Cumitech 5A: Practical Anaerobic Bacteriology

5A

CUMITCCH

Cumitech 1A Blood Cultures II June 1982 Cumitech 2A Laboratory Diagnosis of Urinary Tract Infections March 1987 Cumitech 3A Quality Control and Quality Assurance Practices in Clinical Microbiology May 1990 Cumitech 4 - Laboratory Diagnosis of Gonorrhea October 1976 Cumitech 5 Practical Anaerobic Bacteriology April 1977 Cumitech 6A New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide February 1991 Cumitech 7A Laboratory Diagnosis of Lower Respiratory Tract Infections September 1987 Cumitech 8 Detection of Microbial Antigens by Counterimmunoelectrophoresis December 1978 Cumitech 9 Collection and Processing of Bacteriological Specimens August 1979 Cumitech 10 Laboratory Diagnosis of Upper Respiratory Tract Infections December 1979 Cumitech 11 Practical Methods for Culture and Identification of Fungi in the Clinical Microbiology Laboratory August 1980 Cumitech 12 Laboratory Diagnosis of Bacterial Diarrhea October 1980 Cumitech 13 Laboratory Diagnosis of Ocular Infections May 1981 Cumitech 14 Laboratory Diagnosis of Central Nervous System Infections January 1982 Cumitech 15 Laboratory Diagnosis of Viral Infections March 1982 Cumitech 16 Laboratory Diagnosis of the Mycobacterioses March 1983 Cumitech 17 Laboratory Diagnosis of Female Genital Tract Infections August 1983 Cumitech 18 Laboratory Diagnosis of Hepatitis Viruses January 1984 Cumitech 19 Laboratory Diagnosis of Chlamydial and Mycoplasmal infections August 1984 Cumitech 20 9 Therapeutic Drug Monitoring: Antimicrobial Agents October 1984 Cumttech 21 Laboratory Diagnosis of Viral Respiratory Disease March 1986 Cumitech 22 Immunoserology of Staphylococcal Disease August 1987 Cumitech 23 Infections of the Skin and Subcutaneous Tissues June 1988 Cumitech 24 Rapid Detection of Viruses by Immunofluorescence August 1988 Cumitech 25 Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing December 1988 Cumitech 26 Laboratory Diagnosis of Viral Infections Producing Enteritis September 1989 l

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Cum&&s should be cited as follows, e,g,: Rodloff, A. C., P. C. Appelbaum, and R. J. Zabransky 1991. Cumitech 5A. Practical anaerobic bacteriology Coordinating ed., A. C. Rodloff. American Society for Microbiology, Washington, D.C.

Editorial Board for ASM Cumitechs: Steven C Specter, Chairman, Mary J R Gilchrist. William J Martone, John E McGowan, Jr, Frederick W Smith, John A Smrth, Thomas J Tinghitella, and Alice S Weissfeld.

Carl Abramson, Ellen Jo Baron, S Nolte, Arne C Rodloff, James

The purpose of the Cumttech series is to provide consensus recommendations state-of-the-art operating procedures for clinical microbiology laboratories which evaluating routine or new methods The procedures given are not proposed as “standard” methods.

Copyright

0 1991 Amerrcan Socrety for Mrcrobrology 1325 Massachusetts Ave, NW Washington, DC 20005

by the authors as to appropriate may lack the facilities for fullv

PRACTICAL ARNE C. RODLOW,

ANAEROBIC

Paul-Ehrlich-Institut,

Paul-Ehrlich-Str.

BACTERIOLOGY 51-59, W- 6070 Langen 1, Germany

PETER C. APPELBAUM, Pennsylvania State University College of Medicine, Mihon S. Hershey Medical Center, Hershey, Pennsylvania 17033 RONALD J. ZABRANSKY, Department of Clinical Microbiology Medical Branch at Galveston, Galveston, Taas 77550 COORDINATING ARNE C. RODLOFF, Paul-Ehrlich-Institut,

1

and Immunology,

The

University of Taas

EDITOR

Paul-Ehrlich-Str.

The first edition of the Cumitech on “Practical Anaerobic Bacteriology” appearedin 1977.This revised edition draws heavily on the early work of S, M. Finegold, W. E. Shepherd, and E. H. Spaulding (24), whom the present authors acknowledge with respect. The scientific definition of anaerobic bacteria is still a matter for discussion. Finegold and George (22) suggestedthat an anaerobe is a bacterium that requires reduced oxygen tension for growth and fails to grow on the surface of solid media in 10% CO2 in air (18% oxygen). This definition still appears to be suitable for clinical laboratories. Anaerobesconstitute a major part of the normal bacterial flora of the human skin, mucousmembranes,andintestinaltract. In the colon, members of the family Enterobacteriaceaesuch asEscherfchia coZiare outnumberedby anaerobesat least 1,000:1. The physiological anaerobic flora is thought to play a significant role in resistance againstcolonization by pathogenicorganisms(54). Thus, normally, anaerobesseemto be beneficial to the host. Nevertheless, anaerobesmay also causesevere, even lethal, infections in humans. Most of these infections are endogenousin nature; they arise when bacteria of the physiological flora gain accessto normally sterile body sites. A loss of integrity of body surface structures may serve as a prerequisite for such a translocation. Less commonly, infecting anaerobes may be acquired from exogenoussources. Classic examples are toxigenic infections such as tetanus or botulism or nosocomialoutbreaks of Clostridium di’ficile infections. Anaerobic infections are relatively common. On average, approximately 5 to 10% of gramnegative blood culture isolates are membersof the family Bacteroidaceae. Knowledge about the pathogenic mechanisms of anaerobicorganismsis incomplete. Normally, anaerobesare found in mixed infections either togetherwith other anaerobicbacteria or in asso-

University Hospital,

51-59, W- 6070 Langen 1, Germany

ciation with aerobic organisms.However, infections (e.g., sepsis)with singleorganisms,particularly with clostridia and gram-negativerods, may occur. The clinical relevance of anaerobic organisms has been demonstratedby the failure of therapeutic regimensnot covering anaerobesto control infectionswith anaerobicetiology. Hence, anaerobicculture is essentialfor satisfactory evaluation of many clinical specimenssubmitted for the diagnosisof infectious agents. During the past decade, the laboratory diagnosisof anaerobicinfections hasbeen facilitated by a number of commercially available products. These include ready-to-use media, new anaerobic incubation systems, and especially kits for identification and susceptibility testing of anaerobes. Still, none of these products can replace the extra attention and special care needed for anaerobic bacteriology. They do, however, make anaerobic bacteriology more amenable to the routine laboratory and may expedite the acquisition and reporting of results. Therefore, anaerobicbacteriology resultsshould be more readily available to the attending physician and may be translated sooner into adequate therapy for the patient. The purpose of this Cumitech is to outline practical procedures that can be used to establish the presence of clinically important anaerobes. It will not address sophisticated techniques necessary, e.g., for oral microbiology or for studiesof fecal or other normal flora. CLINICAL

CONSIDERATIONS

Anaerobic infection may occur in any organ or tissue of the body. Figure 1 illustrates the variety of sites that may be affected. A number of factors are known to predisposeto anaerobic infections. Because anaerobesare prevalent in large numbers on all mucosal surfaces, this endogenousflora is commonly the source of infection, especially in locations near these surfaces. Accidental or surgical wounds are easily

2

RODLOFF

ET AL.

CUMITECH

SA

2-4

5 ~6 7 8 9 10

11

12 13 14 15

FIG. 1. Location of anaerobic infections. 1, brain abscess; 2-4, dental infection, chronic otitis, mastoiditis, sinusitis, tonsillitis; 5, aspiration pneumonia, necrotizing pneumonitis, lung abscess, empyema; 6, endocarditis; 7, breast abscess; 8, liver abscess; 9, wound infection, peritonitis, retroperitoneal abscess, appendicitis; 10, pseudomembranous colitis; 11, nonspecific vaginosis, bartholinitis, endometritis, salpingitis, tubo-ovarial abscess, septic abortion; 12, nonspecific urethritis, prostatitis; 13, perianal abscess; 14, osteomyelitis; 15, cellulitis, necrotizing fasciitis, gas gangrene.

contaminated with these bacteria, which may give rise to infection. A reduction in Oxygen supply to the tissue damaged by hemostasis, wound necrosis, foreign body implantation, diabetes, etc., facilitates the establishmentof anaerobic organisms.Infections with aerobic bacteria that lower the redox potential of the damaged tissue may serve the same purpose. All types of immunosuppressionmay also aggravate anaerobicinfections. Accordingly, clinical signs of anaerobic infections are often infection in the proximity of mucosal surfaces, tissue necrosis, and abscess formation. Volatile amines and short-chain fatty acids produced by anaerobes may result in foul odor of infected tissue or discharge and in turn further lower the redox potential. The formation of gas in the tissue is less specific, unless the classic picture of clostridial myonecrosis(“gas gangrene”) is present. Infections with pigmentedanaerobes,Porphyomonas or Prevotella spp., may lead to

black discolorations of the exudate. Finally, certain types of infections should always be suspectedto be at least in part anaerobic, e.g., brain, lung, or liver abscesses,aspiration pneumonia, and peritonitis. COLLECTION AND TRANSPORT OF SPECIMENS Since anaerobic bacteriology is a time-consuming, expensive process,it is most important that collection and transport of appropriate specimensare done with great care. Specimens must be obtainedfrom the infected sitesby using procedures that avoid contamination with endogenousflora. In general, this is achieved by needle aspiration of the material or by biopsy under appropriate conditions (after proper decontamination with antiseptics). Acceptable procedures include percutaneous transtracheal needleaspiration for the diagnosisof pulmonary infections and culdocentesis for patients with

CUMITEXH

5A

pelvic inflammatory disease. Experience suggests that material obtained from the lung with a double-lumen catheter or protected brush may yield acceptable specimens for aerobic and anaerobic culture (79). However, sometimes contamination can occur despite employment of these techniques. Swabs with purulent material are frequently used but are less desirable, since the recovery rate of bacteria is decreased and material might be less representative of the actual infectious process. All specimens that are inevitably contaminated with endogenous flora (e.g., sputum, tracheal secretions, bronchial lavages, contents of the digestive system, vaginal or cervical swabs, voided urine) should be excluded from anaerobic culture. The most sophisticated cultural techniques can only demonstrate infectious agents that reach the laboratory in a viable state. In the case of anaerobic bacteria, this not only calls for protection from desiccation and overgrowth by other less fastidious organisms, but also necessitates protection from toxic oxygen. A number of commercially available transport systems can support the survival of anaerobes and should be used if the native specimen cannot reach the laboratory within 15 min. However, any transport system can only prolong the survival of anaerobes for a limited period of time, depending on the type and 0, sensitivity of the specific organism. Therefore, it is nevertheless necessary to transport as rapidly as possible. Moreover, it is mandatory that the laboratory be informed of the time of collection of a particular specimen. As a general rule, we suggest that a transportation time of 24 h should not be exceeded. Specimens for anaerobic culture should never be refrigerated. DIRECT DETECTION OF ANAEROBES The detection of anaerobic organisms in clinical specimens follows the same principles as for any other bacteria, as discussed below. Microscopy Aerobes and anaerobes are not always distinguishable by Gram stain. However, anaerobes may yield typical microscopic patterns that are recognized by the experienced eye, Hence, it cannot be emphasized too strongly that a Gram stain may provide the earliest microbiological evidence of anaerobic infection. Anaerobes may not always take up the stain evenly, some species have a tendency to stain gram variably (e.g., cocci and Clostridium spp.), and some grampositive species may stain negatively (e.g., Clostridium spp.). Other microscopic procedures include acridine orange staining and darkfield microscopy.

PRACTICAL

ANAEROBIC

BACTERIOLOGY

3

Other Direct Methods Other direct methods include radioimmunoasor say, coagglutination, and countercurrent crossed immunoelectrophoresis (21, 41, 67). Currently, they are not suitable for the routine laboratory. CULTURE TECHNIQUES We suggest that all suitable clinical specimens be cultured for anaerobic organisms rather than a decision being made for each specimen individually, since it cannot be expected that clinicians will always indicate a specific request for anaerobic culture. Media Primary isolation of anaerobes involves liquid and solid media. Broth media are usually employed to back up plating. Supplemented thioglycolate medium is widely accepted because it does not have to be incubated anaerobically, but other media may be used instead (e.g., choppedmeat-glucose medium). A number of different formulations meet the requirements for nonselective solid media (e.g., CDC anaerobe blood agar, Columbia blood agar, Schaedler blood agar, brucella blood agar); however, all media should contain hemin and vitamin K,. Since anaerobes are often found in association with facultatively anaerobic organisms, the use of selective solid media is helpful to speed up isolation procedures. The addition of aminoglycosides and vancomycin (and possibly nystatin) to enriched formulations results in media selective for anaerobic gram-negative rods. The species of the Bacteroides Ji-agilis group are often involved in severe infections causing significant therapeutic problems; consequently, their early detection may be particularly helpful. For this purpose, a supplemented bile esculin agar as a selective and indicative medium for these organisms is often used. As a minimal set of media, we recommend the inoculation of a supplemented thioglycolate broth and an enriched blood agar plate with and without the addition of kanamycin and vancomycin (67). The selective media should be inoculated heavily; however, it is helpful to dilute the sample while spreading it on the nonselective agar. Inoculation Procedures for inoculation and incubation depend on the availability of an anaerobic chamber. These devices require high initial investment costs and additional laboratory space. Processing of specimens in a chamber is less convenient and more time-consuming. However, especially in conjunction with prereduced, anaerobically sterilized (PRAS) media (46), the

4

RODLOFF

ET AL.

yield of anaerobic organisms from clinical specimens seems to be optimal. Alternatively, clinical specimens may be plated on the open laboratory bench. In this case, exposure of the plated specimens to air should be kept to a minimum. If necessary, inoculated plates can be kept (up to 4 h) in an anaerobic holding jar (with an N2 or CO2 atmosphere) until it is feasible to incubate them. Incubation An anaerobic atmosphere may be conveniently established by using jars and commercially available gas generator kits. The latter operate by generating H, and CO, after water is added to sodium borohydrate and citric acidsodium bicarbonate. Palladium-coated aluminum pellets constitute the necessary catalyst responsible for converting oxygen in the jar together with the generated H, into water. The catalyst becomes inactivated by moisture or H,S; therefore, it has to be replaced after each incubation or regenerated by heating the pellets to 160°C for approximately 2 h. If individual plates need anaerobic incubation, the use of plastic bags with specially designed gas generator kits may be efficient (e.g., BioBag, Becton Dickinson, Cockeysville, Md.; Anaerobic Pouch, Difco Laboratories, Detroit, Mich. [ 181). Alternatively, the jars may be evacuated and the air replaced by a gas mixture consisting of 5 to 10% H,, 5 to 10% CO,, and 80 to 90% N,. The evacuation-replacement procedure should be repeated three times to ensure anaerobiosis. Again, a catalyst should be used to convert 0, to H,O. Specially designed incubators can also be used with the evacuation-replacement procedure. Finally, an incubator may be included inside an anaerobic chamber. This form of incubation has the advantage of low running expenses. The anaerobiosis in the respective incubation devices should be confnmed with suitable indicators that are available commercially. Normally, cultures should be incubated for at least 48 h. Exceptions may be made if clostridia are suspected, since these organisms may form colonies within 8 to 12 h. Some anaerobes require >3 days for visible growth (e.g., some Fusobacterium spp. and anaerobic cocci). For all practical purposes, cultures may be checked for growth after 48 h and immediately reincubated for an additional 48 to 72 h. Anaerobic chambers have the advantage that cultures may be viewed every day without interrupting the anaerobiosis. The anaerobe laboratory at Virginia Polytechnic Institute (VPI) has popularized a culture method, using stoppered tubes that are coated with agar (“roll tubes” [36]). Tubes are prereduced bv inserting a cannula carrying a stream

CUMITECH

5A

of anaerobic-grade CO, and inoculated while the tube is being turned. The restoppered tube then serves as its own anaerobic culture chamber. Subculture Bacteria from one specimen that are growing on plates incubated anaerobically but not aerobically are almost certainly anaerobes. However, because media used for parallel aerobic and anaerobic cultures are often of different composition (e.g., regular blood agar versus Columbia agar), nutritional requirements of fastidious aerobic organisms may in some cases result in growth on anaerobically incubated plates only. Therefore, subcultures should be done using the same medium for anaerobic and control aerobic incubation in 5 to 10% CO, to ensure the obligately anaerobic nature of the organism. Inclusion of a chocolate agar plate, incubated in 5% C02, detects Haemophilus spp. that might grow well on some anaerobic blood agars but not aerobically on the same medium. If mixed cultures are found after primary incubation, one should consider that the colonial morphology of one bacterial strain may vary markedly with the medium and incubation procedure. Again, proper subcultures are helpful in determining the nature of a particular organism. IDENTIFICATION PROCEDURES Any identification procedure of anaerobic bacteria should start with a careful examination of colonial morphology and a Gram stain. Not all anaerobes require extended biochemical testing for identification, and in most clinical laboratories it is neither practical nor feasible to use the large number of tests required by standard manuals. Fortunately, certain characteristics (Table 1) are useful in preliminary identification of many anaerobic isolates (41). Several “shortcuts” (Table 2) are available to aid in the preliminary identification of many groups. Moreover, in recent years a number of commercial products have become available that facilitate identification procedures for the routine laboratory. Dowel1 and colleagues at the Centers for Disease Control (CDC) have devised a modified conventional plate system (Presumpto method [2, 41, 441) for the identification of anaerobes (Table 3). The method consists of a three-plate system; each plate is divided into quadrants containing different components in LombardDowel1 agar base (Remel Inc., Lenexa, Kans.; Carr-Scarborough Inc., Atlanta, Ga.). This method should be used together with a blood agar plate and thioglycolate broth. Identification tables for use with this system are available (2, 41, 44). Other commercially available systems make use of miniaturized biochemical tests. API 20A (Analvtab Products, Plainview, N-Y.) con-

CUMITECH

TABLE

SA 1. Some cardinal identification anaerobes”

PRACTICAL criteria for

O2 sensitivity Colonial characteristics Swarming Pigment Hemolysis Pitting of medium Motility Gram stain reaction Morphology Flagella Miscellaneous: growth in thioglycolate broth, catalase production, reactions on liquid and solid milk medium; production of indole; hydrolysis of starch, esculin, and gelatin; reduction of nitrate; fermentation of key carbohydrates (e.g., glucose, mannitol, lactose, rhamnose); growth in the presence of bile, penicillin, rifampin, and kanamycin; inhibition by sodium polyanetholsulfonate; metabolic products “Modified after Koneman et al. (41).

sistsof a plastic strip with microcupulescontaining dehydrated substrates.Strips are inoculated with turbid suspensionsof fresh growth, and reactions are read after 24 h of anaerobic incubation. Carbohydrate fermentations are read after the addition of a bromcresol purple indicator but are sometimes equivocal and difficult to read. Additionally, the system requires supplemental tests, including gas-liquid chromatography (GLC), for the correct identification of nonsaccharolytic or weakly saccharolytic organismssuch as somestrains of fusobacteria and anaerobic cocci. Results of published evaluations of API 20A are presentedin Table 4 (5,28, 30, 39, 49). The Minitek (Becton Dickinson) system utilizes paper disks impregnated with various biochemical substrates. Disks are dispensed into wells of specialized plastic plates and inoculated. Reactions are read after 48 h of anaerobic incubation. Color reactions of carbohydrate fermentation tests are developed with phenol red indicator. These may be difficult to read, but interpretation may be facilitated by the addition of one extra drop of phenol red to wells exhibiting equivocal reactions. As with API 2OA, supplemental tests, including GLC, are required for less reactive organisms.Published resultsfor the Minitek system are given in Table 4 (5, 9, 37). Allen and coworkers (1, 2) have developed a microtube plate system which permits the use of up to 50 differential tests for expandedbiochemicalcharacterization of anaer; obes.A computerized data basehasbeen developed. Biochemical tests show a high degree of correlation with conventional procedures, and the method may be combinedwith microdilution antimicrobial susceptibility tests. This systemis

ANAEROBIC

BACTERIOLOGY

5

suitable for laboratories with resourcesto prepare and store their own plates. An innovative new approachto clinical anaerobic bacteriology is the development of commercially available packaged microsystems for detection of preformed enzymes a few hours after inoculation (60). Thesetests usually rely to a great extent on the detection of glycosidases and aminopeptidases.API ZYM (Analytab) has been available for someyears as a researchtool. Studies have shown that enzyme profiles of clinically isolated and reference anaerobic strains are well circumscribed and distinctive, but the method lacks a computerized data base (47). The AnIdent (Analytab [9, 11, 49, 57, 60]), RapID ANA (Innovative Diagnostic Systems, Inc., Atlanta, Ga. [4, 9, 11, 16, 30, 37, 39, 49]), ATB 32A (API System, Montalieu-Vercieu, France [40, 45]), Vitek AN1 (Vitek Systems, Inc., Hazelwood, MO. [61]), and MicroScan (American Microscan, Inc., Sacramento, Calif. [65]) systems are all commercially available same-daysystems(usually 4 h). Resultsof evaluations of these methodsare presentedin Table 4. The RapID ANA method has recently been modified (RapID ANA II) to contain a revised test selectionand an updated code book, Advantages include easier interpretations of results, improved microcode entry, the flexible (4- to 6-h) incubation time, and an overnight refrigeration option prior to the addition of reagents (12). With the exception of Vitek ANI, which yields a high proportion of mis- and nonidentifications, and MicroScan, which requires modifications of existing testscombinedwith improvements in the data base, these systems provide accurate identification of clinically encountered anaerobes, including weakly saccharolytic and nonsaccharolytic strains, without GLC. Additional tests required in somecasesfor accurate identification are simpleand within the scopeof smaller microbiology laboratories. Problems may arisein identification of bile-sensitivegramnegative rods and non-C. perjkingens clostridia, especially C. di#kiZe. Color reactions are sometimes difficult to interpret. This may account for the variance in results of somepublishedstudies (3, 4, 49, 60). Finally, it should be pointed out that studies evaluating the efficacy of commercially available identification kits need to be carefully read with respect to (i) the conventional identification method employed and (ii) the numbersand types of organismsbeing used. With this proviso, however, AnIdent, RapID ANA II, and ATB 32A all provide accurate anaerobicidentification and are suitable for routine use. The “gold standard” for laboratory identification of anaerobesincludes reactions in a series of conventional carbohydrates and other bio-

6

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ET AL. TABLE

5A

CUMITIXH

2. Shortcuts for preliminary

identification

of anaerobes

Method

Purpose

Special-potency antimicrobial susceptibility disks (kanamycin, 1 mg; vancomycin, 5 pg; colistin, 10 pg; rifampin, 15 pg; penicillin, 2 U)

Preliminary differentiation between gram-positive and -negative nonsporeforming rods; differentiation between some Bacteroides spp. and fusobacteria Differentiation between Bacteroides spp. (sensitive) and fusobacteria (resistant) Screening for fermentative Bacteroides species Preliminary identification of B. f+agiZis group Preliminary differentiation between pigmented Prevotella or Porphyromonas spp.: P. intermedia, brillant red; P. melaninogenica, red-orange; P. asaccharolytica, yellow or red; P. gingivaks, negative Typical of fusobacteria; Bacteroides and Prevotella spp. possess the enzyme Presumptive identification of Bilophila wadkwotihia Preliminary identification of VeiZZoneZZaspp.

67

Preliminary identification of Peptostreptococcus anaerobius (resistant) Preliminary identification of P. anaerobius

74

Susceptibility to phosphomycin

(200-

500pglml)=

Fermentation of glucose l-phosphate Bile and kanamycin disks Fluorescence with long-wave UV light

Lack of constitutive &#.zosidase (esculinase) Growth on bile-containing media; catalase and urease positive Red fluorescence under long-wave (366~nm) UV light Polyanetholsulfonate disk test GLC of fatty acids after growth in modified norleucine-tyrosine broth” Growth pattern on egg yolk agar Rapid lipase test Synergistic hemolysis on blood agar Disk test for nitrate reduction Spot esculin test Spot indole test with p-dimethylaminocinnamaldehyde” Addition of 3% KOH to culture

Rapid glutamic acid decarboxylase test? Slide catalase with 15% H,02 and added Tween 80 Rapid urease testing

Wltemative

Clostridia Clostridia Differentiation of C. perfringens, bifermentans, C. sordellii, C. [email protected] Useful for all groups Useful for all groups Useful for all groups

References

C.

Differentiation between gram-positive (no stringing) and gram-negative (stringing) organisms in cases when Gram stain yields equivocal results Preliminary identification of B. fragiZis group, clostridia, E. Zentum More sensitive for detection of catalasepositive organisms Identification of C. sordeZZii, Actinomyces spp.,Bacteroides ureotyticus, gram-positive cocci

59 78 71 62

19 7 13

70 67 35 29 73 56 43 31

6, 38 32 48

method; not readily available.

chemical tests in combination with GLC. Three procedure manualsare available, one published by VP1 to be usedin conjunction with the latest update furnished by VP1 only to attendees of their anaerobe course (36), another one from CDC (17), and a third from the Wadsworth Anaerobe Laboratory (67). Both the VP1 system and Wadsworth Anaerobe Laboratory use PRAS media that may be prepared in-house or purchasedcommercially. Originally, PRAS media were marketed in wide-mouthedtubes with butyl rubber stoppers(36) that had to be inoculated with a special gassingapparatus or in an anaerobic glove box. Scott Laboratories (now

Adams Scientific, Fiskeville, R.I.) has developed the PRAS II system in shorter and narrower tubes, which may be inoculated with a needle and syringe through a rubber diaphragm. In comparisonwith conventional and commercial systems, PRAS II has been found to correctly identify >93% of strains to the genusand specieslevel (11, 39). Biochemical data can be interpreted with the aid of a computerized data base.Citron and colleagueshave devised a short biochemical scheme using PRAS biochemicals for the identification of bile-resistant Bacteroides spp. (14). The CDC system utilizes aerobically prepared and inoculated thioglycolate

PRACTICAL

SA

CUMITEEH

TABLE

ANAEROBIC

BACTERIOLOGY

7

3. Media and characteristics of cultures that can be determined by the differential agar medium (Presumpto) system for identifying anaerobes”

Blood agar

Relation to 0,” colonial characteristics, hemolysis, pigment, fluorescence with UV light (Wood’s lamp), pitting of agar, cellular morphology, Gram stain reaction, spores, motility (wet mount); inhibition by penicillin, rifampin, or kanamycin

Enriched thioglymlate medium

Growth, rapidity of growth, gas production, odor, cellular morphology

Presumpto 1 plate L;Dc agar LD esculin agar LD egg yolk agar LD bile agar

Indole, growth on LD medium, catalased Esculin hydrolysis, H,S, catalase Lipase, lecithinase, proteolysis Growth in presence of 20% bile (2% oxgall), insoluble precipitate under and immediately surrounding growth

Presumpto 2 plate LD glucose agar LD starch agar LD milk agar LD DNA agar

Glucose fermentation: stimulation of growth by fermentable carbohydrate Starch hydrolysis Casein hydrolysis Detection of DNase activity

Presumpto 3 plate LD mannitol agar LD lactose agar LD rhamnose agar LD gelatin

Mannitol fermentation Lactose fermentation Rhamnose fermentation Gelatin hydrolysis

“Modified from Koneman et al. (41). bBy comparing growth on an anaerobe plate with that on blood agar (or chocolate agar) incubated in a 5 to 10% COz incubator (or candle jar) or in room air. ‘LD, Lombard-Dowel1 agar. dThe catalase test can be performed by adding 3% hydrogen peroxide to the growth on LD agar, but reactions after the addition of HzOz to catalase-positive cultures are more vigorous on LD esculin agar.

media. It is very important to note that when employing any one of these three manuals, it is necessaryto use the sugarbasesrecommended for the specific system. Failure to do so may invalidate results (2). GLC analysis for volatile and nonvolatile acids of broth cultures in peptone-yeast-glucose (PYG) or chopped meat-carbohydrate (CMC) forms an integral part of the speciesidentificaTABLE System

tion of anaerobes. The reader is referred to standardmanualsfor descriptionsof the method (36, 67). Briefly, turbid broth cultures are acidified with H,SO, and volatile fatty acids are extracted with ether. Extraction of nonvolatile acids requires derivatization with methanol and extraction with chloroform. Pankuch and Appelbaum have devised a method for the direct extraction of volatile and nonvolatile acids (55).

4. Evaluations of commercial kits for the identification % Correct (no additional tests)

% Correct (with additional tests)=

of anaerobes to the species level

% Incorrect or no identification

API 20A 54-8Sb 68-95 Minitek 66-76 86-93 AnIdent F-91 88-98 72-98 RapID ANA 62-100 97 RapID ANA 87 II Vitek AN1 70 83 API 32A 68-88 95-96 MicroScan Visual 70 80 Autoscan 66 77 “As prescribed by the manufacturer or investigator. “Percent agreement with reference as described by the investigator. cC. dificile only tested.

References

5-46 5-14 2-12 3-28 3

5, 28, 30, 39, 49 5, 9, 37 9, 11, 49, 57, 60 4, 9, 11, 16, 30, 37, 39, 49 12

17 4-5

61 40,45

20 23

65 65

8

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ET AL.

TABLE Former

CUMITECH

5. Taxonomic

changes of and additions to anaerobic gram-negative rods

designation

B. fragilis group Other Bacteroides spp. B. melaninogenicus-B. asaccharolyticus-B. groups

oralis

Bacteroides 3452A B. amylophilus B. furcosus B. hypermegas B. micro+us B. multiacidus B. praeacutus B. succinogenes B. tennitidis New Bacteroides spp. New Fusobacterium spp. New Selenomonas spp. Other new species

5A

New

Bacteroidesfiagilis, B. distasonis, B. eggetihii, B. ovatus, B. thetaiotaomicron, B. uniformis, B. vulgatus (new additions: B. caccae, B. merdae, B. stercoris [see below]) Bacteroides capillosus, B. coagulans, B. gracilis, B. levii, B. macacae, B. pneumosintes, B. putredinis, B. salivosus, B. splanchnicus, B. ureolyticus Prevotella melaninogenica, P. bivia, P. buccae, P. buccalis, P. corporis, P. denticola, P. disiens, P. heparinolytica, P. intermedia, P. loescheii, P. oralis, P. oris, P. oulora, P. ruminicola, P. veroralis, P. zoogleoformans Porphyromonas asaccharoEytica, P. endodentalis, P. gingivalis Bacteroides caccae Ruminobacter amylophilus Anaerorhabdus furcosus Megamonas hypermegas Rikinella micro*us Mitsuokella multiacida Tissierella praeacuta Fibrobacter succinogenes Sebaldella termitidis Bacteroides forsythus, B. galacturonicus, B. merdae, B. pectinophilus, B. salivosus, B. stercoris, B. tectum Fusobacterium aloicis, F. sulci, F. ulcerans Selenomonas artemidis, S. dianae, S. fluggei, S. infelix, S. noxia Bilophila wadsworthia, Centipeda periodontii, Fibrobacter intestinalis, Mitsuokella dentalis, Wolinella curva

A block of agar with pure growth is removed, dissolvedwith acid, and extracted by standard procedures with modified amounts of reagents. In 84% of cases,patterns of volatile and nonvolatile acids from agar correspondedwith those in PYG and CMC. The majority of discrepancies were seen with organisms usually producing major lactate peaks(Actinomyces spp., Streptococcus intermedius), which produced only trace or minor amounts on agar as compared with broth. With this caveat, direct GLC in conjunction with additional simple tests, such as Gram stain, colonial morphology, and spot indole and catalasetesting, may result in rapid preliminary genus and possibly species identification of many anaerobes(55). Direct extraction of acids from clinical specimensis also feasible and has been shown to be accurate in the rapid preliminary identification of organismgroups. This procedure may be directly applied to purulent or serous specimens(26, 64, 69), blood (20), and vaginal (63) or amniotic (27) fluids. The method hasalsobeen successfullyappliedto swabs(58). Although the presenceof volatile fatty acids in specimensis a reliable indicator of the presence of anaerobes, there is incomplete agreement concerning the presence of specific acids and their correlation with specific anaerobes(26, 64, 69). Spiegel and coworkers (63) have demonstrated chromatographicchangesin vaginal fluid from patients with nonspecific vaginitis (bacte-

rial vaginosis) for which culture is not indicated. Fluids from such patients demonstrated higher succinate/lactate ratios than fluids from normal cases. Gravett and coworkers have also used direct GLC of amniotic fluid as a method for rapid diagnosisof chorioamnionitis (27). In recent years, a number of taxonomic changeshave occurred especially with anaerobic gram-negativerods; someguidanceis offered in Table 5. EXTENT OF ANAEROBE IDENTIFICATION IN CLINICAL LABORATORIES Wounds or abscessesoften contain multiple speciesof aerobic and anaerobicbacteria. There are no clear-cut rules as to how many isolatesto identify in suchcases.The relative proportion of epithelial cells to polymorphonuclear leukocytes and the relative number of different morphotypes in the Gram-stained smear help to determine specimen quality. When numerous morphological forms are seen but no pus cells are present, the physician should be contacted to discuss the clinical situation in an attempt to determinethe specimenquality. In suchcases,it is probably acceptable to limit the extent of workup and preliminary identification basedon Gram stain and colonial morphology. When the direct smear suggeststhat the specimen is of poor quality, no useful information may be gained from extensive identification and testing.

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In certain clinical situations (bacteremia; endocarditis; meningitis; liver, brain, tubo-ovarian, and lung abscesses;serious skin, soft tissue,bone, and joint infections; infections which do not respond to antimicrobial therapy), however, all anaerobespresent should be identified as fully as possible. Cultures reported preliminarily at the level of the group of the organism shouldbe held anaerobically for several days to allow for more complete identification should the need arise. ANTIMICROBIAL SUSCEPTIBILITY TESTS While the history of the susceptibility testing of anaerobesgoesback to the middle 1950s(25), attempts at standardizing testing procedures were made formal by the National Committee for Clinical Laboratory Standards (NCCLS) only in 1973,when a working group to study this particular problem was established.That group publishedtheir first recommendationsfor a standard reference agar dilution (AD) technique in 1979(50). Those recommendationswere based on an extensive multicenter study in which a number of candidate control organismswere tested against antibiotics known to be effective againstanaerobesat that time (66). One of the key findingsof that study was the reproducibility of the AD procedure with the relatively defined medium of Wilkins and Chalgren (76). Another major development was the identification of the three control strains B. frasilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741,and C. perfkingens ATCC 13124. NCCLS recognized at that time that the reference AD procedure was too cumbersome for routine use in clinical laboratories; its primary purposewas to serve as a method with which other techniques as they were developed could be compared. Since then, a multitude of reports have appeared describing other procedures, comparing them with the reference method and with each other as well as evaluating new antibiotics as they have been developed. Two approachesthat gainedrecognition were the broth microdilution (BMD) technique and the broth disk elution (BDE) procedure (42, 72, 76). A number of variations of these procedures have been described, most frequently with differencesin test medium, method of inoculation, inoculum size, and incubation conditions. In 1985, NCCLS published guidelinesfor performing these alternative procedures (51), trying to bring some organization to what wasvery confusingto small and average-sizeclinical laboratories. Despite these approachesat standardization, numerous investigators continued to use their own procedures for determining the antimicrobial susceptibility of anaerobesand, despite this variation, several factors became evident: (i)

PRACTICAL

ANAEROBIC

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9

surveys illustrated that specific speciesand genera had predictable susceptibility patterns; (ii) repeated surveys from the samelocales showed trends in increasingresistancewith someorganism-antibiotic combinations; (iii) a higher inoculum size (comparedwith the aerobic procedures) is required; (iv) not all organismswill grow on any one medium; (v) the addition of blood to a mediumusually haslittle effect on MICs; (vi) the rapidly growing anaerobes(B. fragilis group and some Clostridium species) need only 24 h of incubation; (vii) certain organism-antibiotic combinationsgive nonreproducible results; (viii) establishedcontrol strains are not suitable for some of the newer antibiotics; (ix) MICs obtained with BMD testing are usually lower than those determined by AD; and (x) the BDE procedure frequently gives unreliable results with g-lactamase-producing anaerobes when tested against g-lactam antibiotics. From the clinical point of view there are other issues: (i) increasing resistance of anaerobic bacteria to antimicrobial agents is a significant clinical problem; (ii) there is disagreementabout the clinical effectiveness of certain agents in anaerobic infection; (iii) variations in in vitro activity as determined by different techniques compromise our ability to predict in vivo response; and (iv) the interpretation breakpoints (e.g., susceptible)usedfor aerobesmay or may not apply to anaerobic organisms. Theselaboratory andclinical issueshave led the current NCCLS Working Group on Anaerobic Susceptibility Testing to study the problem and makerecommendations on the indicationsfor susceptibility testing of anaerobesand the reliability of current test methodsand their correlation with clinical outcome. This group publishedits recommendationsas a review in 1988 (23) and as a foreword in the latest edition of the NCCLS standard for susceptibility testing of anaerobes(53). This new standard(Mll-A2) combinesall of the methodsinto one documentand makesa number of specific recommendations.For instance,these recommendationsstate that susceptibility testing of anaerobesshouldbe done under the following limited circumstances:(i) to determinepatterns to new agents,(ii) to periodically monitor patternsin hospitalsandlocally throughout the United States and other countries, and (iii) to assist in the managementof individual patients. There are indicationsfor testingin clinical situationswhere the choice of an agent is critical becauseof (i) the failure of the usual antimicrobial regimen and persistenceof infection, (ii) the pivotal role of an agent in determiningoutcome, or (iii) where the choice of an agentbasedon precedentis difficult. Although uniform agreement on breakpoints for susceptibility of anaerobesto various drugs is lacking, NCCLS has tentatively made some

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6. MIC breakpoints indicating susceptibility”

Antimicrobial

agent

Amoxicillin-clavulanate Ampicillin Ampicillin-sulbactam Carbenicillin Cefamandole Cefmetazole Cefoperazone Cefotaxime Cefotetan Cefoxitin Ceftizoxime Ceftriaxone Chloramphenicol Clindamycin Imipenem Metronidazole Mezlocillin Moxalactam Penicillin G Piperacillin Tetracycline Ticarcillin Ticarcillin-clavulanate

MIC(P&bw S/4 4= 16/S 128 16 32 32 32 32 32 64 (32 in broth)d 32 16 4 8 16 64 32= 4= 64 8 64

6412

“Modified from reference 53. ‘MICs at or lower than those listed indicate “susceptible.” Interpretation comprises the definitions for “susceptible, ” “moderately susceptible,” and “intermediate” categories as defined for aerobes (except for metronidazole). Interpretation guidelines are based on pharmacokinetic data from maximum dosage and optimum route of administration as available in drug package inserts or other sources, population distribution of MICs, and studies of clinical efficacy. =Higher levels in blood are achievable so that infection with non-p-lactamase producers requiring higher MICs might be treatable. dFor AD testing, the breakpoint is 64 pg/ml. However, 32 rJLg/ml is used if tested by BMD. “Breakpoint may be lower with maximal doses of 4

g/day. recommendations(Table 6). At present, these are based on levels achieved in serum with maximum recommended dosageand a limited number of clinical studies comparing susceptibility and clinical effectiveness. No moderately susceptibleor intermediate categories are suggested because maximum dosage is generally recommendedfor anaerobic infections. Three methodswith several variations for the susceptibility testing of anaerobes have been described in detail in earlier NCCLS standards (51,52): AD, BMD, and BDE. The last method had the immediate attraction of being easy to perform and providing a “yes or no” type of answer. When the BDE method was first described (77), the limited number of antibiotics that were available for the treatment of anaerobic infections were either highly active or inactive againstanaerobes,and the test results cor-

5A

related well with MICs. Even though the advanced-generation cephalosporins used today are clinically active, their MICs are frequently near the currently used breakpoints for resistance (Table 6). In addition, P-lactamase-producing organisms yield difficult-to-read endpoints, and the BDE test has not been adequately standardized. The increasingfrequency of reports substantiating the lack of reproducibility and lack of accuracy of this method (80) hascausedthe NCCLS working group to reconsider the utility of the method; consequently, it was abandoned completely in the most recent edition of the NCCLS standard (53). This was done despite the immense popularity of the method throughout the United States. Therefore, the BDE method will not be described in this Cumitech. Also, the laboriousAD technique will not be considered further here, except to say that if an AD method is still preferred, then the limited dilution method of Hauser, Johnston, and Zabransky should be considered (33,53). Although not advocated for all anaerobes,a modification of the disk diffusion method has been describedfor the rapidly growing and easyto-propagate anaerobessuch as membersof the B. j?-a&& group (67, 68). Anaerobe strains which are slow growing, produce exceedingly largezones of inhibition, or swarmshouldnot be tested by this approach. Newer approachesinclude the spiral gradient test (34), a method that hasbeen investigated by a limited group of workers. A spiral inoculator dispensesa decreasingvolume of a liquid sample (antibiotic) on an agar plate in a concentric spiral, resulting in a decreasingconcentration of the drug; the test organismis inoculated at right anglesto the spiral as spokes in a wheel. The length of the radius from the center along the spoketo the point where growth of the organism begins correlates directly with an MIC. The investigators have developed standard curves for interpretation. Still another innovative approach has been evaluated by Citron and colleagues(15). It consists of a plastic strip (Epsilometer/E test, AI3 Biodisk, Solna, Sweden) coated with a gradient of antibiotic. The strip is placed on the surface of an inoculated agar plate, and after incubation, the MIC is read at the intersection of the zone of inhibition and the interpretation scale on the strip. When rapidly growing anaerobes were tested, only the advanced-generation cephalosporinsgave lessthan 90% agreementcompared with standard MICs. Despite their attractiveness in terms of easeof performance, the three aforementioned techniques have not been adequately assessedby a broad group of workers; consequently, NCCLS recommendsthe use of microdilution testing as

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an economical and utilitarian approach to the susceptibility testing of anaerobes. One tray can contain a complete and varied array of drugs and concentrations. Prepared trays, frozen or lyophilized, are available from several commercial sources (Becton Dickinson; American MicroScan; Microtech Medical, Aurora, Colo.; Sensititre/Radiometer, Westlake, Ohio), but there are very few published studies concerning the accuracy and reproducibility of these products. The trays may also be prepared by the user and then frozen for subsequent use. Test Media For BMD testing, a broth medium of the same formulation as Wilkins-Chalgren agar (76) with the agar omitted is recommended. This medium is available commercially (Anaerobe Broth MIC, Difco Laboratories). Other broth media may be used: Schaedler, Wilkins-West (72), and brain heart infusion broths. The wells of microdilution trays should contain or be able to be filled with 0.1 ml of broth; smaller volumes have been shown to yield inconsistent results because of evaporation and the low inoculum/volume ratio (53). Antibiotics Laboratory standard powders (for dilution procedures) are available from drug manufacturers. They should be stored and diluted in accordance with the manufacturer’s specifications or as described in the NCCLS standard (53). Usually, stock solutions are prepared to contain 2,560 pg of drug per ml of water or other appropriate diluent. Care should be taken in preparing stock solutions and dilutions, accounting for the standard units of activity and proper storage conditions (-60°C or, for imipenem and clavulanate, - 70°C). Thawed stock solutions should not be refrozen or reused. Storage Unless lyophilized, all tubes and microdilution trays prepared in advance for later use should be frozen at -60°C or, if imipenem or clavulanate is included, at -70°C. Storage time should not exceed the manufacturer’s recommendations or the expiration date of the antibiotic standard powder or 6 months, whichever comes first. Commercially prepared lyophilized plates usually have a longer shelf life. Inoculum The final inoculum density should be approximately lo6 CFU/ml (lo5 CFU per well) for microdilution. The organisms are grown in enriched thioglycolate broth (BBL 13X [Becton Dickinson] [or equivalent] plus 5 rJLgof hemin per ml, 0.1 pg of vitamin K, per ml, and 1 mg of

PRACTICAL

ANAEROBIC

BACTERIOLOGY

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NaHC03 per ml [or three to four marble chips]). The medium should be boiled and cooled just prior to use; if NaHCO, is used, it must be added after cooling. The inoculated medium is incubated for 6 to 24 h or until maximum turbidity is obtained and then adjusted to a no. 0.5 McFarland standard, using an appropriate clear broth which has been boiled and cooled just before use. An alternate and easier method is to make a direct suspension of colonies in a suitable diluent broth to achieve the same concentration(s) as above. The source agar should be no more than 72 h old and should not remain in an aerobic atmosphere for more than 30 min before colonies are picked. When microdilution trays are being inoculated, attention must be given to the volume of inoculum such that the final concentration of the drug is not affected and that the final concentration of organisms in each well is approximately 10’ CFU. Incubation Microdilution trays should be incubated anaerobically at 35 to 36°C for 48 h. Results The MIC to be reported is the lowest concentration of antibiotic that completely inhibits the growth of the organism. Control wells are used for comparison and must have discernible growth. Trailing endpoints may be observed with some media, drugs, and organism-antibiotic combinations. In these situations the endpoint is where the most significant reduction of growth is observed. Other Methods The p-lactamase test can be used as a rapid supplement to, but not a replacement for, conventional tests (53). Although the nitrocefin (chromogenic cephalosporin) test (Cefinase, Becton Dickinson) is simple and readily detects p-lactamases produced by Bacteroides spp. and other anaerobes, resistance to p-lactam agents is not always mediated by l3-lactamase production. Furthermore, hydrolysis of drugs such as imipenem and advanced-generation cephalosporins is not always predictable by the test, and some p-lactam agents are resistant to the enzymes which may be detected by the test. A positive test, however, can reliably predict resistance to penicillin G, ampicillin, amoxicillin, and firstgeneration cephalosporins.

QC A properly designed quality control (QC) program will monitor the accuracy of the test method, the performance of reagents, and the performance of individuals who conduct the tests. A QC program for anaerobe susceptibility

12

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ET AL.

agent

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Bacteroides ATCCR

fiagi1i.s 25285

Bacteroides thetaiotaomicron ATCCR

29741

Clostridium P+w~~ ATCCR

13124

Eubacterium lentum ATCCR

43055

Amoxicillin-clavulanate 0.25-l OS-2 Ampicillin l&64 16-64 NRC Ampicillin-sulbactam 0.5-2 OS-2 Carbenicillin 1664 16-64 0.25-l Cefamandole 32-128 32-128 0.06-0.25 Cefmetazole 8-32 32-128 NR “16 Cefoperazone 32-128 32-128 NR 32-128 Cefotaxime &32 16-64 0.06-0.25 64-256 Cefotetan 4-16 32-128 NR 32-128 Cefoxitin “16 8-32 0.25-l 4-16 Ceftizoxime 32-128 NR NR 16-64 Ceftriaxone 32-128 64-256 NR Chloramphenicol 2-8 4-16 2-8 Clindamycin O-5-2 2-8 0.03-0.12 0.06-0.25 Imipenem 0.03-0.12 O-06-0.25 0.03-0.12 0.25-1.0 Metronidazole 0.25-l 0.5-2 0.12-0.5 Mezlociliin 16-64 8-32 0.06-0.25 8-32 Moxalactam 0.25-l 4-16d 0.03-0.12d 64-256 Penicillin 16-64 16-64 0.06-0.25 Piperacillin 2-8 8-32 0.06-0.25 8-32 Tetracycline 0.12-0.5 8-32 0.03-0.12 Ticarcillin 16-64 16-64 0.25-l Ticarcillin-clavulanate NR 0.5-2 0.12-O-5 “Adapted from NCCLS Mll-A2 (53). Results with media containing blood tend to be one twofold step higher and results from broth dilution tests one twofold step lower than above. ‘MIC in micrograms per milliliter. “NR, No MIC is recommended with this organism-antibiotic combination. dValues valid for AD reference method only; different results may be obtained -with other methods, media, or enrichments.

tests must demonstratethat each lot of agar or broth will support the growth of organisms,that antimicrobial agents have not deteriorated, and that the QC strains produce the expected results. Table 7 lists the acceptable MIC ranges that shouldbe received with the four QC strains. The reading of endpoints is one of the more variable stepsin susceptibility testing of anaerobes; therefore, this step needsparticular attention within each laboratory. It must also be rememberedthat BMD MICs may be one dilution higher than those received with AD. Because of the problems recently observed with the C. persringens QC strain with some of the newer antibiotics, a new strain, Eubacterium lent-urn ATCC 43055, is now recommended along with more specific guidelineson QC recommendations(53). If susceptibility tests are performed on a regular basisand consistency in test results of the reference organismshas been established,it seemssufficient to evaluate QC strains once weekly and possibly limit the strains to the type of organismsbeing tested (e.g., Bacteroides spp.). There are a number of clinical issuesthat need to be resolved so that a proper interpretation

may be placed on the laboratory result. In clinical laboratories, the frequency of conducting anaerobesusceptibility tests should be limited. In addition to the circumstancesdescribed above, other considerations that indicate that such testing is warranted include situations where the anaerobe is the sole isolate in a clinical specimen, is from specific specimens such as blood and normally sterile body fluids, and has a known resistance pattern or known mechanismof resistance(e.g., B. frasilis group) and asa meansof monitoring resistancepatterns within institutions. A number of drugs with known anaerobic activity are available (e.g., metronidazole, imipenem,and sulbactamor clavulanate combinations) and probably need testing only in broad surveillance programs. CLOSTRIDIA AND CLOSTRIDIAL TOXINS Clostridia are at the same time among the oldest and newest pathogensknown in medical history. Although the clinical features of tetanus were already described in great detail by Hippocrates, the role of C. di!ciZe in antibioticassociatedpseudomembranouscolitis was only elucidatedin 1977(8). Clostridia show numerous

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PRACTICAL TABLE

Bacteria

Tetanospasmin

c. botulinum

Tetanolysin Toxins A, B, C,, C,, D, E, F, G

C. pemngens

Alpha Beta Epsilon Iota Theta Kappa Enterotoxins

C. septicurn C. hktolyticum C. sordellii

Alpha Gamma Epsilon Alpha Alpha Beta Alpha LT

C. bijknentans C. difficile

BACTERIOLOGY

13

8. Important clostridial toxins

Toxin(s)

C. tetani

C. novyi

ANAEROBIC

A B

properties that set them apart from other anaerobes and warrant specialconsideration. Clostridia are gram-positive organismsbased upon cell wall structure; however, Gram stainsof older cultures often appearto be gram negative. They are capableof forming spores;this allows them to survive in the presenceof oxygen or in a bacteriologicallyhostile environment. Hence, unlike other anaerobic organisms,they are found ubiquitously in nature, e.g., in the soil. Some speciesare rather aerotolerant (e.g., C. per+ gens and C. tertium); in thesecases,the catalase test is usually helpful in discrimination(Clostridium spp. = negativeandBacillus spp. = positive). A number of well-identified toxins are of major importancefor the pathogenic@of clostridia (Table 8). Therefore, identification of specific toxins in clinical materialsis often more important than cultivation of infecting organisms.

Mode of action

Inhibition of inhibitory interneurons causes spastic paralysis Hemolysin Pathogenic for humans: A, B, E, F, inhibition of release of acetylcholine causes flaccid paralysis Lecithinase C, activated by Ca*+ and M$+, lyses cell membranes and thus causes necrosis, hemolysis Necrotizing Necrotizing, permease Necrotizing (Qto-) hemolysin Collagenase, gelatinase Inhibition of transport mechanisms?, cytotoxic Necrotizing, altering permeability of vessels Lecithinase that is necrotizing, hemolysing Lipase Necrotizing, hemolysing Similar to C. septicum alpha Necrotizing, edema forming Similar to C. pe@zgem alpha Equivalent to C. di’ile A Equivalent to C. diflcik B Similar toxins as in C. sordellii Enterotoxin Cytotoxin

from incompletely cooked food that is contaminated with the toxin or the organismsor both (food-borne botulism), during a wound infection with C. botulinurn (wound botulism), or as a result of inadequate development of normal bowel flora (infant botulism). Tetanus develops as a consequenceof a wound infection (often minor) with C. tetani alone in mixed infection. The tetanus toxin is generated at the site of injury and distributed throughout the body tissues.The toxins of both organismsare similar in structure and primarily affect the nervous system. Botulinurn toxin interferes with the release of acetylcholine at the myoneural junction and thus resultsin muscleparalysis. Airway obstruction and respiratory paralysis finally cause the death of the patient. In contrast, tetanus toxin interferes with the action of inhibitory interneurons, thus causing muscle spasticity. Respiratory and cardiac disturbances may lead to the Neurotoxic Clostridia (Botulism, Tetanus) death of the patient. Botulism and tetanus are well-defined clinical Adequate specimens for demonstrating the entities that are causedby C. botulinum and C. presence of toxins include serum, specimens tetani, respectively. The clinical manifestations obtained from a debridedwound, and in the case are determinedby the action of singleneurotox- of suspected botulism, gastric washings, stool ins produced by these organisms.C. botulinurn (infant botulism), and incriminated food. Specistrains may elaborate eight serologically differ- mens should be collected prior to therapy with ent toxins designatedA, B, Ca, C,, D, E, F, and specific antisera and refrigerated until testing is G, of which A, B, E, and F are found in human arranged. The classic method of demonstrating disease.The heat-labile toxin may be acquired clostridial neurotoxins calls for inoculation of

14

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mice with clinical specimens. The animals develop characteristic symptoms such as spasticity of extremities and convulsions (tetanus) or gasping breathing (botulism) prior to their death. Specificity of the test is ensured by administering an appropriate antitoxin to control mice that are subsequently protected from disease. Since most laboratories are not experienced with these test procedures, state or local health departments or CDC should be contacted. Alternative methods have been described (enzyme immunoassay) but do not seem to be equally sensitive. Culture of the infecting organisms may be attempted but is often less reliable. If the clinical diagnosis is highly suggestive of the diseases discussed here (especially in the case of tetanus), animal tests should be avoided. Histotoxic Clostridia (Gas Gangrene) Gas gangrene is a clinical syndrome that is often caused by histotoxic clostridia such as C. pe#n..ens, C. novyi, C. septicum, and others. Clostridial myonecrosis is a severe disease rapidly leading to the death of the affected patient. The organisms produce a variety of histotoxins, of which the most prominent are listed in Table 8. These toxins are responsible for the gross tissue destruction caused by the clostridia. While the therapy of clostridial myonecrosis calls for extensive surgical intervention, other etiologic agents of gas gangrene-like infections (e.g., streptococci, E. coli, and B. fragilis) are generally more amenable to antibiotic therapy and require less intensive surgery. Hence, a fast presumptive diagnosis, by Gram stain of appropriate material, of the etiologic agents should be attempted prior to surgical intervention. Smears should be viewed carefully, bearing in mind that mixed infections (with staphylococci, streptococci, members of the Enterobacteriaceae, and other anaerobes) are common. Culture, identification, and testing of histotoxic clostridia follow the principles outlined above with the proviso that, in general, they are fast-growing organisms and thus primary cultures may be read for the first time after 12 h of incubation. Enterotoxic Clostridia Antibiotic therapy is often accompanied by gastrointestinal disorders of variable severity. Selection and overgrowth of C. di$kiZe have been found to be responsible in the majority of the severe cases, including pseudomembranous enterocolitis. Two antigenically distinct toxins (A and B) have been described and clinical symptoms are attributed to these toxins. In most cases, the clinical presentation of the patient will initiate laboratory consultation. However, it might be helpful to monitor some patients who require longer courses of antibiotic therapy

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(e.g., bone marrow transplant patients) regularly (once per week) for these organisms and their toxins. An agar medium containing cefoxitin and cycloserine is suitable for selective culture of C. dificile, and GLC is especially helpful in identifying this organism. Nontoxigenic C. dificile strains may be part of the gut flora; therefore, demonstration of toxin B production is mandatory. This may be achieved by testing fluid medium in which the organism was grown for at least 24 h or by employing fecal material directly. Serially diluted, sterile filtered specimens are placed onto cell cultures in tubes or microtitration plates (e.g., McCoy cells or human embryonic fibroblasts) and cytotoxicity is recorded after 48 h of incubation. Specificity of the cytotoxin should be demonstrated by neutralization of the effects with antibodies (raised against C. sordellii toxin). If toxin cannot be demonstrated after the initial 24 h of incubation, the test should be repeated 4 to 7 days later. Commercially prepared agglutination tests are available. These tests detect antigens common to both cytotoxigenic and noncytotoxigenic strains of C. dicfficile as well as to other species of clostridia and other bacterial genera. Recent controversy involving the identity of antigens detected by the latter methods has made decisions on their use difficult (10). Recently, enzyme-linked immunosorbent assay (ELISA) tests for detection of C. difcile toxin have been introduced (Meridian Diagnostics, Cincinnati, Ohio; Baxter-Bartels Diagnostic Division, Bellevue, Wash.; Techlab, Inc., Blacksburg, Va. [distributed by Whittaker Bioproducts, Inc., Walkersville, Md.]; Cambridge Bioscience, Worcester, Mass.). Preliminary findings indicate that the ELISA test is a reliable non-tissue-culture-based detection method (75). CDC reports indicate that the third most common cause of food poisoning is the ingestion of food contaminated with large amounts of C. perfringens type A. The protein enterotoxin of this organism is a structural component of the spores and is elaborated in the process of sporulation (Table 8). At present, commercially prepared test kits for detection of the enterotoxin are under evaluation. CONCLUDING REMARKS Today, the technology available to clinical laboratories is sufficient to allow detection of even the more fastidious anaerobic organisms involved in clinical infections. While some anaerobes are relatively aerotolerant and others may be extremely sensitive to oxygen, the oxygen sensitivity of most species is unknown. Therefore, all diagnostic procedures, such as collection of the specimen, transport of the material, isolation of the organisms in the labo-

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ratory, identification, and susceptibility testing of the anaerobic bacteria, should always be done with the aim of minimizing exposure to air. The use of modern technology for the detection and characterization of anaerobes significantly increases our diagnostic capabilities for identifying the etiology of clinical infections. However, results obtained with this technology often require special attention to ensure the appropriate appreciation by many practicing clinicians. While the pathogenic potential of many clostridia, including the more recently recognized toxigenic C. dificile, is now well understood, work on the pathogenic mechanismsof membersof the Bac~eroidaceae is making slow progress and very little is known about the pathogenicity of anaerobic cocci. The correct identification of anaerobic organismsas agents of infectious diseasewill further increase the awarenessof such infections and thus be directly beneficial for affected patients.

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