonstrate the viral antigen directly from specimens using direct and indirect fluorescent staining, ELISA testing, and Koplik's spots

DNA probes. Some newer treatments include Synagis, a monoclonal antibody that blocks viral attachment to cells, and RSV immunoglobulin obtained from people with high RSV antibody titers. Both can

greatly reduce complications and the need for hospitalization. An antiviral drug, ribavirin (Virazole), can also be administered as an inhaled aerosol. Supportive measures include drugs to reduce fever, providing ventilation, and treatment for secondary bacterial infection ifpresent.

Rhabdoviruses The most conspicuous rhabdovirus* is the rabies* virus, genus Lyssavirus.* The particles of this virus have a distinctive bulletlike appearance, round on one end and flat on the other. Additional features are a helical nucleocapsid and spikes that protrude through the envelope (figure 25.8). The family contains approximately 60 different viruses, but only the rabies lyssavirus infects humans'

Epidemiology of Robies Rabies is a slow, progressive zoonotic disease characteizedby a fatal meningoencephalitis. It is distributed nearly worldwide, ex-

cept for 34 countries that have remained rabies-free by practicing rigorous animal control. The primary reservoirs of the virus are wild mammals such as canines, skunks, raccoons, badgers, cats, and bats. These can spread the infection to domestic cats and dogs. Cats are so important in spreading the virus that most states require vaccination of pet cats as well as dogs. Both wild and domestic mammals can spread the disease to humans through bites, scratches, and inhalation of droplets. The annual worldwide total for human rabies is estimated at about 30,000 cases, but only a tiny number of these cases occur in the United States. Most U.S. cases of rabies

Flgure 25.7

Signs and symptoms of measles. (a) Koplik's spots, named for the physician who described them in the 1800s, are tiny white lesions with a red border that form on the inside of the mouth adiacent to the molars. (b) Appearance of measles rash' Individual lesions are flat or slightly raised bumps (maculopapular) distributed over most of the body.

The epithelia of the nose and eye are the principal portals of entry, and the nasopharynx is the main site of RSV replication' The first symptoms of primary infection are fever that lasts for 3 days, rhinitis, pharyngitis, and otitis. Infections ofthe bronchial tree and lung parenchyma give rise to symptoms of croup that

occur in wild animals (about 5,000-6,000 cases per year), while dog rabies has declined. The epidemiology of animal rabies in the United States varies with geographic region and vector. The most common wild animal reservoir hosts have changed from foxes to skunks to raccoons' Regional differences in the dominant reservoir also occur. Bats,

skunks. and bobcats are the most cornmon carriers of rabies in California, raccoons are the predominant carriers in the East, and coyotes dominate in Texas (figure 25.9). * dyspnea (dysp'-nee-ah) Gr. dyspnoia, difficulty in breathing'

* rhabdovims (rab'-doh-f'-rus) Gt. rhabdos, rod. In reference to its bullet, or bacillary, form. a rabies (ray'-beez)L. rabidus, rage or fury. * Lyssavirus (lye'-suh-vy''-rus) Ctr. lyssa, madness.

Chapter

760

25

The RNA Viruses That Infect Humans

Matrix protein Nucleocapsid

Figure

25.E

The structure of the rabies virus.

(a) Color-enhanced virion shows internal serrations, which represent the tightly coiled nucleocapsid (36,700x). (b) A schematic model of the virus, showing its major features.

Frsure 2s.ro

;;";;:,;':'11",ff'ff::

After an animal bite, the virus spreads to the nervous system and multiplies. From there, it moves to the salivary glands and other organs.

replicates in the salivary glands and is shed into the saliva. Clinical rabies proceeds through several distinct stages and inevitably ends in death.

Clinicol

Phoses

of Robies

is I to 2 months or more, depending upon the wound site, its severity, and the in_ oculation dose. The incubation period is shorter in facial, scalp, or neck wounds because of greater proximity to the brain. The prodromal phase begins with feveq nausea, vomiting, headache, fatigue, and other nonspecific synptoms. Some patients continue to experience pain, burning, prickling, or tingling sensations at the wound site. The average incubation period of rabies

@MlSkunk @ Raccoon

lFox

ECoYote

Flgure 25,9 Distribution of rabies in the United Rabies is

States. found in 10 distinct geographic areas. In each area, a

particular animal is the reservoir as illustrated by four different colors. Not shown is the occurrence of insectivorous bats that cause sporadic cases of rabies in wild animals throughout the country.

lnfection ond Diseqse Infection with rabies virus typically begins when an infected animal's saliva enters a puncture site (figure 25.10). Occasionally, the virus is inhaled or inoculated orally. The virus remains up to a week at the trauma site, where it multiplies. It then gradually enters nerve endings and advances toward the ganglia, spinal cord, and brain. Viral multiplication throughout the brain is eventually followed by migration to such diverse sites as the eye, heart, skin, and oral cavity. The infection cycle is completed when the virus

In the furious phase of rabies, the first acute signs of neurological involvement are agitation, disorientation, seizures, and twitching. Spasms in the neck and pharyngeal muscles lead to severe pain upon swallowing. As a result, attempts to swallow or even the sight of liquids brings on hydrophobia (fear of water). Throughout this phase, the patient is fully coherent and alert. When rabies enters the dumb phase, a patient is not hyperactive but paratyzed, disoriented, and stuporous. Ultimately, both forms, when untreated. progress to the coma phase, resulting in death from cardiac or respiratory arrest. In the past, humans were never known to survive a full-blown case of rabies. But recently, three patients recovered after receiving intensive, long-term treafinent.

Diognosis ond Management

of

Robies

When symptoms appear after a rabid animal attack, the disease is readily diagnosed. But the diagnosis can be obscured when contact with an infected animal is not clearly defined or when symptoms

25.3

Other Enveloped RNAViruses: Coronaviruses, Togaviruses, and Flaviviruses

are absent or delayed. Anxiety, agitation, and depression can pose as a psychoneurosis; muscle spasms resemble tetanus; and encephalitis with convulsions and paralysis mimics a number of other viral infections. Often the disease is diagnosed at autopsy. Criteria

indicative of rabies are intracellular inclusions (Negri bodies) in nervous tissue, isolation rabies virus from saliva or brain tissue, and demonstration of rabies virus antigens in specimens of the brain, serum, cerebrospinal flui4 or cornea using immunofluorescent methods.

Rabies Prevention and

Control A bite from a wild

or stray

animal demands assessment of the animal, meticulous care of the wound, and a specific treatment regimen. A wild mammal, especially a skunk, raccoon, coyote, or bat that bites without provocation, is presumed to be rabi{ and therapy is immediately commenced. If the animal is captured, brain samples and other tissue are examined for verification of rabies. Healthy domestic animals are observed closely for signs of disease and sometimes quarantined. Preventive therapy is initiated ifany signs ofrabies appear. After an animal bite, the wound should be scrupulously washed with soap or detergent and water, followed by debridement and application ofan antiseptic such as alcohol or peroxide. Rabies is one of the few infectious diseases for which a combination of passive and active postexposure immunization is indicated and successful. Initially the wound is infused with human rabies immune globulin (HRIG) to impede the spread of the virus, and globulin is also injected intramuscularly to provide immediate systemic protection. A full course of vaccination is started simultaneously. The current vaccine of choice is the human diploid cell vaccine (HDCV). This potent inactivated vaccine is cultured in human embryonic fibroblasts. The routine in postexposure vaccination entails intramuscular or intradermal injection on the lst, 3rd,7lh, l4th,28th, and 60th days, with two boosters. High-risk groups such as veterinarians, animal handlers, and laboratory personnel should receive three doses to protect against possible exposure. Control measures such as vaccination of domestic animals, elimination of strays, and strict quarantine practices have helped reduce the virus reservoir. In recent years, the United States and other countries have utilized a live oral vaccine made with a vaccinia virus that carries the gene for the rabies virus surface antigen' The vaccine has been incorporated into bait (sometimes peanut butter sandwiches!) placed in the habitats of wild reservoir species such as skunks and raccoons.

€

€

s

The enveloped" nonsegmented, single-stranded group of RNA viruses includes the Paramyxoviridae and the Rhabdoviridae. Paramyxoviruses cause respiratory infections characterizedby the formation of multinucleate giant cells. Paramyxovirus is the cause of parainfluenza and mumps; Mo rbillivirus causes red measles; and Pneumovirus is the agent in respiratory syncytial disease. All are transmitted through respiratory droplets. The most serious of the frhabdoviridae is the Lyssavirus, which causes rabies, a slow progressive, usually fatal, zoonotic disease of the CNS acquired by bites and other close bontact with infected mammals.

761

25.3 Other Enveloped RNA Viruses: Coronaviruses, Togaviruses, and Flaviviruses Coronaviruses Coronavirusesl are relatively large RNA viruses with distinctive, widely spaced spikes on their envelopes. These viruses are cofilmon in domesticated animals and are responsible for epidemic respiratory; enteric; and neurological diseases in pigs, dogs, cats, and poultry. Thus far, three types of human coronaviruses have been characterized. One of these is an etiologic agent of the common cold (see Insight 25.4).The same virus is also thought to cause some forms of viral pneumonia and myocarditis. Another human coronavirus is very closely associated with the intestine and may have a role in some human enteric infections.

Severe Acute Respi ratory Syn d rome-Associoted

Coronovirus The third coronavirus is the agent of a newly emerging disease. Up until recently, most coronaviruses were considered mildly pathogenic and the infections tended to be relatively innocuous in healthy persons. ln 2002, reports of an acute respiratory illness started to filter in fromAsia. The disease was given the name SARS for severe acute respiratory syndrome. By ear$ 2003, the World Health Organization issued a global health alert about the new illness. In a short time, scientists had sequenced the entire genome of the causative virus and made diagnostic tests possible. The epidemic was contained by mid-year, but in less than a year it had sickened more than 8,000 people. About 9% of those died. Most cases were concentrated in China and Southeast Asia. Several dozen countries, from Australia and Canada to the United States, reported cases, many of which originated in people who had traveled to Asia or had contact with travelers. Close contact (direct or droplet) is involved in its transmission. Eventually, no further cases were reported and the virus had evidently disappeared. The most recent research has linked its origin from fruit bats in Southeast Asia. Symptoms begin with a fever of above 38"C (100.4'F) and progress to body aches and an overall feeling of malaise. Early in the infection, there seems to be little virus in the patient and a low probability of transmission. Within a week, viral numbers surge and transmissibility is very high. After 3 weeks, viral levels decrease

significantly and symptoms subside. Patients may or may not experience classical respiratory symptoms, but severe cases ofthe illness can result in respiratory distress and death. Diagnosis of the disease relies first on exclusion of other likely agents, using a Gram stain (for bacterial pneumonia) and identification of influenza and RSV viruses. Acute and convalescent sera should be collected to document a rise in antibodies against the coronavirus. Specimens can be sent to reference labs where PCR will be performed to confirm the diagnosis. There is no specific treatment other than supportive care.

l.

Named for the resemblance of the viral spikes to a crown.

762

Chapter

25

The RNA Viruses That Infect Humans

Rubivirus: The Agent

of Rubella

Togaviruses are nonsegmented, single-stranded RNA viruses with a loose envelope.' There are several important members, including Rubivirus, the agent of rubella, and certain arboviruses. Rubella, or German measles, was first recognized as a distinct clinical entity in the mid-1Sth century and was considered a benign childhood disease until its teratogenic effects were discovered. It was first observed that cataracts often developed in neonates born to mothers who had contracted rubella in the first trimester. Epidemiological investigations over the next generation confirmed the link between rubella and many other congenital defects as well.

Epidemiology of Rubello Rubella is an endemic disease with worldwide distribution. Infection is initiated primarily through contact with respiratory secretions. The virus is shed during the prodromal phase and up to a week after the rash appears. Because the virus is only moderately communicable, close living conditions are required for its spread. Although epidemics and pandemics of rubella once regularly occurred in 6- to 9-year cycles, the introduction of vaccination has essentially stopped this pattern in the United States. Most cases are reported among adolescents and young adults in military training camps, colleges, and summer camps. The greatest concern is that nonimmune women of childbearing age might be caught up in this cycle, raising the prospect of congenital rubella.

lnfection ond Disease TWo forms of rubella can be distinguished: Postnatal infection develops in children or adults, and congenital (prenatal) infection of the fetus is expressed in the newborn as various types ofbirth defects.

Postnatal Rubella During an incubation period of 2 to 3 weeks, the virus multiplies in the respiratory epithelium, infiltrates local lymphoid tissue, and enters the bloodstream. Early symptoms include malaise, mild fever, sore throat, and lymphadenopathy. The rash of pink macules and papules first appears on the face and progresses down the trunk and toward the extremities, advancing and resolving in about 3 days. Adult rubella is often accompanied by joint inflammation and pain rather than a rash. Except for an occasional complication, posfiratal rubella is generally mild and produces lasting immunity.

Congenital Rubella Transmission of the rubella virus to a fetus in utero can result in a serious complication called congenital rubella (figure 25.11), The mother is able to transmit the virus even if she is asymptomatic. Fetal injury varies according to the time of infection. It is generally accepted that infection in the first trimester is most likely to induce miscarriage or multiple permanent defects in the newborn such as cardiac abnormalities, ocular lesions, deafness, and mental and physical retardation. Less drastic sequelae that usually resolve in time are anemia, hepatitis, pneumonia, carditis, and bone infection. 2. Reminiscent of

a toga.

Figure 25.11 An infant born with congenital rubella can manifest a papular and confluent rash. Courtesy Kenneth Schiffer, from AJDC118:25, luly 1969. Medical Association.

@

American

Diagnosis of Rubello Rubella mimics other diseases and is often asymptomatic, so it should not be diagnosed on clinical grounds alone. The confirmatory methods of choice are serological testing and virus isolation systems. IgM tests can determine recent infection, and a rising titer is a clear indicator of continuing rubella infection. Several serological tests for IgM are used, including complement fixation, ELISA, fluorescent assay, and hemagglutination. The latex-agglutination card, a simple, miniatwized test, is a more rapid method for IgM assay.

Prevention of Rubella Posfiratal rubella is generally benign and requires only symptomatic treatrnent. Because no specific therapy for rubella is available, most

control efforts are directed at maintaining herd immunity with attenuated rubella virus vaccine. This vaccine is usually given to children in the combined form (the MMR vaccination) at 12 to 15 months and a booster at 4 or 6 years of age. One complication regarding this method ofprotection is that the rubella vaccine does not always provioe lasting immunity. Because congenital rubella is such serious concern, testing of adult women is often recommended to confirm prior infection and protective antibodies. The current recommendation for nonpregnant, antibody-negative women is immediate immunization. Because the vaccine contains live virus and a teratogenic effect is possible, sexually active women must utilize contraception for 3 months after the vaccine is administered. Antibody-negative pregnant women should not be vaccinated and must be monitored for rubella infection.

Hepatitis C Virus Hepatitis C is

a

type of hepatitis caused by an RNA virus called a

flavivirus. Hepatitis C is sometimes referred to as the ,.silent epidemic" because more than 4 million Americans are infected with the virus, but it takes many years to cause noticeable symptoms. In the United States, epidemiologists estimate that at least 35,000 new

infections occur every year. Liver failure from hepatitis C is one of the most common reasons for liver transplants in this country. See table 24.3 for a complete comparison of the viral hepatitides.

25.4 Transmission and EpidemiologY This virus is acquired in ways similar to hepatitis B (HBV). It is most commonly transmitted through blood contact such as in blood transfusions or needle sharing by injecting drug users. It may occasionally be acquired through transfer of other body fluids or across the placenta. Unlike HB! sexual contact is not a major mode of transmission for HCV

Pothogenesis ond Virulence Foctors Hepatitis C virus is so adept at establishing chronic infections that researchers are studying the ways that it evades immunologic detection and destruction. The virus's core protein seems to play a role in the suppression of cell-mediated immunity as well as in the production of various cytokines. Before a test was available to test blood products for this virus, it seems to have been frequently transmitted through blood transfusions. Hemophiliacs who were treated with clotting factor prior to 1985 were infected at a high rate with HCV Once blood began to be o'non-A non-B" hepatitis, tested for HIV and screened for so-called the risk of contracting HCV from blood was greatly reduced. The current risk for transfusion-associated HCV is thought to be 1 in I 00,000 units transfused. Because HCV was not recognized sooner, a relatively large percentage of the population is infected. Eighty percent of the 4 million carriers in the United States are suspected to have no symptoms. It also has a very high prevalence in parts of South America and CentralAfrica and in China.

Signs ond Symptoms People have widely varying experiences with this infection. It shares characteristics of hepatitis B disease, but it is much more likely to become chronic. Without treatment, 75o/o to 85% will remain infected for life. (In contrast, only about 67o of persons who acquire hepatitis B after the age of 5 will be chronically infected.) While it is possible to have severe symptoms without permanent liver damage, it is more common to have chronic liver disease without overt symptoms. As the infection progresses, HCV disease may give rise to liver cancer. Infection is usually diagnosed with a blood test for antibodies to the virus. P revention a

nd Treotment

There is currently no vaccine for hepatitis C. Various treatment regimens have been attempted. One is the use of a derivative of interferon called pegylated interferon that has sustained activity. Some clinicians also prescribe ribavirin to try to suppress viral multiplication. The treaunents are not curative, but they may prevent or lessen damage to the liver.

25.4 Arboviruses: Viruses SPread by Arthropod Vectors

Arboviruses: Viruses Spread by Arthropod Vectors

(l

763

havirus), flaviviruses (F/av ivirus),some bunyaviruses (Bunyavirus and Phlebwirus), and reoviruses (Orbivirus). The chief vectors are blood-sucking arthropods such as mosquitoes, ticks, flies, and gnats. Most types of illness caused by these viruses are mil4 undifferentiated fevers, though some cause severe encephalitides (plural of encephalitis) and life-threatening hemonhagic fevers. Although these viruses have been assigned taxonomic names, they are more often lcirown by their common names, which are based on geographic location and primary clinical profile.

togaviruses

lp

Epidemiology of Arbovirus Disease Because arthropod vectors are found worldwide, so too are the arboviruses they carry. The vectors and viruses tend to be clustered in the

tropics and subtropics, but many temperate zones report periodic epidemics (figure 25.12).A given arbovirus type may have very restricted distribution, even to a single isolated region; some range over several continents; and others can spread along with their vectors.

The lnfluence of the Vector All aspects of the arbovirus life cycle

are closely tied to the ecology

of the vectors. Factors that weigh most heavily are the arthropod's life span, the availability of food and breeding sites, and climatic influences such as temperature and humidity. Most arthropod vectors become infected by feeding on the blood of hosts' Infections show a peak incidence from late spring through early fall, when the arthropod is actively feeding and reproducing. Warm-blooded vertebrates also maintain the virus during the cold and dry seasons. Humans can serve as dead-en{ accidental hosts, as in equine encephalitis and Colorado tick fever, or they can be a maintenance reservoir, as in dengue fever and yellow fever. Risk ofarboviral infection is greatest in wilderness areas where encounters with arthropod vectors are frequent. Clearing remote forest habitats for human colonization greatly increases this form of contact. Arboviral diseases have a great impact on humans. Although exact statistics are unavailable, there is a consensus that millions of people acquire infections each year and thousands ofthem die. The uncertain nature ofvector and viral cycles frequently results in sudden, unexpected epidemics, sometimes with previously unreported

viruses. Travelers and military personnel entering endemic areas are at special risk because, unlike the natives of that region, they have no immunity to the viruses.

General Characteristics of Arbovirus Infections Otr

coverage of the arboviruses

will

be focused on these main areas:

1. the manifestations of disease in humans;

2. aspects of the most important North American viruses; and 3. methods of diagnosis, treatment, and control'

Febrile lllness ond Encepholitis One type of disease elicited by arboviruses is an acute, undifferenti-

Vertebrates are hosts to more than 400 viruses transmitted primarily by arthropods. To simplifypresentation, these viruses are often lumped together in a loose grouping called the arboviruses (arthropod-borne viruses). The major arboviruses pathogenic to humans belong to the

ated fever, often accompanied by rash. These infections' typified by dengue fever and Colorado tick fever, are usually mild and leave no long-term effects. Prominent symptoms are fever, prostration, headache, myalgia, orbital pain, muscle aches, and joint stiffitess.

764

Chapter

25

The RNA Viruses That Infect Humans

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Colorado tick fever

encephalitis '\) EEE VEE Congolian

Kyasanur fever

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West Nile fever

WEE Western equine encephalitis

m

Dengue fever

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EEE Eastern equine encephalitis VEE Venezuelanequineencephalitis

St. Louis encephalitis Japanese encephalitis

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(outlined areas) Yellow fever

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Shows relatively restricted distribution

Figure 25.12 worldwide distribution of major arboviral

diseases.

Notice that the geographic location of the arthropod-borne viruses is correlated with climates supportive of insect and tick vectors. The colder northern and southern regions are free of these diseases in general.

About midway through the illness, a maculopapular or petechial rash can erupt over the trunk and limbs. When the brain, meninges, and spinal cord are invaded symptoms of viral encephalitis occur. The most conrmon American types are eastern equine, St. Louis, and California encephalitis. The viruses cycle between wild animals (primarily birds) aad mosquitoes or ticks, but humans are not usually reseryoir hosts. The disease begins with a bite, which releases the virus into the tissues and nearby lyrnphatics. Prolonged viremia establishes the virus in the nervous system, where inflammation can cause swelling and damage to the brain, nerves, and meninges. Symptoms are exhemely variable and can include coma, conwlsions, paralysis, tremor, loss of coordination, memory deficits, changes in speech and personality, and heaxt disorders. In some cases, survivors experience some degree of permanent brain damage. young children and the elderly are most sensitive to injury by arboviruses. Colorado tick fever (CTF) is the most common tick-borne viral fever in the United States. Restricted in its distribution to the Rocky Mountain states, it occurs sporadically during the spring and summer, corresponding to the time of greatest tick activity and human contacts. Two hundred to three hundred endemic cases are reported each year, and deaths are rare. Western equine encephalitis (WEE) occurs sporadically in the western United States and Canada, first in horses and later in humans. The mosquito that carries the virus emerges in the early summer when irrigation begins in rural areas and breeding sites are abundant. The disease is extremely dangerous to infants and small children, with a case fatality rate of 3o/o to iYo. Eastern equine encephalitis (EEE) is endemic to an area along the eastern coast of North America and Canada. The usual pattern is sporadic cases, but occasional epidemics can occur in humans

and horses. High periods of rainfall in the late summer increase the chance of an outbreak, and disease usually appears first in horses and caged birds. The case fatality rate can be very high (70%) in pregnant women. A disease known commonly as California encephalitis is caused by fwo different viral strains. The California strain occurs occasionally in the western states and has little impact on humans. The LaCrosse strain is widely distributed in the eastern United States and Canada and is a prevalent cause of viral encephalitis in North America. Children living in rural areas are the primary target group, and most of them exhibit mild, transient symptoms. Fatalities arc rare. St. Louis encephalitis (SLE) is the most common of all American viral encephalitides. Cases appear throughout North and South America, but epidemics occur most often in the midwestern and southern states. Inapparent infection is very colnmon, and the total number of cases is probably thousands of times greater than the 50 to 100 reported each year. The seasons ofpeak activity are spring and summer, depending on the region and species of mosquito. In the East, mosquitoes breed in stagnant or polluted water in urban and suburban areas during the summer. In the West, mosquitoes frequent spring floodwaters in rural areas. A close relative of the St. Louis virus, the West Nile virus, has received a great deal of media attention since it emerged as a serious pathogen in the United States (Insight 25.2).

Hemorrhagic Fevers Certain arboviruses, principally the yellow fever and dengue fever viruses, cause capillary fragility and disrupt the blood clotting system. These effects lead to localized bleeding, fever, and shock, which explains the common name for the disease. Hemorrhasic

25.4

765

Arboviruses: Viruses Spread by Arthropod Vectors

A Long Way frorn Egypt: West Nile Virus in the United States 1999 the first cases of West Nile fever in the United States appeared in several northeastern states. Within four years, West Nile virus has already spread westward to the other coast, resulting in 25'000 human infections and around 1,125 deaths by 2008. The arrival ofa deadly new disease, especially at a time of public nervousness concerning potential

In

biological warfare, led to a great deal of media attention concerning West Nile virus, much of it sensational even if not entirely accurate. West Nile virus is an arbovirus commonly found inAfrica, the Middle East, and parts ofAsia but until 1999 had not been detected in the Americas. The virus is known to infect birds, mosquitoes, and a host of mammals including humans. Mosquitoes generally become infected when they feed on birds infected with the virus. Infected mosquitoes can then bite and transmit the virus to humans. Ifinfection results, the illness is generally characterized by flulike symptoms that last just a few days and have no long-term consequences. Less than 1% ofinfected persons will suffer the potentially lethal inflammation of the brain known as "West Nile encephalitis." Because transmission of the virus depends on the presence of mosquitoes to act as vectors, viral connol is synonymous with veclor confol. Insect repellent, long-sleeved shirts and long pants, and conhol ofmosquito breeding grounds (primarily stagaant water) all decrease the spread ofthe virus. Because the virus is blood-borne, transmission through person-to-person contact is unlikely. A greater concem has been fansmission by transfusions. Thousands of samples of donated blood have tested positive for the virus, and a number of infections were reported before testing became routine.

Epidemiologists are also closely monitoring the magnitude of the disease in animals. Both wild and domesticated animals' called sentinels, are routinely tested for evidence ofinfection. So far, the virus has been isolated in nearly 200 bird species as well as cats, dogs; rodents, horses, and even alligators. This not only increases the potential sources ofdiseaseo but it can cause massive die-offs in the animals and may wipe out endangered populations, such as the native Hawaiian birds.

fevers are caused by a variety ofviruses, are carried by a variety of vectors, and are distributed globally. Reservoir animals are usually

small mammals, although yellow fever and dengue fever can be harbored in the human population. The best-known arboviral disease is yellow fever. Although this disease was at one time cosmopolitan in its distribution, mosquito control measures have eliminated it in many countries, including the United States. Two patterns of transmission occur in nature. One is an urban cycle between humans and the mosquito Aedes aegypti, which reproduces in standing water in cities. The other is a sylvan cycle, maintained between forest monkeys and mosquitoes. Most cases in the Western Hemisphere occur in the jungles of Brazil, Peru, and Colombia during the rainy season. Infectionbegins acutely with fever, headache, and muscle pain. In some patients the disease progresses to oral hemorrhage, nosebleed" vomiting, jaundice, and

liver and kidney damage with significant mortality rates. Dengue* fever is caused by a flavivirus and is also carried by the Aedes mosquitoes. Although mild infection is the usual pattern, a *

dengue (den'-gha) A corruption of Sp. "dandy'' fever. Other synonl'rns are dengue shock syndrome and stifhreck fever.

is screened for the presence of West Nile virus. Epidemiologists rely on sentinel animals to monitor this pathogen and trace its spread.

What are some likely factors to account for the rapid spread West Nile virus across the United States in just four years? Answer available at http ://www.mhhe.com/talaroT

of

form called dengue hemorrhagic shock syndrome can be lethal. This disease is also called "breakbone fever" because ofthe severe pain it induces in the muscles and joints. The illness is endemic to Southeast Asia and India, and several epidemics have occurred in South and Central America, the Caribbean, and Mexico. It is a serious risk factor for about 40o/o of rhe world's population. The WHO has estimated that more than 50 million cases occur every year, with about 400,000 hemorrhagic fever cases. It is thought that climate change, including heavy rains and warmer weather that favor the mosquito life cycle, is a contributing factor in several major epidemics across Asia. Concern over the possible spread of the disease to the United States led the CDC to survey mosquito populations in states along the southern border. In several states, they discovered theAsian tiger mosquito (Aedes albopictus), a potential vector of dengue fever virus. Contact between infected human carriers could establish the virus in these mosquito populations. So far, no cases of hemorrhagic dengue fever have been reported in the United States, and it is hoped that continued mosquito abatement will block its spread. One group of researchers is currently testing a method to genetically engineer mosquitoes so that they are resistant to infection and no longer capable ofspreading the virus.

766

Chapter

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The RNA Viruses That Infect Humans

Diagnosis, Treatment, and Control of Arbovirus Infection Except during epidemics, detecting arboviral infections can be difficult. The patient's history of travel or contact with vectors, along with serum analysis, are highly supportive of a diagnosis. Treatment of the various arbovirus diseases relies completely on support measures to control fever, convulsions, dehydration, shock, and edema. The most reliable arbovirus vaccine is a live, attenuated yellow fever vaccine that provides relatively long-lasting immunity. Vaccination is a requirement for travelers in the tropics and can be administered to entire populations during epidemics. Live, attenuated vaccines for WEE and EEE are administered to laboratorv workers. veterinarians, ranchers, and horses.

The most prominent human retroviruses are HIV and the T-cell lymphotropic viruses I and II (HTLV I and HTLV II). The two types of HIV are HIV-I, which is the dominant form in most of the world, and HIV-2. HTLV type I is associated with leukemia and lymphoma (discussed in a later section).

HIV and other retroviruses display structural features typical of enveloped RNA viruses (figure 25.13a).The outermost component is a lipid envelope with hansmembrane glycoprotein spikes and knobs that mediate viral adsorption and fusion to the host cell. HIV can only infect host cells that present the required receptors, which is a combination receptor consisting of the CD4 marker plus a coreceptor. The virus uses these receptors to gain enfrance to several types ofleukocytes and tissue cells (figure 21.l3b).

Epidemiology of HIV lnfection E

Other enveloped RNA viruses include the Coronaviridae, agents of the common cold; Togaviridae, agents of rubella and hepatitis C; and arboviruses, a collection ofviruses that are spread by arthropod vectors. Examples are togaviruses that cause St. Louis and equine encephalitis; bunyaviruses, agents ofhemorrhagic fever; and flaviviruses, which cause yellow and dengue fevers.

25.5 HIV Infection and AIDS The sudden emergence ofAIDS in the early 1980s focused an enormous amount of public attention, research studies, and financial resources on the virus and its disease. The first cases ofAIDS were seen by physicians in Los Angeles, San Francisco, and NewYork City. They observed clusters of young male patients with one or more of a complex of symptoms: severe pneumonia caused by Pneumocystis (carinii) jiroveci (ordinaily a

harmless fungus); a rare vascular cancer called Kaposi,s sarcoma; sudden weight loss; swollen lymph nodes; and general loss ofimmune

function. Eventually, virologists at the Pasteur Institute in France isolated a novel retrovirus, later named the human immunodeficiency virus (HIV). The cluster of symptoms was therefore clearly a communicable infectious disease, and the medical community termed it acquired immunodeficiency syndrome, or AIDS.

Causative Agent HIV is a retroviruso in the genus Lentivirus. Most retroviruses have the potential to cause cancer, produce dire, often fatal diseases, and can alter the host's DNA in profound ways. They are named ,.retroviruses" because they reverse the usual order of transcription. They contain an enrpe called reverse transcriptase @T) that catalyzes the replication of double-stranded DNA from single-stranded RNA. The association of retroviruses with their hosts can be so intimate that viral genes are permanently integrated into the host genome. In fact, as the technology of DNA probes for detecting retroviral genes is employed it becomes increasingly evident that retroviral sequences are integral parts of host chromosomes. Not only can this retroviral DNA be incorporated into the host genome as a provirus that can be passed on to progeny cells, but some retroviruses also transform cells and regulate certain host genes.

HIV seems to have been answered: l(here did it come from? Researchers have been comparing the genetics of HIV with various African monkey viruses called simian immunodeficiency viruses or SIVs. The genetic sequences in these viruses led researchers to conclude that HIV is a hybrid virus that has evolved from two separate monkey SIVs. One of the SIVs has as its natural host the greater spot-nosed monkey, and the other infects red-capped mangabeys. Apparently, where chimpanzees became coinfected with the two monkey viruses, a third type of virus emerged that contained genetic sequences from both SIVs. This new type of SIV was probably transmitted to humans, possibly when they used the chimps for food. The crossover into humans probably occurred in the early part ofthe 1900s; the earliest record we have of human infection is a blood sample preserved from an African man who died in 1959. HIV probably remained in small isolated villages, causing sporadic cases and mutating into more virulent strains that were readily transmitted from human to human. When this pattern was combined with changing social and sexual practices and increased immigration and travel, a pathway was opened up for rapid spread of the virus to the rest of the world. One important question about

Modes of Tronsmission HIV transmission occurs mainly through two forms of

contact: sexual intercourse and transfer ofblood or blood products. Babies can also be infected before or during birth, as well as through breast feeding. The mode of transmission is similar to that of hepatitis B virus, except that the AIDS virus does not survive for as long outside the host and it is far more sensitive to heat and disinfectants. And HIV is not transmitted through saliva, as hepatitis B can be. In general, HIV is spread only by direct and rather specific routes (figure 25.14). Because the blood of HlV-infected people harbors high levels of free virus in both very early and very late stages ofinfection, and high levels ofinfected leukocytes throughout infection, any form of intimate contact involving transfer of blood

(trauma, needle sharing) can be a potential source of infection. Semen and vaginal secretions also harbor free virus and infected white blood cells and account for a major source in sexual transmission. The virus can be isolated from urine, tears, sweat, and saliva but in such small numbers (less than I virion per cubic millimeter) that these fluids are not considered sources ofinfection.

25.5 Blood exposure through needles -120 GP-41

Protease molecule RNA strands Capsid Integrase molecules Reverse

transcriptase molecules

HIV Infection and AIDS

Direct blood exposure, during sexual intercourse or other intimate

contact

767

Semen, vaginal fluid exposure during sexual intercourse

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Flgure 25.14

Primary sources and suggested routes of

infection by HlV.

(b)

ftgure 25.13

The general structure of HlV.

fhe envelope contains two types of glycoprotein (GP) spikes, two identical RNA strands; and several molecules of reverse transcriptase, protease, and integrase encased in a protein capsid. (b) The snug attachment of HIV glycoprotein molecules (CP-41 and GP-l20) to their specific receptors on a human cell membrane. These receptors are CD4 and a co-receptor called CCR-5 (fusin), which permit docking with the host cell and fusion with the cell membrane. 1a)

AIDS

Morbidity

AIDS first became a notifiable disease at the national level in 1984, and it has continued in an epidemic pattern, although the number of people developing the disease in the United States has decreased since 1994. This situation is due to the advent ofeffective therapies that prevent the progression of HIV infection to full-blown AIDS. But in developing countries, which are hardest hit by the HIV epidemic, access to these lifesaving drugs has been more limited. And even in the United States, despite keatment advances, HIV infection and AIDS are the sixth most common cause of death among people ages 25 to 44, although they have

fallen out of the top ten list for causes of death overall.

Unfortunately, new HIV infections are remaining at a steady rate of35,000 to 37,000 per year. Figure 25.15 contrasts the numbers of new infections broken down by gender, risk group, and race. Men still account fot 70o/o of new infections. Forty-seven percent of all new infections are acquired through male homosexual activity. Men having sex with other men (a group labeled MSM) have increased susceptibility because of the practice of anal sex, which is known to lacerate the rectal mucosa and can provide an entrance for viruses from semen into the blood. The receptive partner (whether male or female) is the more likely of the two to become infected. In addition, bisexual men ire a significant factor in spreading the virus to women' In large metropolitan areas especially, as many as 600/o of intravenous drug users (IDUs) can be HIV carriers. Infection from contaminated needles is growing more rapidly than any other mode of transmission, and it is another significant factor in the spread of HIV to the heterosexual population. HIV infection and AIDS have been reported in every country.

ofAfrica andAsia have the highest case rates. In 2007, the WHO revised its estimates to show a decline in the number of AIDS cases and HIV infections worldwide from 45 million down to 33 million. Even at this lower number, the disease continues to have devastating Parts

768

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25

The RNA Viruses That Infect Humans

(a) New HIV diagnoses, comparison by age group, 2001 to 2006*

A NOTE ABOUT SOME HARSH STATISTICS OF AIDS

53% Age 35-49

26% Age 20-34

18% Age

A powerful way to drive home the staggering effect of A|DS is to examine some of its statistics in everyday terms (data generated by WHO and CDC up to 2007):

50-64

. 1%Age 13-19

.

2o/" Age 65

*Numbers

are rounded.

(b) Estimated HIV diagnoses

(c) Diagnosis rate Per 100,000

by transmission method,

population, by racelethnicity in 33 states,2001-2006

33 states,2001-2006

I f I I

Male-to-male sex Heterosexual contact

lv drugs Male-to-male sex and lV druos

(in thousands)

I white I ff Black I Hispanic

Native American

20 18 16

16

14

14

12

IZ 10

8

10 8

b

6

4

4

z

2

0

0

r

Asian

22

18

r .

Figure 25.15 Patterns of HIV infections. Data compare the changes occurring between 2001 and 2006. (a) Percentages of new infections by age group. (b) Numbers of new infections by transmission. (c) Numbers of new infections bv race/ethnicity.

repercussions. Reports from the CDC estimate approximately 1.2 million people in the United States living with HIV/AIDS. A number of these people may be in the latent phase of the disease and not yet aware of their infection. In most parts of the world, heterosexual intercourse is the primary mode of transmission. In the industrialized world the overall rate ofheterosexual infection has increased dramatically in the past several years, especially in adolescent and young adult women. In the

United States, about 33% of HIV infections arise from unprotected sexual intercourse with an infected parher of the opposite sex. Now that donated blood is routinely tested for antibodies to the AIDS virus, transfusions are no longer considered a serious risk. Because there can be a lag period of a few weeks to several months before antibodies appear in an infected person, it is remotely possible to be infected through donated blood. Rarely, organ transplants can carry HI! so they too are tested. Other blood products (serum, coagulation factors) were once implicated in AIDS. Thousands of hemophiliacs died from the disease in the 1980s and 1990s. It is now standard practice to heat-treat any therapeutic blood products to destroy all viruses.

By most recent estimates, worldwide, about 35 million people are infected with HIV or have AIDS. Every I0 seconds another person becomes infected with the virus. Every day about 6,000 people die from AIDS.

Since the beginning of the epidemic, about 35 million people have died from it. lt is the cause of about 5olo of deaths from all causes worldwide, rivaled among infectious agents only by malaria and tuberculosis.

Parts of Africa are hit harder than other regions. Consider: Nearly 70o/o of cases occur in sub-Saharan Africa.

. r

e

c . .

In this part of the world, three-quarters of deaths are caused by AIDS.

ln this region, anywhere from

1o/o

to

3oo/o

of adults

are

infected.

of childhood deaths are caused by AIDS. By 2A10, 18.5 million African children could be orphaned by AIDS. Only 1 in 10 people have been tested and are aware of their 35o/o

infection. The United States has its own disturbing set of statistics: Over'l million people are infected with HlV. About 36,000 new cases of AIDS are reported every year.

. . t . .

71o/o

of cases occur in males.

African Americans are 8.4o/o more likely to become infected than other ethnic groups. Nearly 20,000 people die each year from AIDS.

A small percentage ofAIDS cases (less thanS%) occur in people without apparent risk factors. This does not mean that some other unknolrm route of spread exists. Factors such as patient denial, unavailability of history death, or uncooperativeness make it impossible to explain every case. We should note that not everyone who becomes infected or is antibody-positive develops AIDS. About 5% of people who are antibody-positive remain free of disease, indicating that some people can develop immunity or are less susceptible to infection. Treatment of HlV-infected mothers with AZT has dramatically decreased the rate of maternal to infant transmission of HIV during pregnancy. Current treatment regimens reduce the transmission rate to 5Voto llYo.Untreated mothers pass the virus to their babies at the rate of 33Yo. The cost of perinatal prevention strategies (approximately $1,000 per pregnancy) and the scarcity of medical counseling in underserved areas have led to an increase in maternal

transmission of HIV in developing parts of the world, at the same time that the developed world has seen a marked decrease. Medical and dental personnel are not considered a high-risk group. A relatively small number of medical and dental workers have acquired HIV or become antibody-positive as a result of clinical accidents. A health care worker involved in an accident in which

25.5

HIV lnfection and AIDS

769

lmmune stimulus

t\ {

ll '=

o o.

c

o (d

J

The virus is adsorbed and fuses with the cell.The twin RNAs are uncoated- Reverse transcriptase catalyzes the synthesis of a single complementary strand of DNA (ssDNA). This single strand serves as a template for synthesis of a double strand (ds) of DNA. In latency, dsDNA is inserted into the host chromosome as a provirus.

@ Rgure 25.16

(b)

(c)

After a latent period, various immune activators stimulate the infected cell, causing reactivation of the provirus genes and production of viral mRNA.

HIV mRNA is translated by the cell's synthetic machinery into virus components (capsid, reverse transcriptase, spikes), and the viruses are assembled. Budding of mature viruses lyses the intected cell.

The general multiplication cycle of HlV.

gross inoculation with contaminated blood occurs (as in the case of a needlestick) has a less than I in 1,000 chance of becoming infected. Experts emphasize that hansmission of HIV will not occur through casual contact or routine patient care procedures, and that universal precautions for infection confol (see chapter 13) provide protection for both worker and patient.

Pathogenesis and Virulence Foctors of HIV As summarized in figure 25.|4,HIV enters a mucoug membranc or the skin and is phagocytosed by a dendritic cell. In the dendritic cell, the virus grows and is shed from the cell without killing it. New viruses are taken up and anrplified by macrophages in the skin, lymph organs, bone marrow, and blood. One of the great ironies of HIV is that it infects and desfroys many of the very cells needed to combat it, especially the helper (CD4 or T4) class of lymphocytes. It also infects monocytes and B lymphocytes.

Once the virus is inside a target cell, its rwerse kanscriptase converts its RNA into DNA. Although initially many viruses produce alytic infection, in some cellsthe DNAbecomes inactive inthe nucleus of the host cell and its DNA becomes integrated into host DNA (figure 25.16). This event accounts for the lenghy course of the disease. Because different host cells are in different stages of infection, some host cells are releasing new viruses and being lysed, and new T cells are constantly being infected. In the absence oftreatment, the host cells ultimately lose this race for survival. The primary effects due directly to HIV infection are extreme leukopenia, with lowered levels of lymphocytes in particular. Both T cells and B cells un&rgo extensive die-offs through programmed cell suicide (apoptosis).The CD4 memory clones and stem cells are among the prime targets. The viruses also cause formation of giant T cells and other syncytia, which allow the spread ofviruses direcfly from cell to cell, followed by mass destruction of the syncytia. The

770

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25

The RNA Viruses That Infect Humans

central nervous system is affected when infected macrophages cross the blood-brain barrier and spread viruses into brain cells. Studies have indicated that some of the viral envelope proteins can have a direct toxic effect on the brain's glial cells and other cells. Other research has shown that some peripheral nerves become demyelinated and the brain becomes inflamed. The secondary effects of HIV infection are the opportunistic infections and malignancies associated with destruction of essential CD4 functions needed to control pathogens. These are summarized in Insight 25.3.

Stages, Signs, and Symptoms of HIV lnfection and AIDS The clinical spectrum of HIV infection ranges from acute early symptoms to the end stage symptoms ofAIDS. To understand the progression, closely follow figures 25.17 and,25.18. Pathology in HIV infection is direct$ tied to two factors: the level of viruses and the level of T cells in the blood. Figure 25.18 presents relative amounts of viruses and T cells over the course of the infection and disease. Note also that the figure is showing the pattern of HIV infection in the absence of medical intervention or chemotherapy.

Period of infectiousness (virus present)

Antibody (+)

The initial infection is acute and often attended by vague, mononucleosis-like symptoms that soon disappear. This phase is marked by high levels of free virus in the blood (figure 25.18, phase I), followedby arapiddrop (figure 25.18, phase II). Observe

that the antibody levels rise (figure 25.18, phase II and III) at the same time that the virus load is dropping. The antibodies are responsible for neutralizing the free viruses in circulation during this stage. One feature of the ongoing HIV infection is a period of mostly asymptomatic disease (sometimes called latency) that varies in length from 2to 15 years, with the average being about l0 years (figure 25.17 , phase III). Another important occurrence during the mid-to-late asynptomatic periods is how the number of T cells in the blood steadily decreases (figure 25.18, IID. Once the CD4 cell levels fall below 200 cells/mm3 (200/1tl) of blood, symptoms of

AIDS appear (figure 25.18, IV).

Initial symptoms of AIDS may be fatigue, diarrhea, weight loss, and neurological changes, but most patients first notice this phase of infection because of one or more opportunistic infections or neoplasms. These AlDS-defining illnesses are detailed in Insight 25.3. Other disease-related symptoms appear to accompany severe immune deregulation, hormone imbalances, and metabolic disturbances. Pronounced wasting ofbody mass is a consequence ofweight loss, diarrhea, and poor nutrient absorption. protracted fever, fatigue, sore throat, and night sweats are significant and debilitating. Both a rash and generalized lymphadenopathy in multiple lymph nodes are presenting symptoms in many AIDS patients.

r 3

Level of viruses/HlV antigen Level of antibodies to one or more antigens Level of CD4 T cells >500 cells/pl

g

o tr o

CL

E

oo

to

.9 @

o tr o G

co

I

________>

IJ

___>il

1 month

_-______> 2-1 5 years

o

Months-years

ll Incubation period

o tr 0

IV_ ,

I

v Symptoms occur

(I) Infection with virus. (II) Appearance of antibodies in standard HIV tests. (III) Asymptomatic HIV disease, which can encompass

an

elitensive time oerioo.

(IV)

Overt symptoms of AIDS include some combination of opportunistic infections, cancers, and general loss of immune function.

Figure 25.17

Time line in HIV infection, asymptomatic

HIV disease, and AIDS.

. Virus levels are high during the initial acute infection and decrease until the later phases of HIV disease and AIDS. . Antibody levels gradually rise and remain relatively high throughout phases lll and IV. . T-cell numbers remain relatively normal until the later phases of HIV disease and full-blown AIDS.

Flgure 25.18 Changes in the quantity of virus antigen, antibody, and T cells in circulation over time.

25.5

HIV Infection and AIDS

771

AIDS-Defining lllnesses (ADls) InAIDS patients who do not receive or do not comply with antiretroviral therapy (and even in some who do), the slow destruction of the immune system results in a wide variety ofinfectious and noninfectious conditions, called AlDS-associated illnesses or AlDS-defining illnesses (ADIs). lt is almost always one or more of these conditions that causes death in AIDS patients. Because the virus eventually destroys an essential immune function, it is not surprising that the body is beset by normally harmless microorganisms, many of which have been living in or on the host for decades without causing disease. The spechum ofAlDS-associated illnesses also provides insight into how vital the immune system is in controlling or mitigating cancerous changes in our cells. AIDS patients are at increased risk for Burkitt lymphoma, Kaposi's sarcoma (KS), and invasive cervical carcinomas, all of which are associated with viral infections. Since the beginning of the AIDS epidemic in the early 1980s, the CDC has maintained a list of conditions that are part of the case definition. The list has been modified periodically over almost 30 years of

its existence. One of the ways that people currently meet the case definition for AIDS is if they are positive for the virus and experience one or more of these ADIs. The ADIs are listed in table 25.A. The diseases are listed according to the organ system where the presenting symptoms might be found. (Some of the conditions may be listed in more than one column.) You can see that many of them---or at least the

Al

Kaposi's sarcoma lesion on the arm. The flat, purple tumors occur in almost any tissue and are frequently multiple. way they occw in AIDS

patients-are very rare in the otherwise healthy

population.

What accounts for the enormous variety in AlDS-defining illnesses? Answer available at http://www.mhhe.com/talaroT

DS-Defining lllnesses Genitourinary and/or

Skin and/or

Mucous Membranes

(lncludes Eyes)

Nervous System

Cytomegalovirus

Cryptococcosis, extrapulmonary

retinitis (with loss

ofvision)

HIV encephalopathy

Cardiovascular and Lymphatic System or Multiple Organ Systems

Respir4tory Tract

Coccidioidomycosis, disseminated or extrapulmonary

Candidiasis of trachea, bronchi, or lungs Herpes simplex bronchitis or pneumonitis

Herpes simplex chronic ulcers (>1 month duration)

Lymphoma, primarily in brain

Cytomegalovirus (other than

Progressive multifocal

Histoplasmosis, disseminated or extrapulmonary

Kaposi's sarcoma

Toxoplasmosis of the

leukoencephalopathy

brain

liver, spleen, nodes)

Burkitt lymphoma lmmunoblastic lymphoma My c o b a c t erium kan

s

as

ii,

disseminated or extrapulmonary M ycobac t eri um

tu bercu I os is,

disseminated or extrapulmonary S almo

nella septicemia,

recurrent wasting syndrome

Mycobacterium avium complex (MAC) Tuberculosis

(I[ycobacterium tuberculosis) Pneumocystis

(carinii) jiroveci pneumonia Pneumonia, recurrent in 12-month period

Gastrointestinal Tract

of

Reproductive Tract

esophagus, GI tract

Invasive cervical carcinoma

Herpes simplex chronic ulceri 1>t month duration) or esophagitis

Herpes slmplex chronic ulcers (>l month duration)

Candidiasis

Isosporiasis, (diarrhea caused by Isospora) chronic intestinal (>1 month duration) Cryptosporidiosis, chronic intestinal (>1 month duration)

772

Chapter

25

The RNA Viruses That Infect Humans

Some of the most virulent complications are neurological. Lesions occur in the brain, meninges, spinal column, and peripheral nerves. Patients with nervous system involvement show some degree of withdrawal, persistent memory loss, spasticity, sensory loss, and progressive AIDS dementia.

Diagnosis of HIV Infection A person is diagnosed

as having HIV infection if he or she has tested positive on follow-up tests for the human immunodeficiency virus. This diagnosis is not the same as havingAIDS.

Most viral testing is based on detection of antibodies specific to the virus in serum or other fluids, which allows for the rapid inexpensive screening of large numbers of samples. Testing usually proceeds at two levels. The initial screening tests include the older ELISA and newer latex agglutination and rapid antibody tests. One initial screening test is an "oral swab test" licensed only for use by health care professionals. Even though saliva can be tested with this kit, blood is more often used in the test because of increased antibody levels of blood relative to saliva. The advantage of these tests is that results are available within minutes instead of the days to weeks previously required for the return of results from ELISA testing. One kit has been licensed by the Food and Drug Administration for home use; in this test, a blood spot is applied to a card and then sent to a testing laboratory to perform the actual procedure. The client can call an automated phone service, enter his or her anonymous testing code, and receive results over the phone. Other home kits have been devised to provide results immediately in the clientb home, much like a home pregnancy test, but there is some concern about their accuracy. Although the approved testsjust described are largely accurate, around l% of results are false positives, and they always require follow-up with a more specific test called Western b/or analysis (see page 525). This test detects several different anti-HIV antibodies and can usually rule out false positive results. Another inaccuracy can be false negative results that occur when testing is performed before the onset of detectable antibody production. To rule out this possibiliry persons who test negative but are at some risk for exposure should be tested a second time 3 to 6 months later.

Blood and blood products are sometimes tested for HIV antigens (rather than for HIV antibodies) to close the window of time between infection and detectable levels of antibodies during which contamination could be missed by antibody tests. The American Red Cross is currently participating in a program to test its blood supply with a DNA probe for HIV For many AIDS patients, it is necessary to check viral loads in order to monitor the effectiveness ofdrugs. In the United States, people are diagnosed with AIDS if they meet the following criteria: (l) they are positive for the virus, and (2) they

fulfill

one of these additional criteria:

o They have a CD4

(helper T cell) count of fewer than 200 cells per milliliter of blood.

r Their CD4 cells o

account

for fewer than l4o/o of all

lymphocytes. They experience one or more of a CDC-provided list ofAIDSdefining illnesses (ADIs) (see Insight 25.3).

Preventing HIV Infection Avoidance ofsexual contact with infected persons is a cornerstone of HIV prevention. Abstaining from sex is an obvious prevention method" although those who are sexually active can also take steps to decrease their risk. Epidemiologists cannot overemphasize the need to screen prospective sex partners and to follow a monogamous sexual lifestyle. The only sure way to avoid infection is for all sexually active people to consider every parher to be infected unless proven otherwise. Barrier protection (condoms) should be used when having sex with anyone whose HIV status is not known with certainfy to be negative. Although avoiding intravenous drugs is an obvious deterrent, many drug addicts do not or will not choose this option. In such cases, risk can be decreased by not sharing syringes or needles or by cleaning needles with bleach and then rinsing before another use. From the very first years of the AIDS epidemic, the potential for creating a vaccine has been regarded as slim, because the virus presents many seemingly insurmountable problems. Among them, HIV becomes latent in cells; its cell surface antigens mutate rapidly. Although it does elicit immune responses, it is apparently not completely controlled by them. In view of the great need for a vaccine, however, none of those facts has stopped the medical community from moving ahead. Currently, dozens of potential HIV vaccines are in clinical trials overseen by an international agency-the HIV Vaccine Trials Network. None of the trials have yet produced an effective vaccine, and even those that showed some promise have been fraught with problems. One of these is based on a viral vector technology, which uses adenoviruses or vaccinia viruses that have low virulence to ferry three HIV genes into the body (figure 25.19). One advantage of this vaccine is that it stimulates the formation of cytotoxic T cells that destroy virally infected cells. It also appears to produce an immune response to a wide variety of HIV strains. Even with the large-scale efforts, HIV specialists do not expect a viable vaccine to be approved and available for several more yea$.

Treating HIV Infection and AIDS It must be forcefully stated: There is no cure for HIV infection or AIDS. None of the therapies do more than slow the progress of the disease or diminish symptoms.

Clear-cut guidelines exist for treating people who test HIVpositive. These guidelines are updated regularly, and they differ depending on whether a person is completely asymptomatic or experiencing some of the manifestations of HIV infection, and also whether they have previously been treated with antiretroviral agents. A person diagnosed with AIDS receives treafirrent for HIV infection as well as drugs to prevent or treat a variety of opportunistic infections and otherADls such as wasting disease. These treatment regimens vary according to each patient's profile and needs. Treatment strategies against HIV aim to disrupt some part of its multiplication cycle (see table 12.6, page 364). The first effective anti-HIV drugs were synthetic nucleoside analogs that inhibit reverse transcriptase, including azidothymidine (AZT), didanosine (ddl), Epivir (3TC), and stavudine (d4T). They interrupt the HIV multiplication cycle by mimicking the structure of actual nucleosides and being added to viral DNA by reverse transcriptase. Because these

25.6

Adult T-Cell Leukemia and Hairy-Cell Leukemia

773

......------Injected into host as vaccine Virus vector (hybrid virus)

Figure 25.79 "Trojan horse," or viral vector technique,

Envelope genes of HIV are expressed, stimulating immunity

a novel technique for making an AIDS vaccine.

The part of the HIV genome coding for envelope glycoproteins is inserted into a harmless carrier virus. This hybrid virus replicates and expresses the HIV genes when it is injected into a host.

drugs lack all of the correct binding sites for further DNA synthesis, viral replication and the viral cycle are terminated (figure 25.20a\. Other reverse transcriptase inhibitors that arc not nucleosides are Nevirapine and Sustiva, both of which bind to the enzyme and restructure it. Another important class of drugs, the protease inhibitors (figure 25.20b), block the action of the HIV enz5rme (protease) involved in the final assembly and maturation of the virus. Examples of these drugs include Crixivan, Norvir, andAgenerase. Newer additions to the arsenal are drugs that block attachment

or fusion. They prevent the virus from attaching to receptors on target cells, thereby preventing infection altogether. Drug companies have also released a new class of anti-AIDS drugs called integrase inhibitors. These act at the stage of viral integration into the host DNA molecule, which effectively ends the stage of infection leading to latency and viral replication. A regimen that has proved to be extremely effective in controlling AIDS and inevitable drug resistance is HAART' short for highly active anti-retrwiral therapy. By combining two reverse transcriptase inhibitors and one protease inhibitor in a single pill, the virus is interrupted in two diferent phases of its cycle. Addition of a receptor blocker and an integrase inhibitor can completely shut down the virus. This therapy has been shown to reduce viral load to undetectable levels and facilitate the improvement of immune function. It has also reduced the incidence of viral drug resistance, because the virus would have to undergo at least two sepaxate mutations simultaneously to develop resistance. Patients who are HlV-positive but asymptomatic can avoidAIDS with this therapy as well. The primary drawbacks to antiHIV drugs are high cost, toxic side effects, drug failure due to patient noncompliance, and an inability to completely eradicate the virus.

25.6 Other Retroviruses: Human T-Cell Lymphotropic Viruses Leukemia is the general name for at least four different malignant diseases of the white blood cell forming elements originating in the bone marrow. Leukemias are all acquired, rather than being inherited; some forms are acute and others are chronic. Leukemias have many causes, some of which are thought to be viral. The human Gcell lymphotropic virus I (HTLV-I) is associated with a form of leukemia called adult T cell leukemia.

Location of reaction

ffi ffi

External to cell Cytoplasm

(a) Reverse transcrlptase blockers. A prominent group of drugs (AZf, ddl, 3TC) act as nucleoside analogs to inhibit reverse transcriptase. They are inserted in place of the natural nucleotide by reverse transcriptase but btock further action of the enzyme and synthesis of viral DNA.

(b) Protease inhibitors that cause abnormal viruses to be released. Protease inhibitors plug into the active sites on HIV protease.This enzyme is necessary to cut elongate HIV protein strands and produce smaller protein units. During budding viruses incorporate this uncut nonfunctioning protein. The resultant viruses are unable to mount an infection.

@ fig,rt" 25,20

Mechanisms of action of anti-HlV drugs (simplified to show basic drug effects).

774

Chapter

25

The RNA Viruses That lnfect Humans

All leukemias first manifest with conditions such as easy bruising or bleeding, paleness, fatigue, and recurring minor infections. Underlying pathologies that account for these effects are anemia, platelet deficiency, and immune dysfunction brought about by the disturbed lymphocyte ratio and function. Some cases of adult T:cell leukemia first present with cutaneous T:cell lymphoma accompanied by dermatitis, with thickened, scaly, ulcerative, or tumorous skin lesions. Other complications are lymphadenopathy and dissemination of the tumors to the lung, spleen, and liver. The possible mechanisms by which this retrovirus induces cancer are not entirely clear. One hypothesis is that the virus carries an oncogene that, when spliced into a host's chromosome and triggered by various carcinogens, can immortalize the cell and deregulate the cell division cycle. One of HTLV-I's genetic targets seems to be the gene and receptor for interleukin-2, apotent stimulator of T cells. Adult Tcell leukemia was first described by physicians working with a cluster of patients in southern Japan. Later, a similar clinical disease was described in Caribbean immigrants. In time, it was shown that these two diseases were the same. Although more common in Japan, Europe, and the Caribbean, a small number of cases occur in the United States. The disease is not highly transmissible; studies among families show that repeated close or intimate contact is required. Because the virus is thought to be transferred in infected blood cells, blood hansfusions and blood products are potential agents oftransmission. Intravenous drug users could spread it through needle sharing. The retrovirus HTLV-I is a close relative of HTLV-I, but is not as clearly associated with a distinct disease. Serological studies suggest that infection with HTLV-II is fairly common among certain populations in Latin America, although its occurrence can be tracked worldwide. The virus is probably transmitted through blood transfusions, needles, and sexual intercourse. Although it is not yet established as a pathogen, some reports suggest it may be involved in certain neurodegenerative conditions such as myelopathy and spastic parapesis. Additional research will be needed to clarifu this possible role of HTLV-[.

coordinate the immune responses to most infections. Following infection, the virus often enters a dormant stage lasting 2 to 15 years.

= AIDS is associated with a number of medical conditions-the

AlDS-defining illnesses-resulting from the destruction of CD4 lymphocytes and stem cel1s. The virus is carried to the CNS, caus-

ing neurological degeneration. Opportunistic infections involve

#

+

u

fungi, protozoa, bacteria, and other viruses. Cancers of importance are Kaposi's sarcoma and lymphoma. Protection against HIV includes following safe sex practices and increased precautions when handling needles and body fluids. Research continues for an effectiveAlDS vaccine, but additional years ofresearch and testing are necessary. Hry infection is diagnosed by ELISA and the Western blot tests, which detect antibodies to HIV There is no cure forAIDS. but improved supportive care and HAART drug therapy have increased the life expectancy for many patients. Drugs are given in combinations to counteract the development of drug resistance. Other retroviruses include the human Aceil lymphotropic viruses (HTLV-I and -II). HTLV-I has been identified as the causative agent ofATL (adult T-cell leukemia) and HTLV-il may be a factor in certain neurological diseases.

25.7 Nonenveloped Nonsegmented Single-Stranded RNA Viruses: Picornaviruses and Caliciviruses As suggested by the prefix, picorna viruses are named for their small (pico) size and their RNA core (figure 25.21).Important representatives include Enteroviras and Rhinovirus, which are responsible for a broad spectrum ofhuman neurological, enteric, and other illnesses (table 25.4); and, Cardiovirus, which infects the brain and heart in humans and other mammals. The following discussion on human picornaviruses centers upon the poliovirus and other related enteroviruses, the hepatitis A virus, and the human

rhinoviruses (HRVs).

Poliovirus and Poliomyelitis sq Retroviruses are unique in their

ability to create a double-stranded

DNA copy from an RNA template using the enzyme

reverse

transcriptase.

* €

Retroviruses infect many birds and mammals. Those infecting humans are named HTLV-I, HTLV-II, and HI! and a1l altack various white blood cells, leading to cancer or immune dysfunction. # AIDS is caused by the human immunodeficiency virus (HIV). It is the most significant viral pandemic of this century; with 35 million people infected worldwide and approximately 800,000 to 2 million in the United States. It is acquired through gross contact with blood or sexual fluids. = Those at greatest risk for infection include homosexual or bisexual males, IV drug users, heterosexual partners ofAIDS carriers, recipients ofblood products or tissue transplants, and neonates from infected mothers. r*: HIV first infects skin phagocytes. where it multiplies and is shed, infecting lymphocytes and other cells that possess the CD4 receptor site. The most significant of these are the T4 (helper) cells that

Poliomyelitis* (polio) is an acute enteroviral infection of the spinal cord that can cause neuromuscular paralysis. Because it often affects small children, it is also called infantile paralysis. No civilization or culture has escaped the devastation ofpolio. The poliovirus has a naked capsid (figure 25.21a) that confers chemical stability and resistance to acid" bile, and detergents. By this means, the virus survives the gastric environment and other harsh conditions. which contributes to its ease in transmission.

Epidemiology of Polio Sporadic cases ofpolio can break out at any time ofthe year, but its incidence is more pronounced during the summer and fall. The vi rus is passed within the population through food water, hands, objects contaminated with feces, and mechanical vectors. Due to worldwide vaccination programs, the number of cases was reduced a

polionl:slit1" (poh"-lee-oh-my'LehJy'-tis)

ilrs, inflamrnation.

Gr.

polios, gray, myelos, medulla, and

25.7

Nonenveloped Nonsegmented Single-Stranded RNA Viruses: Picornaviruses and Caliciviruses

775

RNA core

Figure 25.21

Typical structure of a picornavirus.

(a) A poliovirus, a type of picornavirus that is one of the simplest and smallest viruses (30 nm). lt consists of an icosahedral capsid shell around a tightly packed molecule of RNA. (b) A crystalline mass of stacked poliovirus particles in an infected host cell (300,000x).

Selected Representatives of the Human Picornaviruses Representative

Primary Diseases

Poliovirus

Poliomyelitis

CoxsackievirusA Coxsackievirus B

Focal necrosis, myositis

Echovirus

Aseptic meningitis, enteritis, others

Myocarditis of newborn

Enterovirus 72

HepatitisA

Rhinovirus

Rhinovirus

Common cold

Cardiovirus

Cardiovirus

EncephalomyocardiJis

Aphthovirus

Aphthovirus

Foot-and-mouth disease (in cloven-foot animals)

from 350,000 in 1988 to a few thousand by 2008. There have been no cases of wild polio in the Americas since I 991 . After being ingested, polioviruses adsorb to receptors of mucosal cells in the oropharynx and intestine (figure 25.22). Here, they multiply in the mucosal epithelia and lymphoid tissue. Multiplication results in large numbers of viruses being shed into the throat and feces. and some of them leak into the blood. Most infections are expressed in a short-term, mild viremia. Some persons develop mild nonspecific symptoms of fever, headache, nausea, sore throat, and myalgia. If the viremia persists, viruses can be carried to the central nervous system through its

Figure 25.22

The stages of infection and pathogenesis

of poliomyelitis. (a) First, the virus is ingested and carried to the throat and intestinal mucosa. (b) The virus then multiplies in the tonsils. Small numbers of viruses escape to the regional lymph nodes and blood. (c) The viruses are further amplified and cross into certain nerve cells of the spinal column and central nervous system. (d) Last, the intestine actively sheds viruses.

blood supply. The virus then spreads along specific pathways in the spinal cord and brain. Being neurotropico* the virus infiltrates the motor neurons ofthe anterior horn ofthe spinal cord, though it can also attack spinal ganglia, cranial nerves, and motor nuclei (figure 25.23). Nonparalytic disease involves the invasion but not the destruction of nervous tissue. It gives rise to muscle pain and spasm, meningeal inflammation, and vague hypersensitivity. The progression of infection and disease is shown in figure 25.24.

Porolytic Diseose Invasion ofmotor neurons causes various degrees offlaccid paralysis over a period ofa few hours to several days. Depending on the level of damage to motor neurons, paralysis of the muscles of the legs, abdomen, back, intercostals, diaphragm, pectoral girdle, and bladder can result. In rare cases of bulbar poliomyelitis' the brain stem. medulla. or even cranial nerves are affected. This situation

* neut"ottopic (nl'-roh-troh'-pik) Having an affinity for the nervous system

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The RNA Viruses That Infect Humans

leads to loss of control of cardiorespiratory centers, requiring mechanical respirators. In time, the unused muscles begin to atrophy, growth is slowed, and severe deformities of the trunk and limbs develop. Common sites of deformities are the spine, shoulder, hips, knees, and feet. Because motor function, but not sensation, is compromised, the crippled limbs are often very painful. In recent times a condition called post-polio syndrome (ppS) has been diagnosed in long-term survivors ofchildhood infection. PPS manifests as a progressive muscle deterioration that develops in about 25o/o to 50% ofpatients several decades after their original

polio attack.

Diagnosis of Polio Polio is mainly suspected when epidemics of neuromuscular disease occur in the summer in temperate climates. Poliovirus can usually be isolated by inoculating cell cultures with stool or throat washings in the early part of the disease. The stage of the patient's infection can also be demonstrated by testing serum samples for the type and amount of antibody.

Meosures for Treatment ond Prevention

of Polio

Treatment of polio rests largely on alleviating pain and suffering. During the acute phase, muscle spasm, headache, and associated discomfort can be alleviated by pain-relieving drugs. Respiratory

failure may require artificial ventilation maintenance. Prompt physical therapy to diminish crippling deformities and to retrain muscles is recommended after the acute febrile phase subsides. The mainstay of prevention is vaccination as early in life as possible, usually in four doses starting at about 2 months of age. Adult candidates for immunization are travelers and members of the armed forces. The two forms of vaccine currently in use are inactivated poliovirus vaccine (IPV), known as the Salk3 vaccine, and oral poliovirus vaccine (OPV), known as the Sabina vaccine. Both are prepared from animal cell culhres and are trivalent (combinations of the three serotlpes of the poliovirus). Both vaccines are effective,

Anterior horn cells, damaged

Flgure 25.2?

Targets of poliovirus.

(a) A cross section of the spinal column indicates the areas (circled) most damaged in spinal poliomyelitis. (b) The anterior horn cells of a monkey before (top) and after (bottom) poliovirus infection (450x).

r Asymptomatic r Minor non-CNS r Aseptic meningitis m Paralytic

40

60

illness

100

but one may be favored over the other under certain circumstances. For many years, the Sabin vaccine was used in the United States because it is easily administered by mouth, but it is not free of medical complications. It contains an attenuated virus that can multiply in vaccinated people and be spread to others. In very rare instances, the attenuated virus reverts to a neurovirulent strain that causes disease rather than protects against it. Numerous instances of paralytic polio have occurred among children with hypogammaglobulinemia who have been mistakenly vaccinated. There is a tiny risk (about one case in 4 million) that an unvaccinated family member will acquire infection and disease from a vaccinated child. For these reasons, public health officials have revised their recommendations and now favor IPV for all immunizations in the United States.

The efforts of a WHO campaign, called National Vaccination Days, have significantly reduced the global incidence of polio. It is the campaign's goal to eradicate all of the remaining wild polioviruses by 2010. The last cases of infection are confined to a few pockets in Nigeria, India, Pakistan, and Afghanistan. Unfortunately,

Percent

Flgure

25.24

Diagrammatic representation of possible outcomes of poliovirus infection.

3. Named for Dr. Jonas Salk, who developed the vaccine in 1954. 4. Named for Dr. Albert Sabin, who had the idea to develop an oral attenuated vaccine in the 1960s.

25.7

Nonenveloped Nonsegmented Single-Stranded RNA Viruses: Picornaviruses and Caliciviruses

777

the success of this campaign has been plagued by outbreaks in countries that barred the use of the oral polio vaccine because of political upheavals and concerns about its safety.

Nonpolio Enteroviruses A few viruses related to poliovirus commonly cause short term, usu-

ally mild infections. The most common of these are coxsackieviruses* A and B, echoviruseso* and nonpolio enteroviruses. They are similar to the poliovirus in many of their epidemiological and infectious characteristics. The incidence ofinfection is highest from late spring to early summer in temperate climates and is most frequent in infants and persons living under unhygienic circumstances.

Specific Types of Enterovirus lnfection Enteroviral infections are either subclinical or fall into the category of "undifferentiated febrile illness," characterized by fever, myalgia, and malaise. Symptoms are usually mild and last only a few days. The initial phase of infection is intestinal, after which viruses enter the lymph and blood and disseminate to other organs. The outcome of this infection largely depends on the organ affected. An overview of the more severe complications follows. I

mporto nt Com plications

Children are more prone than adults to lower respiratory tract illness,

namely bronchitis, bronchiolitis, croup, and pneumonia'

All

ages,

however, are susceptible to the "common cold syndrome" of enteroviruses (see Insight 25.4). Pteurodynia* is an acute disease characterized

25.25 Acute hemorrhagic coniunctivitis caused enterovirus. by an

Ffgure

In the early phase of disease, the eye is severely inflamed, with the sclera bright red owing to subconiunctival hemorrhage. Later, edema of the lids causes complete closure of the eye.

B or C viruses, it shares their tropism for liver cells. Otherwise, the two viruses are different in almost every respect (see table 24.3). The hepatitis A virus is a cubical picornavirus relatively resistant to heat and acid but sensitive to formalin, chlorine, and ultraviolet radiation. There appears to be only one major serotype of this virus.

by recurrent sharp, sudden intercostal or abdominal pain accompanied by fever and sore throat. Although nonpolio enteroviruses are less virulent than the polioviruses, rare cases of coxsackiwirus and echo-

virus paralysis, aseptic meningitis, and encephalitis occur' Even in severe childhood cases involving seizures, ataxia, coma, and other cental nervous system symptoms, recovery is usually complete. Eruptive skin rashes (exanthems) that resemble the rubella rash are other manifestations of enterovirus infection. Coxsackievirus can cause a peculiar pattern oflesions on the hands, feet, and oral mucosa (hand-foot-mouth disease), along with fever, headache, and muscle pain. Acute hemorrhagic conjunctivitis is an abrupt inflammation associated with subconjunctival bleeding, serous discharge, painful swelling, and sensitivity to light (figure 25.25\. Spread of the virus to the heart in infants can cause extensive damage to the myocardium, leading to heart failure and death in nearly half of the cases. Heart involvement in older children and adults is generally less serious, with symptoms of chest pain, altered heart rhythms, and pericardial infl ammation.

One enterovirus that tends to affect the digestive fract alone is the hepatitisA virus (HAV; enterovirus 72),the cause of infectious, or short-term, hepatitis. Although this virus is not relatedto the hepatitis

virus, * pleurodynia (plul'-oh-din'-ee- ah) Gr. pleum, rib, side, and odyne, pain'

of Hepatitis A

Hepatitis

A virus is spread

ers or people with clinical disease.

The Course of HAV Infection Swallowed hepatitis A virus multiplies in the small intestine during an incubation period of 2 to 6 weeks. The virus is then shed in the feces, and in a few days, it

Hepatitis A Virus ond lnfectious Hepatitis

* coxsackievirus (kok-sak'-ee-vy''-rus) Named for Coxsackie, NewYork, where viruses were first isolated. * echovims (ek'-oh-vy''-rus) An acronym for enteric cytopathic human orphan

Epidemiology

through the oral-fecal route, but the details of transmission vary from one ateato another. In general, the disease is associated with deficient personal hygiene and lack ofpublic health measures. In countries with inadequate sewage control, most outbreaks are associated with fecally contaminated water and food. The United States has a yearly reported incidence of 4,000 to 5,000 cases. Most of these result from close institutional contact, unhygienic food handling, eating shellfish, sexual fansmission, or travel to other countries. Occasionally, hepatitis A can be spread by blood or blood products. In developing countries, children account for most cases due to exposure early in life, whereas in NorthAmerica and Europe, more cases appear in adults. Because the virus is not carried chronically, the principal reservoirs are asymptomatic, short-term carri-

tle

enters the blood and is carried to the liver. Most infections are either

subclinical or accompanied by yague, flulike symptoms. In more overt cases, the presenting symptoms are loss of appetite, nausea, diarrhea, fever, pain and discomfort in the region of the liver, and darkened urine. Jaundice is present in only about one in l0 cases. Occasionally, hepatitisA occurs as a fulminating disease and causes liver damage, but this manifestation is quite rare. Because the virus is not oncogenic and does not predispose to liver cancer, complete uncomplicated recovery results.

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The RNA Viruses That Infect Humans

Uncommon Facts about the Common Cold The common cold touches the lives of humans more than any other viral infection. afllicting at least halfthe population every year and accounting for millions of hours of absenteeism from work and school. The reason for its widespread distribution is not that it is more virulent or transmis-

sible than other infections but that symptoms of colds are linked to hundreds of different viruses and viral strains. Among the known causative viruses. in order ofimportance. are rhinoviruses (which cause about half of all colds), paramyxoviruses. enteroviruses. coronaviruses, reoviruses. and adenoviruses. A given cold can be caused by a single virus type or it can result from a mixed infection. The name implies a condition caused by cold weather or drafts. But studies in which human volunteers with wet heads or feet chilled or ex-

posed to moist. frigid air have faited to show that cold weather alone causes colds. Most colds occur in the late autumn, winteg and early spring-all periods of colder weather-but this seasonal connection has more to do with being confined in closed spaces with carriers than with

Finding a cure for the common cold has been a long-standing goal of medical science. This quest is not motivated by the clinical nature of a cold, which is really a rather benign infection. A more likely reason to

for a "magic cold bullet" is productivity in the workplace and schools, as well as the potential profits from a truly effective cold drug. One nonspecific approach has been to destroy the virus outright and search

halt its spread. Special facial tissues impregnated with antiviral compounds have been marketed for use during the cold season. A recent study has finally shown that the herb echinacea can help prevent colds and zinc lozenges may also help slow the onset of cold symptoms.

The important role of natural interferon in controlling many cold viruses has led to the testing and marketing ofa nasal spray containing recombinant interferon. One therapy uses antibodies raised to the site on the human cell (receptor) to which the rhinovirus attaches. In theory these antibodies should occupy the cell recepror, competitively inhibit viral attachment, and prevent infection. Experiments in chimpanzees and hu-

temperature.

mans showed that, when administered intranasally, this antibody

The most significant single factor in the spread of colds is contamination of hands with viruses in mucous secretions. The viruses usually

preparation delayed symptoms and reduced their severity.

invade through the mucous membranes of the nose and eyes. The moit comrnon symptom is a nasal discharge, and the least common is fever, except in infants and children.

Diagnosis and Control of Hepatitis

A A patient's history,

liver and blood tests, serological tests, and viral identification all play a role in diagnosing hepatitis A and differentiating it from the other forms of hepatitis. Certain liver enz)'rnes are elevated, and leukopenia is common. Diagnosis is aided by detection of antiHAV IgM antibodies produced early in the infection and tests to identify HA antigen or virus directly in stool samples. There is no specific treatment for hepatitis A once the symptoms begin. Patients who receive immune serum globulin early in the disease usually experience milder symptoms than patients who do not receive it. Prevention of hepatitis A is based primarily on immunization. An inactivatedviral vaccine (HAVRAX) is currently approved, and an oral vaccine based on an attenuated strain ofthe virus is in development. Vaccination is recommended for children and adults with increased risk of exposure to HAV Pooled immune serum globulin is also recommended for travelers and armed forces personnel planning to enter endemic areas, contacts ofknown cases, and occupants of various institutions during epidemics. Control of this disease can be improved by sewage treatment, hygienic food handling and preparation, and adequate cooking of shellfish.

Humon Rhinovirus (HRV) The rhinoviruses* are a large group of picornaviruses (more than 110 serotypes) associated with the common cold (Insight 25.4). Although the majority of characteristics are shared with other picornaviruses, two distinctions setrhinoviruses apart. First, they are sensitive

' rhi'torin,.' (ry'-noh-ry'-rus) Cr. rhinos. nose

Propose some reasons that cold temperatures could make people more susceptible to cold viruses. Answer available at http://www. mhhe.com/talaroT

to acidic environments such as that of the stomach. and second their optimum temperature of multiplication is not normal body temperature but 33'C, the average temperature in the human nose.

Portrait of Rhinovirus Virologists have subjected rhinovirus (type 14) to a detailed structural analysis. The results of this study provided a striking three-dimensional view of its molecular surface and also explained why immunity to the rhinovirus has been so elusive. The capsid subunits are of two types: protuberances (knobs), which are antigenically diverse among the rhinoviruses, and indentations (pockets), which exist only in two forms (figure 25.26). Because the antigens on the surface are the only ones accessible to the immune system, a successful vaccine would have to contain hundreds ofdifferent antigens, so it is not practical. Unfortunately, the recessed antigens are too inaccessible for immune actions. These factors make the development of a vaccine unlikely, but it could provide a basis for developing drugs to block the pocket receptors.

Epidemiology and Infection of Rhinoviruses Rhinovirus infections occur in all areas and all age groups at all times of the year. Epidemics caused by a single type arise on occasion, but usually many strains circulate in the population at one time. As mutations increase and herd immunity to a given type is established, newer types predominate. People acquire infection from contaminated hands and fomites, and to a lesser extent from droplet nuclei. Children are highly susceptible to colds and often transmit the virus to the rest of a family. Although other animals have rhinoviruses, interspecies infections have never been reported. After an incubation

25.8

Nonenveloped segmented Double-Stranded RNAViruses: Reoviruses

779

Caliciviruses Antibody

Knob antigen

Pocket antigen

Caliciviruses* are an ill-defined group of enteric viruses found in humans and mammals. The best-known human pathogen is the Norwalk agento also known as norovirus, named for an outbreak of gastroenteritis that occurred in Norwalk, Ohio, from which a new type of virus was isolated. The Norwalk virus is now believed to cause one-third of all cases of viral gastroenteritis. It is transmitted orofecally in schools, camps, cruise ships, and nursing homes and through contaminated water and shellfish. Infection can occur at any time of the year and in people of all ages. Onset is acute, accompanied by nausea, vomiting, cramps, diarrhea, and chills; recovery is rapid and comPlete.

25.8 Nonenveloped Segmented Double-Stranded RNA Viruses: Reoviruses

(b)

Flgure 25.26 Antigen structure of a rhinovirus. (a) The surface of a rhinovirus is composed of knobs and pockets. The antigens on the knobs are extremely variable in shape among the scores of viral strains, and those in the pockets do not vary among the strains. (b) The knobs are readily accessible to the immune system, and antibodies formed against them will neutralize the virus (circle). Unfortunately being fully protected against rhinoviruses would require making antibodies that react with 100 different knob antigens. lt is true that reactions against pocket antigens would be universal, but these molecules are too inaccessible for immune reactions'

period of 1 to 3 days, the patient can experience some combination ofheadache, chills, fatigue, sore throat, cough' a mild nasal drainage, and afypical pneumonia. Natural host defenses and nasal antibodies have a beneficial local effect on the infection, but immunity is shortlived.

Control of Rhinoviruses The usual therapy is to force fluids and relieve symptoms with various cold remedies and cough syrups that contain nasal decongestants, antihistamines, and analgesics. The actual effectiveness of most of these remedies (of which there are hundreds) is rather questionable. Owing to the extreme diversity of rhinoviruses, prevention of colds through vaccination will probably never be a medical reality' Some protection is afforded by such simple measures as hand washing and care in han-

dlins nasal secretions.

Reoviruses have an unusual double-stranded RNA genome and both an inner and an outer capsid (see table 25.1). Two of the beststudied viruses of the group are Ro tavirus and Reovirus. Named for its wheel-shaped capsomer, Rotsvirus* (figure 25.27a) is a significant cause of diarrhea in newborn humans, calves, and piglets. Because the viruses are transmitted via fecally contaminated food, water, and fomites, disease is prevalent in areas of the world where access to adequate sanitation is lacking. Globally, Rotairus is the primary viral cause of mortality and morbidity resulting from di' arrhea. It accounts for near$ 50% ofall the cases, resulting in the deaths of over 600,000 children. The effects of infection vary with the nutritional state, general health, and living conditions of the infant. Babies from 6 to 24 months of age lacking maternal antibodies have the greatest risk for fatal disease. These children present with symptoms of watery diarrhea, feveq vomiting, dehydration, and shock. The intestinal mucosa can be damaged in a way that chronically compromises nutrition, and long-term or repeated infections can retard growth (figure 25.27b). In the United States, rotavirus infection is relatively common' but its course is generally mild. Children are treated with oral replacement fluid and electrolytes. Two vaccines (RotaTeq, Rotarix) based on various versions of live, attenuated viruses are currently available. Initial results indicate that the vaccines reduced serious illness by 85Yo to 95o/o inthe groups of infants tested. They are both easily administered oral vaccines but rather costly. Reovirus* is not considered a significant human pathogen. Adults who were voluntarily inoculated with the virus developed coldlike symptoms. The virus has been isolated from the feces of children with enteritis and from people suffering from an upper respiratory infection and rash, but most infections are asymptomatic. Although a link to biliary meningeal, hepatic, and renal syndromes has been suggested, a causal relationship has never been proved. +

calicivirus (kal'-ih-sih-vy''-rus)L, calix, the cup of

a flower. These viruses have

cup-shaped surface depressions.

* Rotavirus (roh'-tah-vy''-rus) L. rota, wheel, a Reovirus (ree'oh-vy''-rus) An acronlrm for respiratory enteric orphan virus'

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The RNA Viruses That Infect Humans

25.9 Prions and Spongiform Encephalopathies Devastating diseases such as subacute sclerosing panencephalitis and progressive multifocal leukoencephalopathy arising from persistent viral infections of the CNS have previously been described. They are characteized by a long incubation period, and they can progress over months or years to a state of severe neurological impairment. These types of chronic infections are caused by known viruses with conventional morphology. But there are other types of CNS infectious diseases for which no traditional microorganism has been isolated. The most serious ofthese are the diseases associated with prions.5 You may recall from chapter 6 that prions are proteinaceous infectious particles that appear to lack genetic material. prions are incredibly hardy 'pathogens." They are highly resistant to chemicals, radiation, and heat. They can withstand prolonged autoclaving (table 25.5). They are known to cause diseases called transmissi-

ble spongiform encephalopathies* (TSEs), neurodegenerative with long incubation periods but rapid progressions once

diseases

theybegin. The humanTSEs are Creutzfeldt-Jakob disease (CJD), kuru, Gerstmann-Strussler-scheinker disease, and fatal familial insomnia, TSEs are also found in animals and include a disease called scrapie in sheep and goats, transmissible mink encephalopathy, and bovine spongiform encephalopathy (BSE). This last disease, cofilmonlyknownasmadcowdisease, hasbeeninthe headlines in recent years due to its apparent link to a variant form of CJD human disease in Great Britain.

Pathogenesis and Effects of

Flgure 25,27

Diagnosing gastroenteritis.

(a) A sample of feces from a child with gastroenteritis as viewed by electron microscopy. Note the unique "spoked-wheel,, morphology that can be used to identify Rotavirus (150,000x). (b) An infant suffering from chronic rotavirus gastroenteritis shows evidence of malnutrition, failure to thrive, and stunted growth.

€ b € e

Picornaviruses are tiny RNA viruses distributed worldwide. The most prominent members of the family arc Enterovirus, the hepatitis A; and. Rhinwirus, the agenr in about :uy^rr:f.ryltg-and half of all cold infections. Other common human enteric viruses are coxsackievirus and echovirus, both of which cause acute intestinal or respiratory infections. Reoviruses are unusual in possessing both double-stranded RNA and two capsids. The two major groups are rotaviruses and reoviruses. Rotavirus is a significant intestinal pathogen for young children worldwide. Reovirus causes an upper respiratory infection similar to the common cold.

Cf

D

The pathogenesis of the prions involve an alteration in the structure of a normal host protein (called PrP) found in mammalian brains. The PrP has acquired a significant change in its shape through mutation or some other means. The effect of this alteration is that the abnormal PrP itself becomes catalytic and able to spontaneously convert other normal human PrP proteins into the abnormal form. This becomes a self-propagating chain reaction that creates a massive 5. A contraction ofproteinaceous infectious particle.

* spongiJbrnr encephalopathies (spunj'-ih-form en-sef,,-uhlop,-uh-theez)

Properties of the Agents of Spongiform Encephalopathies Very resistant to chemicals, radiation, and heat (can withstand autoclaving) Do not present virus morphology in electron microscopy of infected brain tissue

Not integrated into nucleic acid of infected host cells Proteinaceous

Do not elicit inflammatory reaction or cytopathic effects in host Do not elicit antibody formation in host Responsible for vacuoles and abnormal fibers forming in brain of host

Transmitted only by close, direct contact with infected tissues and secretions

25.9

Prions and Spongiform Encephalopathies

781

Glial cells

Glial cells

fgure 25.2a The microscopic effects of spongiform encephalopathy. fl(a) Normal patient shows numerous holes. The open cerebral cortex section, showing neurons and glial cells. (b) section of cortex in cJD This gives the tissue a spongy appearance. spaces correspond to sites once occupied by brain cells that have been destroyed. (750x). disease in the outcome deadly loss of neurons and glial cells that gives rise to to the

accumulation of altered PrP that results in nerve cell death, spongiform damage (that is, holes in the brain), and severe loss of brain function. Autopsies of the brain of CJD patients reveal spongiform lesions (figure 25.28) as well as tangled protein fibers (neurofibrillary tangles) and enlarged astroglial cells. These changes occur primarily in the gray matter of the CNS. The altered PrPs are not apparently antigenic and do not stimulate an immune response. Symptoms ofCJD include alteredbehavior, dementia, memory loss, impaired senses, delirium, and premature senility. Uncontrollable muscle contractions continue until death, which usually occurs within 1 year of diagnosis.

Transmission and EPidemiology The transmissible form of CJD is not a highly communicable disease in that ordinary contact with infected people is not known to transmit the prions. Direct or indirect contact with infected brain tissue or cerebrospinal fluid is one factor in its acquisition. The classic form of CJD is considered endemic and occurs at the rate of one case per million persons in the United States per year, mainly in the elderly. Using the term transmissible agent may be a bit misleading, because some cases of CJD arise through genetic mutation of the PrP gene rather than through contact with infectious material. In fact, up to 15% of CJD cases appear to develop after inheritance of a mutated gene. In the late 1990s, it became apparent that humans were con-

tracting a variant form of CJD (vCJD) after ingesting meat from cattle that had been afflicted by bovine spongiform encephalopathy. Presumably, meat products had been contaminated with fluid or tissues infected with the prion, although the exact food responsible for the transmission has not been pinpointed' When experimenters purposely infect cattle with prions, they subsequently detect the agent in the retina, dorsal root ganglia, parts ofthe digestive tract, and the bone marrow of the animals. Evert so, the risk of contracting vCJD from the ingestion of meat is extremely small, even in

countries such as Great Britain, where a significant number of livestock have been found to have BSE, There the risk of infection

lt

is such massive

is estimated to be one case per 10 billion meat servings. As of 2007, a total of 210 cases of vCJD had occurred worldwide. The median

age at death of patients with vCJD is 28 years. In contrast, the median age at death of patients with classic CJD is 68 years. Heatth care professionals should be aware of the possibility of CJD in patients, especially when surgical procedures are performed, as cases have been reported of transmission of CJD via contaminated surgical instruments. Due to the heat and chemical resistance of prions, normal disinfection and sterilization procedures are usually not sufficient to eliminate them from instruments and surfaces. The latest CDC guidelines for handling of CJD patients in a health care environment should be consulted. CJD has also been transmitted through corneal grafts and administration of contaminated human growth hormone. Currently, no cases have been documented of fansmission through blood products, although scientists have shown that it is possible in laboratory experiments. Experiments suggest that vCJD seems to be more transmissible through blood than is classic CJD. For that reason, blood donation prograrns screen for possible exposure to BSE by asking about travel and residence history.

Culture and Diagnosis It can be very difficult to diagnose CJD. Definitive diagnosis requires examination of biopsied brain or nervous tissue, and this procedure is usually considered too risky because of both the trauma induced in the patient and the undesirability of contaminating surgical instruments and operating rootns. Electroencephalogmms and magnetic resonance imaging can provide important clues. A new test has been developed that can detect prion proteins in cerebrospinal flui{ but it is not yet widely used because it has a relatively high error rate.

Prevention and/or Treatment Prevention of either type of CJD relies on avoiding any contact with infected tissues. No known treatment exists for CJD, and patients inevitably die. Medical intervention focuses on easing symptoms and making the patient as comfortable as possible.

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25

The RNA Viruses That Infect Humans

Long-term degenerative and incurable CNS infections that cannot prions, infectious proteins with atypical biological properties that are un_

be linked to any known microbe are thought to be caused by

usually resistant to destruction.

Creutzfeldt-Jakob disease (CJD) and kuru are two ofseveral TSEs (transmissible spongiform encephalopathies) identified to date. Disease results from the accumulation of abnormal proteins and death ofbrain cel1s, creating empty spaces.

Chapter Summary with Key Terms 25.1 Enveloped Segmented Single-Stranded RNA Viruses

A.

B.

the presence of antibodies to

HIV along with some combination of opportunistic infections, fever, weight loss, and other symptoms. 1. HIV is found in blood, semen, and vaginal secretions and can be transmitted by any activity that causes these fluids to be exchanged (sex, sharing needles, childbirth, etc.) 2. HIV attacks the cells of the immune system, beginning with T lymphocytes and macrophages. 3. The first signs ofAIDS are the AlDS-defining illnesses (ADIs), opportunistic infections such as Pneumocystis (carinii) jiroveci pneumonia (pCp) and cancers such as Kaposits sarcoma. 4. Treatment for HIV infection andAIDS centers on drug cocktails containing inhibitors ofthe HIV life cycle such as azidothymidine (AZT), ddl, 3TC, and protease inhibitors. Drugs are also given to prevent/treat opportunistic infections.

Orthomyxoviruses: Influenza viruses have a segmented genome that encourages antigenic shift and drift and glycoprotein spikes (hemagglutinin and neuraminidase), which are needed to bind to host cells. Bunyaviruses: Zoonotic viruses. Hantavirus is responsible for Korean hemorrhagic fever and hantavirus pulmonary syndrome.

C. Arenaviruses:

Lassa virus causes hemorrhagic fever and is spread by rodent secretions.

25.2 Enveloped Nonsegmented Single-Stranded RNA Viruses

A.

Paramyxoviruses: Nonsegmented genome. paramyxovirus causes parainfluenza and mumps. Morbillivirus is responsible for measles (roseola). paramyxovirus and.

Morbillivirus are prevented through vaccination (MMR). Pneamoviras (respiratory syncytial virus, RSV) is the causative agent of croup and is treated with aerosol

B.

ribavirin. Rhabdovirtsest Lyssaviru,s is the cause of rabies, a lethal zoonosis. Treatment requires both active and passive

immunization.

25.3 Other Enveloped RllA Viruses: Coronaviruses, Togaviruses, and Flaviviruses A. Coronaviruses are one cause of the common cold. B. Togaviruses: Rubella (German measles) is of minor concern to healthy adults but can be teratogenic (cause birth defects) in pregnant women. Control is through

C.

immunization (MMR). Flaviviruses: Hepatitis C virus is a major cause of hepatitis transmitted through blood contact. Chronic infection may cause

liver damage and cancer.

25.4 Arboviruses: Viruses Spread byArthropodVectors

A.

B.

Arboviruses: large group (400+) of viruses spread by arthropod vectors and harbored by birds and other animals. Examples of viruses include togaviruses, bunyaviruses, and reoviruses; diseases include western and eastern equine encephalitis, yellow fever, and dengue fever.

25.5 HIV Infection and AIDS

A.

B.

Retroviruses are enveloped, single-stranded RNA viruses that encode the en4lme reverse transcriptase, which allows the viruses to convert their single-stranded RNA genome to a double-stranded DNA genome. The most prominent retrovirus is human immunodeficiency

virus (HIV types 1 and 2), the causative agent ofacquired immunodeficiency syndrome (AIDS). AIDS is marked by

25.6 Other Retroviruses: Human T:cell Lymphotropic Viruses Other retroviruses include HTLV-I and HTLV-[. The former causes adult T:cell leukemia while the latter may be involved in some forms of brain disease.

25.7 Nonenveloped Nonsegmented Single-Stranded RNA Viruses: Picornaviruses and Caliciviruses A. Picornaviruses are the smallest human viruses and include I . Poliovirus, the causative agent ofpolio. 2. Coxsackievirus, which is responsible for respiratory infections, hand-foot-mouth disease, and conjunctivitis. 3. Hepatitis A virus, which is responsible for the mildest form ofhepatitis. 4. Rhinovirus, the most prominent agent of the common cold.

B.

Caliciviruses, the most cofirmon of which, Norwalk agento (norovirus) is a common cause of viral gastroenteritis.

25.8 Nonenveloped Segmented Double-Stranded RNA Viruses: Reoviruses Reoviruses, the most important of which is Rotavirus, a leading cause ofsevere infantile diarrhea worldwide.

25.9 Prions and Spongiform Encephalopathies Prions are infectious agents consisting exclusively ofprotein. They are responsible for transmissible spongiform encephalopathies such as mad cow disease, scrapie in animals, and Creutzfeldt-Jakob disease in humans.

M u ltiple-Choice

M

783

Questions

ultiple-Choice Questions

the with blanks, choose the combination of answers that most accurately completes Select the correct answer from the answers provided For ouestions statement.

l.

which receptors of the influenza virus are responsible for binding to

a. arbo b. enteric

the host cell?

c. typeA d. capsid proteins

a. hemagglutinin b. neuraminidase 2.

c. skin a. small intestine b. respiratory epithelium d' meninges 3.Whichofthesehappen(s)inthecaseofantigenicshiftininfluenzaA? a. single mutations in hemagglutinin b. recombination of RNA segments between bird and human strains change from influenzaA to influenza B a and b

d. both

4. Infections with

_

virus cause the development of multinucleate

giant cells.

c. pneumo d. corona

a. rabies

b. influenza 5. Which virus is responsible for Korean hemorrhagic fever? c. arenavirus a. Ebolavirus d. flavivirus b. hantavirus 6. For which disease is an exanthem (skin rash) not a symptom?

c. coxsackievirus infection

a. measles b. rubella

d. parainfluenza

c. d.

SARS

g. viruses that cause serious diseases in infants are a. mumps, calicivirus

- mumps virus -HIVmeasles virus - hepatitis A virus - hantavirus - western equine - encephalitis virus - enterovirus

and

-

-.

c. coxsackievirus, HTLV-II d. bunyavirus, cardiovirus 9. Rabies virus has an average incubation period of

a.2to3weeks

b. lto2years

c.4to5days d. lto2months

10. For which disease are active and passive immunization given simultaneously? c. measles a. influenza d. rabies b. yellow fever II

. What property of

t9

12. Which of the following cells is atarget of HIV infection? c. helperT cells a. dendritic cells d. all ofthese can support infection b. monocytes 13. Which of the following conditions is nol associated with AIDS? a. Pneumocystis (cariniil jiroveci pneumonia b. Kaposi's sarcoma c. dementia d. adult T:cell leukemia

poliovirus rabies virus rubella virus

a. brain

b. parotid gland

c. respiratory tract d. white blood cells e. kidney

f.

spinal cord

g. liver h. intestinal tract

i.

j.

heart eye

- rotavirus the virus with its primary mode of transmission' -Matching. Match may have more than one mode. Some viruses

SARS

the retroviruses enables them to integrate into the

host genome? a. the RNA they carrY b. presence of glycoprotein receptors c. presence of reverse transcriptase d. a positive-sense genome

of

17. Multiple Matching. Select all descriptions that fit each type of virus' a. enveloped orthomyxovirus b. double-strandedRNA rhabdovirus - paramyxovirus c. single-stranded RNA - reovirus d. genome in segments - morbillivirus e. icosahedral caPsid - poliovirus f. helical nucleocaPsid - retrovirus g. nonenveloped - hantavirus 18. -Multiple Matching. Match the virus with its pnmary target organ or site of attack. Some viruses may attack more than one organ'

sore throat Koplik's spots

b. respiratory syncytial virus, rotavirus

d. syncytial

16. Which of the followingis not a characteristic of the agents spongiform encePhaloPathies? a. highly resistant b. associated with tangled protein fibers in the brain c. are naked fragments of RNA d. cause chronic transmissible disease

7. A common, highly diagnostic sign of measles is a. vrremra b. red rash

c. cold

15. Rhinoviruses are the most common cause of c. hand-foot-mouth disease a. conjunctivitis the common cold d. b. gastroenteritis

The primary site of attack in influenza is the

c.

viruses'

14. Polio and hepatitis A viruses are

- mumps virus -HIVmeasles virus - hepatitis A virus - hantavirus - western equine - encephalitis virus - enterovirus -

poliovirus rabies virus rubella virus rotavirus

a. respiratory droPlets

b. sexual transmission c. ingestion (oral-fecal) d. arthropod bite e. contact with mammals

f.

blood transfusion

g. fomites

Chapter

784

25

The RNA Viruses That Infect Humans

W#w€insto'riarn These questions are suggested as a

writing-to-learn experience. For each question, compose

a one- or two-paragraph answer that includes the factual

information needed to completely address the question. General page refeiences for these topics are given in parentheses.

l.

a. Describe the structure and functions of the hemagglutinin and neuraminidase spikes in influenza virus. (750,751) b. What is unusual about the genome of influenza virus? (750,751,752)

c.

11. a. Differentiate between HIV infection, HIV disease, and

ArDS.

Use drawings and words to explain the concepts of antigenic drift and antigenic shift, and explain their impact on the epidemiology

of this disease . ('751,752)

d. How do the names for the types of flu originate? (751,752) 2. a. Explain the course of infection and disease in influenza. ,J53\ b. What are the complications? (753) c. Explain generally how the flu vaccines are prepared, and for which groups is vaccination indicated? (753,754) 3. a. Give examples of bunyaviruses and arenaviruses. (754) b. Briefly describe the nature ofthe diseases they cause. (j54) c. Describe their modes of transmission. (754) 4. a. Describe the steps in the production of multinucleate giant cells during a viral

infection. (756)

b. Which viruses have this effect, and what impact does cell fusion have on the spread

ofinfection? (756)

c. Name two examples of Paramyxovirus and describe the diseases they cause. (756,757) 5.

Describe the progress of measles symptoms. (757 ,7 58) What is the cause of death in measles, and what are the most severe complications of measles infections? (758) c. Describe methods of treatrnent and prevention for measles. (75S)

a.

b.

6.

Describe the epidemiological cycle in rabies. (759\ b. Which animals in the United States are most frequently involved

a.

as

carriers? (759,760)

c. Describe

the route of infection and the virus' pathologic

effects. (760) d. Why is rabies so uniformly fatal? (i60) e. Describe the indications for pre- and postexposure rabies treatment and vaccination. (761)

f.

What is given in postexposure treatment that is not given in preexposure treatmentofrabies? (761)'

7. a. What is a teratogenic virus? (762) b. Which RNA virus has this potential? (762) c. In what ways is rubella different from red measles? (75g,762) d. What is the protocol to prevent congenital rubella? (762') 8. a. What are the principal carrier arthropods for arboviruses? (763) b. How is the cycle of the virus maintained in the wild? '763.) c. Describe the symptoms of the encephalitis type of infection (WEE, EEE) and the hemorrhagic fevers (yellow fever). (763,764,765) 9. a. What are retroviruses, and how are they different from other

viruses? (766) b. Give examples of some human retroviruses and the diseases they

cause. (766) c. Give a comprehensive definition ofAIDS. (769,772)

d. Overview the statistics for AIDS on worldwide and U.S.

levels. (767,768) 10. a. Briefly explain the activities most likely to spreadAlDS and what factors increase the relative risk among certain populations. (766-:768) b. Discuss some ways that HIV has notbeetdocumented to

spread. (769)

(770,772)

b. What are the primary target tissues and pathologic effects over time? (767,769) c. Explain why only certain T cells are targeted by HIV and what it does to them. (766,767) d. Howisthebrainaffectedbyinfection? (770,772) e. What major factors cause the long latency period in

ArDS? (769,770)

12. a. Describe the changes over time in virus antigen levels, antibody levels, and CD4 T cells in the blood of an HlV-infected

individual. (770\ b. Relate these changes to the progress ofthe disease, the infectiousness of the person, and the effectiveness of the immune response to the I

virus.

(770)

3. a. List the major opportunistic bacterial, fungal, protozoan, and viral diseases used to define

AIDS.

(771, j72)

b. Describe four other secondary diseases or conditions that accompanyAIDS. (772) c. Explain which effects HIV has that create vulnerability to these

conditions. ,769) for HIV testing. (772)

particularpathogens and the other

14. a. Describe the rationales b. What is the most sensible interpretation for a seropositive or seronegative

result? (772)

c. What is meant by the "window" regarding antibody presence in the

blood?

(772)

15. a. How do AZT and other nucleotide analogs control theAIDS virus? How do protease and fusion inhibitors work? (772,7l.3) b. Are there any actual cures for AIDS? (j72) c. What is the rationale behind the new,.drug cocktail"

treatment? (773\ d. Explain strategies for vaccine development; include in your answer the primary drawbacks to development of an effective

vaccine. (772,773) 16. a. Describe the epidemiology and progress ofpolio infection and disease. (774, 775,'7'76) b. What causes the paralysis and deformity? (775,776) Compare and contrast the two fypes ofvaccines. (776) d. What characteristics of enteric viruses cause them to be readilv

transmissible? (774\ 17. a. Describe some common ways that hepatitis A is spread. (77.7) b. What are its primary effects on the body? (777) c. How is it different from hepatitis C? (762,763,7jj) 18. a. What is the common cold, and which groups of viruses can be involved in its transmission? (778\ b. Why is it so difficult to control? (i78,779) c. Discuss the similarities in the symptoms of influenza, parainfluenza, mumps, measles, rubella, RS! and (7 53,756, 7 57 ,7 58, 762, 779)

rhinoviruses.

19. a. Explain how bovine spongiform encephalopathy can be transmitted to humans. (781) b. Make a flowchart to explain the mechanism of how prions can cause a progressive effect, even though they cannot divide by

themselves. (780,781) c. What measures would be necessary to control infections with these agents? (781)

Critical Thinking Questions

20. a. Forwhich ofthe RNAviruses are vaccines available? (472) b. For which of the RNA viruses are there specific drug

21

.

785

Describe the involvement of RNA viruses in cancers. (726' 727 , 7

63,774)

treatments? (363,364)

concept Mappins

Wf

Appendix E provides guidance for working with concept maps. as many links as you linking words or phrases.

2. Provide the missing concepts in this map. Supply

1. Construct a concept map using the following words as the concepts. Supply the linking words between each pair of concepts.

can among all the concepts, and provide

HIV T cells macrophages

Enveloped singlestrand€d nonsegmented genome

Enveloped single-

standed segmentd

antibodies to HIV

9enome

AlDS-defining illnesses low CD4 levels as1'rnptomatic disease

nonprogression

AIDS

HIV disease

-l l= ":",t;j,:'|"'

f;"'j;ril t'*.*

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l*";:';*fl F-"'"' -"-l

F;f

GL g Critical - .. Thinking Questions iffiE6 ,z-/F

ryi

Critical thinking is the ability to reason and solve problems using facts and concepts. These questions can be approached from a number ofangles, and

in most cases, they do not have a single correct answer. Arboviral. California Serogroup. Number of reported cases, by month of onset-United States, 1998-2003

1. a. Explain the relationship between herd immunity and the development of influenzal pandemics. b. Why will herd immunity be lacking if antigenic shift or drift

50

occurs?

2. a. Explain how the antibody content of a patient's

sera could be used to predict which strain of influenza predominated during a previous epidemic. b. Explain the precise way that cross-species influenza infections give rise to new and different strains ofvirus. c. Relate this answer to the avian influenza virus and how it could cause a human pandemic. d. Explain why influenza RNA must enter the cell nucleus during its multiplication cycle whereas many RNA viruses do not.

3. a. Why do infections such

as mumps, measles,

diseases possible and

and host make zoonotic

difficult to control.

b. Make a diagram that details the progression of arboviruses from deep in the Amazon Forest to Florida or from Central Africa to Seattle, Washington (as happened with West Nile fever).

c.

35

-30 o

Eru

z2Q 15

't0 5

0

polio, rubella, and

RSV regularly infect children and not adults? b. Measles is considered to be a highly contagious infection. Explain how it is possible to acquire measles from a person who has only been exposed to this infection.

4. a. Explain what characteristics of virus

40

Observe the graph showing case reports for arboviral encephalitis from 1998-2003. Describe the general patternthat is evident and analyze the factors that would cause it.

1998 1999 2000 2001 2002 2003 Year and month

5.

a.

Can you think of a plausible series of events that could explain how HIV came to be so widespread? b. Explain why there can be such a discrepancy between the total number of reported cases of AIDS and the projected number of cases estimated by health authorities. c. Using figxe25.16 and table 12.6 as references, outline the strategies of anti-HIV drugs and their effects as correlated with the virus cycle.

likely possible transmission pathways of HIV (source in first person, mode of infection in second person, portals of exit and entry) for each of the following: a. From one infected homosexual man to another homosexual man

6. Trace the

b. From one drug user to another drug user c. From a bisexual man to his wife

Chapter

786

25

The RNA Viruses That Infect Humans

d. From

a monogamous man to his monogamous

e. From

a

wife of 50 years

11

mother to a child

inhibit

or not? Support your answer.

a

12. Case study I . Late in the spring, a young man

from rural Idaho developed fever, loss of memory, difficulty in speech, convulsions, and tremor and lapsed into a coma. He tested negative for bacterial

for HIV infection would be preferable to a serological test for antibodies. b. What factors have prevented the development of an effective AIDS vaccine? c. Give a plausible explanation for the fact that live, attenuated (or even dead)

remedies control or

inflammation and depress the symptoms of colds. Is this beneficial

7. Explain the characteristics of HIV and infection that make it such difficult target for the immune system and for drugs. 8. a. Explain why

. Various over-the-counter cold

a direct antigen test

HIV vaccines pose a risk.

9. a. What precautions can a person take to prevent himself or herself from contracting HIV infection? b. How can a health care worker prevent possible infection? 10. a. If wild type polio had disappeared from the Western Hemisphere by 1991, how do you explain the I I cases ofpolio reported in the United States in 2000? b. Provide an explanation for changing the guidelines for using the Salk (IPV) vaccine instead of Sabin (OPV) vaccine for routine

meningitis and had no known contact with dogs or cats. He survived but had long-term neurological disabilities. What are the possible diseases he might have had, and how would he have contracted them?

13. Case study 2. Biopsies from the liver and intestine of an otherwise asymptomatic 35-year-old male show masses of acid-fast bacilli throughout the tissue. Explain the pathology going on here and provide a preliminary diagnosis. 14. Look at figures 25.2 and,25.26, and, compare and contrast how both viruses use surface receptors to evade an immune reaction and neutral izing antibodies.

vaccination.

Visual Understanding l.

From chapter l2rtable 12.6. Use this table to review drug targets for viruses to correlate which stages in the multiplication cycle of the influenza virus (figure 25 .l) are targeted by drugs and how the drugs stop multiplication.

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Internet Search Topics Examine the results when the phrase 'Norwalk virus and cruise ships" is entered into a search engine. 2. You be the detective. Surf the Web to

find some possible answers to

these mysteries:

a. How did West Nile fever spread from Africa to the rest of the world? b. What appears to be the origin of the SARS coronavirus and the Ebola virus? c. What factors gave rise to the severity of the influenza pandemic

of 1918?

d.

Search using keywords "avian inflienza" and "history" to develop a time line for the emergence of avian flu H5N1.

J.

Go to: http://rvww.mhhe.com ltalaroT. Go to chapter 25, Internet Search Topics, and 1og on to the available websites to locate the lists of category A, B, and C pathogens and determine which viruses from this chapter are on this list.

information about the study in which polioviruses were synthesized in a test tube. Answer questions about the why, how, and potential dangers ofthis technology.

4. Find

Go to this website for an excellent overview on guidelines for control and prevention of influenza. http://wwrv.cdc.gov/fl u/

professionals/antivirals/

'::l'''

Environmental

CASE FILE

26

n spring of 1989, the oiltanker ExxonVoldez ran aground on Bligh Reef in Prince William Sound in Alaska. Almost 41 million liters of crude oil were spilled into the beautiful, pristine wilderness of the Sound. The news media rushed to the scene to cover this tragic event, the largest oil spill in U.S. history. Americans watched in horror as about 2,000 kilometers of some of the most spectacular shores in the country were reduced to oil-covered graves for indigenous flora and fauna. Rapid cleanup was imperative to minimize further negative impacts of the spill on the ecology of this fragile environment. As anyone who has washed clothes knows, oil stains can be difficult to remove. Extricating spilled oil from the natural environment is a far more arduous and complex task. Similar to cleaning heavily stained clothes, though, hot water was used to remove oil from the shoreline of the Sound. Specifically, steam was applied under high pressure, a technique that initially seemed to work. Another part of the cleanup strategy relied on the presence of natural communities of microorganisms that could help degrade the oil. Many microorganisms-even those inhabiting the rocks on the shores of the Sound-can use oil as a source of carbon and energy, simultaneously transforming it into harmless water and carbon dioxide. This process is called bioremediation. Crude oil is indeed a rich source of carbon for microorganisms; however, microorganisms require nutrients other than carbon to survive. In fact, without added nutrients, bacterial metabolism and bioremediation often do not proceed very quickly. Today, the shores superficially appear as they were before the oil spill. Closer examination unfortunately reveals that oil still remains. The high-pressure steam cleaning forced it deeper into the rocky shores, inaccessible to microbial action. The tragedy of the Exxon Valdez serves as a forceful reminder of the impact of humans on nature and the limitations inherent in microbial cleanup of toxic substances.

) ) )

Do you think steom cleoning wos ultimately beneficial to the cleanup process? Outtine some of the opproaches environmentol microbiologists might have used to speed up bioremediation in Prince Williqm Sound. Predict the types of microbes that would be involved in "cleoning up" toxic spills. Cose File

CHAPTER OVERVIEW

26 Wrop-Up appeors on poge 801

.

Earth is a microbial planet. Hence, microorganisms contribute in profound ways to its structure and function and, therefore, to the survival of all other life forms. Microorganisms exist in complex associations with both living and nonliving components of their environment. Microbes have adapted to specific habitats and niches from which they derive food, energy, shelter, and other essential components of the biosphere. 787

788

Chapter

26

Environmental Microbiology

Microbes maintain and cycle the biologically important elements, such as carbon, nitrogen, and phosphorus, that exist only in certain reservoirs. Microbes constitute the beginning and the end of every energy pyramid as primary producers and as decomposers, respectively. The soil forms complex ecosystems that support a wide variety of microorganisms involved in decomposition, plant nutrition, and soil structure. Aquatic ecosystems are influenced by temperature, sun, and geologic actions to create numerous adaptive zones for microorganisms.

26.', Ecology: The Interconnecting Web of Life The study of microbes in their natural habitats is known as microbial ecology; the study ofthe practical uses ofmicrobes in food processing, industrial production, and biotechnology is known as industrial or applied microbiology (chapter 27).The two areas actually overlap to a considerable degree-largely because most natural habitats have been altered by human activities. Human intervention in natural settings has changed the earth's warming and cooling cycles, increased wastes in soil, polluted water, and altered some of the basic relationships between microbial, plant, and animal life. Now that humans are also beginning to release new, genetically recombined microbes into the environment and to alter the genes of plants, animals, and even themselves, what does the future hold? Although this question has no clear answer, we know one thing for certain: Microbes-the most vast and powerful resource of allwill be silently working in nature. In chapter 7, we first touched upon the widespread distribution of microorganisms and their adaptations to most habitats of the world, from extreme to temperate. Regardless of their exact location or type of adaptation, microorganisms necessarily are exposed to and interact with their environment in complex and extraordinary ways. Microbial ecology studies interactions between microbes and

their environment and the effects of those interactions on the earth. Unlike studies that deal with the activities of a single organism or its individual characteristics in the laboratory, ecological sfudies are aimed at the interactions taking place between organisms and their environment atmany levels at any given moment. Therefore, ecology is a broad-based science that merges many subsciences of biol-

ogy as well as geology, physics, and chemistry. Ecological studies deal with both the biotic and the abiotic components of an organism's environment. Biotic* factors are defined as any living or dead organismsr that occupy an organism's habitat. Abiotic* factors include nonliving components such as atmospheric gases, minerals, water, temperatue, and light. You may recall these from chapter 7 as the major factors affecting microbial growth. A collection of organisms and its surrounding physical and chemical factors are defined as an ecosystem.

l.

Biologists make a distinction between nonliving and dead. A nonliving thing has never been alive, whereas a dead thing was once alive but no longer is.

* biotic (by-ah'-tik) Gr. Bms, living. + abidic (a\/'-by-ah'-tik) Gr. c, not, and bios, living.

The Organization of Ecosystems The earth initially may seem like a random, chaotic place, but it is actually an incredibly organized, fine-tuned machine. Ecological relationships exist at several levels, ranging from the entire earth all the way down to a single organism (figure 26.1). The most all-encompassing of these levels, the biosphere, contains all physical locations on earth that support life, including the thin envelope of life that surrounds the earth's surface and extends some distance into the crust. This global ecosystem comprises the hydrosphere (water), the lithosphere (3 to 6 miles into the earth's crust), and the atmosphere (a few miles into the air), The biosphere maintains or creates the conditions of temperature, light, gases, moisture, and minerals required for life processes.

The biosphere can be naturally subdivided into terrestrial and aquatic realms. The terrestrial realm is usually distributed into particular climatic regions called biomes (by'-ohmz), each of which is characterized by a dominant plant form, altitude, and latitude. Particular biomes include grassland, desert, mountain, and tropical rain forest. The aquatic biosphere is generally divisible into freshwater and marine realms. We have also recently learned that the earth's crust also supports a vast and diverse number of life forms, estimated to be equal to or even greater than life as we know it in aquatic and terrestrial realms. Biomes and aquatic ecosystems are generally composed of mixed assemblages of organisms that live together at the same place and time and that usually exhibit well-defined nutritional or behavioral interrelationships. These clustered associations are called communities. Although most communities are identified by their easily visualized dominant plants and animals, they also contain a complex assortment of bacteria, fungi, algae, protozoa, and even viruses. The basic unit of community structure is the population, defined as a group of organisms of the same kind. For organisms with sexual reproduction, this level is the species. In contrast, prokaryotes are classified using taxonomic units such as strain or colony. The organizational unit of a population is the individual organism, and each multicellular organism, in furn, has its own levels of organ izalion (organs, tissues, cells). Ecosystems are generally balanced, with each organism existing in its particular habitat and niche. The habitat is the physical location in the environment to which an organism has adapted. In the case of microorganisms, the habitat is frequently a microenvironment, where particular qualities of oxygen, light, or nutrient content are somewhat stable. The niche is the overall role that a species (or population) serves in a community. This includes such

26.1

Ecology: The lnterconnecting Web of Life

Biosphere

Hydrosphere

it

eats), position in the community structure (what is eating it), and rate of population growth. A niche can be broad (such as scavengers that feed on nearly any organic food source) or nanow (microbes that decompose cellulose in forest litter or that fix nitrogen).

activities as nutritional intake (what

Lithosphere

789

Note that microbes exist as communities in and on plants and animals as well, including humans. Pure cultures are seldom found anywhere in nature.

Atmosphere

Energy and Nutritional Flow in Ecosystems All living things must obtain nutrients and a usable form of energy from their abiotic and biotic environments. The energy and nutritional relationships in ecosystems can be described in a number of convenient ways. An energy pyramid or food chain provides a simple summary of the general trophic (feeding) lev-

els, designated as producers, consumers, and decomposers, and traces the flow and quantity of available energy from one level to another (figure 26.2\.lt is worth noting that microorganisms are the only living beings that exist at all three major trophic levels. The nutritional roles of microorganisms in ecosystems are summarized in table 26.1.

tf

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26.1 Levels of organization in an ecosystem, ranging from the biosphere to the individual organism. Flgure

Energy Source

Flgure 26,2

+ CO.

A trophic and energy pyramid.

The relative size of the blocks indicates the number of individuals that exist at a given trophic level. The arrow on the right indicates the amount of usable energy from producers to top consumers. Both the number of organisms and the amount of usable energy decrease with each trophic level. Decomposers are an exception because they can feed from all trophic levels (gray arrows). Blocks at the left indicate the general nutritional types and levels that correspond with the pyramid.

Chapter26 Environmental Microbiology

790

:::;:t;

,::a-1.'

'

,.a::-a.=

The Major Roles of Microorganisms in Ecosystems

Role Primary producers

Consumers

Description of-'-'--'--t Activitv

Examples of Microorganisms Involved

Photosynthesis

Algae. bacteria. sulfur bacteria

Chemosynthesis

Chemolithotrophic bacteria in thermal vents

Predation

Free-living protozoa that feed on algae and bacteria; some fungi that prey upon nematodes

Decomposers

Degrading dead organisms and wastes

Soil saprobes (primarily bacteria and fungi) that degrade cellulose, lignin, and other complex macromolecules

Mineralization of organic compounds

Soil bacteria that reduce organic compounds to inorganic compor-mds such as CO2 and minerals

Cycling agents for biogeochemical cycles Parasites

Recycting compounds containing carbon, nitrogen, phosphorus, sulfur

Specialized bacteria that transform elements and keep them cycling from the biotic to the abiotic and back to the biotic phases ofthe biosphere

Living and feeding on hosts

Viruses, bacteria, protozoa, fungi, and worms that play a role in population control

Life would not be possible without producers, because they provide the fundamental energy source that drives the trophic pyramid. Producers are the only organisms in an ecosystem that can produce organic carbon compounds such as glucose by assimilating (fixing) inorganic carbon (CO2) from the atmosphere. If CO2 is the sole source from which they can obtain carbon for growth, these organisms are called autotrophs. Most producers are photosynthetic organisms, such as plants and cyanobacteria, that convert the sun's energy into chemical bond energy (covered in chapter 8). A smaller but not less important amount of CO2 assimilation is brought about by bacteria called lithotrophs. These organisms derive energy from simple inorganic compounds such as ammonia, sulfides, and hydrogen by using redox reactions. In certain ecosystems (see thermal vents, Insight 7.3), lithotrophs are the sole snpporters of the energy pyramid as primary producers. Consumers feed on other living organisms and obtain energy from bonds present in the organic substrates they contain. The cat-

Tertiary

Cyclops (crustacean)

consumer

consumer

Secondary

egory includes animals, protozoa, and some bacteria and fungi. A pyramid usually has several levels of consumers, ranging from primaty consumers (grazers or herbivores), which consume producers; to secondary consumers (carnivores), which feed on primary consumers; to tertiary consumers, which feed on secondary consumers; and up to quaternary consumers (usually the last level), which feed on tertiary consumers. Figures 26,3 and 26.4 show specific organisms at these levels.

Decomposers, primarily microbes inhabiting soil and water, break down and absorb the organic matter of dead organisms, including plants, animals, and other microorganisms. Because of their biological function, decomposers are active at all levels of the food pyramid. Without this important nutritional class of saprobes, the biosphere would stagnate and die. The work of decomposers is to reduce organic matter into inorganic minerals and gases that can be cycled back into the ecosystem, especially for the use of primary producers. This process, also termed mineralization, is so efficient that almost all biological compounds can be reduced by some type of decomposer. Numerous microorganisms decompose cellulose and lignin, polysaccharides from plant cell walls that account for the vast bulk of detritus in soil and water. Complex

Insect tarva

ffi

Didinium (protozoan)

Primary consumer

i I

Diatoms

Producer

,@h

*-%b Figure

26.3

Food chain.

A food chain is the simplest way

to present specific feeding relationships among organisms, but it may not reflect the total nutritional interactions in

a

community (figure not to scale).

26.1

Ecology: The Interconnecting Web of Life

79r

Food Web

macromolecules from animal bodies are also broken down by an assortment of bacteria and fungi. Surprisingly, decomposers can also break down most man-made compounds

that are not naturally found on earth. This process is referred to as bioremediation. Often, bioremediation involves more than one kind of microbe; the collection of participating microbes in this process is known as a consortium. The pyramid in figure 26.2 illustrates several limitations of ecosystems with regard to energy. Unlike nutrients, which can be passed among trophic levels, recycle4 and reused, energy does not cycle. Maintenance of complex interdependent trophic relationships such as those shown in figures 26.3 and 26.4 requires a constant input ofenergy at the

producer level. As energy is transferred to the next level, a large proportion (as high as 90%) of the energy will be lost in a form (primarily heat) that cannot be fed back into the system. Thus, the amount of energy available decreases at each successive trophic level. This energy loss also decreases the actual number of individuals that can be supported at each successive level. The most basic image of a feeding pathway can be provided by a food chain. Although it is a somewhat simplistic way to describe feeding relationships, a food chain helps identi$ the types of organisms that are present at a given trophic level in a natLlral setting (see figure 26.3). Feeding relationships in communities are more accurately represented by a multichannel food chain, or a food web (see figure 26.4). A food web reflects the actual nutritional structure of a community. It can help to identify feeding patterns typical of herbivores (plant eaters), carnivores (flesh eaters), and omnivores (feed on both plants and flesh).

:_)% Paramecium

Elgure 26.4

Food web.

More complex trophic patterns are accurately depicted by a food web, which traces the multiple feeding options that exist for most organisms. Note: Arrows point toward the consumers. Compare this pattern of feeding with the chain in figure 26.3 (organisms not to scale).

Ecological lnteractions between Organisms in a Community Whenever complex mixtures of organisms associate, they develop various dynamic interrelationships based on nutrition and shared habitat. These relationships, some of which were described in earlier chapters, include mutualism, commensalism, parasitism, competition, slmergism (cross-feeding), predation, and scavenging. Mutually beneficial associations (mutualism), such as that of protozoans living in the termite intestine, are so well evolved that the two members require each other for survival. In contrast, commensalism is one-sided and independent. (These terms describing relationships.between organisms echo the terms we described in chapter 7.) Although the action of one microbe favorably affects another, the

first microbe receives no benefit. Many commensal unions involve co-metqbolism, meaning that the waste products of the first microbe are useful nutrients for the second one, a process called syntrophy.

In synergism, two organisms that are usually independent cooperate to break down a nutrient neither one could have metabolized alone. Parasitism is an intimate relationship whereby a parasite derives its nutrients and habitat from a host that is usually harmed in the process. In competition, one microbe gives off antagonistic substances that inhibit or kill susceptible species sharing its habitat. A predator is a form of consumer that actively seeks out and ingests live prey (protozoa that prey on algae and bacteria). Scavengers are nutri-

tional jacks-of-all-trades; they feed on a vaiety of food sodrces, ranging from live cells to dead cells and wastes.

Chapter26 Environmental Microbiology

792

w The study of ecology includes both living (biotic) and nonliving (abiotic) components of the earth. Applied microbiology studies their utilization for commercial purposes. ,r,r Ecosystems are organizations of living populations in specific habitats. Environmental ecosystems require a continuous outside source of energy for survival and a nonliving habitat consisting of soil" water. and air. u,rr A lMng community is composed of populations that show a pattem of energy and nutritional relationships called a food web. Microorganisms are essential producers and decomposers in any ecosystem. lr: The relationships between populations in a community are described according to the degree ofbenefit or harm they pose to one another. These relationships include mutualism, commensalism, predation, parasitism, synergism, scavenging, and competition.

environment, the atmosphere, and even the soil would not exist as they do without the actions of living things. For billions of years, microbes have played prominent roles in the formation and maintenance of the earth's crust, the development of rocks and minerals, and the formation of fossil fuels. This revolution in understanding the biological involvement in geologic processes has given rise to a new field called geomicrobiology.

A NOTE ABOUT BIOGEOCHEMICAL CYCLES, BIOFILMS, AND THE LIVING EARTH We started this textbook marveling at the wondrous involvement of microbes in the dynamics of planet earth, and are finally at a point at which we can explore these roles in depth. Because the earth is an ancient place and microbes are the most ancient life forms, it is not surprising that they have essentially coevolved over several billion years. This is especially true of the lithosphere and hydrosphere, where microbes penetrate miles below the earth's surface and oceans. In fact, microbes dominate in most habitats, where they form stable and usually long-lasting communities. In most cases, these communities are composed of microbial biofilms that colonize and shape the composition of their microenvironments. 5o when we discuss the geochemical cycles, realize that most participants have adapted to a particular niche in a shared habitat, where they cooperate in processing elements through their various phases as a survival mechanism. Ceomicrobiologists are actively studying these communities and so far have documented hundreds of bacterial species that participate in transforming bioelements of organic molecules as well as minerals such as iron, manganese, and other met-

26.2 The Natural Recycling of Bioelements Environmental ecosystems are exposed to the sun, which constantly infuses them with a renewable source of energy. In contrast, the bioelements and nutrients that are essential components of cells and multicellular organisms are supplied exclusively by sources somewhere in the biosphere and are not being continually replenished from outside the earth. In fact, the lack ofa required nutrient in the immediate habitat is one ofthe chief factors limiting organismic and population growth. It is for these reasons that there must be continuous and sustained recycling of elements and nutrients in the biosphere. Essential elements such as carbon, nitrogen, sulfur, phosphorus, oxygen, and iron are recharged through biological, geologic, and chemical mechanisms called biogeochemical cycles. Although these cycles vary in certain specific characteristics, they share several general qualities, as summarized in the following list:

o All o o

.

o

elements ultimately originate from a nonliving, long-term

reservoir in the atmosphere, the lithosphere, or the hydrosphere. They cycle in pure form Q.{) or as compounds (POo). Elements make the rounds between the abiotic environment and the biotic environment. Recycling maintains a necessary balance of nutrients in the biosphere so that they do not build up or become unavailable. Cycles are complex systems that rely upon the interplay of producers, consumers, and decomposers. Often the waste products of one organism become a source of energy or building material for another. All organisms participate directly in recycling, but only certain categories of microorganisms have the metabolic pathways for converting inorganic compounds from one nutritional form to another.

The English biologist James Lovelock has postulated a concept called the Gaia (guy'-uh) theory, after the mythical Greek goddess of earth. Its primary idea proposes that the biosphere contains a diversity of habitats and niches favorable to life because living things have made it that way. Not only does the earth shape the characler of living things, but living things shape the character of the earth. After all, we know that the compositions of the aquatic

afs

(figure 26.5).

In the next several sections, we examine how, jointly and over period of time, the varied microbial activities affect and are themselves affected by the abiotic environment.

a

Atmospheric Cycles The Cqrbon Cycle Because carbon is the fundamental atom in all biomolecules and accounts for at least one-half of the dry weight of biomass, the carbon cycle is more intimately associated with the energy transfers and trophic patterns in the biosphere than are other elements. Carbon exists predominantly in the mineral state and as an organic reservoir in the bodies of organisms. A much smaller amount of carbon also exists in the gaseous state as carbon dioxide (COz), carbon monoxide (CO), and methane (CHa). In general, carbon is recycled through ecosystems via carbon fixation, respiration, or fermentation of organic molecules, limestone decomposition, and

methane production.

A

convenient starting point from which to

trace the movement of carbon is with carbon dioxide, which occupies a central position in the cycle and represents a large common pool that diffrrses into all parts ofthe ecosystem (figure 26.6). As a general rule, the cycles ofoxygen and hydrogen are closely allied to the carbon cycle.

26.2

The Natural Recycling of Bioelements

793

Organic carbon taken in by consumers (animals, protozoa)

Process Flgure

26.6

The carbon cycle.

This cycle traces carbon from the CO2 pool in the atmosphere to the

@ Figure 26.5

A sample of water from ai imaged by scanning electron microscopy.

a deep cavern

This view shows a bacterial biofilm (blue) that actively forms mineral deposits of zinc and sulfate (light green and yellow).

The principal users ofthe atrnospheric carbon dioxide pool are photosynthetic autotrophs (photoautotrophs) such as plants, algae, and bacteria. An estimated 165 billion tons of organic material per

year arc produced by terrestrial and aquatic photosynthesis. Although we don't yet know exactly how many autotrophs exist in the earth's crust, a small amount of CO2 is used by these bacteria (chemolithoautotrophs) that derive their energy from bonds in inorganic chemicals. A review of figure 8.28 reveals that phototrophs use energy from the sun to fix CO2 into organic compounds such as glucose that can be used in slmthesis. Photosynthesis is also the primary means by which the atmospheric supply of 02 is regenerated. Just as photosynthesis removes CO2 from the atmosphere, other modes of generating energy, such as respiration and fermentation, can be used to remove and return it. As you may recall from the discussion ofaerobic respiration in chapter 8, in the presence of 02, organic compounds such as glucose are degraded completely to CO2, with the release of energy and the formation of H2O. Carbon dioxide is also released by anaerobic respiration and by certain types of fermentation reactions. A small but important phase of the carbon cycle involves certain limestone deposits composed primarily of calcium carbonate (CaCO3). Limestone is produced when marine organisms such as

mollusks, corals, protozoans, and algae form hardened shells by combining carbon dioxide and calcium ions from the surrounding

primary producers (green) where it is fixed into protoplasm. Organic carbon compounds are taken in by consumers (blue) and decomposers (yellow) that produce CO2 through respiration and return it to the atmosphere (pink). Combustion of fossil fuels and volcanic eruptions also add to the CO2 pool. Some of the COr is carried into inorganic sediments by organisms that synthesize carbonate (CO3) skeletons. In time, natural processes acting on exposed carbonate skeletons can liberate CO2. water. When these organisms die, the durable skeletal components

accumulate in marine deposits. As these immense deposits are gradually exposed by geologic upheavals or receding ocean levels, various decomposing agents liberate CO2 and return it to the CO, pool of the water and atrnosphere. The complementary actions of photosynthesis and respiration, along with other natural CO2-releasing processes such as limestone erosion and volcanic activity, have maintained a relatively stable atmospheric pool of carbon dioxide. Recent figures show that this balance is being disturbed as humans barr. fossil fuels and other organic carbon sources. Fossil fuels, including coal, oil, and nafural gas, were formed through millions of years of natural biological and geologic activities. Humans are so dependent upon this energy source that, within the past 25 yearc, the proportion of CO2 in the atmosphere has steadily increased from 32 ppm to 36 ppm. Although this increase may seem slight and insignificant, most scientists now feel it has begun to disrupt the delicate temperature balance ofthe biosphere (Insight 26.1).

Compared with carbon dioxide, methane gas (CHo) plays a it can be a significant produqt in anaerobic ecosystems dominated by methanogens (methane producers). In general, when methanogens reduce CO2 by means of various oxidizable substrates, they give offCH.. The practical applications of methanogens are covered in chapter 27 in a section on sewage treatment, and their contribution to the greenhouse effect is also discussed in Insisht 26.1 . secondary part in the carbon cycle, though

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Chapter26 Environmental Microbiology

Greenhouse Gases, Fossil Fuels, Cows, Termites, and Global Warming The sun's radiant energy does more than drive photosynthesis; it also helps maintain the stability of the earth's temperature and climatic conditions. As radiation impinges on the earth's surface, much of it is absorbed, but a large amount ofthe infrared theatl radiation bounces back into the upper levels of the atmosphere. For billions of years, the atmosphere has been insulated by a layer of gases (primarily COr; CHa; water vapor; and nitrous oxide, N2O) formed by natural processes such as respiration and decomposition, which are part of biogeochemical cycles. This layer traps a certain amount of the reflected heat yet also allows some of it to escape into space. As long as the amounts ofheat entering and leaving are balanced the mean temperature of the earth will not rise or fall in an erratic or life-threatening way. Although this phenomenon, called the greenhouse effect, is popularly viewed in a negative light, it must be emphasized that its function for eons has been primarily to foster life. The greenhouse effect has recently been a matter ofconcern because greenhouse gases appear to be increasing at arate that could disrupt the temperature balance. In effect, a denser insulation layer will trap more heat energy and gradually heat the earth. The amount of CO2 released collectively by respiration, anaerobic microbial activiry fuel combustion, and volcanic activity has increased more than 30% since the beginning ofthe industrial era. By far the greatest increase in CO, productionresults from human activities such as combustion of fossil fuels, burning forests to clear agricultural lan4 and manufacturing. Deforestation has the added impact of removing large areas of photosynthesizing plants that would otherwise consume some of the COr.

I E F

Originally, experts on the greenhouse effect were concerned primar-

ily about increasing CO2 levels, but it now appears that the other greenhouse gases combined may have as great a contribution as CO2, and they, too, are increasing. One of these gases, methane (CHa) released from the

gastrointestinal tract ofruminant animals such as cattle, goats. and sheep, has doubled over the past century. The gut oftermites also harbors wooddigesting bacteria and methanogenic archaea. Even the human intestinal tract can support methanogens. Methane traps 2l times more heat than does carbon dioxide. Other greenhouse gases such as nilrous oxide and sulfur dioxide (SO2) are also increasing through automobile and indus-

trial pollution. There is little doubt in the scientific community that global warming is a reality. It has been documented that the mean temperature of the earth

by -f 1.0oC since 1860. Ifthe rate ofincrease continues, by 2050 a rise in the average temperature of 4oC to 5'C will escalate the melting ofthe polar ice caps and raise the levels ofthe ocean 2to3 feet. Some experts predict extreme weather and climate change, inctuding massive flooding ofcoastal regions, changes in rainfall patterns, expansion of deserts, and long-term climatic disruptions. Some of these effects have already been set in motion. has increased

Throughout this text, we have emphasized the adaptability of microbes. Why is global warming considered such a serious problem even for them? Answer available at http://www.mhhe.com/talaroT

Global Mean Temperature over Land & Ocean ou o.+

o.z

T

:i'

o.o

:-n2

e

-0.4 E = (t

&-o.o o

The Nitrogen Cycle

from plants or other animals; however, microorganisms can use all

Nitrogen (].{u) gas is the most abundant component of the atrnosphere, accounting for nearly 19Vo of atr volume. As we will see, this extensive reservoir in the air is largely unavailable to most organisms. Only about 0.03% of the earth's nitrogen is combined (or fixed) in some other form such as nitrates (NOr), nitrites (NOz), ammonium ion (NHo-), and organic nitrogen compounds (proteins, nucleic acids). The nitrogen cycle is relatively more intricate than other cycles because it involves such a diversity of specialized microbes to maintain the flow of the cycle. In many ways, it is actually more of a nitrogen "web" because of the anay of adaptations that occur. Higher plants can utilize NO3 and NHa+; animals must receive nitrogen in organic form

only process that can remove N, from the air and convert it to a form usable by living beings. This process, called nitrogen fixation, is the beginning step in the synthesis ofvirtually all nitrogenous compounds. Nitrogen fixation is brought about primarily by nitrogen-fixing bacteria in soil and wateq though a small amount is formed through nonliving processes involving lightning. Nitrogenfixing microbes have developed a unique enzyme system capable

forms of nitrogen: NO2-, NOr-, NI{4+, \, and organic nitrogen. The cycle includes four basic types of reactions: nitrogen fixation, alnmonification, nitrification, and denitrification (figure 26.7).

Nitrogen Fixation The biosphere is most dependent upon the

26.2

The Natural Recycling of Bioelements

795

Legume root

Nodules Early nodule

NHa'

tV (a)

ft

Nitrification

tIY|

Ammonification

+

*1'-

Figure

26.8

Nitrogen fixation through symbiosis.

(a) Events leading to formation of root nodules. Cells of the bacterium Rhizobium attach to a legume root hair and cause it to curl. nvasion of the legume root proper by Rhizobium initiates the formation of an infection thread that spreads into numerous adjacent cells. The presence of bacteria in cells causes nodule formation. (b) Mature nodules that have developed in a sweet clover plant. f

ruo.I

Root Nodules: Natural Fertilizer Factories A significant symbiotic association occurs between rhizobia* (bacteria in the genera such as Rhizobium, Bradyrhizobium, and Azorhizobium) and legumes* (plants such as soybeans, peas, alfalfa, and clover

I

I Plants, algae, other bacteria

\.-__--., @

Process Figure

Organic nitrogen

Animals, protozoa

@

26.7

Simplified events in the nitrogen

cycle. (1) In nitrogen fixation, gaseous nitrogen (Nr) is acted on by nitrogenfixing bacteria, which give off ammonia (NHo). (2) Ammonia is converted to nitrite (NOzl and nitrate (NOr-) by nitrifying bacteria in nitrification. (3) Plants, algae, and bacteria use nitrates to synthesize nitrogenous organic compounds (proteins, amino acidl nucleic acids). (4) Organic nitrogen compounds are used by animals and other consumers. (5) ln ammonification, nitrogenous macromolecules from wastes and dead organisms are converted to NHa* by ammonifying bacteria. NHa* can be either directly recycled into nitrates or (6) returned to the atmospheric N2 form by denitrifying bacteria (denitrification).

of breaking the triple bonds of the Nt molecule and reducing the N atoms, an anaerobic process that requires the expenditure ofconsiderable ATP. The primary product of nitrogen fixation is the ammonium ion, NHo+. Nitrogen-fixing bacteria live free or in a symbiotic

relationship with plants. Among the common free-living nitrogen fixers are the aerobic Azotobqcter and Azospirillum, certainmembers of the anaerobic genus Clostridium, and the cyanobacteria Anabaena and Nostoc.

that characteristically produce seeds in pods). The infection oflegume roots by these gram-negative, motile, rod-shaped bacteria causes the formation of special nitrogen-fixing organs called root nodules (figure 26.8). Nodulation begins when rhizobia colonize specific sites on root hairs. From there, the bacteria invade deeper root cells and induce the cells to form tumorlike masses. The bacterium's enzyme system supplies a constant source of reduced nitrogen to the plant, and the plant furnishes nutrients and energy for the activities of the bacterium. The legume uses the NHo+ to aminate (add an amino group to) various carbohydrate intermediates and thereby synthesize amino acids and other nitrogenous compounds that are used in plant and animal synthesis. Plant-bacteria associations have great practical importance in agriculture, because an avallable source ofnitrogen is often a limiting factor in the growth of crops. The self-fertilizing nature of legumes makes them valuable food plants in areas with poor soils and in countries with limited resources. It has been shown that crop health and yields can be improved by inoculating legume seeds with pure cultures of rhizobia, because the soil is often deficient in the proper strain of bacteria for forming nodules (figure 26.9).

* rltizobia (ry-zoh'-bee-uh) Gr. rhiza, root, * legume (leg' -yoom) L. legere, to gather.

and 6los, to live.

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Chapter26 Environmental Microbiology

Sedimentary Cycles The Sulfur Cycle

Flgure 26.9 Inoculating legume

seeds

with Rhizobium

bacteria increases the plant's access to nitrogen. The legumes in (a) were inoculated and are healthy. The poor growth and yellowish color of the uninoculated legumes in (b) indicate a lack of nitrogen.

Ammonification, Nitrification, and Denitrification Inanotherpart of the nitrogen cycle, nitrogen-containing organic matter is decomposed by various bacteia (Clostridium, Proteus, for example) that live in the soil and water. Organic detritus consists of large amounts of protein and nucleic acids from dead organisms and nitrogenous animal wastes such as urea and uric acid. The decomposition of these substances splits off amino groups and produces NH4+. This process is thus known as ammonification. The ammonium released can be reused by certain plants or converted to other nitrogen compounds, as discussed next. The oxidation of NIIa+ to NO2- and NO3- is called nitrilication. It is an essential conversion process for generating the most oxidized form of nitrogen (NOr). This reaction occurs in two phases and involves two different kinds of lithohophic bacteria in soil and water. In the first phase, certain gram-negative genera such as Nitrosomonas, Nitrosospira, andNitrosococcus oxidize NH, to NO2- as a means of generating energy. Nihite is rapidly acted upon by a second group of nitrifiers, including Nitrobacter, Nitrosospira, and Nitrococcus, whichperformlhefina/ oxidation ofNOr- to NOr-. Nitrates can be assimilated through several routes by a variety of organisms (plants, firngi, and bacteria). Nitrate and nitrite are also important in anaerobic respiration where they serve as terminal electron acceptors; some bacteria use them as a source of oxygen as well. The nitrogen cycle is completed through a process of denitrification. This occurs when nitrogen compounds undergo a series ofreactions that convert NO3- through intermediate steps to atmospheric nitrogen. The fint step, which involves the reduction of nitrate to nitite, is so common that hundreds of different bacterial species can do it. Several genera such as Bacillus, Pseu.domonas, Spirillum, and Thiobacillus can carry out denitrification to completion as follows: NO3- -+ NO2- -> NO -+ NrO -- Nz (gas) The final product of this series returns nitrogen gas to its primary reservoir. Along the way, other gases-notably, nitrous oxide (N2O)-are given offduring incomplete denitrification. This is the main source ofN2O in the atrnosphere and an important contributor to the greenhouse effect.

The sulfur cycle resembles the phosphorus cycle in that both elements exist mostly in solid form and originate from natural sedimentary deposits in rocks, oceans, and lakes and not from the atmosphere. Sulfur exists in the elemental form (S) and as hydrogen sulfide gas (I{2S), sulfate (SOo), and thiosulfate (SzOr). Most of the oxidations and reductions that convert one form of inorganic sulfur to another are accomplished by bacteria. Plants and many microorganisms can assimilate only SOa, and animals must have an organic source. Organic sulfur occurs in the amino acids cystine, cysteine, and methionine, which contain sulflrydryl (-SH) groups and form disulfide (S-S) bonds that contribute to the stability and configuration ofproteins. One of the most remarkable contributors to the cycling of sulfur in the biosphere are the thiobacilli. These gram-negative, motile rods flourish in mud, sewage, bogs, mining drainage, and brackish springs that can be inhospitable to organisms that require complex organic nutrients. But the metabolism of these specialized lithotrophic bacteria is adapted to extracting energy by oxidizing elemental sulfur, sulfides, and thiosulfate. One species, Ihiobacillus thiooxidans, is so efficient at this process that it secretes large amounts of sulfuric acid into its environment, as shown bv the following equation: Na2S2O3

+

H2O

*

02 -+ Na2SOa+H2SOa (sulfuric acid)

+ 45

The marvel of this bacterium is its ability to create and survive in the most acidic habitats on the earth. It also plays an essential part

in the phosphorus cycle, and its relative, T. ferrooxidans, participates in the cycling of iron. Other bacteria that can oxidize sulfur to sulfates are the photosynthetic sulfir bacteria mentioned in the section on photosynthesis.

The sulfates formed from oxidation of sulfurous compounds are assimilated into biomass by a wide variety of organisms. The sulfur cycle reaches completion when inorganic and organic sulfur componnds are reduced. Bacteria in the genera Desulftvibrio and Desulfuromonqs anaercbically reduce sulfates to hydrogen sulfide (H2S) or metal sulfide as the final step in electron transport. Sites in ocean sediments and mud where these bacteria live usually emanate a strong, rotten-egg stench from HrS and may be blackened by the iron thev contain.

The Phosphorus Cycle Phosphorus is an integral component of DNA, RNA, andAIP, and all life depends upon a constant supply ofit. It cycles between the

abiotic and biotic environments almost exclusively as inorganic phosphate (POa) rather than its elemental form (figure 26.10). The chief inorganic reservoir is phosphate rock, which contains the insoluble compound fluorapatite, Ca5(POa)3F. Before it can enter biological systems, this mineral mustbe phosphatized---converted into more soluble POa'- by the action of acid. Phosphate is released naturally when the sulfuric acid produced.by Thiobacil/ns dissolves phosphate rock. Soluble phosphate in the soil and water is the principal source for autotrophs, which fix it onto organic molecules and pass it on to heterotrophs in this form. Organic phosphate is returned to the pool ofsoluble phosphate by decomposers, and it is

26.3

Microbes on Land and in Water

797

mercury, as well as hundreds of thousands of synthetic chemicals introduced into the environment over the past hundred years, are readily caught up in cycles by microbial actions. Some of these chemicals will be converted into less harmful substances, but others, such as PCB and heavy metals, persist and flow along with nutrients into all levels of the biosphere. If such a pollutant accumulates in living tissue and is not excretedo it can be accumulated by living things through the

I-ry

such as

.F (u o)

E

E

o

U)

One example ofthis is mercury compounds used in household antiseptics and disinfectants, agriculture, and industry.(Elemental mercury precipitates proteins by attaching to functional groups and is most toxic in the ethyl or methyl mercury form. Recent studies have disclosed increased mercury content in fish taken from oceans and freshwater lakes in North America and even in canned tuna, adding to the risk in consumption of these products. This is another global problem that is being addressed by bioremediation (Insight 26.2).

s

€

c

Flgure 26.10

Nutrients and minerals necessary to communities and ecosystems must be continuously recycled. These biogeochemical cycles involve transformation of elements from inorganic to organic forms usable by many populations in the community and back again. Specific types of microorganisms are needed to convert many nutrients from one form to another. The sun is the primary energy source for most surface ecosystems. Photosynthesis captures this energy, which can be used for carbon fixation by producer populations. Elements of critical importance to all ecosystems thatcycle through various forms are carbon, nitrogen, sulfur, phosphorus, and water. Carbon and nifrogen are part of the afiriospheric cycle. Sulfur and phosphorus are part of the sedimentary cycling of nutrients.

The phosphorus cycle.

The pool of phosphate existing in sedimentary rocks is released into the ecosystem either naturally by erosion and microbial action or artificially by mining and the use of phosphate fertilizers. Soluble phosphate (POo3-) is cycled through producers, consumers, and decomposers back into the soluble pool of phosphate, or it is returned to sediment in the aquatic biosphere.

finally cycled back to the mineral reservoir by slow geologic proclow phosphate content of many soils can limit productivity, phosphate is added to soil to increase agricultural yields. The excess runoff of fertilizer into the hydrosphere is often responsible for overgrowth of aquatic pests (see eutrophication in a subsequent section on aquatic habitats). esses such as sedimentation. Because the

Other Forms of Cycling The involvement of microbes in cycling elements and compounds can be escalated by the introduction oftoxic substances into the environment. Such toxic elements as arsenic, chromium. lead. and

26.3 Microbes on Land and in Water Soil Microbiology: The Composition of the Lithosphere Descriptions such as "soiled" or "dirtyo' may suggesf to some that soil is an undesirable, possibly harmful substance; or its appearance

might suggest a somewhat homogeneous, inert substance. At the microscopic level, however, soil is a dynamic ecosystem that supports complex interactions between numerous geologic, chemical, and biological factors. This rich region, part of the lithosphere, teems with microbes, serves a dynamic role in biogeochemical cycles, and is an important repository for organic detritus and dead terrestrial organisms. The abiotic portion of soil is a composite of mineral particles, water, and atmospheric gas. The development of soil begins when geologic sediments are mechanically disturbed and exposed to weather and microbial action.

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Chapter26 Environmental Microbiology

Bioremediation: The Pollution Solution? The soil and water ofthe earth have long been considered convenient repositories for solid and liquid wastes. Humans have been burying solid wastes for thousands ofyears, but the process has escalated in the past 50 years. Every year, about 300 metric tons of pollutants, industrial wastes, and garbage are deposited into the natural environment. Often, this dumping is done with the mistaken idea that naturally occurring microbes will eventually biodegrade (break down) waste material. Landfills currently serve as a final resting place for hundreds of castoffs from an affluent society, including yard wastes, paper, glass, plastics, wood, textiles, rubber, metal, paints, and solvents. This conglomeration is dumped into holes and is covered with soil. Although it is true that many substa.nces are readily biodegradable, materials such as plastics and glass are not. Successful biodegradation also requires a compost containing specific types of microorganisms, adequate moisture, and oxygen. The environment surrounding buried trash provides none of these conditions. Large, dry, anaerobic masses of plant materials, paper, and other organic materials will not be successfully attacked by the aerobic microorganisms that dominate in biodegradation. As we continue to fill up hillsides with waste, the future of these landfills is a prime concern. One of the most serious of these concerns is that they will be a source oftoxic compounds that seep into the ground and water. Pollution of groundwater, the primary source of drinking water for 100 million people in the United States, is an increasing problem. Because of the extensive cycling of water through the hydrosphere and lithosphere, groundwater is often the final collection point for hazardous chemicals released into lakes, streams. oceans. and even garbage dumps. Many of these chemicals are pesticide residues from agriculture (dioxin. selenium, 2,4-D), industrial hydrocarbon wastes (PCBs), and hydrocarbon solvents (benzene, toluene). They are often hard to detect, and, if detected" are hard to remove. For many years, polluted soil and water were simply sealed off or dredged and dumped in a different site, with no attempt to get rid of the pollutant. But now, with greater awareness of toxic wastes, many Americans are adopting an attitude known as NIMBY (not in my backyard!),

This marsh had been used to dump oil refinery waste. The level of certain pollutants was over 130,000 ppm.

and environmentalists are troubled by the long-term effects of contaminating the earth. In a search for solutions. waste management has turned to bioremediation-using microbes to break down or remove toxic wastes in water and soil. Some of these waste-eatins microbes are natural soil and water residents with a surprising capacit! to decompose even artificial substances. Because the natural, unaided process occurs too slowly, most cleanups are accomplished by commercial bioremediation services that treat the contaminated soil with oxygen, nutrients, and water to increase the rate of microbial action. Through these actions, levels of pesticides such as 2,4-D can be reduced to 960/o of their original levels, and solvents can be reduced from I million parts per billion lppb) to l0 ppb or less. Bacteria are also being used to help break up and digest oil from spills and refineries. Among the most importaat bioremedial microbes are species of Pseudomonas and Bacillus and various toxin-eating fungi. Although much recent work has focused on creating "superbugs" through genetic engineering, public resistance to releasing genetically modified organisms (or GMOs) in the environment is high. Thankfully, naturally occurring biodegraders are plentiful, and efforts to optimize their performance are also very successful. So far, about 35 recombinant microbes have been created for biore-

mediation. Species of Rhodococcus and Burkholderia have been engineered to decompose PCBs, and certain forms of Pseudomonds now contain genes for detoxifring healry metals, carbon tetrachloride, and naphthalene. With over 3,000 toxic waste sites in the United States alone, the need for effective bioremediation is a top priority. The genome sequence of the high-powered PCB degrader Burkhold-

eria xenovorans LB400 has just been worked out. This knowledge will provide additional tools for the cleanup ofpolluted environments. Name some other solutions to cleaning up or getting rid of wastes and pollutants, besides microbial action. Answer available at http : //www.

m h h e.

com/t al aro 7

After bioremediation with nutrients and microbes, the levels were reduced to less than 300 ppm in 4 months. lt is bioremediated to the point that the land may be used for growing plants.

26,3

Microbes on Land and in Water

799

also an important habitat for microbes that decompose the complex litter and recycle nutrients. The humus content varies with climate, temperature, moisture and mineral content, and microbial action. Warm, tropical soils have a high rate of humus production and microbial decomposition. Because nutrients in these soils are swiftly released and used up, they do not accumulate. Fertilized agricultural soils in temperate climates build up humus at a high rate and are rich in nutrients. The very low content of humus and moisture in desert soils greatly reduces its microbial biota, rate of decomposition, and nutrient con-

tent. Bogs are likewise nutrient-poor due to a slow rate of decomposition of the humus caused by high acid content and lack of oxygen. Humans can artificially increase the amount of humus by mixing plant refuse and animal wastes with soil and allowing natural decomposition to occur, a process called composting. Composting is a very active metabolic process that generates a great deal of heat. The temperature inside a well-maintained compost can reach 80oC to 100'C.

Living Activities in Soil I e'f *"m"**-

Figure 26.11

The soil habitat.

A typical soil habitat contains a mixture of clay, silt and sand along with soil organic matter. Roots and animals (e.9., nematodes and mites), as well as protozoa and bacteria, consume oxygen, which rapidly diffuses into the soil pores where the microbes live. Note that two types of fungi are present: mycorrhizal fungi, which derive their organic carbon from

plant roots; and saprophytic fungi, which help degrade organic material.

Rock decomposition releases various-size particles rangrng from rocks, pebbles, and sand grains to microscopic morsels that lie in a loose aggregate (figure 26.11). The porous structure ofsoil creates various-size pockets or spaces that provide numerous microhabitats. Some spaces trap moisture and form a liquid phase in which mineral ions and other nutrients are dissolved. Other spaces trap air that will provide gases to soil microbes, plants, and animals. Because both water and air compete for these pockets, the water content of soil is directly related to its oxygen content. Water-saturated soils contain less oxygen, and dry soils have more. Gas tensions in soil can also vary vertically. In general, the concentration ofO2 decreases and that of CO, increases with the depth of soil. Aerobic and facultative organisms tend to oocupy looser, drier soils, whereas anaerobes would adapf b waterlogged, poorly aerated soils. Within the superstructure of the soil are varying amounts of humuso* the slowly decaying organic litter from plant and animal tissues. This soft, crumbly mixture holds water like a sponge. It is * humus (hyoo'-mts) L., earth.

The rich culture medium of the soil supports a fantastic array of microorganisms (bacteria, fungi, algae, protozoa, and viruses). A gram of moist loam soil with high humus content can have a microbe count as high as 10 billion, each competing for its orvn niche and microhabitat. Some of the most distinctive biological interactions occur in the rhizosphere, the zone of soil surrounding the roots of plants, which contains associated bacteia, fungi, and protozoa (see figure 26.11). Plants interact with soil microbes in a truly synergistic fashion. Studies have shown that a rich microbial community grows in a biofilm around the root hairs and other exposed surfaces. Their presence stimulates the plant to exude growth factors such as carbon dioxide, sugars, amino acids, and vitamins. These nutrients are released into fluid spaces, where they can be readily captured by microbes. Bacteria and frmgi likewise contribute to plant survival by releasing hormonelike growth factors and protective substances. They also convert minerals into forms usable by plants. We saw numerous examples in the nitrogen, sulfur, and phosphorus cycles. We previously observed that plants can form close symbiotic associations with microbes to fix nitrogen. Other mutualistic partnerships between plant roots and microbes are mycorrhizae.* These associations occur when various species of basidiomycetes, ascomycetes, or zygomycetes attach themselves to the roots of vascular plants (figure 26.12). The plant feeds the fungus through photosynthesis, and the fungus sustains the relationship in several ways. By extending its mycelium into the rhizosphere, it helps anchor the plant and increases the surface area for capturing water from dry soils and minerals from poor soils. Plants with mycorrhizae caninhabit severe habitats more successfully than plants without them. The topsoil, which extends a few inches to a few feet from the surface, supports a host of burrowing animals such as nematodes, termites, and earthworms. Many of these animals are decomposerreducer organisms that break down organic nutrients through digestion and also mechanically reduce or fragment the size of particles

so that they are more readily mineralized by microbes. Aerobic bacteria initiate the digestion of organic matter into carbon dioxide * mycorrhizae (my''-koh-ry'-zee)

C:r. mykos,

fvngx,

and rhiza, root.

800

Chapter

26

Environmental Microbiology

Evaporation

Figure 26.12 Mycorrhizae, symbiotic

Evaporation

associations

between fungi and plant roots, favor the absorption of water and minerals from the soil. and water and generate minerals such as sulfate, phosphate, and nitrate, which can be further degraded by anaerobic bacteria. Fungal enzymes increase the efficiency of soil decomposition by hydrolyzing complex natural substances such as cellulose, keratin, lignin, chitin, and paraffin. The soil is also a repository for agricultural, industrial, and domestic wastes such as insecticides, herbicides, fungicides, manufacfuring wastes, and household chemicals. Applied microbiologists, using expertise from engineering, biotechnology, and ecology, work to explore the feasibility of harnessing indigenous soil microbes to break down undesirable hydrocarbons and pesticides (see Insight 26.2).

Figure 26.13

through respiration.

Distribution of Water on Earth's Surface

Aquatic Microbiology Water occupies nearly three-fourths of the earth's surface. In the same maffler as minerals, the earth's supply of water is continuously cycled between the hydrosphere, atmosphere, and lithosphere (figure 26.13). The hydrologic cycle begins when surface water (lakes, oceans, rivers) exposed to the sun and wind evaporates and enters the vapor phase of the atmosphere. Living beings contribute to this reservoir by various activities. Plants lose moisture through transpiration (evaporation through leaves), and all aerobic organisms give off water during respiration. Airborne moisture accumulates in the atmosphere, most conspicuously as clouds. Water is returned to the earth through condensation or precipitation (rain, snow). The largest proportion of precipitation falls back into surface waters, where it circulates rapidly between running water and standing water. Only about 2Yo of water seeps into the earth or is bound in ice, but these are very important reseryoirs. Table 26.2 shows how water is distributed in the various surface compartments. Surface water collects in extensive subterranean pockets produced by the underlying layers of rock, gravel, and sand. This process forms a deep groundwater source called an aq{l&r. Jbe water in aquifers circulates very slowly and is an imfri tant replenishing source for surface water. It can resurface through springs, geysers, and hot vents, and it is also tapped as the primary supply for one-fourth of all water used by humans.

The hydrologic cycle.

The largest proportion of water cycles through evaporation, transpiration, and precipitation between the hydrosphere and the atmosphere. Other reservoirs of water exist in the groundwater or deep storage aquifers in sedimentary rocks. Plants add to this cycle by releasing water through transpiration, and heterotrophs release it

Water Volume, in Water Source

Cubic Miles

Percentage of Total Water

317,000,000

97.24

Icecaps, glaciers

7,ooo,ooo

2.t4

Groundwater

2,ooo,ooo

0.61

Oceans

Freshwater lakes

30,000

0.009

Inland seas

25,000

0.008

Soil moisture

16,000

0.005

Atmosphere

3,100

0.001

Rivers

300

0.0001 100

Source: U.S. Geological Survey.

Although the total amount of water in the hydrologic cycle has not changed over millions of years, its distribution and quality have been greatly altered by human activities. Two serious problems have arisen with aquifers. First, as a result of increased well drilling, land development, and persistent local droughts, the aquifers in many areas have not been replenished as rapidly as they have been depleted. As these reserves are used up, humans will have to rely on other delivery systems such as pipelines, dams, and reservoirs, which can further disrupt the cycling of water. Second, because

26.3

ofthe greatest challenges ofthis century.

801

Blue-green bacteria

water picks up materials when falling through air or percolating through the ground, aquifers are also important collection points for pollutants. As we will see, the proper management of water resources is one

Microbes on Land and in Water

Phytoplankton

#;e ii€r Aquatic

CASE FILE

26

Wrop-Up

Bioremediation, as discussed in lnsight 26.2, relies upon microorganisms to mineralize pollutants, such as oil spills and pesticides. As with all microbial processes, those involved in bioremediation require basic nutrients. Oil, being a hydrocarbon, is a rich carbon source, but it lacks other essential nutrients, such as nitrogen and phosphorus. Understanding this, environmental microbiologists attempted to accelerate bioremediation of the oil spill by applying fertilizers containing nitrogen and

phosphorus. Approximately 50,000 kilograms of nitrogen '1989 and 5,000 kilograms of phosphorus were applied between and 1992. Overall, these enormous applications appeared to have the desired effect: Bacteria from fertilized beaches mineralized components of oil up to 18 times faster than bacteria from beaches that did not receive fertilizer. The primary benefit of steam cleaning the shores was quick removal of large quantities of oil. But an adverse effect of the cleaning was to wipe out many of the bacteria that could have facilitated a more complete removal of the oil. ond R. M. Atlas, "Effectiveness of Bioremediation for the ExxonYaldez Oil Spilt," Nature 368 (1994): 413-18. See: J. R. Biragg, R. C. Prince, E. J. Horner,

The Structure of Aquatic Ecosystems Surface waters such as ponds, lakes, oceans, and rivers differ to a considerable extent in size, geographic location, and physical and chemical character. Although an aquatic ecosystem is composed primarily of liquid it is predictably structured" and it contains significant gradients or local differences in composition. Factors that contribute to the development of zones in aquatic systems are sunlight, temperature, aeration, and dissolved nutrient content. These

variations create numerous macro- and microenvironments for communities of organisms. An example of zonation can be seen in the schematic section of a freshwater lake in figure 26.14. A lake is stratified vertically into three zones, or strata. The uppermost region, called the photic zone, extends from the surface to the lowest limit of sunlight penetration. Its lower boundary (the compensation depth) is the greatest depth at which photosynthesis can occur. Directly beneath the photic zone lies the profundal* zoneo which extends from the edge ofthe photic zone to the lake sediment. The sediment itself, or benthic* zone, is composed of

organic debris and mud" and it lies directly on the bedrock that forms the lake basin. The horizontal zonation includes the shoreline, or littoral* zone, an area ofrelatively shallow water. The open, deeper water beyond the littoral zone is the

limnetic* zone.

prafundal (proh-fun'-dul) L. pro, before, andfundus, bottom. b en thic (ben'thik) Gr. benthos, deprh of the sea. r' littoruI (lit'-or-u1) L. /ltus, seashore.

'N

*

* linnetic (lim-neh'-tik)

Gr. limne, marsh.

Figure 26.14 Stratification in a freshwater

lake.

Depth changes in this ecosystem create significant zones that differ in light penetration, temperature, and community structure. Plankton suspended in upper zones float with the currents, whereas benthic organisms remain in or near the sediments.

Morine Environments The ocean exhibits extreme variations in salinity, depth, temperature, hydrostatic pressure, and mixing. Even so, it supports a greet abundance of bacteria and viruses, the extent of which has only been appreciated in very recent years. It contains a uni{ue zone where the river meets the sea called an estuary. This region flucfuates in salinity, is very high in nutrients, and supports a specialized microbial community. It is often dominated by salt-tolerant species of Pseudomonas and Vibrio. Another important factor is the tidal and wave action that subjects the coastal habitat to alternate periods of submersion and exposure. The deep oeean, or abyssal zoneo is poor in nutrients and lacks sunlight for photosynthesis, and its tremendous depth (up to 10,000 meters) makes it oxygen-poor and cold (average temperature 4'C). This zone supports communities with extreme adaptations, including halophilic, psychrophilic, barophilic, and anaerobic lifestyles.

802

Chapter

26

Environmental Microbiology

Aquotic Communities The freshwater environment is a site of tremendous microbiological

activity. Microbial distribution is associated with sunlight, temperature, oxygen levels, and nutrient availability. The uppermost portion is the most productive self-sustaining region because it contains large numbers of plankton,* a floating microbial community that drifts with wave action and currents. A major member of this assemblage is the phytoplankton, containing a variety of photosynthetic algae and cyanobacteria. The phytoplanktonprovide nuhition for zooplankton, microscopic consumers such as protozoa and invertebrates that filter, feed, prey, or scavenge. The plankton supports numerous other trophic levels such as larger invertebrates and fish. With its high nutrient content, the deeper regions also support an extensive variety and concentration of organisms, including aquatic plants, aerobic bacteria, and anaerobic bacteria actively involved in recycling organic detritus. Larger bodies of standing water develop gradients in temperature or thermal sfiatification, especially during the summer (figure 26.15). The upper region, called the epilimnion, is warmest, and the deeper hypolimnion is cooler. Between these is a buffer zone, the thermocline, that ordinarily prevents the mixing of the two. +

Twice a year, during the warming cycle of spring and the cooling cycle of fall, temperature changes in the water column break down the thermocline and cause the water from the two strata to mix. Mixing disrupts the stratification and creates currents that bring nutrients up from the sediments. This process, called upwelling, is associated with increased activity by certain groups of microbes and is one explanation for the periodic emergence of red ti.des in oceans (figure 26.16) caused by toxin-producing dinoflagellates. A recent outbreak of fish and human disease on the eastern seaboard has been attributed to the overgrowth of certain species of these algae in polluted water. These algae produce a potent muscle toxin that can be concentrated by shellfish through filtration feeding. When humans eat clams, mussels, or oysters that contain the toxin, they develop paralytic shellfish poisoning. People living in coastal areas are cautioned not to eat shellfish during those months ofthe year associated with red tides (varies from one area to another). Because oxygen is not very soluble in water and is rapidly used up by the plankton, its concentration forms a gradient from highest

planhon Siang'-nn) G. planlros, wandering.

Figure 26.15

Profiles of a lake. (a) During summer, a lake becomes stabilized into three major

temperature strata. (b) During fall and spring, cooling or heating of the water disrupts the temperature strata and causes upwelling of nutrients from the bottom sediments.

Figure 26.16

Red tides. (a) Single-cefled red algae called dinoflagellates (Gymnodinium shown here) bloom in high-nutrient, warm seawater and impart a noticeable red color to it as shown in (b). (b) An aerial view of California coastline in the midst of a massive red tide.

26.3

803

Microbes on Land and in Water

Water Monitoring tci Prevent Diseose Microbiology of Drinking Water Supplies

We do not have

to look far for overwhelming reminders of the importance of safe water. Worldwide epidemics of cholera have killed thousands of people, and an outbreak of Cryptosporidium in Wisconsin affecting 370,000 people was traced to a contaminated municipal water supply. In alarge segment of the world's population, the lack of sani-

tary water is responsible for billions of cases of diarrheal illness that kills 3 million children each year. In the United States, nearly I million people develop water-borne illness every year. Good health is dependent upon a clean, potable (drinkable)

Figure 26.17

Heavy surface growth of algae and cyanobacteria in a eutrophic pond. in the epilimnion to lowest at the bottom. In general, the amount of oxygen that can be dissolved is dependent on temperature. Warmer strata on the surface tend to carry lower levels ofthis gas. But ofall the characteristics of water, the greatest range occurs in nukient levels. Nutrient-deficient aquatic ecosystems are called oligotrophic.* Species that can make a living on such starvation rations are Hyphomicrobium and CaulobacterThese bacteria have special stalks that capture even minuscule amounts of hydrocarbons present in oligotrophic habitats. At one time, it was thought that viruses

were present only in very low levels

in aquatic habitats, but re-

searchers have now discovered that there are anywhere from 2 to l0 times as many viruses as bacteria in marine and freshwater communities. Oceans and lakes contain anywhere from I to 125 viruses per milliliter. Most of these viruses pose no danger to humans, but as parasites ofbacteria, they appear to be a natural control mecha-

nism for these populations. At the other extreme are waters overburdened with organic matler and dissolved nutrients. Some nutrients are added naturally through seasonal upwelling and disasters (floods or typhoons), but the most significant alteration of natural waters comes from efflu-

ents from sewage, agriculture, and industry that contain heavy loads oforganic debris or nitrate and phosphate fertilizers. The addition of excess quantities of nutrients to aquatic ecosystems, termed eutrophicationr* often wreaks havoc on the communities involved. The sudden influx of abundant nutrients, along with warm temperatures, encourages a heavy surface growth ofcyanobacteria andalgae called a bloom (figure 26.17). This heavy mat of biomass effectively shuts offthe oxygen supply to the lake below The oxygen content below the surface is further depleted by aerobic heterotrophs that actively decompose the organic matter. The lack of oxygen greatly disturbs the ecological balance of the community. It causes massive die-offs of strict aerobes (fish, invertebrates), and onlv anaerobic or facultative microbes will survive. * oligotrophic (ahl"-ih-goh-trof ik) Gt. oligo, small, and troph, to feed. * eutrophication (yoo"-txoh-fih-kay'-shun) Gr. eu, good.

water supply. This means the water must be free of pathogens; dissolved toxins; and disagreeable turbidity, odor, color, and taste' As we shall see, water of high quality does not come easily, and we must look to microbes as part of the problem and part of the solution. Through ordinary exposure to air, soil, and effluents, surface waters usually acquire harmless, saprobic microorganisms. But along its course, water can also pick up pathogenic contaminants. Among the most prominent water-borne pathogens of recent times are thefrgtozgans @fia and CrrypspollliumJ the bacteria Campylobacter, Salmonella, Shigella, Vibrio, and Mycobacterium ; and hepatitis A and Norwalk viruses. Some of these agents (especially encysted protozoans) can survive in natural waters for long periods without a human host, whereas others are present only transiently and are rapidly lost. The microbial content of drinking water must be continuously monitored to ensure that the water is free of infectious agents. Attempting to survey water for specific pathogens can be very difficult and time-consuming, so most assays of water purity are more focused on detecting fecal contamination. High fecal levels can mean the water contains pathogens and is consequently unsafe to drink. Thus, wells, reservoirs, and other water sources can be analyzed for the presence ofvarious indicator bacteria. These species are intestinal residents of birds and mammals, and they are readily identified using routine lab procedures. Enteric bacteria most useful in the routine monitoring of microbial pollution are gram-negative rods called coliftrms and en-

teic streptococci, which survive in natural waters but do not multiply there. Finding them in high numbers thus implicates recent or high levels of fecal contamination. Environmental Protection Agency standards for water sanitation are based primarily on the levels of coliforms, which are described as gram-negative, lactose-fermenting, gas-producing bacteria such as Escherichia coli. Enterobacter and Citrobacter Fecal contamination of marine waters that poses a risk for gastrointestinal disease is more readily correlated with gram-positive cocci, primarily inthe genus Enterococcus. Occasionally, coliform bacteriophages and reoviruses (the Norwalk virus) are good indicators of fecal pollution, but their detection is more difficult and more technically demanding.

Water Quality Assays A rapid method for testing

the total bac-

terial levels in water is lhe standard plate count.In this technique, a small sample of water is spread over the surface of a solid medium. The numbers of colonies that develop provide an estimate of the total viable population without differentiating coliforms from other species. This information is particularly helpful in evaluating the effectiveness of various water purification stages. Another general

804

Chapter26 Environmental Microbiology

The Waning Days of a Classic Test? Keeping water and the food we harvest from it free from fecal contamination is absolutely imperative in making it safe to ingest. In the late 1800s. it was suggested that a good way to determine lf water or its products had been exposed to feces was to test for E. coli. Although most E'. coli strains are not pathogenic, they almost always come from a mammal's intestinal tract so their presence in a sample is a clear

commonly found growing in nonfecal environments

such as fresh water and plants that eventually become food. In other words, ifthe tests are not specifically for E. coli, you can't be sure that feces are present. Recently, there was a minor panic when media outlets reported that iced tea from restaurants contained significant numbers of "fecal coliforms." The public was outraged. One headline read, "Iced Tea Worse Than indicator of fecal contamination. River Water." Restaurants were named, and their repuAt one time, it was too difficult to differentiate tations were damaged. When scientists did more deE. coli from the closely related species of Citrobacter, tailed testing, they found that the predominant species 19S?g€:{y.,iarr,r,::t*tl}€1:g{Xxe:st' Klebsiella, and, Enterobacter so laboratories instead found were K/ebsiella and, Enterobacter. both of which simply reported whether a sample contained any of these isolates. All of are normal colonizers ofplants, such as tea leaves. Furthermore, despite these bacteria ferment lactose and are phenotypically similar. The termithe reports of widespread contamination with large numbers of "fecal nology adopted was "col;form-" meaning E. coti-Iike positive or negacoliforms," no one ever became sick from drinking iced tea. tive. In other words, such bacteria were present in the sample but not Microbiologists are now advocating that E. coli alone-'not just any necessarily E col/. coliforms-be used as an indicator of fecal contamination. Newer idenThe use ofthis coliform assay has been the standard procedure since tification techniques make this as simple, if not simpler, than the standard 1914, and. it is still in widespread use. Pick up a newspaper in the sumcoliform tests. But old habits die hard, and regulatory and public laboramer, and you will likely find a report about a swimming pool or a river tories are proving slow to convert to the,E coli standard. with a high coliform count. Coliform counts are also used to regulate lf you read about non-.E coli coliforms in chapter 20, you will find food production and to trace the causes of food-borne outbreaks. Rethat they da cause infections. Give some possible reasons that they cently, microbiologists have noted serious problems with the use of colididn't cause any problems when ingested with iced tea. Answer form counts to indicate fecal contamination. The main issue is that the available at http : //www.mhhe.com/talaroT three other bacterial genera



%$already mentioned, among others, are

indicator of water quality is the level of dissolved oxygen it contains called the biological oxygen demand (BOD).It is established that water containing high levels of organic matter and bacteria will have a lower oxygen content because of consumption by aerobic respiration.

Coliform Enumeration Water quality departments employ some standard assays for routine detection and quantification of coliforms. The techniques available are

o

.

simple tests, such as presence-absence broth, that detect coliform activity but do not quantify it; rapid tests that isolate coliform colonies and provide quantities

.

rapid tests that identi$ specific coliforms and determine

of coliforms present; and numbers.

In many circumstances (drinking water, for example), it is important to dffirentiate between facultative coliforms (Enterobacter) that are often found in other habitats (soil, water) and true fecal coliforms that live mainly in human and animal intestines. Microbiologists are considering the discontinuation of the use of coliforms alone as an indicator of fecal contamination (Insight 26.3). Some of the tests are still widespread, so we briefly cover their principles here. The membranefilter method is a widely used rapid method that can be used in the field or lab to process and test larger quantities of water. This method is more suitable for dilute fluids, such as

drinking water, that are relatively free ofparticulate matter, and it is less suitable for water containing healy microbial growth or debris. This technique is related to the method described in chapter 11 for sterilizing fluids by filtering out microbial contaminants, except that in this system, the filter containing the trapped microbes is the desired end product. The steps in membrane filtration are diagrammed in figure 26.18a,b. After filtration, the membrane filter is placed in a Petri dish containing selective broth. After incubation, both nonfecal and fecal coliform colonies can be counted and often presumptively identified by their distinctive characteristics on these media (figure 26.18c,d). Another more time-consuming but useful technique is the most probable number (MPI$ procedure, which detects coliforms by a series ofpresumptive, confirmatory, and completed tests. The presumptive test involves three subsets of fermentation fubes, each containing different amounts of lactose or lauryl tryptose broth. The three subsets are inoculated with various-size water samples. After 24 hours of incubation, the tubes are evaluated for gas production. A positive test for gas formation is presumptive evidence of coliforms; negative for gas means no coliforms. The number of positive tubes in each subset is tallied, and this set of numbers is applied to a statistical table to estimate the most likely or probable concentration of coliforms (see appendix C and table C.1). It does not specifically detect fecal coliforms. When a test is negative for coliforms, the water is considered generally fit for human consumption. But even slight coliform levels are allowable

26.3

w

Microbes on Land and in Water

805

(a) Membrane filter technique. The water sample is filtered through a sterile membrane filter assembly and collected in a flask.

re I

M

(b) The filter is removed and placed in a small Petri dish containing a ditferential selective medium such as M-FD endo agar and incubated.

(c) On M-FD endo medium, colonies of Escherichia coli often yield a noticeable metallic sheen. The medium permits easy differentiation of various genera of coliforms, and the grid pattern can be used as a guide for rapidly counting the colonies.

(d) Some tests for water-borne coliforms are based on formation of specialized enzymes to metabolize lactose. The Ml tests shown here utilize synthetic substrates that release a colored substance when the appropriate enzymes are present. The total coliform count is indicated by the plate on the left; fecal coliforms (E. coli) are seen in the plate on the right. This test is especially accurate with surface or groundwater samples. under a black light.

Flgure 26.1A

under natural light

Rapid methods of water analysis for coliform contamination.

under some circumstances. For example, municipal waters can have a maximum of 4 coliforms per 100 ml; private wells can have an even higher count. There is no acceptable level for fecal coliforms, enterococci, viruses, or pathogenic protozoans in drinking water.

will not be consumed but are used for fishing or swimming are permitted to have counts of 70 to 200 coliforms per 100 ml. If the coliform level of recreational water reaches 1,000 coliforms per 100 ml, health departments usually bar its usage. Waters that

The lithosphere, or soil, is an ecosystem in which mineral-rich

Significant water-borne pathogens include protozoans, bacteria,

rocks are decomposed to organic humus, the base for the soil community. Soil ecosystems vary according to the kinds of rocks and amount of water, air, and nutrients present. The rhizosphere is the most ecologically active zone of the soil. The food web of the aquatic community is built on phytoplankton and zooplankton. The nafure of the aquatic community varies with the temperature, depth, minerals, and amount of light present in eachzooe. Aquatic ecosystems are readily contaminated by chemical pollutants and pathogens because of industry agriculture, and improper disposal of human wastes.

and viruses. Giardia and Cryptosporidium are lbe most significant protozoan pathogens. Campylobacter, Salmonella, and Vibrio are the most significant bacterial pathogens. Hepatitis A and Norwalk virus are the most significant viral pathogens. Water quality assays assess the most probable number of microorganisms in a water sample and screen for the presence of enteric pathogens using coliforms as the indicator organisms.

Other methods for assessing water quality are t}re standard plate count, biological oxygen deman4 and membrane filter.

Chapter

806

26

Environmental Microbiology

Chapter Summary with Key Terms 26.1 Ecology: The Interconnecting Web of Life

a. In nitrogen fixation, atmospheric N, gas (the

This chapter emphasizes microbial activities that help maintain, sustain, and control the life support systems on the earth. This includes the natural roles of microorganisms in the environment and their contributions to the ecological balance, including soil, water, and mineral cycles. A. Microbial ecology deals with the interaction between the environment and microorganisms. The environment is composed of biotic (living or once-living) and abiotic (nonliving) components. The combination of organisms and the environment make up an ecosystem.

B.

primary reservoir) is converted to NOr-, NO,-, or NHo- salts.

b. Ammonification

B.

EcosystemOrganization 1. Living things inhabit only that area ofthe earth called the biosphere, which is made up ofthe hydrosphere (water), the lithosphere (soil), and the atmosphere (air). 2. The biosphere consists of terrestrial ecosystems (biomes) and aquatic ecosystems. Biomes contain communities, assemblages of coexisting organisms. 4. Communities consist of populationso groups of like organisms of the same species. 5. The space within which an organism lives is its habitat; its role in community dynamics is its niche. Energy and Nutrient Flow 1. Organisms derive nutrients and energy from their habitat. 2. Their collective trophic status relative to one another is summarized in a food or energy pyramid. 3. At the beginning of the chain or pyramid are producers-

3.

C.

4. 5.

is a stage in the degradation of nitrogenous organic compounds (proteins, nucleic acids) by bacteria to ammonium. c. Some bacteria nitrify NHa+ by converting it to NOr-and to NO3 d. Denitrification is a multistep microbial conversion of various nitrogen salts back to atmospheric Nr. Sedimentary Cycles 1. In the sulfur cycle, environmental sulfurous compounds are converted into useful substrates and returned to the inorganic reservoir through the action ofmicrobes. 2. The chiefcompound in the phosphorus cycle is phosphate (POo) found in certain mineral rocks. Microbial action on this reservoir makes it available to be incorporated into organic phosphate forms. 3. Microorganisms often cycle and help accumulate heavy metals and other toxic pollutants that have been added to habitats by human activities.

26.3 Microbes on Land and in Water

A.

Soil Microbiology Soil is a dynamic, complex ecosystem that accommodates a vast array of microbes, animals, and plants coexisting among rich organic debris, water and air spaces, and minerals.

B. AquaticMicrobiology 1. The surface water, atmospheric moisture, and

organisms that slmthesize large, complex organic compounds from small, simple inorganic molecules. The levels above producer are occupied by consumers, organisms that feed upon other organisms.

Decomposers are consumers that obtain nutrition from the remains of dead organisms and help recycle and

2.

mineralize nutrients.

6. Bioremediation

is the process by which microbes, or communities of microbes, decompose chemicals that are harmful to the environment and its inhabitants.

3.

26,2 The Natural Recycling of Bioelements

A.

groundwater are linked through a hydrologic cycle that involves evaporation and precipitation. Living things contribute to the cycle through respiration and transpiration. The diversity and distribution of water communities are related to sunlight, temperature, aeration, and dissolved nutrients. Phytoplankton and zooplankton drifting in the uppermost zone constitute a microbial community that supports the aquatic ecosystem. Water Monitoring

a.

Atmospheric Cycles

b.

Key compounds in the carbon cycle include carbon dioxide, methane, and carbonates.

1. Carbon is fixed when autotrophs (photosynthesizers)

2.

c.

add carbon dioxide to organic carbon compounds. The nitrogen cycle requires four processes and several

tvoes of microbes.

YK#et

u

Providing potable water is central to prevention of water-borne disease. Water is constantly surveyed for indicator bacteria such as E. coli, coliforms, and enterococci, that signal fecal contamination. Assays for possible water contamination include the standard plate count and membrane filter tests to enumerate coliforms.

hiPle=€hoice Questions

Select the correct answer from the answers provided. For questions with blanks, choose the combination of answers that most accurately completes the statement. 1

. Which of the followin g is not a major subdivision of the a. hydrosphere c. stratosphere b. lithosphere

2.

biosphere?

d. atmosphere

Alan is defined as a collection ofpopulations sharing a given habitat. a. biosphere c. biome

-

b. community

d. ecosystem

3

. The quantity of available nutrients energy pyramid to the higher ones. a. lncreases

b.

decreases

from the lower levels of the

-

c. remains stable d. cycles

4. Which of the following is/are considered a greenhouse a. CO2 c. N2O b. cH4 d. all ofthese

gas?

Concept Mapping

which can

5. Root nodules contain a. Azotobacter, fixN2

-,

b. Nitrosomoncs, nitriS NH3-

c. rhizobia, fix N2 -. d. Bacillus, denitrify NOI

6. Which element(s) has/have an inorganic reservoir that exists primarily in sedimentary deposits?

c. sulfur

a. nltrogen b. phosphorus

d. both b and c

7. Which of the following bacteria would be the most accurate indicator of fecal contamination? a. Enterobacter aerogenes

b. Thiobacillus

acidophilus

c. Escherichia coli d. Staplrylococcus aures

807

8. The floating assemblage of microbes, plants, and animals that drifts on community. or near the surface of large bodies of water is the

c. littoral

a. abyssal b. benthic

-

d. plankton

9. An oligotrophic ecosystem would be most likely to exist in a/an c. tropical pond a. ocean d. polluted river b. high mountain lake 10. Which of the following does not vary predictably with the depth the aquatic environment? c. penetration by sunlight a. dissolved oxygen d. salinity b. temperature

of

writins to Learn

Wf

the factual These questions are suggested as a writing-to-learn experience. For each question, compose a one- or two-paragraph answer that includes parentheses. given in page are references question. General the information needed to completely address

1. a. Present in outline form the levels oforganization in the biosphere. Define the term biome. (788)

b. Compare autotrophs and heterotrophs; producers

b. What form ofnitrogen is requiredbyplants? By animals? (794,795)

6. Summarize

and

consumers. (789,790) c. Where in the energy and trophic schemes do decomposers

7.

(figure 26.1) as an example. (788, 789)

2. a. Using figures 26.3 and26.4,pontout specific secondary, and tertiary camivores; and

for

it?

a.

9.

a.

omnivores. (790,791)

(790)

3. a. Outline the general characteristics ofa biogeochemical cycle. (791) b. What are the major sources of carbon, nitrogen, phosphorus, and 4. a. In what natural forms is carbon found? Name three ways carbon is returned to the atmosphere. ('792,793) b. Name a way it is fixed into organic compounds. (793) c. What form is the least available for the majority of living

things? (793)

humus? (799)

Outline the modes of cycling water through the lithosphere, hydrosphere, and ahnosphere. (800) b. What are the roles of precipitation, condensation, respiration, transpiration, surface water, and aquifers? (800)

8.

examples of producers;

sultur? (792)

Describe the structure of the soil and the rhizosphere. (797 ,799) Compare and contrast root nodules with mycorrhizae. (795,799)

primary, secondary, and tertiary consumers, herbivores; primary.

b. What is mineralization, and which organisms are responsible

a.

b. What is

enter? (790) d. Compare the concepts of habitat and niche usirtg Chlamydomonas

the main stages in the cycling of sulfur and

phosphorus. (796,797)

10.

What causes the formation of the epilimnion, hypolimnion, and thermocline? (801, 802) b. What is upwelling? (802) c. How are red tides and algal blooms similar and different? (802, 803)

a. Why must water be subjected to microbiological analysis? (802,803) b. What are the characteristics of good indicator organisms, and why are they monitored rather than pathogens? (803)

c. Give specific

examples of indicator organisms and water-borne

pathogens. (803,804) d. Describe two methods of water analysis. (804, 805)

5. a. Describe nitrogen fixation, ammonification, nitrification, and

denitrification. (794, 796)

@ff

concept Mapping

Appendix E provides guidance for working with concept maps.

l.

Supply your own linking words or phrases in this concept map, and provide the missing concepts in the empty boxes.

Soil

moisture Lakes

Groundwater

,:..

Oceans Seas Rivers

lcecaps Glaciers

Chapter

808

26

Environmental Microbiology

critical Thinkins Questions

&#

Critical thinking is the ability to reason and solve problems using facts and concepts. These questions can be approached flom a number ofangles, and in most cases, they do not have a single correct answer. 1. a. What factors cause energy to decrease with each trophic level? b. How is it possible for energy to be lost and the ecosystem to still run efficiently? c. Are the nutrients on the earth a renewable resource? Whv. or why not?

2.

Give specific examples from biogeochemical cycles that support the

c. What d.

6. Why are organisms in the abyssal zone of the ocean necessarily

Gaia theory.

halophilic, psychrophilic, barophilic, and anaerobic?

3. Biologists can

set up an ecosystem in a small, sealed aquarium that continues to function without maintenance for years. Describe the minimum biotic and abiotic components it must contain to remain

7. a. What eventually happens to the nutrients that run off into the ocean with sewage and other effluents?

b. Why can high mountain communities usually dispense with water

balanced and stable.

4.

occurrence has made them dangerous to the global ecosystem? What could each person do on a daily basis to cut down on the potential for disrupting the delicate balance ofthe earth's ecosystems?

treatment?

Observe the carbon and nitrogen cycles and explain those places in the cycles where interactions in biofilms would be important factors.

8. a. If we are to rely on microorganisms to biodegrade wastes in

b.

5. a. Is the greenhouse effect harmful under ordinary circumstances? b. List the primary greenhouse gases and explain their effects on the

c.

earth's temperature.

1. From chapter 3, figure 3.90. If this MacConkey agar plate was inoculated with well water, would you report that coliforms were present in the well? Is it safe to drink the water?

landfills, aquatic habitats, and soil, list some ways that this process could be made more efficient. Because elemental poisons (heavy metals) cannot be further degraded even by microbes, what is a possible fate of these metals? Provide some possible solutions for this form of pollution.

chapter 8, figure 8.28. What process does this represent? How does it link to the biogeochemical cycles from this chapter?

2. From

{-

Wlnternet l.

search ropics

Go to: http://www.mhhe.com/talaro7. Go to chapter 26,lntemet Search Topics, and log on to the available websites to: a. Look up information on techniques for testing water. Explain how several ofthe tests work and their uses. b. Find information on red tide outbreaks and illness in humans.

2. Conduct

a search using the term "interplanetary transfer

(of)

microbes." What role does this phenomenon play in the science of astrobiology?

J. Search

for "iced tea coliforms" and critically analyze what you find.

Be sure to examine multiple sites. 1

Research the subject of bioremediation. What sorts of toxic substances are being cleaned up and what types of microbes are

involved?

t,

€-.-Y g*?

'$.ie:E :, .:

Industrial y' 't,,

CASE FILE

27

n'1998, a multistate outbreak of intestinal illness occurred between August and December. It eventually affected 108 persons in 24 states and resulted in 14 deaths and 4 miscarriages or stillbirths. Most patients reported with symptoms of fever and gastrointestinal distress. Elderly patients, pregnant women, neonates, and immunosuppressed patients experienced more severe effects, including septicemia, meningitis, and encephalitis. It took nearly a month before departments of health in several states were able to complete traceback surveillance to determine the cause and source of infection. Assuming it was an outbreak of food-borne disease, they tested food samples from patients who had saved suspected

food as well as clinical specimens (blood, spinal fluid, placenta). They performed traditional bacteriologic isolation techniques and serotying to determine the identity of the isolates. In every case, the microbe of interest turned out to be Listeria monocytogenes. The same organism was discovered in hot dogs and deli meats and samples taken from patients. But it was not entirely clear that every person was infected from this same source. For this, more sophisticated molecular techniques were needed. Samples were sent to the Centers for Disease Control and Prevention (CDC) to arrive at more definitive confirmation. The CDC had been applying a rapid molecular technique for identifying and typing pathogens in food called pulse-field gel electrophoresis (PFGE). With this technique, a quick, specific DNA fingerprint unique to a microbe and its exact genetic strain can be made. PFCE had first been used to trace an outbreak of E. coli 0157:H7 infection to hamburger patties. Since that time, the technique had become the basis for a new database called PulseNet. All of the processed meat was eventually traced back to a single plant. The genetic fingerprint of bacteria cultured from a floor drain at the plant was identical to that seen in the patient isolates. Plant records indicated that all poultry was held at or below 4oC at all times. On October 12,2002, the company that owned the processing plant in Franconia, Pennsylvania, recalled 27.4 million pounds of poultry processed in the plant, one of the largest meat recalls in U.S. history.

2

Why is it importont to be oble to verify that the sqme stroin of Listeria monocytogenes wos involved in all cases?

) )

The food had been properly stored. Why was

it

still oble to moke people sick?

Why did it affect primarily elderly ond weokened potients? Case File

27 Wrap-Up appeorc on page 819.

809

810

Chapter

27

Applied and Industrial Microbiology

CHAPTER OVERVIEW

Microbes play significant roles in practical endeavors related to agriculture, food production, industrial processes, and waste treatment. Water quality is greatly dependent on its microbial and chemical content. Water is made safe by treatment methods that remove pathogenic microbes and toxic wastes. Biotechnology creates industrial, agricultural, nutritional, or medical products through

microbial activities. Food fermentations are used to make a variety of milk products (cheeses, yogurt), alcoholic beverages (beer, wine, spirits), and pickles. Large-scale industrial fermentations employ microbial metabolism

hormones, enzymes, vaccines, and vitamins.

27;l Applied Microbiology and Biotechnology In this chapteq we address the diverse ways microbes are used to perform specific metabolic tasks useful to humans. One area of interest, called applied microbiology, takes advantage of the m! crobes living in natural habitats to treat wastewater and bioremediate damaged environments. Because of this, it correlates directly with several concepts introduced in the previous chapter on envi-

ronmental microbiology. Another area-industrial microbiology-explores the use of microbes in making a wide variety of food, medical, manufacturing, and agricultural products. Although this may be an "artificial" use of microbes, it applies many of the principles we learned about the natural metabolic and adaptive qualities of microorganisms covered in chapters 7,8, and9. In the previous chapter, we witnessed the profound and sweep-

ing involvement of microbes in the natural world. Although our daily encounters with them usually go unnoticed, human and microbial life are clear$ intertwined on many levels. It is no wonder that long ago humans rcalizedthe power of microbes and harnessed them for practical applications. These efforts gave rise to the large and diverse area known as biotechnotogy. Biotechnology has an ancient history dating back nearly 6,000 years to those first observant humans who discovered that grape juice left sitting produced wine or that bread dough properly infused with a starter would rise. Today, biotechnology has become a fertile ground for hundreds of applications in industry, medicine, agriculture, food sciences, and environmental protection, and it has even come to include the genetic alterations of microbes and other organisms. Most biotechnological systems involve the actions of bacteria, yeasts, and molds that are able to slmthesize a certain food, drug, organic acid, alcohol, or vitamin. Many such end products are obtained through fermentation, a general term used here to refer to the mass, controlled culture of microbes to produce desired organic compounds. It also includes the use of microbes in sewage control, pollution control, metal mining, and bioremediation. These subjects could easily be the inspiration for a whole book, let alone a single chapter. Our coverage ofnecessity only touches on some basic concepts important for rounding out your background in microbiology. The rest of section 27 .I is devoted to management of water and sewage; section 27 .2 covers food microbiology; and section 27.3 concerns industrial biotechnology. Bioremediation was discussed in chapter 26 (case file and Insight 26.2), along with soil and water microbiology.

to manufacture drugs,

Microorganisms in Water and Wastewater Treatment Most drinking water comes from rivers, aquifers, and springs. Only in remote, undeveloped, or high mountain areas is this water used in its natural form. In most cities, it must be treated before it is supplied to consumers. Wetersupplies such as-glggy4lls that are relatively clean and free of contaminants require less treatment than those from surface sources laden with wastes. The stepwise process in water purification as carried out by most cities is shown in figu,re27.l. Treatment begins with the impoundment of water in a large reservoir such as a dam or catch basin that serves the dual purpose of storage and sedimentation. The access to reservoirs is controlled to avoid contamination by animals, wastes, and runoff water. Igaddition, overgrowth of cyanobacteria and algae that add undesirable qualities to the water is prevented by preheatment with :gqp.t *]}te (0.3 ppm). Sedimentation to remove large particulate matter is also encouraged during this storage period. Next, the water is pumped to holding ponds or tanks, where it undergoes further settling, aeration, and filtration. The water is filtered fint through sand beds or pulverized diatomaceous earth to remove residual bacteria, viruses, andprotozoans and then through activated charcoal to remove undesirable organic contaminants. Pipes coming from the filfration beds collect the water in storage tanks. The final step in treatrnent is chemical disinfection by bubbling chlorine gas through the tank until it reaches a concentration of I to 2 ppm (some municipal plants use chloramines for this purpose) (see chapter 11). A few pilot plants in the United States are using ozone or peroxide for final disinfection, but these methods are expensive and carmot sustain an antimicrobial effect over long storage times. The final quality varies, but most tap water has a slight odor or taste from disinfection. In many parts of the world, the same water that serves as a source of drinking water is also used as a dump for solid and liquid wastes (figure 27.2), Continued pressure on the finite water resources may require reclaiming and recycling of contaminated water such as sewage. Sewage is the used wastewater draining out of homes and induskies that contains a wide variety of chemicals, debris, and microorganisms. The dangers of fyphoi{ cholera, and dysentery linked to the unsanitary mixing of household water and sewage have been attxeat for centuries. In current practice, some sewage is treated to reduce its microbial load before release, but a large quantity is still being emptied raw (untreated) into the aquatic environment primarily because heavily contaminated waters require far more stringent and costly methods of treatment than are currently available to most cities.

27.1 Applied Microbiology and Biotechnology Primary

Figure

Stage

27.7

Secondary

Stage

811

Teniary Stage

The primary secondary and tertiary stages

in sewage treatment.

To consumer through

domestic water pipes

tl

Flgure 27.1 The maior steps in water purification cairied out by a modern municipaltreatment plant.

Flgure

27.2

Water: one source, many uses.

as

Sewage contains large amounts of solid wastes, dissolved organic matteq and toxic chemicals that pose a health risk. To remove all potential health hazards, treatrnent fypically requires three phases: The primary stage separates out large matter; the secondary stage reduces remaining matter and can remove some toxic substances; and the tertiary stage completes the purification of the water (figure 27.3). Microbial activity is an integral part of the overall process. The systems for sewage treatment are massive engineering marvels. In the primary phase of treatment, floating bulkier materials such as paper, plastic waste, and bottles are skimmed off. The remaining smalleq suspended particulates are allowed to settle. Sedimentation in settling tanks usually takes2 to 10 hours and leaves a mixture rich in organic matter. This aqueous residue is carried into a secondary phase of active microbial decomposition, or biodegradation. In this phase, a diverse community of natural bioremediators (bacteria, algae, and protozoa) aerobically decomposes the remaining particles of wood, papeq fabrics, petroleum, and organic molecules inside a large digester tank (figure 27. ).Tltts forms a suspension of material called sludge that tends to settle out and slow the process. To hasten aerobic decomposition of the sludge, most processing plants have systems to activate it by injecting air, mechanically stirring it, and recirculating it. A large amount of organic matter is mineralized into sulfates, nitrates, phosphates, carbon dioxide, and water. Certain volatile gases such as hydrogen sulfide, ammonia, nitrogen, and methane may also be released. Water fiom this process is siphoned offand carried to the tertiary phase, which involves further filtering and chlorinating prior to discharge. Such reclaimed sewage water is usually used to water golfcourses and parks rather than for drinking, or it is gradually released into large bodies of water. In some cases, the solid waste that remains after aerobic decomposition is harvested and reused. Its rich content of nitrogen, potassium, and phosphorus makes it a useful fertilizer. But if the waste contains large amounts of nondegradable or toxic substances,

812

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Applied and Industriat Microbiology

27.2 Microorganisms and Food All human food-from

vegetables to caviarto cheese-comes from some other organism, and rarely is it obtained in a sterile, uncon_ taminated state. Food is but a brief stopover in the overall scheme of biogeochemical cycling. This means that microbes and humans

are in direct competition for the nutrients in food, and we must be constantly aware that microbes'fast growth rates give them the winning edge. Somewhere along the route ofprocurement, process-

ing, or preparation, food becomes contaminated with microbes from the soil, the bodies ofplants and animals, water, air, food handlers, or utensils. The final effects depend upon the types and numbers of microbes and whether the food is cooked or preserved. In some cases, specific microbes can even be added to food to obtain a desired effect. The effects of microorganisms on food can be clas-

sified as beneficial, detrimental, or neutral to humans. as summarizedby the following outline: Beneficial effects Food is fermented or otherwise chemically changed by the addition of microbes or microbial products to alter or improve flavor, taste, or texture. Microbes can serve as food.

Detrimental effects Food poisoning or food-borne illness Food spoilage

Growth of microbes makes food unfit for consumption; adds undesirable flavors, appearance, and smell; destroys food value

Neutral effects The presence or growth ofmicrobes that do not cause disease or change the nature ofthe food

Flgure 27,4 Treatment of sewage and wastewater. (a) Digester tanks used in the primary phase of treatment; each tank can process several million gallons of raw sewage a day. (b) View inside the secondary reactor shows the large stirring paddle that mixes the sludge to aerate it to encourage microbial decomposition.

it

must be disposed of properly. In many parts of the world, the sludge, which still contains significant amounts of simple but useful organic matter, is used as a secondary source ofenergy. Further digestion is carried out by microbes in sealed chambers called bioreactors, or anaerobic digesters. The digesters convert components of the sludge to swamp gas, primarily methane with small amounts ofhydrogen, carbon dioxide, and othervolatile compounds. Swamp gas can be burned to provide energy to run the sewage processing facility itself or to power small industrial plants. Recently, scientists found a way to harness the bacteria found in sewage to construct a microbial fuel cell to produce usable energy. In these experiments, wastewater bacteria form biofilms on special rods inserted in the sewage that is being treated. These biofilms generate electrons that are transferred via copper wires to cathodes, producing electricity. Considering the mounting waste disposal and energy shortage problems, these technologies should gain momenfum.

As long as food contains no harmful substances or organisms, its suitability for consumption is largely a matter of taste. But what tastes like rich flavor to some may seem like decay to others. The test ofwhether certain foods are edible is guided by culture, experience, and preference. The flavors, colors, textures, and aromas of many cultural delicacies are supplied by bacteria and fungi. Chocolate, pickled cabbage, fermented fish, and Limburger cheese are notable examples. If you examine the foods of most cultures, you will find some foods that derive their delicious flavor from microbes.

Microbial Fermentations in Food Products from Plants In contrast to methods that destroy or keep out unwanted microbes,

many culinary procedures deliberately add microorganisms and encourage them to grow. Common substances such as bread, cheese,

beer, wine, yogurt, and pickles are the result of food fermentations. These reactions actively encourage biochemical activities that impart a particular taste, smell, or appeaf,ance to food. The microbe or microbes can occur naturally on the food substrate, as in sauerkraut, or they can be added as pure or mixed samples of known bacteia, molds, or yeasts called starter cultures. Many food fermentations are syrergistic, with a series of microbes acting in concert to convert a starting substrate to the desired end product.

27.2

Microorganisms and Food

813

Because large-scale production of fermented milk, cheese, brea4 alcoholic brews, and vinegar depends upon inoculation with starter cultures, considerable effort is spent selecting, maintaining, and preparing these cultures and excluding contaminants that can spoil

the fermentation. Most starting raw materials are of plant origin (grains, vegetables, beans) and, to a lesser extent, ofanimal origin (milk, meat).

Breod Microorganisms accomplish three functions in bread making:

1. leavening the flour-based dough,

2. imparting flavor and odor, and 3. conditioning the dough to make it workable. Leavening is achieved primarily through the release of gas to produce a porous and spongy product' Without leavening, bread dough remains dense, flat, and hard. Although various microbes and leavening agents can be used, the most common ones are various strains of the baker's yeast Saccharomyces cerevisiae. Other gas-forming

microbes such as coliform bacteria, certain Clostridium species, heterofermentative lactic acid bacteria, and wild yeasts can be employed" depending on the type ofbread desired. Yeast metabolism requires a source of fermentable sugar such as maltose or glucose. Because the yeast respires aerobically in bread dough, the chief products of maltose fermentation are carbon dioxide and water rather than alcohol (the main product in beer and wine). Other contributions to bread texture come from kneading, which incorporates air into the dough, and from microbial enzymes, which break down flour proteins (gluten) and give the dough elasticity. Besides carbon dioxide production, bread fermentation generates other volatile organic acids and alcohols that impart delicate flavors and aromas. These are especially well developed in homebaked bread. which is leavened more slowly than commercial bread. Yeasts and bacteria can also impart unique flavors, depending upon the culture mixture and baking techniques used' The pungent flavor of rye bread, for example, comes in part from starter cultures of lactic acid bacteria such as Lactobacillus plantarum, L. brevis, L. bulgaricus, Leuconostoc mesenteroides, and Streptococcus thermophilus. Sourdough bread gets its unique tang from L ac t o

b a

ci

I lus s

anfranc

i s c o.

Beer and Other Alcoholic Beverages The production ofalcoholic beverages takes advantage ofanother useful property of yeasts. By fermenting carbohydrates in fruits or grains anaerobically, they produce ethyl alcohol, as shown by this equation:

-->2crH5OH +

(Yeast

c6H12o6 Sugar : Ethanol

*

*

2CO2

Carbon dioxide)

Depending upon the starting materials and the processing method, alcoholic beverages vary in alcohol content and flavor. The principal types of fermented beverages are beers' wines, and spirit

liquors. The earliest evidence ofbeer brewing appears in ancient tablets by the Sumerians and Babylonians around 6000 BC. The starting ingredients for both ancient and present-day versions ofbeer, ale,

Flgure

27.5

Female flowers

Hops. of hops, the herb that gives beer some of its flavor

and aroma.

stout, porter, and other variations are water, malt (barley graln), hops, and special strains of yeasts. The steps in brewing include malting, mashing, adding hops, fermenting, aging, and finishing' For brewer's yeast to convert the carbohydrates in grain into ethyl alcohol, the barley must first be sprouted and softened to make its complex nutrients available to yeasts. This process, called maltingr* releases amylases that convert starch to dextrins and maltose, and proteases that digest proteins. Other sugar and starch supplements added in some forms of beer ilre corn' rice, wheat, soybeans, potatoes, and sorghum. After the sprouts have been separated, the remaining malt grain is dried and stored in preparation for mashing. The malt grain is soaked in warm water and ground up to prepare a mash. Sugar and starch supplements are then introduced to the mash mixture, which is heated to a temperature of about 65oC to 70oC. During this step, the starch is hydrolyzed by amylase and simple sugars

are released. Heating this mixture to 75"C stops the activity of the enzymes. Solid particles are next removed by settling and filtering. Wort * the clear fluid that comes off, is rich in dissolved carbohydrates. It is boiled for about 2.5 hours with hopso the dried scales of the female flower of Humulus lupulus (figure 27.S\rto exfract the bitter acids and resins that give aroma and flavor to the finished product.'

Boiling also caramelizes the sugar and imparts a golden or brown color, desfoys any bacterial contaminants that can destoy flavoq and concentrates the mixture. The filtered and cooled supernatant is then ready for the addition of yeasts and fermentation. Fermentation begins when wort is inoculated with a species of Saccharomyces that has been specially developed for beer making. Top yeasts such as Saccharomyces cerevisiae function at the sur-

face and are used to produce the higher alcohol content of ales. Bottom yeasts such as S' uvarum (carlsbergensls) function deep in the fermentation vat and are used to make other beers. In both cases, the initial inoculum ofyeast starter is aerated briefly to promote rapid growth and increase the load of yeast cells. Shortly

* malting (mawlt'-ing) Gr. nalz, to soften + worr (wurt) O.E. uyrr, a sPice. 1. This substance, called humulus, provides some ratlter interesting side effects besides flavor. It is a moderate sedative, a diuretic, and a mild antiseptic.

814

Chapter

27

Applied and Industrial Microbiology

Grapes are harvested when their sugar content reaches l5o/o to 25o/o,

depending on the type of wine to be made. Grapes from the field

carry a mixed biofilm on their surface called the bloom that can serye as a source ofwild yeasts. Some winemakers allow these natu_ ral yeasts to dominate, but many wineries inoculate the must with a

special strain of Saccharomyces cerevisiae, variety ellipsoideus. To discourage yeast and bacterial spoilage agents, winemakers some_ times treat grapes with sulfur dioxide or potassium metabisulfite.

Flgure 27.6 Anaerobic conditions in homemade beer

production.

A layer of carbon dioxide foam keeps oxygen out.

thereafter, an insulating blanket offoam and carbon dioxide devel-

ops on the surface of the vat and promotes anaerobic conditions (figure 27.6).Durng 8 to 14 days of fermentation, the wort sugar is converted chiefly to ethanol and carbon dioxide. The diversity of flavors in the finished product is partly due to the release of small amounts of glycerol, acetic acid, and esters. Fermentation is selflimited, and it essentially ceases when a concentration of 3%i to 6yo ethyl alcohol is reached. Freshly fermented" or'ogreen," beer is lageredr* meaning it is held for several weeks to months in vats near 0oC. During this maturation period" yeast, proteins, resin, and other materials settle, leaving behind a clear, mellow fluid. Lager beer is subjected to a final filtration step to remove any residual yeasts that could spoil it. Finally, it is carbonated with carbon dioxide collected during fermentation and packaged in kegs, bottles, or cans.

Wine ond Liquors Wine is traditionally considered any alcoholic beverage arisrng from the fermentation of grape juice, but practically any fruit can be rendered into wine. The essential starting point is the preparation of must, the juice given off by crushed fruit that is used as a sub-

strate for fermentation. In general, grape wines are either white or red. The color comes from the skins of the grapes, so white wine is

prepared either from white-skinned grapes or from red-skinned grapes that have had the skin removed. Red wine comes from the red- or purple-skinned varieties. Major steps in making wine include mustpreparation (crushing), fermentation, storage, and aging

(frgtre27.7).

For proper fermentation, must should contain 12% to 25% ghtcose or fructose, so the art of wine making begins in the vineyard. * lagered (law' -gtxd) Gl laget; to

store or age.

The inoculated must is thoroughly aerated and mixed to promote rapid aerobic growth ofyeasts, but when the desired level ofyeast growth is achieved, anaerobic alcoholic fermentation is begun. The temperature of the vat during fermentation must be care_ fully controlled to facilitate alcohol production. The length of fer_ mentation varies from 3 to 5 days in red wines and from 7 to 14 davs in white wines. The initial fermentation yields ethanol concentrations reaching 7oh to l5Yo by volume, depending upon the type of yeast, the source ofthe juice, and ambient conditions. The fermented juice (raw wine) is decanted and transferred to large vats to settle and clarify. Before the final aging process, it is flash-pasteurized to kill microorganisms and filtered to remove any remaining yeasts and sediments. Wine is aged in wooden casks for varying time periods (months to years), after which it is bottled and stored for further as_ ing. During aging, nonmicrobial changes produce aromas and flivors (the bouquet) characteristic of a particular wine. The fermentation processes discussed thus far can only achieve a maximum alcoholic content of l7o/o, because concentrations above this level inhibit the metabolism of the yeast. The fermentation product must be distilled to obtain higher concentrations such as those found in liquors. During distillation, heating the liquor separates the more volatile alcohol from the less volatile aqueous phase. The alcohol is then condensed and collected. The alcohol content of distilled liquors is rated by proof a measurement that is usually two times the alcohol content. Thus, 80 proof vodka contains 40oh ethyl alcohol. Distilled liquors originate through a process similar to wine making, although the starting substrates can be extremely diverse. In addition to distillation, liquors can be subjected to special treat_ ments such as aging to provide unique flavor or color. Vodka, a colorless liquor, is usually prepared from fermented potatoes, and rum is distilled from fermented sugarcane. Assorted whiskeys are derived from fermented grain mashes; rye whiskey is produced from rye mash, and bourbon from corn mash. Brandy is distilled grape, peach, or apricot wine.

Other Fermented Plont Products Fermentation provides an effective way ofpreserving vegetables, as well as enhancing flavor with lactic add and salt. During pickling fermentations, vegetables are immersed in an anaerobic salty solution (brine) to extract sugar and nutrient-ladenjuices. The salt also disperses bacterial clumps, and its high osmotic pressure inhibits proteolytic bacteria and sporeformers that can spoil the product. Sauerkraut is the fermentation product ofcabbage. Cabbage is washed, wilted, shredded, salted, and packed tightly into a fermentation vat. Weights cover the cabbage mass and squeeze out its juices. The fermentation is achieved by natural cabbage microbiota or by an added culture. The initial agent of fermentation is Leuconostoc mesenteroides, which grows rapidly in the brine and produces

27.2

lactic acid.

It is followed by Lactobacillus

plantarum, which

continues to raise the acid content to as high as 2o/o (pH 3'5) by the end of fermentation. The high acid content restricts the growth of spoilage microbes.

'

FJrmented cucumber pickles come chiefly in salt and dill varieties. Salt pickles are prepared by washing immature cucumbers, placing them in barrels of brine, and allowing them to ferment for 6 to 9 weeks. The brine can be inoculated with Pediococcus cerevisiae and Lactobacillus plantarum to avoid unfavorable Outcome

Processing SteP

Formation of must with fruit sugars

Grape pressing

Microorganisms and Food

815

qualities caused by natural microbiota and to achieve a more consistent product. Fermented dill pickles are prepared in a somewhat more elaborate fashion, with the addition of dill herb, spices, garlic, onion, and vinegar. Natural vinegar is produced when the alcohol in fermented plantjuice is oxidized to acetic aci4 which is responsible for the p*g"nt odor and sour taste. Although a reasonable facsimile of urr"g* could be made by mixing about 4o/o acetic acid and a dash of sugar in water, this preparation would lack the traces of various esteri alcohol, glycerin, and volatile oils that give natural vinegar its pleasant character. Vinegar is actually produced in two stages' the first stage is similar to wine or beer making, in which a plant juice is fermented to alcohol by Saccharomyces' The second stage involves an aerobic fermentation carried out by acetic acid bacteria

inthegeneraAcetobacterandGluconobacterThesebacteriaoxidize the ethanol in a two-step process' as shown here:

* Ethanol

2C2H5OH

Heat sterilization

Elimination of contaminants

Yeast inoculation

Addition of desired organisms

tfzO2-+ CH3CHO + H2O AcetaldehYde

* tfzO2+ CH3COOH AcetaldehYde Acetic acid CH3CHO

The abundance of oxygen necessary in commercial vinegar making is furnished by exposing inoculated raw material to air by arranging

it in thin layers in open trays,

allowing it to trickle over loosely

packed beechwood twigs and shavings, or aerating it in a large vat. biff"r"rrt types of vinegar are derived from substrates such as apple juice cider (cider vinegar), malted grains (malt vinegar), and grape Alcohol production from sugars

Fermentation of must

(wine vinegar).

Tank

t Development of tinal wine bouquet

Storage in barrels to age

Barrel

3 r$ I

Y

Filtration and collection

Bottling

(a)

Flgure 27.7 Wine making.

Removal of yeast and particles

winery. (a) General stePs in wine making. (b) wine fermentation vats in a large commercial

816

Chapter

27

Applied and Industrial Microbiology

the milk proteins to coagurate into a solid mass cailed the curd. curdling also causes the separation of a watery liquid called whey on the surface. Curd can be produced by microbial action or by rennin (casein coagulase), which is isolated from the stomach oiunweaned calves.

^iru.*,,

Cheese Since 5000 BC, various forms of cheese have been produced by spontaneous fermentation of cow, goat, or sheep milk. present_day, large-scale cheese production is carefully controlled and uses only freeze-dried samples of pure curtures. These are first inoculatei into a small quantity of pasteurized milk to form an active starter

culture. This amplified culture is subsequently inocurated into a large vat of milk, where rapid curd devel_ Desirable opment takes place. Such rapid growth is Undesirable desired because it promotes the overgrowth o Streptococcus E Pseudomonas of the desired inoculum and prevents the o lactis Lactobacilli Yeasts tr sporeformers, etc. _ pH7 6 activities of undesirable contaminants. 9: €* 6-Rennin is usually added to increase the rate iir I of curd formation. .=tl =G II After its separation from whey, the oc I pH S curd is rendered to produce one of the 20 o 3t major types of soft, semisoft, or hard E Lactic acid f cheese (figure 27.9).The composition of z cheese is varied by adjusting water, fat, acid, and salt content. Cottage and cream pH 3 Time i cheese are examples of the soft, more per_ (b) ishable variety. After light salting and the optional addition of cream, they are ready Flgure Microbes at work in milk products. for consumption without further process(a) Litmus milk is a medium used to indicate pH and consistency changes in milk resulting from ing. Other cheeses acquire their character microbial action. The first tube is an uninoculated, unchangeO iontrol.-tne second tube has a from "ripening," a complex curing process white, decolorized zone indicative of litmus reduction. The third tube has become acidified (pink), and its proteins have formed a loose curd. In the fourth involving bacterial, mold, and enzyme tube, digestion of milk proteins

=tr

27.8

has caused complete clarification or peptonization of the milk. The fifttitube shows a welldeveloped solid curd overlaid by a clear fluid, the whey. (b) chart depicting spontaneous changes in the number and type of microorganism and the pH of raw milkis it incubates. Source chart from Philip L. carpenter, Microbiologla 4/E, copyright @ 1977. Reprinted by permission of Brook/Cole, a division of Thomson Learning: wrfr.thornpsonrights.com.

Microbes in Mirk and Dairy

products

reactions that develop the

final flavor, of

aroma, and other features characteristic

particular cheeses.

The distinctive traits of soft cheeses such as Limburger, Camembert, and Liederkranz are acquired by ripening with

il:T*;Htr,;;;:',t:H:??"",i:"#:#J[i,],?",lffiLfiit: teins, carbohydrates, and other substrates. This process leaves assorted acids and other by-products that give the finished cheese powerful aromas and delicate flavors. Semisoft varieties of cheese such as Roquefort, bleu, or Gorgonzola are infused and aged with a

Milk has a highly nutritious composition. It contains an abundance of water and is rich in minerals, protein (chiefly casein)" butterfat, sugar (especially lactose), and vitamins. tt'starts iis journey in the udder of a mammal as a sterile substance, but as itpasses out of the teat, it is inoculated by the animal,s nor-ut strainof Penicilliumroquefortimold. Hardcheeses such-as Swiss, cheddar, and Parmesan develop a sharper flavor by aging with sebiota. other microbes can be introducei by milking il;ii;. lected bacteria. The pockets in Swiss iheese come from entrapped Because milk is a nearly perfect culture medium, it i. ;ishb;rcarbon dioxide formed by Propionibacterium, which is also ceptible to microbial growth. When raw milk is fet r."o- t"-"t produce responsible for its bittersweet taste. perature, a series of bacteria ferment the lactose, acid, and alter the milk's content and texture (figure ZZ.Sa). fhis progression can occur naturally, or it can be iiduce4 Other Fermented Milk Products

production of cheese and yogurt' "rilri; Yogurt is formed by the fermentation of milk by Lactobacillus butIn the initial stages of milk fermentation, lactose is.rapidly aG garicus and streptococcus thermophilus. Theseorganisms produce tacked by streptococcus lactis and Lactobacil/zs species (figure organic acids and other flavor components and can grow in such 27'8b)' The resultant lactic acid accumulation and lowered prl cause numbers that a gram of yogurt regularly contains 100 million

27,2

Microorganisms and Food

817

Microbial Involvement in Food-Borne Diseases Diseases caused by ingesting food are usually referred to as food poisoning, and although this term is often used synonymously with microbial food-borne illness, not all food poisoning is caused by microbes or their products. Several illnesses are caused by poisonous plant and animal tissues or by ingesting food contaminated by pesticides or other poisonous substances. Table 27.1 summarizes the major types ofbacterial food-borne disease.

Food poisoning of microbial origin can be divided into two general categories2 (figure 27.L}).Food intoxication results from

the ingestion of exotoxin secreted by bacterial cells growing in food. The absorbed toxin disrupts a particular target such as the

Flgure 27.9

Cheese making.

The curd-cutting stage in the making of cheddar cheese.

bacteria. Live cultures of Lactobacillus acidophilus ate an important additive to acidophilus milk, which is said to benefit digestion and to help maintain the normal biota of the intestine. Fermented milks such as kefir, koumiss, and buttermilk are a basic food source in many cultures.

Microorganisms as Food algae, and yeasts may macroscopic relatives their eat We do seem odd or even unappetizing. we are used to thinkbut seaweed, such as mushtooms, truffles, and and disease or, at decay of agents ing of the microscopic forms as

At first, the thought of eating bacteria, molds,

most, as food flavorings. The consumption of microorganisms is not a new concept. In Germany during World War II, it became necessary to supplement the diets of undernourished citizens by adding yeasts and molds to foods. At present, most countries are able to produce enough food for their inhabitants, but in the future, countries with exploding human populations and dwindling arable land may need to consider microbes as a significant source of protein, fat, and vitamins. Several countries already commercially mass-produce food yeasts; bacteria; an4 in a few cases, algae. Although eating microbes has yet to win total public acceptance, their use as feed supplements for livestock is increasing. A technology that shows some promise in increasing world food productivity is single-cell protein (SCP)- This material is produced from waste materials such as molasses from sugar refining, petroleum by-products, and agricultural wastes. In England, an animal feed called pruteen is produced by mass culture

of the bacteriwn Methylophilus methylotrophus. Mycoprotein,

a

product made from the fungus Fusarium gruminearum, is also sold there. The filamentous texture of this product makes it a likely candidate for producing meat substitutes for human consumption. Health food stores carry bottles of dark green pellets or powder

that are a culture of a spiral-shaped cyanobacterium called Spirutina. This microbe is harvested from the surface of lakes and ponds, where

it grows in great mats' In some parts of Africa

and

Mexico, Spirulina has become a viable alternative to green plants as a primary nutrient source. It can be eaten in its natural form or added to other foods and beverages.

intestine (ifan enterotoxin) or the nervous system (ifa neurotoxin). The symptoms of intoxication vary from bouts of vomiting and diarrhea (staphylococcal intoxication) to severely disrupted muscle function (botulism). In contrast, food infection is associated with the ingestion of whole, intact microbial cells that target the intestine. In some cases, they infect the surface of the intestine, and in

others, they invade the intestine and other body strucfures' Most food infections manifest some degree of diarrhea and abdominal distress (see Insight 20.2). lt is important to realize that disease symptoms in food infection can be initiated by toxins but that the toxins are released by microbes growing in the infected tissue rather than in the food. Reports of food poisoning are escalating in the United States and worldwide. Outbreaks attributed to common pathogens (Salmonella, E. coli, Vibrio, hepatitis A, and various protozoa) have doubled in the past 25 years. The CDC estimates that near$ onefourth of the population suffers each year from some form of food poisoning. A major factor in this changing pattern is the mass production and distribution ofprocessed food such as raw vegetables, fruits, and meats. Improper handling can lead to gross contamination of these products with soil or animal wastes. Estimated Incidence of Food-Borne lllness in the United States* Illnesses

76,000,000 cases 323,000 cases

Hospitalizations

5,200 cases

Deaths *One-third ofall reported

cases result

from eating restaurant food.

Many reported food poisoning outbreaks occur where contaminated food has been served to large groups of people, but most cases probably occur in the home and are not reported. On occasion, even commercially prepared foods can be a source of infec-

tions (Insight27.l). Data collected by food microbiologists indicate that the most common bacterial food-borne pathogens ate Campylobacter Salmonella, Shigella, Clostridium, and Staphylococcus aureus. The dominant protozoa causing food infections are Giardia, Cryptosporidium, and Toxoplasma. The top viruses are Norwalk and hepatitis A viruses. These microbes and their diseases were previously discussed in chapters 18, 19, 20,21,23 and25. these categories are useful for clarifying fie general forms offood poisoning, few diseases fall in between. For example, perfringens intoxication is caused by a toxin, but the toxin is released in the intestinal lumen rather than in the food.

2. Although a

Chapter

818

27

Applied and Industrial Microbiology

Baby Food and Meningitis It will come as no surprise to you that there are potentially dangerous bacteria in a wide variety of

are sterile. The scientists who conducted the infant food study also investigated ideal conditions

foods we consume. Cases of E coli Olli.H7 disease associated with hamburgers and hepatitis contracted by customers of an upscale restaurant get a lot of media attention. It may surprise you to leam that pathogenic bacteria have been found in

for preparing and storing the products.

They

noted that the bacterial doubling time in prepared

formula is 10 hours when refrigerated, and 30 minutes at room temperature. This means that leaving prepared formula in your diaper bag or

dried infant formula and dried baby food, as we1l.

on the kitchen counter for even a few hours could lead to high levels ofbacteria in the bottle.

One such outbreak occurred in a neonatal intensive care unit in Tennessee. It was traced to a batch of powdered formula that had to be recalled after the Centers for Disease Control and prevention issued a warning. In 2004, scientists in England investigated 1 l0 different tlpes ofbaby foods. Ten percent of the powdered formula samples and25yo of the dried infant

foods contained intestinal bacteria. In many ofthe samples they found a bacterium called Enterobacter sakazakir, an intestinal bacterium that has been linked to several fatal outbreaks ofmeningitis in children,s hospitals. The disease is rare but almost always associated with infant foods and, has a 3 3oh fatality rate, with up to 8 0% of infants suffering permanent neurological damage. So what is to be done? In this case, it is ,.consumer beware." Manufacturers have never claimed that their formulas and foods

Powdered formula is made by manufacturing the nutritious liquid and then freeze-drying it. It is sterile as a liquid but bacteria can be introduced during the freezedrying and packaging phases. Since the outbreaks, the FDA has recom_ mended that the powder be reconstituted with boiling water. The CDC has not supported this recommendation because of many problems with it including the risk ofdestroying important nutrients and the concern that boiling would not be sufficient to kill E. sakazakii. Hospitals are advised to use ready-to-feed or concentrated Iiquid formulas.

Propose several ways that potential pathogens can enter processed Answer available at http://www.mhhe.com/talaroT

foods.

Maior Forms of Bacterial Food poisoning Disease

Microbe

Foods Involved

Comments

Food Intoxications: caused by Ingestion of Foods containing preformed roxins Staphylococcal enteritis

Staphylococcusaureus Custards,cream-filledpastries,ham, oresslngs

Botulism

Clostridium botulinum

Perfringens

enterotoxemia

Clostridium

perfringens

Very common; symptoms come on rapidly; usually nonfatal

Home-canned or poorly preserved low-acid foods

Recent cases involved vacuum-packed foods; can be fatal

Inadequately cooked meats

Vegetative cells produce toxin within the

intestine B ac i I lus

c e reu

s enterilis

Bacillus cereus

Reheated rice, potatoes, puddings, custards

Food Infections: caused by Ingestion of Live Microbes That lnvade the lntestine Campylobacter jejuni Raw milk; raw chicken, shellfish

Campylobacteriosis

and meats

Salmonellosis

Salmonella and S.

Shigellosis

typhimurium

species

species

Very common; can be severe and life-threatening

Unsanitary cooked food; fish, shrimp,

Carriers and flies contaminate food; the cause of bacillary dysentery

potatoes, salads

parahaemolyticus

Very common; animals are carriers of other

Poultry eggs, dairy products, meats

enteriditis

Various Shigella

Mimics staphylococcal enteritis; usually self-limited

Vibrio ententis

Vibrio

Listeriosis

Listeria

Escherichia enteritis

Escherichia coli

Contaminated raw vegetables, cheese

Various strains can produce infantile and traveler's diarrhea.

E. coli O157 H7

Raw or rare beef, vegetables, water

The cause of hemolytic uremic syndrome (see chapter 20)

monocytogenes

Raw or poorly cooked seafoods

Microbe lives naturally on marine animals

Poorly pasteurized milk, cheeses

Most severe in fefuses, newborns, and the

immunodeficient

27.2

and bread. As the procedures become more widespread, microbiol-

Food lnfection

Food Intoxication

819

Microorganisms and Food

ogy-trained HACCP coordinators will be in high demand' Some 2005 data show that some food-borne illnesses have begun to decline, due in part to implementation of HACCP procedures'

CASE F:|LE

(4 tb

27

Wrop-Up

The food industry is increasingly global. Contaminated food can be rapidly shipped around the country and world and eaten before its risk has been determined. A rapid, real-time system

.t#

will favor tracing food-borne disease and allow authorities to stay on top of outbreaks and stop them before they spread'

PulseNet laboratories all around the world can compare PFGE patterns they obtain from patients or suspected foods to patterns in the centralized database. In this way, outbreaks that are geographically dispersed can be identified quickly' When new patterns come in, they are also archived, so that other laboratories submitting the same patterns will quickly realize

,f Ingestion

*l*

&B

&

W

lntestinal infection

(b)

Figure 27.1O Food-borne

that the cases are related. This pathogen is a psychrotroph that grows readily at refrigerator temperatures, so that even if the hot dogs had low numbers at the time of processing, the pathogen would continue to grow to a level that it could infect. Listerio monocytogenes is opportunistic and can invade the gastrointestinal epithelium of compromised patients. From here, it may cross into the circulation and invade the brain and spinal column. Pregnant women are particularly susceptible to infection, and they transmit the infection across the placenta to the fetus. See: CDC, "Public Health Dispatch: Outbreok of Listeriosis-N ortheastern united States, 2002." Morbidity and Mortality Weekly Report 57 (2002): 9s0-s|.

illnesses of microbial origin.

(a) Food intoxication. The toxin is released by microbes growing in the food. After the toxin is ingested, it acts upon its target tissue and causes symptoms. (b) Food infection' The infectious agent comes from food or is introduced into it through poor food processing and storage. After the cells are ingested, they invade the intestine and cause symptoms of gastroenteritis.

Prevention Measures for Food Poisoning and SPoilage Growing concerns about food safety led to a new approach to regulating the food industry. The system is called HazardAnalysis and Critical Control Point, or HACCP' and it is adapted from procedures crafted for the space program in the 1970s' It involves principles that are more systematic and scientific than previous random-sampling quality procedures. The program focuses on the identification, evaluation, control, and prevention ofhazards at all stages of the food production process. Since 1998, HACCP has been phased in by the U.S. Department ofAgriculture for meat and poultry processing plants and by the Food and Drug Administration for seafood and juice plants. Pilot HACCP projects are taking place in facilities that process cheese, breakfast cereals, salad dressings,

It will never be possible to avoid all types of food-borne illness because of the ubiquity of microbes in air, wateq food and the human body. But most types of food poisoning require the growth of microbes in the food. In the case of food infections, an infectious dose (sufficient cells to initiate infection) must be present, and in food intoxication, enough cells to produce the toxin must be present' Thus, food poisoning or spoilage can be prevented by proper food handling, preparation, and storage. The methods shown in figure 27.11 are aimed at preventing the incorporation of microbes into food removing or destroying microbes in food, and keeping microbes from multiplying.

Preventi ng the I ncorPorotion of Microbes into Food Most agricultural products such as fruits, vegetables, grains, meats, eggs, and milk are naturally exposed to microbes. Vigorous washing reduces the levels of contaminants in fruits and vegetables, whereas meat, eggs, and milk must be taken from their animal source as aseptically as possible. Aseptic techniques are also essential in the kitchen. Contamination of foods by fingers can be easily remedied by hand washing and proper hygiene, and contamination

820

Chapter

27

Applied and Industrial Microbiology

Care in Harvesting, Preparation

by flies or other insects can be stopped by covering foods or eliminating pests from the kitchen. Care and common sense also apply in

managing utensils. It is important to avoid cross-contaminating food by using the same cutting board for meat and vegetables without disinfecting it between uses.

Preventing the Survival or Multiplication of Microbes in Food Because it is not possible to eliminate all microbes from certain types of food by clean techniques alone, amore efficient approach is to preserve the food by physical or chemical methods. Hygieni_ cally preserving foods is especially important for large commercial companies that process and sell bulk foods and must ensure that

products are free from harmful contaminants. Regulations and Destruction of Microbes Heat

Canning

Pasteurization

Cooking

dq-.'

w ffi=

EN

I-

GW

Radiation

Filtration

W Prevention of Growth Maintenance temperature

'")(** Preservative additives

W..@*

fi*{^.-p

Flgure 27,11

standards for food processing are administered by two federal agen_ cies: the Food and Drug Administration (FDA) and the U.S. Depart_ ment of Agriculture (USDA).

The primary methods to prevent food poisoning and food spoilage.

Temperoture ond Food Preservotion Heat is a common way to destroy microbial contaminants or to reduce the load of microorganisms. Commercial canneries pre_ serve food in hermetically sealed containers that have been ex_ posed to high temperatures over a specified time period. The temperature used depends upon the type offood, and it can range from 60"C to l2l"C, with exposure times ranging from 20 min_ utes to 115 minutes. The food is usually processed at a thermal death time (TDT; see chapter I l) that will destroy the main spoilage organisms and pathogens but will not alter the nutrient value or flavor of the food. For example, tomato juice must be heated to between l2l"C and 132"C for 20 minutes to ensure destruc_ tion of the spoilage agent Bacillus coagulans. Most canning methods are rigorous enough to sterilize the food completely, but some only render the food "commercially sterile,,, which means it contains live bacteria that arc unable to grow under normal

conditions ofstorage. Another use of heat is pasteurization, usually defined as the application ofheat below 100"c to destroy nonresistant bacteria and yeasts in liquids such as milk, wine, and fruit juices. The heat is applied in the form of steam, hot water, or even electrical cur_ rent. The most prevalent technology isthe high+emperature short_ time (HTSI), or flash method, using extensive networks of tubes that expose the liquid to 72oC for 15 seconds (figure 27.12), An alternative method" ultrahigh-temperature (UHT) pasteuriza_ tion, steams the product until it reaches a temperature of 134"C for at least I second. Although milk processed this way is not ac-

tually sterile, it is often marketed as sterile, with a shelf life of up to 3 months;tOlder methods involve large bulk tanks that hold the fluid at a lower temperature for a longer time, usually 62.3"C for 30 minutes.

Cooking temperatures used to boil, roast, or fry foods can render them free or relatively free of living microbes if carried out for sufficient time to destroy any potential pathogens. A quick warming of chicken or an egg is inadequate to kill microbes such as

Salmonella. In fact, any meat is a potential source of infectious agents and should be adequately cooked. Because most meat_ associated food poisoning is caused by nonsporulating bacteria,

27.2

Flgure 27.12 A modern flash pasteurizeq

Microorganisms and Food

821

a system

used in dairies for high-temperature short-time (HTST)

pasteurization. Source: Photo taken at Alta Dena Dairy, City of Industry, California.

heating the center of meat to at least 80"C and holding it there for 30 minutes is usually sufficient to kill pathogens. Roasting or frying food at temperatures of at least 200oC or boiling it will achieve a satisfactory degree of disinfection. Any perishable raw or cooked food that could serve as a growth medium must be stored to prevent the multiplication ofbacteria that have survived during processing or handling. Because most foodborne bacteria and molds that are agents of spoilage or infection can multiply at room temperature, manipulation of the holding temperature is a useful preservation method (figure 27.13). A good general directive is to store foods at temperatures below 4oC or

fgure 27.1t

Temperatures favoring and inhibiting

th-e growth of microbes in food. Most microbial agents of disease or spoilage grow in the temperature range of 15'C to 40oC. Preventing unwanted growth in foods in longterm storage is best achieved by refrigeration or freezing (4"C or lower). Preventing microbial growth in foods intended to be consumed warm in a few minutes or hours requires maintaining the foods above 60'C. Source: From Ronald Atlas, Microbiology: Fundomentols and Applicotions, 2nd ed', O 1988, p. 475. Electronically reproduced by permission of Pearson Education, Inc., Upper Saddle River, New lersey.

above 60oC.

Regular refrigeration reduces the growth rate of most mesophilic bacteria by 10 times, although some psychrotrophic microbes can continue to grow at arate that causes spoilage. This factor limits the shelf life of milk, because even at 1oC, a population could go from a few cells to a billion in l0 days. Pathogens such as Listeria monocytogenes and Sqlmonella can also continue to grow in refrigerated foods. Freezing is a longer-term method for cold preservation. Foods can be either slow-frozenfor 3 to72

-15"C to -23"C or rapidly frozen for 30 minutes at -17"C to -34"C. Because freezing cannot be counted upon to

hours at

kill microbes, rancid" spoiled, or infectious foods will still be unfit to eat after freezing and defrosting; Salmonella is known to survive several months in frozen chieken and ice cream' and Vibrio parahaemolyticus can survive in frozen shellfish. For this reason' frozen foods should be defrosted rapidly and immediately cooked or reheated. However, even this practice will not prevent staphylococcal intoxication ifthe toxin is already present in the food before it is heated. Foods such as soups, stews, gravies, meats, and vegetables that are generally eaten hot should not be maintained at warm or room

temperatures, especially in settings such as cafeterias, banquets, and picnics. The use ofa hot plate, chafing dish, or hot water bath will maintain foods above 60oC, well above the incubation tempemture of food-poisoning agents. ' As a final note about methods to prevent food poisoning, ooWhen in doubt, throw it out." remember the simple axiom:

Rodiation Ultraviolet (nonionizing) lamps are commonly used to destroy microbes on the surfaces of foods or utensils, but they do not penetrate far enough to sterilize bulky foods or food in packages.

Food preparation areas are often equipped with UV radiation devices that are used to destroy spores on the surfaces ofcheese, breads, and cakes and to disinfect packaging machines and storage areas. Food itself is usually sterilized by giilnma or cathode radiation because these ionizing rays can penetrate denser materials' It must also be emphasized that this method does not cause the targets of irradiation to become radioactive.

822

Chapter

27

Applied and Industrial Microbiology

Concerns have been raised about the possible secondary effects ofradiation that could alter the safety and edibility offoods. Experiments over the past 30 years have demonstrated some side reactions that affect flavor, odor, and vitamin content, but it is currently thought that irradiated foods are relatively free oftoxic byproducts. The government has currently approved the use of radiation in sterilizing beef, pork, poultry, fish, spices, grain, and some fruits and vegetables. Less than l0% of these products are sterilized this way, but outbreaks offood-borne illness have increased its desirability for companies and consumers. It also increases the shelf life of perishable foods, thus lowering their cost.

s e

Organic acids, including lactic, benzoic, and propionic acids, are among the most widely used preservatives. They are added to baked goods, cheeses, pickles, carbonated beverages, jams, jellies, and dried fruits to reduce spoilage from molds and some bacteria. Nitrites and nitrates are used primarily to maintain the red color of

cured meats (hams, bacon, and sausage). By inhibiting the germination of Clostridium botulinum spores, they also prevent botulism intoxication, but their effects against other microorganisms are limited. Sulfite prevents the growth of undesirable molds in dried fruits, juices, and wines and retards discoloration in various foodstuffs. Ethylene and propylene oxide gases disinfect various dried foodstuffs. Their ussis restricted to fruit, cereals, spices, nuts, and cocoa. The high osmotic pressure contributed by hypertonic levels

of

salt plasmolyzes bacteria and fungi and removes moisture from food thereby inhibiting microbial growth. Salt is commonly added to brines, pickled foods, meats, and fish. However, it does not retard

the growth of pathogenic halophiles such as Staphylococcus errreus, which grows readily even in 7.5oh salt solutions. The high sugar concentrations ofcandies,jellies, and canned fruits also exert an osmotic preservative effect. Other chemical additives that function in preservation are alcohols and antibiotics. Alcohol is added to flavoring extracts, and antibiotics are approved for treating the carcasses ofchickens, fish, and shrimp.

Food can also be preserved by desiccation, a process that removes moisture needed by microbes for growth by exposing the food to dry warm air. Solar drying was traditionally used for fruits and vegetables, but modern commercial dehydration is carried out in rapid-evaporation mechanical devices. Drying is not a reliable microbicidal method, however. Numerous resistant microbes such as micrococci, coliforms, staphylococci, salmonellae, and fungi survive in dried milk and eggs, which can subsequently serve as agents ofspoilage and infections. In 2006, the Food and Drug Administration approved the spraying ofbacteriophages onto ready-to-eat meat products. The bacteriophages are specific for Listeria and will kill this pathogen in cold cuts and poultry that are usually not cooked before consumption.

Wastewater or sewage is treated in tlrree stages to remove organic material, microorganisms, and chemical pollutants. The primary phase removes physical objects from the wastewater. The seiondary phase removes the organic matter by biodegradation. The tertiary phase dis-

infects the water and removes chemical pollutants. Microorganisms can compete with humans for the nutrients in food. = Their presence in food can be beneficial, detrimental, or ofneutral consequence to human consumers. ffi Food fermentation processes utilize bacteria or yeast to produce desired components such as alcohols and organic acids in foods and beverages. Beer, wine, yogurt, and cheeses are examples of

Other Forms of Preservotion The addition of chemical preservatives to many foods can prevent the growth of microorganisms that could cause spoilage or disease. Preservatives include natural chemicals such as salt (NaCl) or table sugar and artificial substances such as ethylene oxide. The main classes of preservatives are organic acids, nitrogen salts, sulfur compounds, oxides, salt, and sugar.

The use of microorganisms for practical purposes to benefit humans is called biotechnology.

such processes.

= +$

€

.

Some microorganisms are used as a source of protein. Examples are single-cell protein, mycoprotein, and Spirulina. Microbial

protein could replace meat as a major protein source. Food-borne disease can be an intoxication caused by microbial toxins produced as by-products ofmicrobial decomposition offood. Or it can be a food infection when pathogenic microorganisms in the food attack the human host after being consumed. Heat, radiation, chemicals, and drying are methods used to limit numbers of microorganisms in food. The type of method used depends on the nature of the food and the type of pathogens or agenK it contains.

..=lllihg"

27.3 General Concepts in Industrial Microbiology Never underestimate the power of the microbe.

W

Foster,

pioneering -Jackson microbiologist in the development of antibiotics

Virtually any large-scale commercial enterprise that enlists microorganisms to manufacture consumable materials is part of the realm of industrial microbiology. Here the term pertains primarily to bulk production of organic compounds such as antibiotics, hormones, vitamins, acids, solvents (table 27.2), and enzymes (table 27.3). Many of the processing steps involve fermentations similar to those described in food technology, but industrial processes usually occur on a much larger scale, produce a specific compound, and involve numerous complex stages. The aim of industrial microbiology is to produce chemicals that can be purified and packaged for sale or for use in other commercial processes. Thousands of tons of organic chemicals worth several billion dollars are produced by this industry every year. To create just one of these products, an industry must determine which microbes, starting compounds, and growth conditions work best. The research and development involved require an investment l0 to 15 years and billions ofdollars.

of

The microbes used by fermentation industries are mutant strains of fungi or bacteria that selectively synthesize large amounts of various metabolic intermediates, or metabolites. Two basic kinds ofmetabolic products are harvested by industrial processes: (1) Primary metabolites are produced during the major metabolic

27.3

ffiffiffiiljffiG

I

nd

u

striaf prod u cts of

M

icroorsan

823

Ceneral Concepts in Industrial Microbiology

is m s

Microbial Source

Substrate

Applications

Cephalosporins

cepnalosportum

Glucose

Pencillins

Penicill ium chry s o ge num

Lactose

Antibacterial antibiotic, broad spectrum Antibacterial antibiotics, broad and narrow spectrum

Vitamin 812

Pseudomonas

Molasses

Dietary supPlement

Steroids

Rh izopus, C unn i nghamel I a

Deoxycholic acid stigmasterol

Treatment of inflammation, allergY; hormone rePlacement theraPY

Acidifier in soft drinks; used to

Chemical Pharmaceuticals

(hydrocortisone)

Food Additives and Amino Acids

set

jam;

Citric acid

Aspergillus. Cundida

Molasses

Xanthan

Xanthomonas

Glucose medium

Food stabilizer; not digested by humans

Acetlc acrd

Acetobacter

Any ethylene source, ethanol

Food acidifer; used in industrial processes

Ethanol

Saccharomyces

Beet, cane, grains, wood" wastes

Acetone

Clostridium

Molasses, starch

Additive to gasoline (gasohol) Solvent for lacquers, resins. rubber, lal. oll

Glycerol

Yeast

By-product ofalcohol fermentation

Explosive (nitroglycerine)

Dextran

Kl e b s i e I I a, Ace tob ac t er,

Glucose, molasses, sucrose

Polymer ofglucose used as adsorbents, blood expanders, and in burn treatment; a plasma extender; used to stabilize ice cream, sugary syrup'

candy additive; fish Preservative; retards discoloration of crabmeat: delays browning of sliced peaches

Miscellaneous

Leuconostoc

candies

ffi

Industrial Enzymes and rheir'uses

Enlyme

Source

Application

Amylase

Aspergillus, Bacillus, RhizoPus

Flour supplement, desizing textiles, mash preparation, synrp manufacture, digestive ai{ precooked foods, spot remover in dry cleaning

Cellulase

As pergil

Hyaluronidase

Various bacteria

Medical use in wound cleansing, preventing surgical adhesions

Keratinase

Streptomyces

Hair removal ffom hides in leather preparation

Pectinase

A sp e rgillus, S cl ero

Proteases

A spergi

Rennet

Mucor

To curdle

Streptokinase

Streptocoecus

Medical use in clot digestion. as a blood thinner

I

us, Trichode

! lus,

rma

tina

B ac i I lus,

Streptomyces

Denim finishing ("stone-washing"). digestive aid. increase digestibility of animal feed degradation of wood or wood by-products

Clarifies wine, vinegar, syrups, and fruitjuices by degrading pectin, a gelatinous substance; used in concentrating coffee To clear and flavor rice wines, process animal feed. remove gelatin from photographic film, recover silver. tenderize meat' unravel silkworm cocoon, remove spots

milk in

cheese making

824

Chapter

27

Applied and Industrial Microbiology

I

()o .9 .o .g

b o

,a

E

z5 ct)

o

Aa-pregnene- 1 1 B,21diol-3,20-dione

Time

t\

Flgur_e 27.14 The origins of primary and secondary microbial metabolites harvested- by industrial processes.

H2?oH

:o :'OH

H2COH

Cortisone

C:O

pathways and are essential to the microbe's function. (2) Second_ ary metabolites are by-products of metabolism that may not be critical to the microbe's function (figure 27.14).In general, pri_ mary products are compounds such as amino acids and organic

acids synthesized during the logarithmic phase of microbial growth; and secondary products are compounds such as vitamins, antibiotics, and steroids synthesized during the stationary phase (see chapter 7). Most strains of industrial microorganism. hurre been chosen for their high production of a particular primary or secondary metabolite. certain mutated strains of yeasts and bacte-

ria can produce 20,000 times more metabolite than a wild strain that same microbe.

of

Industrial microbiologists have several hicks to increase the

amount of the chosen end product. First, they can manipulate the

growth environment to increase the synthesis of a metabolite. For instance, adding lactose instead of glucose as the fermentation substrate increases the production of penicillinby penicittium. Another sfrategy is to select microbial shains that genetically lack a feedback system to regulate the formation ofend products, thus enco'raging mass accumulation of this producl Many syntheses occur in

sequential fashion, wherein the waste products of one organism become the building blocks of the next. During these biotransformations, the substrate undergoes a series of slight modificatilns, each ofwhich gives off a different by-product (figure 27.15). The

production of an antibiotic such as tetracycline requires several microorganisms and 72 separate metabolic steps.

From Microbial Factories to Industrial Factories Industrial fermentations begin with microbial cells acting as living factories. When exposed to optimum conditions, they multiply ii massive numbers and syrthesize large volumes ofa desiredproduct.

/,i\v(9

1l-detrydrocortisone

Flgure 27.15 An example of biotransformation by

microorganisms in the industrial production of steroid

hormones.

Desired hormones such as cortisone and progesterone can require several steps and microbes, and rarely can a single microbe cairy out all the required synthetic steps. (a) Beginning with starting

compound Aa-pregnene-3,20-dione (blue). (b) li -dehydrocor-tironu (orange) is synthesized in three stages by two species of mold. (c) Cortisone (yellow) is synthesized in two stages by a pair of molds. (d) 1Sa-hydroxyprogesterone (green) is synthesized in a single step by a pair of molds.

Producing appropriate levels of growth and fermentation requires cultivation of the microbes in a carefully conholled environment. This process is basically similar to culturing bacteria in a test tube

of nutrient broth. It requires a sterile medium containing appropri_ ate nutrients, protection from contamination, provisions roi i"Lo-

duction

of sterile air or total exclusion of air, and a suitable

temperature and pH (figure 27.16).

Many commercial fermentation processes have been worked out on a small scale in a lab and then scqled up to a large com_ mercial venture. An essential component for scaling up is a fermentor, a device in which mass cultures are grown, reactions take place, and product develops. Some fermentors are large tubes, flasks, or vats, but most industrial types are metal cylindeis with built-in mechanisms for stirring, cooling, monitoring, and harvesting product (figure 27.17). Fermentors are made of

27.3

General Concepts in lndustrial Microbiology

825

materials Ihat can withstand pressure and ate rustproof, nontoxic, and leakproof. They range in holding capacity from small, 5-gallon systems used in research labs to larger, 5,000- to 100,000-gallon vessels and, in some industries, to tanks of 250

million to 500 million gallons. For optimum yield, a fermentor must duplicate the actions occurring in a tiny volume (a test tube) on a massive scale. Most microbes performing fermentations have an aerobic metabolism, and the large volumes make it difficult to provide adequate oxygen' Fermentors have a built-in device called a sparger that aerates the medium to promote aerobic grol /th. Paddles (impellers) located in the central part of the fermentor increase the contact between the

microbe and the nutrients by vigorously stirring the fermentation mixture. Their action also maintains its uniformity.

Substance Production Figure 27.76 A cell culture

vessel used

to mass-produce

pharmaceuticals. Such elaborate systems require the highest levels of sterility and clean

techniques.

The general steps in mass production of organic substances in a fermentor are illustrated in figure 27.18. These can be summarized as

inhoduction of microbes and sterile media into the reaction

)

chamber;

fermentation;

3. downstream processing (recovery purification, and packaging ofproduct); and 4. removal of waste.

All

lmpellers

Cooling

iacket

phases of production must be carried out aseptically and moni-

tored (usually by computer) for rate of flow and quality of product. The starting raw substrates include crude plant residues, molasses, sugars, fish and meat meals, and whey. Additional chemicals can be added to control pH or to increase the yield. In batch fermentation, the substrate is added to the system all at once and taken through a limited run until product is harvested. In continuous feed systems, nutrients are continuously fed into the reactor and the product is siphoned offthroughout the run. Ports in the fermentor allow the raw product and waste materials to be recovered from the reactor chamber when fermentation is complete. The raw product is recovered by settling, precipitation, centrifugation, filtration, or cell lysis. Some products come from this process ready to package, whereas others require further purification. extraction, concentration, or drying. The end product is usually in a powder, cake, granular, or liquid form that is placed in

Such instruments are equipped to add nutrients and cultures; to remove product under sterile or aseptic conditions; and to aerate,

sterilized containers. The waste products can be siphoned offto be used in other processes or discarded, and the residual microbes and nutrients from the fermentation chamber can be recycled back into the system or removed for the next run' Fermentation technology for large-scale cultivation of microbes and production of microbial products is versatile' Table 27 .2 itemizes some of the major pharmaceutical substances, food additives, and solvents produced by microorganisms. Some newer technologies employ extremophiles and their enzymes to run the processes at high or low temperatures or in high-salt conditions. Hyperthermophiles have been adapted for high-temperature detergent and enzyme production. Psychrophiles are used for cold processing of reagents for molecular biology and medical tests. Halophiles are effective for processing ofsalted foods and dietary

stir, and cool the mixture automatically.

supplements.

Air in