Listeria, Listeriosis, and Food Safety, Third Edition (Food Science and Technology)

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Cover photo: An immunofluorescent image of L. monocytogenes (red) showing cell-to-cell spread via polymerized actin tails (green). Photo courtesy of Dr. Pascale Cossart, Institut Pasteur, Paris.

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2007 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8247-5750-5 (Hardcover) International Standard Book Number-13: 978-0-8247-5750-2 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Listeria, listeriosis, and food safety / editors, Elliot T. Ryser and Elmer H. Marth. -- 3rd ed. p. ; cm. -- (Food science and technology ; 160) Includes bibliographical references and index. ISBN-13: 978-0-8247-5750-2 (hardcover : alk. paper) ISBN-10: 1-4200-1518-4 (hardcover : alk. paper) 1. Listeriosis. 2. Listeria monocytogenes 3. Foodborne diseases. 4. Food--Microbiology. I. Ryser, Elliot T., 1957- II. Marth, Elmer H. III. Title. IV. Series: Food science and technology (Taylor & Francis) ; 160. [DNLM: 1. Listeria Infections--etiology. 2. Food Contamination--prevention & control. 3. Food Microbiology. 4. Listeria monocytogenes--pathogenicity. W1 FO509P v.160 2007 / WC 242 L773 2007] QR201.L7R9 2007 615.9’52937--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

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IN MEMORIAM

After conceiving the idea for a third edition of Listeria, Listeriosis and Food Safety in June 2002, Elmer Marth and I were in biweekly contact by phone to ensure continued progress toward its completion. This daunting project involved 33 contributors, 3 of whom in addition to myself (Robert E. Brackett, Jeffrey L. Kornacki, and Ahmed E. Yousef) earned their doctorates in the laboratory of Elmer Marth. As the book was rapidly nearing completion in Spring 2006, I was deeply saddened to learn that Elmer had become seriously ill. On June 19, 2006, Emeritus Professor Elmer H. Marth died in Madison, Wisconsin at age 78 while I was editing the chapter proofs for the book. Consequently, it is only fitting that the third edition of Listeria, Listeriosis and Food Safety be dedicated in memory of Dr. Elmer H. Marth who grew up on a small dairy farm in Grafton, Wisconsin to become one of the most preeminent dairy microbiologists of our time.

Elliot T. Ryser

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Preface to the Third Edition Since the second edition of Listeria, Listeriosis and Food Safety was published in 1999, the United States has seen a 40% decrease in the incidence of listeriosis, with the current annual rate of illness rapidly approaching the 2010 target of 2.5 cases per million population. This reduction in the rate of listeriosis would not have been possible without the combined effort of researchers, academicians, commodity organizations, and various regulatory authorities. We would like to believe that the first and second editions of our book also played a role in this outcome by providing needed information to concerned persons. Despite considerable progress in understanding the sources, spread, control, and pathogenicity of Listeria monocytogenes, research on this foodborne pathogen has continued unabated, with more than 5,000 publications on Listeria and foodborne listeriosis appearing in the scientific literature since the second edition of this book was published. A portion of this work was fueled by a series of widely publicized outbreaks of listeriosis involving ready-to-eat meat products in the United States that began in the late 1990s. Over the last 5 years, increasing emphasis has been given to development of risk assessments that can be used to focus limited financial resources on certain high-risk foods, such as soft cheeses, ready-to-eat meats, and smoked fish, that support growth of the pathogen. Efforts ultimately have had an impact on public policies regarding allowable levels of L. monocytogenes in these and other foods. The third edition of Listeria, Listeriosis and Food Safety again summarizes much of the newly published literature and integrates this information with earlier knowledge to present readers with a complete and current overview of foodborne listeriosis. The 17 chapters in the second edition have been retained; all are updated and expanded as appropriate. A total of 33 authors have lent their expertise to preparing this book, with new authors contributing to Chapters 1, 2, 4, 6, 7, 8, 10, 13, and 17. Sometimes these authors have collaborated with the original authors to develop the revised chapter. Two new chapters, Chapter 18 and Chapter 19, have been added to the book. Chapter 18 deals with risk assessment, cost of foodborne listeriosis outbreaks, and regulatory control of the Listeria problem in various countries. In Chapter 19, four experts point out specific data gaps and where, in their view, research efforts should be directed. As was true for the earlier editions, this book will be useful to advanced undergraduate students in food science, microbiology, and public health; graduate students in these same disciplines; and practitioners in food or dairy microbiology, food or dairy science, bacteriology or microbiology, public health, epidemiology, risk assessment, meat science, animal science, and veterinary medicine. It will also be helpful to personnel in the food and dairy industries and the food service industry, and to researchers in industrial, governmental, and university laboratories. Elliot T. Ryser Elmer H. Marth

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Preface to the Second Edition Listeriosis and Listeria monocytogenes continue to be of worldwide interest to the food industry and regulatory agencies, scientists in various disciplines, and consumers of food. Such interest is prompted by the occasional appearance of L. monocytogenes in ready-to-eat foods, leading to the removal of these products from the marketplace. Furthermore, sporadic cases of listeriosis continue to occur and several food-associated outbreaks of the disease have occurred since the first edition of this book was published. Scientists in several disciplines are still studying different aspects of the listeriosis problem. Their efforts have resulted in the development of much new information that has appeared in hundreds, if not thousands, of papers published since the first edition of this book was completed in 1990. This explosion of information warranted publication of a second edition. The second edition differs markedly from the 1991 edition. Whereas we were the authors of the earlier edition, chapters in this edition have been prepared by various experts in the field. We now serve as editors, although one of us (ETR) has revised several chapters. The contributions of this edition’s authors have resulted in an improved book that contains timely topics. The chapters in the first edition have been retained; each has been revised and expanded with new information when appropriate. Two new chapters deal with typing methods and pathogenesis. Thus, this book contains 17 chapters addressing the following topics: description of L. monocytogenes occurrence and behavior of this pathogen in various natural environments animal and human listeriosis pathogenesis of L. monocytogenes characteristics of L. monocytogenes important to food processors conventional and rapid methods to isolate, detect, and identify L. monocytogenes strain-specific typing of L. monocytogenes foodborne listeriosis incidence of behavior of L. monocytogenes in unfermented and fermented dairy products, meat, poultry (including eggs), fish and seafood, and products of plant origin incidence and control of this pathogen within various types of food-processing facilities This book will be useful to advanced undergraduate students, graduate students, and practitioners in food or dairy microbiology, food or dairy science, bacteriology or microbiology, public health, dietetics, meat science, poultry science, and veterinary medicine. It also will be helpful to personnel in the food and dairy industries and regulatory agencies, as well as researchers in industrial, governmental, and university laboratories. Elliot T. Ryser Elmer H. Marth

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Preface to the First Edition Interest in the occurrence of Listeria in food, particularly Listeria monocytogenes, escalated rapidly during the 1980s and continues unabated as a result of several major outbreaks of foodborne listeriosis. The first of these occurred during 1981 and involved consumption of contaminated coleslaw. In 1983, the reputation of the American dairy industry for producing safe products suffered when epidemiological evidence showed that 14 of 49 people in Massachusetts died after consuming pasteurized milk that was supposedly contaminated with L. monocytogenes. Two years later, consumption of contaminated Mexican-style cheese manufactured in California was directly linked to more than 142 cases of listeriosis, including at least 40 deaths. Heightened public concern regarding the prevalence of L. monocytogenes in food prompted the United States Food and Drug Administration to initiate a series of Listeria surveillance programs. Subsequent discovery of this pathogen in many varieties of domestic and imported cheese, in ice cream, and in other dairy products prompted numerous product recalls, which in turn have led to staggering financial losses for the industry, including several lawsuits. These listeriosis outbreaks, together with a subsequent epidemic in Switzerland involving consumption of Vacherin Mont d’Or soft-ripened cheese and discovery of L. monocytogenes in raw and ready-to-eat meat, poultry, seafood, and vegetables, have underscored the need for additional information concerning foodborne listeriosis. In 1961, Professor H. P. R. Seeliger, now retired from the University of Würzburg, published his time-honored book, Listeriosis. His monograph has provided scientists, veterinarians, and the medical profession with much needed information regarding Listeria and human and animal listeriosis, as well as pathological, bacteriological, and serological methods to diagnose this disease. However, documented cases of foodborne listeriosis were virtually unknown 30 years ago. Although much information in his book is still valid today, some of the knowledge regarding media and methods used to isolate, detect, and identify L. monocytogenes in clinical and, particularly, nonclinical specimens is now largely out of date. The emergence of L. monocytogenes as a serious foodborne pathogen together with the virtual flood of Listeria-related papers that have appeared in scientific and trade journals as well as numerous conference proceedings, prompted us to review and summarize the current information so that food industry personnel, public health and regulatory officials, food microbiologists, veterinarians, and academicians have a ready source of information regarding this now fully emerged foodborne pathogen. This book consists of 15 chapters that address the following topics: L. monocytogenes as the causative agent of listeriosis occurrence and survival of this pathogen in various natural environments human and animal listeriosis characteristics of L. monocytogenes important to food processors conventional and rapid methods for isolating, detecting, and identifying L. monocytogenes in food recognition of cases and outbreaks of foodborne listeriosis incidence and behavior of L. monocytogenes in fermented and unfermented dairy products, meat, poultry (including eggs), seafood, and products of plant origin incidence and control of this pathogen within various types of food-processing facilities

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It is evident that major emphasis has been given to information directly applicable to food processors. Information concerning the bacterium and the disease has been admirably reviewed by Professor Seeliger and others, so our discussion of these topics should not be considered exhaustive. Thus, the first four chapters of this book supply only pertinent background information to complement our discussion of foodborne listeriosis. Although many in the scientific community must be commended for the extraordinary progress made since 1985 toward understanding foodborne listeriosis, the continuing “explosion” of information concerning Listeria and foodborne listeriosis has made the 3-year task of compiling an upto-date review of this subject quite difficult. Therefore, to produce as current a document as possible, we have included a bibliography of references that have appeared since the book was completed. We acknowledge with gratitude the many investigators whose findings made this book necessary and possible. Special thanks go to individuals who shared unpublished information with us so that we could make the book as up to date as possible. Our thanks also go to the scientists who provided photographs or drawings; each person is acknowledged when the appropriate figure appears in the book. We thank Barbara Kamp, Pat Gustafson, Beverly Scullion, and Judy Grudzina for typing various parts of the manuscript. Illustrations were prepared by Jennifer Blitz and Suzanne Smith; their help is acknowledged and appreciated. Special thanks to Dr. Ralston B. Read, Jr., former director of the Microbiology Division of the Food and Drug Administration and now deceased, who in 1984 encouraged development of a research program on foodborne Listeria at the University of Wisconsin–Madison, and to Dr. Joseph A, O’Donnell, formerly with Dairy Research, Inc. and now director of the California Dairy Foods Research Center, for his early interest in and support of research on behavior of L. monocytogenes in dairy foods. Research done in the Department of Food Science at the University of Wisconsin-Madison and described in this book was supported by the U.S. Food and Drug Administration; National Cheese Institute; the National Dairy Promotion and Research Board; the Wisconsin Milk Marketing Board; Kraft, Inc.; Carlin Foods; Chr. Hansen’s Laboratory, Inc.; the Aristotelian University of Thessaloniki, Greece; the Cultural and Educational Bureau of the Egyptian Embassy in the United States; the Malaysian Agricultural Research and Development Institute; the Korean Professors Fund; and the College of Agricultural and Life Sciences, the Center for Dairy Research, and the Food Research Institute—all of the University of Wisconsin. We thank these agencies for their interest in and support of research on L. monocytogenes. Our book is dedicated to all persons who have contributed to a better understanding of foodborne listeriosis so that control of this disease is facilitated. Elliot T. Ryser Elmer H. Marth

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Editors Elmer H. Marth, Ph.D., (1927–2006), a native of Jackson, Wisconsin, was emeritus professor of food science and bacteriology at the University of Wisconsin–Madison. He earned his B.S. (1950), M.S. (1952), and Ph.D. (1954) from the University of Wisconson–Madison in bacteriology with an emphasis on food and dairy bacteriology. After 3 years as instructor of bacteriology, he joined the R&D Division of Kraft Foods in Geneva, Illinois, in 1957. He rose through the ranks and in 1966 was named associate manager of the microbiology laboratory. Also in 1966, Dr. Marth returned to the University of Wisconsin–Madison as associate professor of food science with joint appointments in bacteriology and food microbiology and toxicology. He was promoted to professor in 1971 and, upon retirement in 1990, was named emeritus professor. In 1981, Dr. Marth was a visiting professor at the Swiss Federal Institute of Technology in Zürich. From 1967 to 1987, he was editor of the Journal of Food Protection. At the University of Wisconsin–Madison, Dr. Marth taught courses in food sanitation, food fermentations, farm bacteriology, and writing scientific reports; he lectured in seven other courses and in five short courses. His research program included studies on food spoilage, food fermentation, and foodborne disease organisms, including Listeria monocytogenes. During his career, he was the author, co-author, editor, or co-editor of more than 660 scientific publications, including research papers, review papers, books, chapters in books, patents, and abstracts of papers given at meetings of professional organizations. Dr. Marth served as major professor for 32 students who received M.S. degrees and 32 students who earned Ph.D. degrees; in addition, he supervised 17 postdoctoral researchers who worked in his laboratory. Dr. Marth was named a fellow of the Institute of Food Technologists (IFT) (1983), the International Association for Food Protection (IAFP) (1998), and the American Dairy Science Association (ADSA) (1998). He was also a member of the American Society for Microbiology and the Council of Science Editors. From the ADSA he received Pfizer (1975), Dairy Research Foundation (1980), Borden (1986), and Kraft Teaching (1988) awards. The IAFP honored him with Educator (1977), Citation (1984), Honorary Life Member (1987), and NFPA Food Safety (2000) awards. The IFT presented him with the Nicolas Appert (1987) and Babcock–Hart (1989) awards. In 2002, the Institute for Scientific Information designated Dr. Marth as a highly cited researcher, worldwide, in the agricultural sciences. The National Cheese Institute presented its highest honor, the Laureate Award, to Dr. Marth in 2004. Elliot T. Ryser, Ph.D., a native of Milwaukee, Wisconsin, is an associate professor in the Department of Food Science and Human Nutrition and the National Food Safety and Toxicology Center at Michigan State University. He earned his B.S. (1979) in biology from Carroll College, Waukesha, Wisconsin, and his B.S. (1980) in bacteriology, and M.S. (1982) and Ph.D. (1990) in food science, from the University of Wisconsin–Madison, with an emphasis on microbial safety of food and dairy products. Following a 1-year appointment as a research scientist at Institute National de la Recherche Agronomique, Station de la Recherches Laitieres, Jouy-en-Josas, France, Dr. Ryser joined Silliker Laboratories Group, Inc. in Chicago Heights, Illinois, where he worked for 2 years as a research project manager. In 1994 he left Silliker and began his academic career as a research associate in the Department of Animal and Food Sciences at the University of Vermont, working in the laboratory of Dr. Catherine Donnelly. Dr. Ryser joined Michigan State University as an assistant professor in 1998 and was promoted to associate professor in 2004. He teaches courses on foodborne diseases, food safety, and HACCP.

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Dr. Ryser’s research program is focused on the incidence, survival, transfer, and eradication of Listeria monocytogenes and other foodborne pathogens from various foods, including dairy products. He has authored, co-authored, or co-edited more than 140 scientific publications, including research papers, review papers, books, chapters in books, patents, and abstracts of work presented at professional meetings. He has served thus far as the major professor for four Ph.D. and six M.S. students and supervised the work of four postdoctoral researchers. Dr. Ryser is a member of the International Association for Food Protection, Institute of Food Technologists, American Society for Microbiology, and American Dairy Science Association. He received the National Milk Producers Federation Richard M. Hoyt Award in 1988. In addition, he served as a scientific editor for the Journal of Food Science from 2000 to 2005 and is currently a scientific editor for the Journal of Food Protection.

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Contributors Robert E. Brackett Centers for Food Safety and Applied Nutrition U.S. Food and Drug Administration College Park, Maryland Christopher R. Braden Enteric Diseases Epidemiology Branch Division of Foodborne, Bacterial and Mycotic Diseases National Center for Zoonotic, Vectorborne and Enteric Diseases Centers for Disease Control and Prevention Atlanta, Georgia Byron F. Brehm-Stecher Department of Food Science and Human Nutrition Iowa State University Ames, Iowa Carmen Buchrieser Laboratoire de Genomique des Microorganismes Pathogenes Institut Pasteur Paris, France Catherine W. Donnelly Department of Nutrition and Food Science University of Vermont Burlington, Vermont Mel W. Eklund U.S. National Marine Fisheries Service (retired) Mel Eklund and Associates, Inc. Seattle, Washington Jeffrey M. Farber Microbiology Research Division Bureau of Microbial Hazards Food Directorate Health Canada Banting Research Centre Ottawa, Ontario, Canada

Werner Goebel Department of Microbiology University of Würzburg Würzburg, Germany Lewis M. Graves Enteric Diseases Laboratory Response Branch Division of Foodborne, Bacterial and Mycotic Diseases National Center for Zoonotic, Vectorborne and Enteric Diseases Centers for Disease Control and Prevention Atlanta, Georgia Joshua Gurtler Department of Food Science University of Georgia Griffin, Georgia Susan B. Hunter Coordinating Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia Karen C. Jinneman Seafood Products Research Center, Pacific Regional Laboratory—Northwest Office of Regulatory Affairs U.S. Food and Drug Administration Bothell, Washington Eric A. Johnson Departments of Food Microbiology and Toxicology and Bacteriology University of Wisconsin–Madison Madison, Wisconsin Sophia Kathariou Department of Food Science North Carolina State University Raleigh, North Carolina Jeffrey L. Kornacki Kornacki Microbiology Solutions, LLC McFarland, Wisconsin

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Michael Kuhn Biozentrum University of Würzburg Würzburg, Germany

Brian D. Sauders Department of Food Science Cornell University Ithaca, New York

Beatrice H. Lado Nestle Research and Development Centre Shanghai Ltd., China

Chris Scherf National HIV and Retrovirology Laboratory Public Health Agency of Canada Ottawa, Ontario, Canada

Elmer H. Marth University of Wisconsin–Madison Madison, Wisconsin

Laurence Slutsker Foodborne and Diarrheal Diseases Branch Centers for Disease Control and Prevention Atlanta, Georgia

Dawn M. Norton Enteric Diseases Epidemiology Branch Division of Foodborne, Bacterial and Mycotic Diseases National Center for Zoonotic, Vectorborne and Enteric Diseases Centers for Disease Control and Prevention Atlanta, Georgia David G. Nyachuba Department of Food Science University of Massachusetts Amherst, Massachusetts Franco Pagotto Listeriosis Reference Service Bureau of Microbial Hazards Health Products and Food Branch Health Canada Ottawa, Ontario, Canada John Painter Foodborne and Diarrheal Diseases Branch Centers for Disease Control and Prevention Atlanta, Georgia Jocelyn Rocourt Institut Pasteur Paris, France Elliot T. Ryser Department of Food Science and Human Nutrition Michigan State University East Lansing, Michigan

Bala Swaminathan Foodborne and Diarrheal Diseases Branch Division of Bacterial and Mycotic Diseases National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia Ewen C.D. Todd National Food Safety and Toxicology Center Michigan State University East Lansing, Michigan R. Bruce Tompkin Food Safety Consultant LaGrange, Illinois Marleen M. Wekell Center for Veterinary Medicine U.S. Food and Drug Administration Laurel, Maryland Irene V. Wesley Enteric Diseases and Food Safety Research Unit National Animal Disease Center Agricultural Research Service, USDA Ames, Iowa Martin Wiedmann Department of Food Science Cornell University Ithaca, New York Ahmed E. Yousef Department of Food Science and Technology The Ohio State University Columbus, Ohio

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Contents Chapter 1 The Genus Listeria and Listeria monocytogenes: Phylogenetic Position, Taxonomy, and Identification.............................................................................................................1 Jocelyn Rocourt and Carmen Buchrieser Chapter 2 Ecology of Listeria Species and L. monocytogenes in the Natural Environment..........................21 Brian D. Sauders and Martin Wiedmann Chapter 3 Listeriosis in Animals ......................................................................................................................55 Irene V. Wesley Chapter 4 Listeriosis in Humans ......................................................................................................................85 John Painter and Laurence Slutsker Chapter 5 Molecular Virulence Determinants of Listeria monocytogenes ....................................................111 Michael Kuhn and Werner Goebel Chapter 6 Characteristics of Listeria monocytogenes Important to Food Processors...................................157 Beatrice H. Lado and Ahmed E. Yousef Chapter 7 Conventional Methods to Detect and Isolate Listeria monocytogenes .........................................215 Catherine W. Donnelly and David G. Nyachuba Chapter 8 Rapid Methods for Detection of Listeria .....................................................................................257 Byron F. Brehm-Stecher and Eric A. Johnson Chapter 9 Subtyping Listeria monocytogenes ................................................................................................283 Lewis M. Graves, Bala Swaminathan, and Susan B. Hunter Chapter 10 Foodborne Listeriosis.....................................................................................................................305 Dawn M. Norton and Christopher R. Braden

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Chapter 11 Incidence and Behavior of Listeria monocytogenes in Unfermented Dairy Products .....................357 Elliot T. Ryser Chapter 12 Incidence and Behavior of Listeria monocytogenes in Cheese and Other Fermented Dairy Products .............................................................................................................405 Elliot T. Ryser Chapter 13 Incidence and Behavior of Listeria monocytogenes in Meat Products ........................................503 Jeffrey M. Farber, Franco Pagotto, and Chris Scherf Chapter 14 Incidence and Behavior of Listeria monocytogenes in Poultry and Egg Products ......................571 Elliot T. Ryser Chapter 15 Incidence and Behavior of Listeria monocytogenes in Fish and Seafood....................................617 Karen C. Jinneman, Marleen M. Wekell, and Mel W. Eklund Chapter 16 Incidence and Behavior of Listeria monocytogenes in Products of Plant Origin ........................655 Robert E. Brackett Chapter 17 Incidence and Control of Listeria in Food Processing Facilities .................................................681 Jeffrey L. Kornacki and Joshua Gurtler Chapter 18 Listeria: Risk Assessment, Regulatory Control, and Economic Impact ......................................767 Ewen C.D. Todd Chapter 19 Perspectives on Research Needs....................................................................................................813 Elmer H. Marth, Robert E. Brackett, R. Bruce Tompkin, Sophia Kathariou, and Ewen C.D. Todd Index ..............................................................................................................................................843

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The Genus Listeria and Listeria 1 monocytogenes : Phylogenetic Position, Taxonomy, and Identification Jocelyn Rocourt and Carmen Buchrieser CONTENTS History ................................................................................................................................................1 Phylogenetic Position of the Genus Listeria .....................................................................................3 Numerical Taxonomy ...............................................................................................................3 Chemotaxonomy.......................................................................................................................3 rRNA Sequencing.....................................................................................................................4 Whole Genome Sequencing .....................................................................................................4 Conclusion ................................................................................................................................4 Taxonomy of the Genus Listeria .......................................................................................................5 L. monocytogenes, L. ivanovii, L. welshimeri, and L. seeligeri ..............................................6 L. grayi (and “L. murrayi”)......................................................................................................7 L. denitrificans/Jonesia denitrificans .......................................................................................8 Present State of the Taxonomy of the Genus Listeria ............................................................8 Identification of Bacteria of the Genus Listeria................................................................................9 Genus Characteristics ...............................................................................................................9 Morphology ..................................................................................................................9 Culture ..........................................................................................................................9 Nutritional Requirements ...........................................................................................10 Metabolism and Biochemical Characteristics............................................................10 Species Identification..............................................................................................................10 Conclusion........................................................................................................................................12 References ........................................................................................................................................12

HISTORY Murray, Webb, and Swann first published a description of Listeria monocytogenes in 1926 [112]. Several earlier reports may have described Listeria isolation [62,156]; the most plausible is certainly that by Hulphers [73]. However, the authors of these reports did not deposit their isolates in a permanent collection, so no subsequent investigations or comparisons with further strains were possible. Murray and colleagues observed six cases of sudden death of young rabbits in 1924 in the animal breeding establishment of the Department of Pathology at Cambridge and many more in 1

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2

Listeria, Listeriosis, and Food Safety

the succeeding 15 months. The interesting characteristics presented by the disease and the increasing mortality prompted investigation. The authors wrote at that time [112]: Both the natural and the experimental disease have interesting and characteristic features and their consideration has forced us to the conclusion that the causative organism either has not been described previously, or has been inadequately described and so cannot be traced in the literature. In either case, we feel justified in naming it. Its salient character is the production of a large mononuclear leucocytosis. This is far the most important and most striking character we have discovered and we name the microorganism we shall describe in this paper “Bacterium monocytogenes.” The question of the generic name is more difficult and we have not succeeded in associating our organism with many other genera proposed in Bergey’s Manual of Determinative Bacteriology (1925). We propose for the present to use the undefined Bacterium ([...], for, if the present chaos is to be resolved and if the classification adopted by the American Society of Bacteriologists is to be improved, it will be achieved only by co-operation and with this end in view we cannot use the term Bacillus.

In 1927, during investigations of unusual deaths observed in gerbils near Johannesburg, South Africa, Pirie [130] discovered a new microorganism, agent of what he called “the Tiger River disease.” He named this new agent Listerella hepatolytica 1: “The causative organism is a Grampositive bacillus for which, from its most striking pathogenic effect, I propose the specific name hepatolytica, and the generic name Listerella, dedicating it in honour of Lord Lister, one of the most distinguished of those concerned with bacteriology whose name has not been commemorated in bacteriological nomenclature.”2 Discoverers Murray and Pirie [130] sent their strains to the National Type Collection at the Lister Institute in London. Dr. Leningham, the director, was struck by the similarity of the two microorganisms and put Murray and Pirie into contact. Because the identity was clear, they decided to call this bacterium Listerella monocytogenes [111,130,159]. However, in 1939, the Judicial Commission of the International Committee on Systematic Bacteriology rejected the generic name Listerella because it had previously been used for a mycetozoan in 1906 in honor of Arthur Lister (younger brother of Lord Lister) and for a species of foraminifer in 1933 in honor of Joseph Jackson Lister (father of Lord Lister). As noted by Gibbons in 1972 [54], it is certainly unique that the same name was chosen for three quite different groups of microorganisms to honor contributions of a father and his two sons. In 1940, Pirie proposed the name Listeria [129]. Before and even after this date, numerous names were used to designate L. monocytogenes: Bacterium monocytogenes hominis and later Listerella hominis by Nyfeldt, who considered that it was the agent of infectious mononucleosis [116,117] Corynebacterium parvulum by Schultz et al. in 1934 [154] Listerella ovis by Gill in 1937 [55] Listerella bovina, L. gallinaria, L. cunniculi, and L. gerbilli by Nyfeldt [117,118] Erysipelothrix monocytogenes by Wilson and Miles in 1946 [187] Corynebacterium infantisepticum by Potel (1951) during his first observations of fetal and neonatal listeriosis in Germany [132] Unlike some pathogenic agents responsible for large outbreaks that have marked the history of humans for centuries, the history of L. monocytogenes and listeriosis is recent: It begins officially in 1924. The first confirmed diagnosis in a human was that of a soldier suffering from meningitis at the end of World War I (retrospective identification of the strain [28]). Before this case, there are no validated observations. Interestingly, however, a historian has suggested that L. monocytogenes could have been the cause of Queen Anne’s 17 unsuccessful pregnancies [150].

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The Genus Listeria and Listeria monocytogenes

3

PHYLOGENETIC POSITION OF THE GENUS LISTERIA The relationships of Listeria to other bacteria remained obscure until the 1970s. Absent from the three first editions of Bergey’s Manual of Determinative Bacteriology published in 1923, 1925, and 1930, the genus Listeria was included in the tribe Kurthia of the Corynebacteriaceae family in the next edition in 1934. In the sixth and seventh editions (published in 1948 and 1957, respectively), Listeria was still a member of the Corynebacteriaceae; however, in the next edition (1974), it was considered a genus of uncertain affiliation and placed with Erysipelothrix and Caryophanon after the family of Lactobacillaceae [3–6]. Finally, Listeria was classified with Lactobacillus, Erysipelothrix, Brochothrix, Renibacterium, Kurthia, and Caryophanon in the section of “regular, nonsporing, Gram-positive rods” in Bergey’s Manual of Systematic Bacteriology [7]. How can these repeated reclassifications be explained? On the basis of morphological resemblances (Gram-positive, non-spore-forming rod), Listeria has long been associated with the coryneform group of bacteria. However, with the successive introduction and development of numerical taxonomy, chemotaxonomy, DNA/DNA hybridization, and, more recently, rRNA (ribosomal RNA) and DNA sequencing, the phylogenetic position of Listeria has been more clearly determined.

NUMERICAL TAXONOMY Numerical taxonomy provided the first attempts to investigate in depth the phylogenetic position of Listeria among Gram-positive bacteria. In the first studies, Listeria was included among coryneform bacteria and actinomycetes and, consequently, was located with the corynebacteria [13,31] or in an indefinite position [18,71]. In contrast, from 1969, more natural relationships were described when Listeria was compared to various representatives of lactic acid bacteria [33,172,173]. In 1975, the close relatedness with these microorganisms was clearly demonstrated by the broader numerical taxonomic survey of Jones, who studied 173 characteristics of 233 strains of various genera, including coryneform and lactic acid bacteria [79]. The refined position of Listeria was later investigated by Wilkinson and Jones in 1977 and Feresu and Jones in 1988 [43,186]. From these works, it became clear that Listeria is distinct from other known genera, including Erysipelothrix and Brochothrix thermosphacta (formerly Microbacterium thermosphactum) and is closely related to Lactobacillus and Streptococcus. Consequently, Wilkinson and Jones [186] suggested that Listeria, Gemella, Brochothrix, Streptococcus, and Lactobacillus be classified in the family Lactobacillaceae. In spite of some imprecision over the exact position with regard to the higher taxonomic relationships—especially with Brochothrix, certain lactobacilli, and Carnobacterium [43,186]—conclusions based on numerical analysis of data for large numbers of phenotypic features were precursors of the current phylogenetic classification of the genus Listeria.

CHEMOTAXONOMY A number of chemotaxonomic markers have been especially useful for solving the phylogenetic position of the genus Listeria, reinforcing its distinctness from coryneform bacteria and its relatedness to the lactic acid bacteria. The G + C percent DNA content of L. monocytogenes isolates ranges from 36 to 42% [43,142,172], indicating that Listeria belongs to the low G + C percent DNA content (100 CFU/g can easily be achieved by direct plating on selective and differential media, which allow specific detection of Listeria spp. (e.g., Oxford). On the other hand, specific enumeration of L. monocytogenes by direct plating is not practically feasible unless plating media are available that allow for specific detection of L. monocytogenes. Vazquez-Boland et al. [188] demonstrated that a method using direct plating on Listeria selective agar followed by 37- to 48-h incubation at 37°C with a subsequent overlay with blood agar allowed for quantification of hemolytic Listeria in silage samples. This method can thus be used to enumerate L. monocytogenes if the presence of hemolytic L. seeligeri and L. ivanovii can be excluded. More recently, several selective and differential chromogenic agars were developed to allow specific detection of L. monocytogenes. L. monocytogenes plating medium (LMPM; Biosynth) contains the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate to allow detection of phosphatidylinositol phospholipase C (PI-PLC) activity, which, among the Listeria spp., is unique to L. monocytogenes and L. ivanovii [157]. In combination with rapid biochemical tests for differentiation of L. ivanovii and L. monocytogenes (e.g., L. monocytogenes confirmation media, LMCM, Biosynth), this selective and differential medium thus provides a tool for more rapid quantification of L. monocytogenes by direct plating or MPN procedures.

LIMITATIONS AND PITFALLS OF CURRENT ENVIRONMENTAL CHARACTERIZATION METHODS FOR UNDERSTANDING THE ECOLOGY OF LISTERIA Despite the continued development of improved selective and sensitive isolation procedures for Listeria spp. and L. monocytogenes, culture-based methods show certain limitations that may affect our ability to understand the ecology of Listeria. There is considerable evidence that use of a single enrichment method may limit recovery of injured Listeria cells (if the medium is too selective) or may not allow for effective recovery of Listeria cells in the presence of high levels of other microorganisms (if less selective compounds are used). Although two-step enrichment procedures may ameliorate these problems to a certain extent, recovery of injured Listeria cells in samples with high levels of competing microorganisms represents a particular challenge. Furthermore, a putative viable but not culturable (VBNC) state was recently proposed for L. monocytogenes [21]; cells in this state are unlikely to be recovered by any currently used enrichment procedures. There also is considerable evidence that many environments as well as samples collected from these environments can contain multiple Listeria species and/or multiple Listeria strains [151,161,162]. For example, Ryser et al. [162] performed enrichment of ground beef, pork sausage, ground turkey, and chicken, followed by ribotyping of up to 10 isolates per sample. Over half of all positive samples contained more than one L. monocytogenes ribotype and the choice of enrichment medium affected the subtypes recovered from a given sample. In some instances, detection of certain L. monocytogenes ribotypes was only possible when 10 isolates from a sample were typed. This clearly shows the challenging nature of characterizing the Listeria microbial diversity using current detection methods. Although it is increasingly recognized that collection and characterization of multiple isolates may be necessary to evaluate the Listeria diversity in a given sample, enrichment methods appear to affect recovery of different Listeria species and strains selectively. In addition to the fact that L. innocua may outcompete L. monocytogenes during enrichment [51,126], it also appears that different enrichment media lead to recovery of different bacterial subtypes from the same sample [161]. Future application of molecular methods will provide an opportunity to improve our ability to probe and understand the ecology of Listeria in natural environments. Similar to commonly used 16S rDNA-based approaches to characterize bacterial population structures, Listeria- and/or L. monocytogenes-specific PCR primers [30,166,194] could be used to detect and characterize

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Listeria isolates and their diversity directly, including through construction and sequencing of clone libraries. The full power of these molecular methods can only be realized as sensitive, inexpensive, and rapid methods for isolation from natural environments of DNA suitable for PCR amplification are developed and applied [39,42,107,155,169].

MOLECULAR DETECTION

AND

SUBTYPING METHODS

Chapter 9 provides a comprehensive summary of subtyping methods for L. monocytogenes; application of molecular subtyping methods is critical to our ability to understand the ecology and transmission of L. monocytogenes in natural environments. Methods commonly used to subtype L. monocytogenes include ribotyping, pulsed-field gel electrophoresis (PFGE), and, more recently, multilocus sequence-based typing (MLST). Ribotyping [204,206] and PFGE [122,190] have been applied in studies of the diversity of L. monocytogenes in natural environments. These DNA-based methods define bacterial subtypes using restriction digestion of bacterial DNA to generate DNA fragment banding patterns. DNA fragment size-based subtyping methods have significant drawbacks. For example, despite the existence of software packages for data normalization and analyses (e.g., Bionumerics, Applied Maths, Sint-Martens-Latem, Belgium), these subtyping methods are often difficult to standardize. As a consequence, the ease of exchanging and comparing subtype data among laboratories can be severely limited. Although DNA fragment size-based subtyping methods have been used for cluster analyses, they generally do not provide information amenable to inference of primary genetic characteristics (i.e., nucleotide sequences) for evolutionary analyses. Long-term studies on the ecology and evolution of Listeria require subtyping data that can be used to infer and quantify the genetic relatedness of isolates, so DNA fragment size-based subtyping methods have limited utility for these applications. DNA sequencing-based methods are being developed and increasingly used for subtyping and characterizing L. monocytogenes [30,165]. With these methods, complete or partial nucleotide sequences are determined for one or more bacterial genes or chromosomal regions, thus providing unambiguous and discrete data. Sequencing can target a single gene (single locus approach) or multiple genes. Advantages of sequencing methods over DNA fragment-sized typing methods include their ability to generate unambiguous data that are portable through Web-based databases and that can be used for phylogenetic analyses [54]. Although a variety of DNA sequence-based subtyping strategies targeting virulence genes, housekeeping genes, or other chromosomal genes and regions is feasible, multilocus sequence typing (MLST), which is an extension of multilocus enzyme electrophoresis (MLEE), represents a widely used strategy [30,48,53,54]. Ultimately, MLST may lead to integration of PCR-based detection and subtyping in a single format, eliminating the need for culturing for certain applications [53]. Molecular subtyping methods have been widely used and developed for L. monocytogenes [78,202], but only limited information is available on molecular subtyping methods for Listeria spp. [154,176,193]. Further development, particularly of MLST-based subtyping methods, is necessary to allow comprehensive studies of the ecology of Listeria spp. in natural environments. Our knowledge of the transmission and ecology of Listeria spp. and L. monocytogenes has critically relied on application of classical and molecular subtyping methods for strain differentiation. For the purpose of studying the ecology of L. monocytogenes in the natural environment or otherwise, it is particularly important to bear in mind the limitations of all subtyping methods currently used for L. monocytogenes strain differentiation and also to consider the advantages and disadvantages of any specific subtyping method. As outlined later, subtyping data using a variety of different methods have, for example, shown that human disease-associated strains can also be found in farm and natural environments (see Figure 2.1 for examples); however, one must bear in mind the relative discriminatory power of different subtyping methods when interpreting these findings.

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A Location (city, state)

Ribotype

1985

Los Angeles, CA

DUP-1038B

Jul 1998

Cortland, NY

DUP-1038B

animal (bovine)

Jul 2001

Brooktondale, NY

DUP-1038B

animal (caprine)

Jan 2002

Burdette, NY

DUP-1038B

food (hummus)

Jun 1997

Albany, NY

DUP-1038B

Strain No.

Source

Date collected

FSL J1-119

human epidemic

FSL C1-057

human sporadic

FSL E1-128 FSL N3-080 FSL J1-179 FSL L4-170

processing plant drain

Oct 2002

Unknown, US

DUP-1038B

FSL N3-796

farm (water trough)

May 2002

Perry, NY

DUP-1038B

FSL S4-049

urban lake water

Jun 2001

Syracuse, NY

DUP-1038B

FSL S4-169

urban playground soil

Aug 2001

Rochester, NY

DUP-1038B

FSL S4-257

urban park A soil

Nov 2001

Albany, NY

DUP-1038B

FSL S4-628

urban park B soil

Jun 2002

Albany, NY

DUP-1038B

FSL S4-774

urban sidewalk

Aug 2002

Syracuse, NY

DUP-1038B

FSL S4-780

urban drain water

Aug 2002

Syracuse, NY

DUP-1038B

FSL S4-926

urban stream water

Oct 2002

Rochester, NY

DUP-1038B

FSL S6-096

urban park vegetation

Nov 2002

Albany, NY

DUP-1038B

FSL S6-116

urban sidewalk

Nov 2002

Albany, NY

DUP-1038B

FSL S6-136

urban runoff water

Nov 2002

Albany, NY

DUP-1038B

FSL S4-941

pristine vegetation

Oct 2002

Tompkins county, NY* DUP-1038B

Strain No.

Source

Date collected

Location (city, state)

Ribotype

FSL C1-103

human epidemic

Oct 1998

Troy, NY

DUP-1044A

FSL J1-193

human sporadic

Jun 1997

Manhasset, NY

DUP-1044A

FSL E1-054

animal (bovine)

Mar 1999

Unknown, NY

DUP-1044A

FSL N3-038

animal (caprine)

Oct 2001

Lake Ariel, PA

DUP-1044A

FSL F2-008

food (hispanic cheese)

Jul 1999

New York, NY

DUP-1044A

FSL H1-139

processing plant drain

Mar 2000

Unknown, US

DUP-1044A

FSL N3-243

farm (haylage)

Mar 2002

Cazenovia, NY

DUP-1044A

FSL S4-728

urban sidewalk

Jul 2002

Albany, NY

DUP-1044A

FSL S6-134

urban river water

Nov 2002

Albany, NY

DUP-1044A

Ribotype Pattern Will be run 4/15/03

B Ribotype Pattern

FIGURE 2.1 Ribotypes of Listeria monocytogenes isolated from epidemic and sporadic listeriosis, foods, foodprocessing environments, and the natural environment. Examples demonstrate that ribotypes that have caused human epidemic listeriosis are temporally and geographically widely distributed among animal cases of listeriosis, foods, food-processing environments, and the natural environment. Ribotype and associated isolate information were obtained from the PathogenTracker database (www.pathogentracker.net). (A) Ribotype DUP-1038B was implicated as cause of a human listeriosis outbreak in Los Angeles, California (1985), linked to consumption of Mexican-style cheese. (Linnan, M. J. et al., 1988, N. Engl. J. Med. 319:823–828.) (B) Ribotype DUP-1044A was implicated as the cause of a multistate human listeriosis outbreak in the United States (1998 to 1999) linked to consumption of contaminated hotdogs. (Anonymous, 1999, MMWR 47:1117–1118.) (*) = Sample collected from the Connecticut Hill Wildlife Management area; county location is provided because city was not applicable.

Although use of less discriminatory methods (e.g., MEE, single enzyme ribotyping) to characterize a given set of isolates may show that the same strains are found among isolates from human clinical cases and natural environments, application of more discriminatory subtyping methods (e.g., PFGE) may further differentiate strains. By no means does this make MEE or ribotyping data meaningless; rather, it indicates different levels of relatedness. We do not yet understand the specific levels of relatedness determined by each subtyping method. However, less discriminatory methods will generally indicate that two indistinguishable isolates may share a less recent common ancestor as compared to two indistinguishable isolates using a very discriminatory method. Subtype characterization by a less discriminatory method thus can still provide a very powerful tool to probe the distribution of different clonal groups (i.e., a group of genetically

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closely related isolates) and to differentiate broadly distributed clonal groups (e.g., clonal groups found in natural environments, among human and animal clinical cases and foods) from clonal groups that occupy a specific niche (e.g., cause disease in only a single specific host species). The power of less discriminatory subtyping methods has been demonstrated by the broad contributions of MEE studies to our understanding of bacterial population genetics [17,24,62,63, 113,142,160].

ECOLOGY OF LISTERIA SPECIES IN DIFFERENT ENVIRONMENTS Listeria spp. are generally considered ubiquitously distributed in the natural environment. Listeria spp. and L. monocytogenes have specifically been isolated from many different environments including soil [196], water [195], vegetation [196,199], sewage [3,4,34,67,195], animal feeds [32,58,60,121,162,175,188,190,203,205], farm environments [45,63,101,102,179,184,192], and food-processing environments [43,89,95,105,112,151,160]. Although many reports indicate that L. monocytogenes and L. innocua may represent the most common Listeria spp. in many natural environments, others have reported considerable prevalence of other Listeria spp. in specific natural environments. For example, a survey of an urban setting showed that L. ivanovii and L. seeligeri represented the most common Listeria spp. isolated from soil samples [127]. Cox et al. (1989) isolated L. monocytogenes and L. innocua from environmental samples collected from a sawmill; however, L. ivanovii represented the most prevalent Listeria spp. isolated [37]. Interestingly, although L. ivanovii, as well as L. welshimeri and L. seeligeri (both of which were not isolated in their study) ferment xylose, a common decomposition product of wood, L. monocytogenes and L. innocua do not ferment xylose. Further studies on the distribution of different Listeria spp. and subtypes in different natural environments will be needed to develop a true understanding of the ecology of Listeria and L. monocytogenes, including the reservoirs and niches of the different Listeria spp. and different clonal groups. This knowledge will also be necessary to understand transmission pathways of the pathogenic Listeria and the sources and spread of human and animal infections.

SURVIVAL AND MULTIPLICATION OF LISTERIA SPP. UNDER ENVIRONMENTALLY RELEVANT STRESS CONDITIONS L. monocytogenes is distinguished among foodborne pathogens in that it can tolerate high (up to 20%) salt concentrations, can multiply over a wide range of temperatures (1 to 45°C), and can adapt to and survive acid stress. In contrast to other non-spore-forming bacteria that cause foodborne illness, L. monocytogenes appears to be able to survive longer under adverse environmental conditions [195]. The ability of L. monocytogenes to colonize, multiply, and persist in the food-processing environment and on food-processing equipment likely also reflects its ability to survive in the natural environment for extended periods. Data available on stress resistance and survival characteristics of Listeria spp. are more limited than those available for L. monocytogenes. In general, it is hypothesized that other Listeria spp. show resistance to environmental stress (acid, salt, temperature, etc.) similar to that observed for L. monocytogenes because L. innocua and other Listeria spp. are commonly found in foods [9,31,149,209] and food-processing environments [152,181]. Experimental studies comparing stress resistance between L. monocytogenes and other Listeria spp., including survival and growth of temperature stress [147], salt stress [18], low water activity [140]; in the presence of various antimicrobial agents [183]; and under various combined stress conditions [114,156,163], also support that all Listeria spp. share similar stress resistance characteristics. Recent sequencing of the genome of one L. monocytogenes strain and one L. innocua strain [76] also showed that these two species share a large number of predicted genes that encode various

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components of potential stress response systems including regulatory proteins, transporters, and transcriptional regulators [29,76]. Further comparative genomic analyses on presence and transcription of various stress response genes in different Listeria spp. and subtypes will provide a unique opportunity to increase our understanding of the stress response pathways that facilitate Listeria survival in the environment and under stress conditions. Laboratory studies on survival under controlled environmental stress conditions can help to understand the biology of Listeria, but data on Listeria survival in natural environments also critically contribute to our understanding of Listeria transmission and ecology. The following sections attempt to summarize our current knowledge on distribution, prevalence, and survival of L. monocytogenes and Listeria spp. in different natural environments. In addition, Table 2.1 provides a summary of data from various studies on the persistence of L. monocytogenes in different natural and farm environments.

LISTERIA

SPP. IN

SOIL

AND

VEGETATION

L. monocytogenes as well as other Listeria spp. appears to be commonly present in soil and vegetation in the natural environment. Most studies on Listeria in the natural environment conducted to date have almost exclusively focused on farm environments and associated croplands. Interestingly, most studies on Listeria in natural environments not associated with farms indicate that prevalence of L. monocytogenes is lower than that of other Listeria spp. [127,166]. MacGowan et al. [127] specifically reported that L. seeligeri was more frequently isolated from soils than L. innocua or L. monocytogenes. In early work Weiss and Seeliger (1975) isolated Listeria spp. from plant samples collected from cornfields (9.7% of samples positive), grain fields (13.3%), cultivated fields (12.5%), uncultivated fields (44%), meadows and pastures (15.5%), forests (21.3%), and wildlife feeding areas (23.1%) in southern Germany [197]. Soil samples taken at a depth of 10 cm showed a significantly lower Listeria spp. prevalence as compared to surface soil samples. Although the original paper by Weiss and Seeliger (1975) reported L. monocytogenes prevalence, only 37 of 103 Listeria isolates elicited disease consistent with listeriosis in a mouse bioassay. This indicates that as many as 64% of these isolates may have been Listeria spp. other than L. monocytogenes or L. ivanovii, thus suggesting that these isolates represented a variety of Listeria spp. Welshimer and Donker-Voet [199] did not isolate L. monocytogenes from soil or dead vegetation sampled from agricultural sites in early autumn, yet soil and decayed vegetation sampled the following spring were nearly all positive for the organism. Listeria isolates from this study were also evaluated for virulence in mice and not all were found to be virulent [199], again suggesting the occurrence of species other than L. monocytogenes. Clearly, changes in the Listeria taxonomy that have occurred over the years may require careful interpretation of older studies. These studies may have used a broader definition of a L. monocytogenes sensu lato, which might include Listeria spp. other than those currently classified as L. monocytogenes. Only very limited recent data are available on the presence and distribution of L. monocytogenes and other Listeria spp. in soils, limiting our understanding of the soil ecology of these organisms [127]. More recently, Fenlon et al. [63] examined samples of grass, leaves, stems, and roots/stems from two crops of growing sward before harvesting. L. monocytogenes was not detected in any of the samples, but L. innocua and L. seeligeri were isolated from 3 of 10 samples from the root/stem area. L. monocytogenes was detected though in 9 of 10 samples of cut grass from the same crops that had wilted for 24 h before ensiling. Whittenbury [201] had demonstrated the importance of the sheath area as a source of lactic acid bacteria, so Fenlon [63] hypothesized that the higher incidence of L. monocytogenes in harvested (processed) grass compared with other plant products could be attributed to the presence of a sheath of decaying plant material at the base of the plant that might act as an inoculum at harvest. Although this process may contribute to contamination of grass with Listeria during the ensiling process, Listeria contamination of plant-based feeds and foods probably more likely results from direct deposition of animal feces, spreading of animal waste and sewage sludge as fertilizer, or from indirect contamination via feces-contaminated soil.

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TABLE 2.1 Survival of L. monocytogenes in Various Environmental Samples Sample Soil Sterile soil (I) Clay soil (I) Sealed tubes Fertile soil (I) Sealed tubes Cotton-plugged tubes Top soil (I) Exposed to sunlight Not exposed to sunlight Moist soil Dry soil Soil Soil

Storage Temperature (°C)

Survival (days)

Outside: winter/spring 24–26 24–26

154 225 67

24–26 24–26

295 67

NG NG NG NG 4–12 18–20

12 182 ~497 >730 240–311 201–271

5 Outside Outside Outside Summer Winter

182–2190 347 730 242 36 106

Sewage Sewage sludge cake (NC) Surface Interior Sprayed on field

28–32 48–56 Outside

35 49 >56

Water Sterilized pond water (I) Unsterilzed pond water (I) Pond water Pond water Pond water/ice Pond/river water Pond/river water Water Distilled water (I)

Outside Outside 35–37 15–20 2–8 37 2–5 Outside 4

7 6 months (OR = 0.34, P = 0.04) were protective factors. In contrast, use of Escherichia coli J5 vaccine (OR = 3.3, P = 0.03) was linked to higher incidence. Regional differences have been documented despite relatively low recovery rates (~5%) of L. monocytogenes in milk. Yet Dominguez-Rodriguez et al. [70] found L. monocytogenes in 45.3% of 95 raw milk samples from a single bulk tank (80,000-liter capacity). The dairy received raw milk from a number of small farms in western and central Spain over a 16-month interval. Seasonal distribution of L. monocytogenes in raw milk, mirroring numerous determinants including a change

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of diet or weather-related stress, has been observed. Lovett et al. [170] surveyed raw milk at various times from three different regions in the United States and found L. monocytogenes in 4.2% of the overall samples. However, the recovery of L. monocytogenes from Massachusetts samples was seasonal with the incidence highest during cooler months and lowest in hot-weather months. A more recent survey of 404 New York dairy farms revealed higher recoveries (odds ratio = 1.8) in spring (18%) than other seasons [115]. In the Pacific Northwest, prevalence varied from 4.7% in November to 7% in June. In that study, PFGE profiles suggested relatively stable clones, which may reflect selection and survival of strains that have adapted to the bovine host [198]. Interestingly, no seasonal trend was exhibited by samples recovered from Ohio, Kentucky, and Indiana [173]. A study of L. monocytogenes in raw milk in Nebraska, a state that experiences severe climatic changes, indicated a seasonal distribution with 6% of raw milk samples harboring L. monocytogenes in February and 2% of samples positive in July [164]. Seasonal variation may be related to silage feeding during the winter. L. monocytogenes was cultured from 4.9% of raw milk samples collected from 70 Irish farms with the incidence higher in the winter when cows were housed indoors than in the summer [224]. In Scotland, a seasonal distribution was indicated for L. monocytogenes, which was present overall on 25 premises of the 160 farms surveyed (16%). Contamination was sporadic, with bacterial titers generally less than one L. monocytogenes per milliliter. Although more raw milk samples were positive for L. monocytogenes in January than at other sampling dates throughout the year, the authors caution that no link to farm management practices was evident [84]. In Ontario, Canada, raw milk recoveries indicated seasonal and geographical differences, with the incidence higher in the eastern region [85,254]. Serosurveys have monitored distribution of L. monocytogenes in dairy cows [71,198]. Infection in cattle can be estimated by measuring antibodies to whole cells and antibodies specifically targeting antibodies to listeriolysin (LLO) [12,32,33] as well as internalin A [32]. Because up to 33% of dairy cattle continued to shed L. monocytogenes despite high serological titers, antibody levels do not appear to modulate recovery of L. monocytogenes from milk [71,257,276]. As in humans [26], sheep [13,163,174], and goats [193], antibodies to listeriolysin O (LLO) have confirmed previous or current infection with L. monocytogenes in cattle [12,32,33]. When antibody titers to LLO and internalin A were correlated with dairy farm practices, a positive correlation was found between feeding corn silage (OR = 6.5). In contrast, use of rubber bedding mats (OR = 0.2) and an electrical device to train cows to defecate and urinate off the bedding area (OR = 0.36) were correlated with low LLO and internalin A titers, suggesting low exposure to L. monocytogenes. Serum levels also imply differences in breed susceptibility [32]. Stress-related immunosuppression associated with change of diet, weather, transportation [83], pregnancy, parturition, and lactation may lower resistance to bovine listeriosis [239]. Dexamethasone mimics the stress-related release of glucocorticoids. In cattle, dexamethasone elevates total white blood neutrophil counts and decreases eosinophil and lymphocyte populations. When administered to cows experimentally infected with L. monocytogenes, dexamethasone increased the shedding by up to 100-fold of L. monocytogenes in milk [291]. The increased levels of L. monocytogenes in the milk may reflect impairment of cell-mediated immune mechanisms and phagocytic cell functions that underlie listerial immunity [269]. Likewise, transport of live animals over long distances may increase the level of fecal excretion of L. monocytogenes. A major concern of bovine listeriosis is the potential risk posed to humans. In Denmark, a case-control study indicated that human listeriosis was frequently linked to consumption of unpasteurized milk (risk factor of 8.6), although other factors, such as immunosuppression and underlying diseases, were regarded as more significant [137]. Furthermore, a comparison of 33 isolates from bovine mastitis and 27 human clinical isolates in Denmark recovered during 1993 was made by sero- and ribotyping. Serotyping showed that all bovine and 63% of human isolates belonged to serogroup 1, whereas 37% of the human isolates were of serogroup 4. DNA fingerprinting by ribotyping indicated that a low but constant percentage of Danish dairy herds have cows infected

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with L. monocytogenes strains similar to human clinical strains [141]. L. monocytogenes ribotypes common to dairy processing and the farm environment (dairy cattle, raw milk, silage) were also reported in the United States. This indicated that the farm may serve as a reservoir for L. monocytogenes strains capable of entering the dairy-processing facility [8]. Alternatively, it verifies the ubiquitous distribution of the pathogen. Although foodborne listeriosis in humans is more frequently linked to consumption of contaminated dairy products than to beef consumption, L. monocytogenes was recovered from 3% of composite fecal samples representing 224 feedlot beef cattle [249]. In a limited study of experimentally infected Holstein cows (n = 4), L. monocytogenes was cultured from muscle, organ, and lymphoid tissues at 2 days after infection; none was recovered at 6 or 54 days after inoculation [142]. This indicates that cattle may be an insignificant source of L. monocytogenes contamination in meat [168]. Epizootics have been observed in feedlot cattle and in beef cattle herds [5,301]. Transport of cattle over long distances increased the level of fecal excretion of L. monocytogenes, but not contamination of carcasses [83]. Yet in this study L. monocytogenes was detected in 91% (21 of 23) of minced beef samples, again demonstrating that postharvest processing significantly increases the level of contamination compared to that of the whole carcass [83]. This hypothesis is further strengthened by tracking L. monocytogenes strains by multilocus enzyme electrophoresis. L. monocytogenes strains of electrophoretic type (ET) I have been implicated in major human foodborne epidemics and are found coincidentally in livestock. L. monocytogenes strains of ET I predominate in cattle at the beginning of slaughter, but are not detected on the carcasses at the end of processing or in the environment of the abattoir [31]. In contrast, environmental strains, such as ET 19, contaminate the carcass during processing. Likewise, ET 19 strains were found on the carcasses of pigs at the end of processing in two slaughterhouses, but not on live animals or at the beginning of slaughter. Taken together, these studies indicate that contamination of meat during processing occurs from L. monocytogenes strains that reside in the packing plant rather than from strains indigenous to animals [31].

SWINE Porcine listeriosis manifests primarily as septicemia. Encephalitis is reported less frequently and abortions are rare [29]. Clinical septicemia is usually observed in the neonate where hepatic necrosis may be a characteristic feature [112,191]. Unlike its frequent occurrence in ruminants (cattle, sheep, and goats), listeriosis is rare in monogastric swine. Slabospits’Kii [253] first reported Listeria infection in young swine raised on a Russian farm and designated the organism as L. suis [29]. The first description of porcine listeriosis in the United States occurred when Biester and Schwarte [28] reported it in Iowa in swine with encephalitis. Later, Kerlin and Graham [150] recovered Listeria from the liver of a pig with no clinical signs of encephalitis. In Norway, Hessen [121] reported listeric septicemia in piglets raised on a farm where sheep had died of listeriosis several weeks earlier. Whether transmission was from the sheep to the pigs or the result of common exposure is unknown. In natural and experimental infections, listeriosis is more severe in young animals [30,42,148]. Piglets succumb to infection whereas adults generally survive. In the neonate, L. monocytogenes may originate from the tonsils of the sow, penetrate the intestinal tract of the piglet, and become systemic [267]. Neonatal listeriosis may be seasonal with cases peaking in early winter [169] and spring. Listeric encephalitis seldom occurs in pigs. Symptoms of central nervous system disturbance, including incoordination and progressive weakness followed by death, are characteristic of listeriosis of the younger animal. Meningoencephalitis in swine begins with a sudden refusal to eat and is typically followed by various neurological disorders including trembling, partial paralysis, incoordination, circling movements, and convulsions. Histopathological findings from meningoencephalitis

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include severe monocytic infiltration. Numerous blood vessels, particularly those in the pons, reveal perivascular cuffing [29,102,219,239]. Although listerial meningoencephalitis in swine is infrequent, several such outbreaks have been reported, including one in India in which 27 of 75 pigs died [179,220]. In England, the Veterinary Investigation Center reported only 14 listeriosis cases in swine between 1975 and 1982 as compared to 666 cases in sheep and 472 cases in cattle [96]. Listeriosis in pigs is also reported to be uncommon in The Netherlands [200]. Porcine listeriosis comprised 1% of listeriosis cases in Western Canada [22]. In Iowa, a major hog-producing state, from 1993 to 2000, of a total of 253 listeriosis submissions to the state veterinary diagnostic laboratory, none were from pigs. In that same interval, 87% of listeriosis cases in Iowa were from cattle [292]. Earlier, Blenden reported that cattle and sheep accounted for 274 of 281, or 98%, of listeriosis cases submitted to the Missouri Diagnostic Laboratory; only 1 case was from pigs [29]. Missouri, like Iowa, is a major hog-producing state. Because few surveys are available describing the prevalence of L. monocytogenes in healthy pigs, its distribution (0 to 16%) may be estimated from surveys of fecal excretors and recoveries from tonsils and carcass swabs collected at slaughter [86,125,203,277]. In a study in the United States of 300 market-weight hogs, L. monocytogenes was recovered overall from 2.4% of hog tissues, including tonsils (7.0%) as well as thoracic (3.5%) and superficial inguinal (1.9%) lymph nodes [146]. For cull sows (n = 181)—animals raised primarily for reproductive purposes and under less dense animal housing conditions—L. monocytogenes was cultured from a single tonsil sample (0.6%) and from none of the rectal content samples (Wesley, unpublished). In a limited study of 131 wild boars in Japan, L. monocytogenes serotype 4b was cultured from 2% of fecal samples. The lower frequency of recovery may be attributed to low population density in the wild. No attempts were made to recover the pathogen from other sites, including tonsils [117]. Asymptomatic carriers of Listeria may be more prevalent in Eastern Europe [221,222]. Ralovich [221] reviewed studies in which fecal recovery rates of 47% in individual animals and in 11 of 12 farms were described. A high infection rate in pigs has led to speculation that swine may be important reservoirs of L. monocytogenes in Eastern Europe [102]. Husbandry practices such as feeding pigs dry feed or silage, rearing in closed houses, maintaining specific-pathogen-free (SPF) herds as well as differences in sampling sites (tonsils vs. feces) and seasonality may account for variation in the incidence of healthy porcine carriers reported. To illustrate, in Yugoslavia, L. monocytogenes was recovered more frequently from tonsils of pigs raised on silage (61%) than animals raised on dry feed (29%). Recoveries from meat generally exceed isolation from live animals, suggesting postslaughter contamination during processing. Skovgaard et al. [252] reported that although only 1.7% of pig fecal samples yielded L. monocytogenes, the pathogen was detected in 12% of ground pork samples in Denmark, indicating dissemination of Listeria during processing—an observation made by other investigators [40]. In France, an outbreak involving 279 human cases incriminated pickled pork tongue as a major vehicle of transmission, although other highly processed, ready-to-eat delicatessen items subject to environmental contamination were also implicated [59,134]. Live hogs were thought to have introduced contamination into the processing plant. Although limited epidemiological data are provided, two cases of human neonatal listeriosis may have been linked indirectly to contact with pigs [256]. Alternatively, they may reflect exposure to a common source of contamination.

FOWL Avian listeriosis was first described in 1935 [238], 3 years after TenBroeck isolated L. monocytogenes (then Bacterium monocytogenes) from diseased chickens. Wild [35,227,288] and domestic avians, including turkeys [24,116,204], ducks [100,239], geese [100,239], and pheasants [100], may be asymptomatic carriers. Up to 33% of all healthy chickens may asymptomatically shed

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L. monocytogenes in fecal material [66,67,252]. Birds most likely become infected by pecking Listeria-contaminated soil, feces, or dead animals; however, contaminated fecal material also may pose a hazard to other livestock. Bovine encephalitic listeriosis developed in four cows housed in stables where chicken litter was used as flooring. L. monocytogenes serogroup 4b was recovered from the bovine brains, litter, and the intestinal contents of 4.1% of the donor birds (n = 2.3 million) [68]. In another study, the increased incidence of L. monocytogenes in rooks coincided with nesting season and the peak of ovine listeriosis, which in turn was linked to consumption of contaminated silage [82]. Despite the many sporadic cases of avian listeriosis that have been documented over the last 60 years, this disease is far less common in birds than in sheep, goats, and cattle [100–102, 199,211,236]. Listeriosis in birds may be a secondary infection associated with viral infections [57] as well as salmonellosis, Newcastle disease, fowl pest, coryza, coccidiosis, worm infestations, mites, enteritis, lymphomatosis, ovarian tumors, and other immunocompromising conditions [102]. In 1988, an encephalitic form of listeriosis was reported in broiler chickens in California [54]. Predisposing conditions that may have precipitated the outbreak included recent debeaking and vaccination with a modified live viral arthritis vaccine given subcutaneously in the neck. L. monocytogenes serotype 4b was recovered from a liver and multiple brain samples. Three years later, a second outbreak occurred in breeder replacement birds, affecting 0.3% of the 54,000 birds in the flock. L. monocytogenes was recovered from soil samples collected near an adjacent dairy, but not from other sites on the premises. The stress associated with the unusually cold climate described in the report coupled with vaccine stress of the 7- to 10-day-old chicks may have led to this outbreak [54]. Septicemia, the most frequent manifestation of listeriosis in domestic fowl, is characterized by focal necrosis within the viscera, particularly the liver and spleen [102]. Although not present in all cases [199], cardiac lesions frequently develop and, in turn, lead to engorgement of cardiac vessels, pericarditis, and increased amounts of pericardial fluid [102]. Other conditions produced by the septicemic form of avian listeriosis have included splenomeglia, nephritis, peritonitis, enteritis, ulcers in the ileum and ceca, necrosis of the oviduct, generalized or pulmonary edema, inflammation of the air sacs, and conjunctivitis. In acute cases, lesions resulting from these conditions may be partially obscured by congestion and hemorrhages throughout the viscera [102]. Unfortunately, domestic fowl that suffer from listeric septicemia normally exhibit few overt signs of disease other than progressive emaciation and usually die 5 to 9 days after infection. Although far less common than the septicemic form of listeriosis, L. monocytogenes also can produce meningoencephalitis in domestic fowl. Domestic birds suffering from listeric meningoencephalitis exhibit several striking behavioral changes, including incoordination, tremors, torticollis, unilateral/bilateral toe paralysis, and dropped wings, all of which directly relate to disturbances of the central nervous system [18]. Such infections are virtually always fatal. Postmortem examination often reveals congestion and necrotic foci in the brain along with many of the aforementioned conditions that are characteristic of listeric septicemia. Microscopically, gliosis and satellitosis in the cerebellum and microabscesses containing Gram-positive bacteria are found in the midbrain and medulla of birds with encephalitic listeriosis [55]. L. monocytogenes colonizes chick embryos and young birds; older birds appear to be more resistant [11,61,102,111,266]. Following oral challenge of chickens with 102 or 106 L. monocytogenes cells, Bailey et al. [15] detected the pathogen more frequently in ceca, spleen, liver, and cloacal swab samples from 1- rather than 14- or 35-day-old chickens. Diarrhea and emaciation have been noted in experimental infection, thus facilitating spread via feces and nasal secretions. In a later study, 2-day-old chicks were experimentally infected with L. monocytogenes. Although most of the inoculated chicks appeared healthy, depression, ruffled feathers, dullness, and diarrhea followed by death were noted 2 to 5 days after inoculation. Milder symptoms such as anorexia and drowsiness were also observed in a few of the animals. At 5 days after infection, 100% of the cecal samples

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yielded L. monocytogenes. However, the percentage of L. monocytogenes-positive birds decreased and by day 28, L. monocytogenes was recovered from the caeca of only 10% of experimentally infected birds [128]. In another report using a smaller number of birds, L. monocytogenes was only found on the first day following infection in 15% of fecal samples [183]. Taken together, these data suggest that L. monocytogenes is cleared rapidly, indicating that birds are at best transiently infected and not likely reservoirs of L. monocytogenes [305]. Interestingly, following artificial infection, Pustovaia [218] found that Columbiformes, Passeriformes (perching birds), and Galliformes were susceptible, while Falconiformes and Strigiformes (owls) were resistant [124]. L. monocytogenes has been used to investigate macrophage function in retrovirus infection and the cell-mediated immune response in susceptible and resistant chickens exposed to Marek’s disease virus [45,57]. Viral infection depressed the resistance of 10-day-old chickens to experimental infection with an avian osteopetrosis virus [57]. When compared to virus-free chickens, the dually infected birds were less efficient in clearing L. monocytogenes from their spleens [57]. L. monocytogenes is present in healthy birds from nondetectable levels to up to 33% [66,67,93,123,132,288]. Yet contamination in retail poultry ranges from 17 to 70%, again suggesting postharvest contamination [14,31,86,93,183,207]. A link between transport stress and fecal shedding of L. monocytogenes has been suggested. In one study L. monocytogenes was found in 33% of pooled fecal samples collected from cages, suggesting recrudescence of L. monocytogenes because of transport stress. But no data on the L. monocytogenes status of these birds before shipment are provided [252]. In an effort to trace the source of L. monocytogenes present in retail poultry, low levels of natural carriage (5%) were reported in cecal samples of parent flocks providing broilers. Yet, L. monocytogenes was not cultured from cecal samples from over 2,000 broilers from 90 flocks in Denmark [208]. However, L. monocytogenes was found in processed poultry. Comparison of DNA fingerprinting patterns by pulsed-field gel electrophoresis (PFGE) indicated that live birds contributed little to the total contamination of the product [207,232].

MINOR SPECIES L. monocytogenes has been diagnosed in a number of minor wildlife and livestock species, such as horses, llamas, animals raised commercially for pelts, companion animals, deer, and primates [195]. The routes of transmission and symptoms parallel those of cattle, sheep, and goats. As with other livestock species, Listeria infection in horses can cause abortion [289], septicemia [25,51,74,106], and encephalitis as well as keratoconjunctivitis [233]. In contrast to cattle and sheep, few cases of equine listeriosis are reported [160,182,185,239,263,264,268]. A survey of fecal samples of 400 German horses indicated a carrier rate of 4.8% for L. monocytogenes, 6% for L. innocua, and 1.5% for L. seeligeri; less than 1% harbor L. welshimeri [288]. The results of a limited number of surveys describing L. monocytogenes-seropositive horses should be interpreted cautiously in light of possible cross-reactivity of antibodies of other bacterial species to Listeria. Previous history of contact with cattle and feeding on silage may explain sporadic cases of equine listeriosis [184]. L. monocytogenes was reported from four Welsh and two Shetland ponies housed together with cattle, one of which was diagnosed with listeriosis, and given poor-quality silage [74]. At necropsy, L. monocytogenes was cultured from the equine liver, spleen, heart, kidneys, and lungs [74]. In Tasmania, abortions occurred in two mares allowed to graze on pasture that had previously been a sheep farm but had most recently served as a dairy farm. L. monocytogenes serogroup 1 was cultured from the lung and stomach of one fetus. Following antibiotic therapy, the mare was bred and later gave birth to a normal live foal, indicating again that L. monocytogenes infection may not impair fertility [180]. Equine abortion, preceded by mild respiratory tract infection, caused by L. monocytogenes serotype 4 was reported in a mare that had wintered with cattle and been on ensilage [289].

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L. monocytogenes was cultured from the equine fetal liver, lung, spleen, and stomach. Neonatal septicemia was documented in a 3-day-old foal whose mare was housed indoors and fed poorquality, contaminated hay [122]. As is true in other species, the origin of infection in equine listeriosis cases may be unknown [233]. To illustrate, L. monocytogenes was recovered from the brainstem of a 16-year-old Welsh pony gelding with signs of ataxia, weakness, and deficits of cranial nerves. No immunological deficit was detected and there was no history of contact with ruminants or access to silage. A 21-day-old Appaloosa filly was examined because of diarrhea of 2 weeks’ duration [284]. Septicemia was diagnosed based on presence of L. monocytogenes cultured from blood. No sources of infection were evident, thus leading to speculation that L. monocytogenes was transmitted to a foal via contaminated mare’s milk. As described in humans and livestock, listeriosis occurs in immunocompromised hosts with defects in the humoral and cell-mediated immune components [269]. Listeriosis has been described in an Arabian foal with combined immunodeficiency [51]. The 1-month-old foal was ataxic, lethargic, failed to nurse, and spent most of the time with its head down. Most strikingly, hoofs were dragged when the animal was exercised. At necropsy widespread lesions were present in the viscera and central nervous system. In the llama, listeriosis occurs as a septicemia with meningitis in neonates, but more commonly causes asymmetric vestibular disease in adults [19]. Most animals affected are weaned and grazing or consuming roughage, but are not on silage. Multifocal suppurative encephalitic listeriosis was diagnosed in two adult llamas that were pregnant. L. monocytogenes was cultured from one of two animals and was visualized in the brainstem lesions of both llamas by fluorescein-conjugated antibody to L. monocytogenes [43]. In another report, L. monocytogenes caused fatal meningo-encephalomyelitis in a 3- to 5-month-old llama. The animal displayed unilateral peripheral disease progressing to encephalitis [270]. The source of infection for cases detailed in these two reports is unknown [19,43]. Listeriosis may have an economic impact in commercial pelt farms. An outbreak of disseminated visceral listeriosis in chinchillas in Nova Scotia [89] incriminated consumption of contaminated sugar beet pulp, although L. monocytogenes was not isolated from the feed. The outbreak occurred in a colony with 23% mortality of breeding chinchillas [300]. Approximately 4 days before death, animals were anorexic and hunched and some had torticollis (twisted necks). However, many animals were found dead without clinical signs [300]. Hay contaminated with rodent, bird, or ruminant feces has been implicated in previous outbreaks of chinchilla listeriosis and removal of contaminated feed often interrupts the cycle of transmission [47,89]. L. monocytogenes could have been transmitted by coprophagia because animals defecated in dust bath pans and the pans were transferred from cage to cage [298]. In an enzootic of listeriosis in a rabbitry, L. monocytogenes 1/2a was cultured from feed samples and from a doe that had died of septic metritis [213]. The early literature describes L. monocytogenes in nondomesticated ruminants, including reindeer, roe deer, antelope, and a Grant’s gazelle with previous contact with listeric sheep [20,23,76, 78,149,204,286]. L. monocytogenes has been recovered from ruminants housed in zoological parks [9,76,78,179,286]. Meningoencephalitis occurred in 42 deer in a flock of 1,800 head in a park during the winter and early spring in Denmark. This was preceded in the previous spring by the death of six deer that exhibited circling and appeared to be blind. The following year, the first sign of illness was a drooping ear, due to paralysis of the facial nerve, and a slight inability to follow the herd. No external source of L. monocytogenes was evident and stress because of a poor beech-mast crop, increased stocking rate of animals resulting in overcrowding with possible introduction of healthy carrier animals, and sudden change in weather were all potential contributing factors [76]. Listeriosis is rarely reported in dogs and has caused encephalitis, including circling, and abortion [261]. In a survey of domestic animals, L. monocytogenes was detected in 1.3% of dog (n = 300) and 0.4% of cat (n = 275) fecal samples [288]. The low recovery may indicate that companion

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animals are not important in the epidemiology of listeriosis in humans [287] or that shedding is sporadic. In contrast, serosurveys indicating that up to 90% of dogs may be seropositive [251] should be interpreted cautiously because of the cross-reactivity inherent in agglutination tests. A single report on the possible transmission to L. monocytogenes from humans to dogs [262] warrants re-evaluation because the isolates were later identified as L. innocua. Nevertheless, recovery of a single species of Listeria from humans and dogs in close proximity could reflect substantial environmental contamination rather than human-to-dog passage. Listeriosis occurs in nonhuman primates, where it manifests as septicemia [48,303], meningoencephalitis [61], and stillbirths [185,273], as documented in a large outdoor breeding colony in California [212]. Transmission may occur by consumption of contaminated foods [273]. Experimental infections were attempted by exposure to aerosols [147] as well as by feeding [79,255]. Cynomolgus monkeys (Macaca fascicularis) shed L. monocytogenes in their feces for up to 21 days after receiving an oral dose of 109 Listeria. Neither septicemia nor encephalitis was reported, indicating that the normal healthy primate is resistant to L. monocytogenes. In contrast, abortion and stillbirths were reported in rhesus monkeys fed 108 CFUs in a high-fat vehicle [255].

FISH

AND

CRUSTACEANS

Consumption of seafood is growing in popularity, and the aquaculture industry is responding by providing fish raised in controlled fish farms rather than depending on the availability of fresh caught products. In 1980 in New Zealand, Lennon et al. [160] reported a cluster of 22 perinatal human listeriosis cases. A weak association between these cases and the consumption of contaminated raw fish and shellfish was established. DNA fingerprinting determined that fish in the patient’s refrigerator were identical. The first report of the occurrence of listeriosis in fish was in 1957 from Romania [259]. In that study, L. monocytogenes was isolated from the viscera of pond-reared rainbow trout that presumably became infected after consuming contaminated donkey meat. Results of current surveys indicate that Listeria is absent from live seawater fish, but is present in live freshwater species. In contrast, L. monocytogenes was recovered from two of five intestinal tracts of market-purchased fresh water fish in India [119]. Channel catfish (Ictalurus punctatus) is the most widely cultured species in the United States; most commercial ponds are located in the southeastern region of the country. A study of catfish, water, and feed collected from university ponds in Alabama indicated presence of L. monocytogenes on the skin and viscera (mean presumptive count = 1.99 log CFU/wet weight). L. monocytogenes was not detected in water or feed. Unfortunately, the authors provide no data on the percentage of L. monocytogenes–positive fish, but conclude that the bacterial concentrations in the viscera suggest that cross-contamination is possible during evisceration [162]. A survey of three rainbow-trout farms in Switzerland showed that L. monocytogenes was present in the feces (40%) and on the skin (33%, 5/15) of fish from one of the farms. Yet L. monocytogenes was detected on the finished product in only 6% of the fish from this farm. In contrast, L. monocytogenes was not found in feces, skin of fish, or the finished product of two of the farms where fish were raised in concrete ponds and starved 3 to 7 days before harvest [136]. Similarly, L. monocytogenes was not recovered from the skin, gills, intestines, tank water, or diet of striped bass grown in recirculating water tanks [202]. Experimental infection of zebrafish (Brachydanio rerio) indicated the LD50 was higher in fish than in mice and that L. monocytogenes did not multiply in fish [189]. Experimental infection stimulated an increase in granulocyte and monocytes. In contrast to L. monocytogenes, strains of L. welshimeri, L. innocua, and L. seeligeri killed more than 50% of fish 7 days postinfection [189]. Brackett [36] proposed that contamination of fish and shellfish through their ambient waters may influence distribution of L. monocytogenes. Surface waters, sewage effluents, and agricultural

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runoff all may potentially contribute Listeria spp. to the aquatic environment. In addition, presence of L. monocytogenes in sea gulls may be another source of shellfish contamination [82]. In 1959, L. monocytogenes was detected in crustaceans gathered from a Russian stream [248]. A more recent survey conducted on the Gulf Coast of the United States examined shrimp, oysters, and estuarine waters for L. monocytogenes [196]. L. monocytogenes was detected in 11% of unprocessed shrimp (n = 74), but not in oysters (n = 75), although some of the oysters were harvested from prohibited shellfish-growing sites. In a parallel study conducted in fresh water tributaries off the Humboldt–Arcata Bay in Northern California, L. monocytogenes was detected in the freshwater samples (61%), some of which received runoff from nearby farms. However, L. monocytogenes was not found in oysters in that study [52] or in oysters kept in live holding tanks in seafood markets in Seattle [53]. Taken together, these data indicate that live fish and shellfish are not likely carriers of L. monocytogenes. PCR tests have been used to detect L. monocytogenes in experimentally contaminated marinated rainbow trout [77]. With the increased interest in L. monocytogenes in fish and seafood [77], this sensitive technique may be useful for the rapid screening of live shellfish and fish for L. monocytogenes. Nevertheless, the paucity of reports documenting L. monocytogenes in live freshwater fish and shellfish suggests that Listeria spp. cultured from the retail product are most likely from postharvest contamination.

TREATMENT Poor husbandry, consumption of contaminated feed, and stress are important in initiating the disease. Thus, identifying and eliminating these factors are critical to preventing recurrences. In general, because antemortem diagnosis is rarely made, treatment is seldom attempted. Because listeric encephalitis is a rapidly debilitating disease in ruminants, treatment must be initiated early during the course of infection if there is to be any reasonable hope of survival. L. monocytogenes is resistant to many drugs but is sensitive to chlortetracycline. The intravenous injection of chlortetracycline (10 mg/kg body weight per day for 5 days) is effective in meningoencephalitis of cattle but less so in sheep [219]. If penicillin is used, high doses are required because of the difficulty of maintaining therapeutic levels in the brain. Penicillin G should be given at 44,000 U/kg body weight, intramuscularly daily for 1 to 2 weeks [90]. If signs of encephalitis are severe, death usually occurs in spite of treatment. Supportive therapy, which is usually reserved for valuable animals, includes fluid and electrolyte replacement and is indicated for animals having difficulty eating and drinking as a result of neural damage. Excessive salivation leads to acidosis, which is remedied by intravenous replacement of bicarbonate ions. Permanent neurological damage often occurs in ruminants, despite proper therapy. In view of the severe economic losses from listeric encephalitis in sheep, it may be prudent to consider vaccinating animals against listeriosis, particularly if they are raised in areas prone to listeric infection [202]. In birds, tetracyclines (5 to 10 mg/kg body weight daily for 1 week) are efficacious in acute and subacute forms. Treatment of the chronic form is unsuccessful. As with other livestock species, rigid sanitation and disinfection procedures with culling and isolation of affected birds may be helpful [91]. A benefit of early treatment of animals with listeriosis has been demonstrated. However, timeliness is most important and recognition of the disease depends upon observation of clinical signs. In cattle and sheep the appearance of clinical signs is an indication of neurological damage and thus of a guarded prognosis for treatment. In all cases, the economics of the attempted treatment must be considered along with the alternative of humane euthanasia.

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272. Vazquez-Boland, H. J. A., L. Dominguez, M. Blanco, J. Rocourt, J. F. Fernandez-Garayzabal, C. B. Gutierrez, R. I. Tascon, and E. F. Rodriquez-Ferri. 1992. Epidemiologic investigation of a silageassociated epizootic of ovine listeric encephalitis, using a new Listeria selective enumeration medium and phage typing. Am. J. Vet. Res. 3:368–371. 273. Vetesi, F., A. Balsai, and F. Kemenes. 1972. Abortion in Gray’s monkey (Cercopithecus mona) associated with Listeria monocytogenes. Acta Microbiol. Acad. Sci., Hung. 19:441–443. 274. Vishinsky, Y., A. Grinberg, and R. Ozery. 1993. Listeria monocytogenes udder infection and carcass contamination. Vet. Rec. 133:484. 275. Visser, I. J. 1996. Pustular dermatitis in veterinarians following delivery in domestic animals: an occupational disease. Ned. Tijdschr. Geneeskd. 140:1186–1190. 276. Vizcaino, L. L., and M. A. Garcia. 1975. A note on Listeria milk excretion in sero-positive apparently healthy cows. In Problems of listeriosis, ed. M. Woodbine. Surrey, England: Leicester University Press, p. 74. 277. Vojinovic, G. 1992. The incidence of Listeria monocytogenes in slaughtered healthy animals and minced meat. Acta Vet. (Belgrad) 42:329–336. 278. von Amtsberg, G., A. Elsner, H. A. Grabbar, and W. Winkenwerder. 1969. Die epidemiologische und lebensmittelhygienische Bedeutung der Listerieninfektion des Rindes. Dtsch. Tierärztl. Wochenschr. 76:497–501. 279. von Amtsberg, G., A. Elsner, H. A. Grabbar, and W. Winkenwerder. 1969. The animal health yearbook. 1986. Food and Agriculture Organization of the United Nations, World Health Organization and the International Office of Epizootics, Rome. 280. von Arda, M., W. Bisping, N. Aydin, E. Istanbulluoglu, O. Akay, M. Izgur, Z. Karaer, S. Diker, and G. Kirpal. 1987. Ätiologische Untersuchungen über den Abort bei Schafen unter besonderer Berücksichtigung des Nachweises von Brucellen, Campylobacter, Salmonellen, Listerien, Leptospiren und Chlamydien. Berl. Münch. Tierärztl. Wochenschr. 100:405–408. 281. von Hartwigk, H. 1958. Zum Nachweis von Listerieninder Kuhmilch. Berl. Münch. Tierärztl. Wochenschr. 71:82–85. 282. von Selbitz, H.-J. 1986. Immunological principles for control of listeriosis. Monatsh. Veterinärmed. 41:217–219. 283. Walker, J. K., and J. H. Morgan. 1993. Ovine ophthalmitis associated with Listeria monocytogenes. Vet. Rec. 132:636. 284. Wallace, S., and T. Hathcock. 1995. L. monocytogenes septicemia in a foal. J. Am. Vet. Med. Assoc. 207:1325–1326. 285. Wardrope, D. D., and N. S. M. Macleod. 1983. Outbreak of Listeria meningoencephalitis in young lambs. Vet. Rec. 113:213–214. 286. Webb, D., and A. Rebar. 1987. Listeriosis in an immature black buck antelope (Antilope cervicapra). J. Wildlife Dis. 23:318–320. 287. Weber, A., C. Datzmann, and J. Potel. 1993. Prevalence of Listeria monocytogenes in fecal samples from dogs and cats. Tierärztl. Umsch. 48:727–730. 288. Weber, A., J. Potel, R. Schäfer-Schmidt, A. Prell, and C. Datzmann. 1995. Investigations on the occurrence of L. monocytogenes in fecal samples of domestic and companion animals. Zentralbl. Hyg. Umweltmed. 198:117–123. 289. Welsh, A. 1983. Equine abortion caused by Listeria monocytogenes serotype 4. J. Am. Vet. Med. Assoc. 182:291. 290. Wesley, I. V., and F. Ashton. 1991. Restriction enzyme analysis of Listeria monocytogenes strains associated with food-borne epidemics. Appl. Environ. Microbiol. 57:969–975. 291. Wesley, I. V., J. H. Bryner, and M. J. van der Maaten. l989. Effects of dexamethasone on shedding of Listeria monocytogenes in dairy cattle. Am. J. Vet. Res. 50:2009–2113. 292. Wesley, I. V., D. J. Larson, K. M. Harmon, J. B. Luchansky, and A. R. Schwartz. 2002. A case report of sporadic ovine listerial meningoencephalitis in Iowa with an overview of livestock and human cases. J. Vet. Diag. Invest. 14:314–321. 293. WHO Working Group. 1988. Foodborne listeriosis. Bull. WHO 66:421–428. 294. Wiedmann, M. 2002. Molecular subtyping methods for Listeria monocytogenes. J. AOAC Int. 85:524–531.

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295. Wiedmann, M. 2003. ADSA Foundation scholar award—an integrated science-based approach to dairy food safety: Listeria monocytogenes as a model system. J. Dairy Sci. 86:1865–1875. 296. Wiedmann, M., T. Arvik, J. Bruce, J. Neubauer, F. Pierro, M. C. Smith, J. Hurley, H. O. Mohammed, and C. A. Batt. 1997. Investigation of a listeriosis epizootic in sheep in New York State. Am. J. Vet. Res. 58:733–737. 297. Wiedman, M., J. L. Bruce, C. Keang, A. E. Johson, P. L. McDonough, and C. A. Batt. 1997. Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential. Infect. Immun. 65:2707–2716. 298. Wiedmann, M., J. L. Bruce, R. Knorr, M. Bodis, E. M. Cole, C. I. McDowell, P. L. McDonough, and C. A. Batt. 1996. Ribotype diversity of Listeria monocytogenes strains associated with outbreaks of listeriosis in ruminants. J. Clin. Microbiol. 34:1086–1090. 299. Wilesmith, J. W., and M. Gitter. 1986. Epidemiology of ovine listeriosis in Great Britain. Vet. Rec. 119:467–470. 300. Wilkerson, M. J., A. Melendy, and E. Stauber. 1997. An outbreak of listeriosis in a breeding colony of chinchillas. J. Vet. Diag. Invest. 9:320–323. 301. Wohler, W. H., and C. L. Baugh. 1983. Pulmonary listeriosis in feeder cattle. Med. Vet. Pract. 64:736–739. 302. Wramby, G. O. 1944. Om Listerella monocytogenes bakteriologi och om forekomst av Listerella infectioner has djur. Skand. Vet. Tskr. 34:278–290. 303. Yndestad, M. 1987. Personal communication. 304. Yousif, Y. A., B. P. Joshi, and H. A. Ali. 1984. Ovine and caprine listeric encephalitis in Iraq. Trop. Anim. Health Prod. 16:27–28. 305. Zhu, M., I. Wesley, R. Nannapaneni, M. Cox, A. Mendonca, M. G. Johnson, and D. U. Ahn. 2003. The role of dietary vitamin E in experimental Listeria monocytogenes infections in turkeys. Poultry Sci. 82:2559–2564. 306. Zwart, P., and J. Donker-Voet. 1959. Listeriosis bij in gevangenschap gehouden dieren. Tijdschr. Diergeneesk. 84:712–716.

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4 Listeriosis in Humans John Painter and Laurence Slutsker CONTENTS Introduction ......................................................................................................................................85 Human Infection and Clinical Manifestations of Listeriosis ..........................................................87 Listeriosis during Pregnancy ..................................................................................................87 Neonatal Disease: Early Onset...............................................................................................87 Neonatal Disease: Late Onset ................................................................................................88 Invasive Disease in Nonpregnant Adults .........................................................................................88 Noninvasive Disease: Gastrointestinal Illness .................................................................................90 Asymptomatic Carriage..........................................................................................................91 Epidemiological Patterns of Listeriosis...........................................................................................94 Epidemic Listeriosis ...............................................................................................................94 Sporadic Disease—Incidence.................................................................................................97 Sporadic Disease—Dietary Risk Factors...............................................................................98 Sporadic Disease—Possible Other Sources.........................................................................100 Diagnosis ........................................................................................................................................100 Treatment........................................................................................................................................101 Prevention.......................................................................................................................................101 References ......................................................................................................................................102

INTRODUCTION Listeria monocytogenes has been recognized as a human pathogen since 1929 [114], but the route of transmission was unclear until the 1980s when a series of outbreaks indicated that L. monocytogenes was transmitted by food [13,43,59,70,89,102,123,130]. It is now recognized that nearly all cases of human listeriosis are foodborne [1,104,112,132,136]. Although listeriosis is a very small fraction of all illness due to known foodborne pathogens, it is an important cause of severe illness, accounting for 3.8% of foodborne disease hospitalizations and 27.6% of foodborne disease deaths [104]. Development of improved laboratory techniques to detect and subtype L. monocytogenes has also contributed to an improved understanding of human listeriosis [9,11,14,125,126]. Listeria monocytogenes is found in multiple ecological sites throughout the environment, including soil [152], water, and decaying vegetation [151,153]. Control of human listeriosis, therefore, relies on improving the understanding of how to control Listeria contamination of food. Human disease caused by L. monocytogenes occurs most frequently in women of childbearing age, infants, and the elderly (Figure 4.1). The risk of listeriosis is greatest among certain welldefined high-risk groups, including pregnant women, neonates, and immunocompromised adults but may occasionally occur in persons who have no predisposing underlying condition (Table 4.1). The ongoing epidemic of acquired immunodeficiency syndrome (AIDS), as well as widespread use of immunosuppressive medications for treatment of malignancy and management of organ transplantation, has expanded the immunocompromised population at increased risk of listeriosis.

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35%

Percent of Total

30% 25% FEMALE

20% MALE

15% 10% 5% 0% =1-2

>=2-5

>=5-15

>=15-25 >=25-35 >=35-45 >=45-55 >=55-65 >=65-75

>=75

age group

FIGURE 4.1 Percent of reported laboratory-confirmed listeriosis within age groups for males and females, United States, 2000 to 2004. Isolation of Listeria monocytogenes reported to CDC through the Public Health Laboratory Information System (PHLIS).

Unlike infection with other common foodborne pathogens such as Salmonella, which rarely result in fatalities, listeriosis is associated with a mortality rate of approximately 20% [53]. The high case-fatality rate, growing awareness of listeriosis as a foodborne disease, and increasing clinical concern about illness in the expanding population of highly susceptible persons have resulted in increased attention to the importance of L. monocytogenes as a human pathogen. In this chapter, we will consider various aspects of listeriosis in humans, including infection and clinical manifestations of disease, epidemiological patterns of disease, diagnosis, treatment, and prevention. Information on the microbiology, ecology, pathogenesis, detection, subtyping, manifestations of infection in other animals, and occurrence of L. monocytogenes in various foods is presented elsewhere in this book. Although some foodborne outbreaks of listeriosis will be discussed here, a more exhaustive treatment of foodborne listeriosis outbreaks can be found in Chapter 10.

TABLE 4.1 Clinical Syndromes Associated with Infection with Listeria monocytogenes Population

Predisposing Condition or Circumstances

Clinical Presentation

Diagnosis

Fever ± myalgia ± diarrhea Preterm delivery Abortion Stillbirth

Blood culture ± amniotic fluid culture

Sepsis, pneumonia Meningitis, sepsis

Blood culture Cerebrospinal fluid culture

Prematurity

Nonpregnant adults

Sepsis, meningitis, focal infections

Culture of blood, cerebrospinal fluid, or other normally sterile site

Immunosuppresion, advanced age

Healthy adults

Diarrhea and fever

Stool culture in selective enrichment broth

Possibly large innoculum

Pregnant women

Newborns: =2-5

>=5-15

>=15-25 >=25-35 >=35-45 >=45-55 >=55-65 >=65-75

>=75

Age Group

FIGURE 4.3 Percent of reported laboratory-confirmed listeriosis by age groups for Hispanic and non-Hispanic ethnicity, United States, 2000 to 2004. Isolation of Listeria monocytogenes reported to the CDC through the Public Health Laboratory Information System (PHLIS).

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turkey frankfurter production facility found that cultures from a conveyor belt transporting finished frankfurters yielded the case strain of L. monocytogenes [155]. Systematic culturing at various points in the production process identified likely points where L. monocytogenes was introduced into the product and suggested appropriate control points for reducing contamination in such foodprocessing facilities. From 1988 to 1990, a larger case-control study of 165 patients and 376 controls was conducted that included microbiological assessment of foods eaten by patients [132]. Case-patients were significantly more likely than controls to have eaten soft cheeses or delicatessen-counter foods. In a separate analysis examining dietary risks among a subset of patients defined as highly immunosuppressed (persons with malignancy, AIDS, or organ transplants or who had received corticosteroids or chemotherapy), consumption of undercooked chicken was associated with a threefold increased risk of listeriosis. Other exposures associated with an increased risk of sporadic disease included recent use of antacids, laxatives, or H2-blocking agents. In the microbiological component of this study, foods were collected from the refrigerators of 123 patients [120]. L. monocytogenes was isolated from at least one food in the refrigerators of 64% of patients. Highest contamination rates among the 2,013 food specimens were seen in beef (36%) and poultry (31%) with 7.6% of ready-to-eat foods (processed meats, raw vegetables, leftovers, and cheeses) also yielding L. monocytogenes. One third of refrigerators contained food isolates of L. monocytogenes that were the same multilocus enzyme elctrophoretic type as those isolated from the patient. In multivariate analysis, foods that were ready to eat, foods that contained high numbers of L. monocytogenes, and foods that yielded serovar 4b were associated with disease. Dietary risk factors for sporadic listeriosis were also examined in a recent study in Denmark; drinking unpasteurized milk or eating pâté were the only risk factors identified [82]. However, one third of cases reported during the study period could not be included in the risk analysis for sporadic disease because the ill persons were infected with an outbreak strain epidemiologically linked to Danish blue-mold cheeses.

SPORADIC DISEASE—POSSIBLE OTHER SOURCES Transmission by routes other than food may play a role in a few cases of sporadic listeriosis. Sexual transmission of L. monocytogenes has been hypothesized as a possible route in perinatal listeriosis; however, there is no evidence to support this [121]. Because L. monocytogenes can cause asymptomatic bacteremia and survives refrigeration, it is theoretically possible that transmission through donated blood could occur. Such transmission has been documented for Yersinia enterocolitica but has not yet been described for L. monocytogenes [144]. Nosocomial transmission of L. monocytogenes has been documented in a variety of settings [34,61,70,73,74,81] in addition to the transmission among newborns in a nursery mentioned earlier.

DIAGNOSIS Diagnosis of invasive listeriosis depends on isolation of L. monocytogenes from a normally sterile site such as blood or cerebrospinal fluid. Because the organism may be mistaken for a diphtheroid contaminant on Gram stain, complete bacteriological evaluation should be done. Recovery of the organism from stool samples is helpful when febrile gastroenteritis is suspected, but isolation from stool by itself is not diagnostic because asymptomatic carriage occurs. To document an outbreak of febrile gastroenteritis, the isolation rate among symptomatic persons should be significantly higher than among asymptomatic persons. L. monocytogenes strains isolated from sterile-site specimens usually grow well in routinely used media. The specimen is directly plated on tryptic soy agar containing 5% sheep, horse, or rabbit blood. The organism is usually identified within 36 hours. Isolation of the organism from other sources such as stool specimens that contain large numbers of competing microorganisms is

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more difficult; these specimens should be selectively enriched for Listeria spp. before being plated on Listeria-selective media. Identification of L. monocytogenes by use of fluorescent antibody methods or approaches that use DNA probes coupled with PCR technology may prove useful for some specimens. Experimental assays for antibody to listeriolysin O have been useful in some epidemiological investigations [37] and have been used to support the diagnosis in culture-negative listeriosis of the central nervous system [51].

TREATMENT Controlled trials to determine the optimal antibiotic therapy for listeriosis have not been done. The treatment of choice is high doses of amoxicillin I.V. (2 to 3 g three to four times a day), with an additional dose of gentamicin (360 mg once a day) for nonpregnant adults [72]. The role of aminoglycosides is poorly understood because they penetrate cells poorly and may be ineffective in the living host. L. monocytogenes has grown in cells despite high extracelluar concentrations of aminoglycosides [69]. However, the majority of Listeria may be extracellular in cases of meningitis [71]. Trimethoprim-sulfamethoxazole readily enters cells and kills L. monocytogenes. This drug combination has proved effective in patients with listeriosis who have hypersensitivity to penicillin [5]. Compared with the aminopenicillins, newer quinolone antimicrobials may have increased bacteridicidal activity, cross the blood–brain barrier into the CNS, and accumulate within host cells, but clinical studies have not been done [72]. The ability of L. monocytogenes to grow and survive within cells probably explains the poor response to bacteriostatic drugs and the slow response to penicillin [141]. Bacteriostatic drugs such as chloramphenicol or tetracycline have been associated with high treatment failure rates, so they are not recommended [141]. Cephalosporins are not recommended for treatment because they have a low affinity for the penicillin-binding protein of Listeria [147]. Relapses have been reported in immunosuppressed patients after 2 weeks of penicillin therapy [150]. Because many immunosuppressed patients have a decreased ability to clear infected cells, antibiotic treatment for 3 to 6 weeks may be prudent [6]. Optimal length of therapy for other groups of patients has not been established. A prudent treatment course may be 2 weeks for listeriosis in pregnancy, 2 to 3 weeks for neonatal listeriosis, 2 to 4 weeks for nonimmunosuppressed adults with meningitis and bacteremia, and longer for complicated infections such as endocarditis.

PREVENTION Recognition that most human listeriosis is foodborne has led to control measures that have reduced the incidence of listeriosis. Healthy People 2010 goals established for the United States called for a reduction of foodborne listeriosis by 50% by the end of the year 2005; by 2004, those goals were nearly met [29]. The observed decrease in perinatal and nonperinatal cases since 1989 is likely the result of enhanced listeriosis prevention efforts by the U.S. food industry, including enforcement by regulatory agencies of a zero-tolerance policy for processed meat and intensified clean-up programs in meat-processing facilities. In 2001 and 2003, the Food and Drug Administration (FDA), CDC, and the U.S. Department of Agriculture (USDA) released a national Listeria Action Plan to help guide control efforts by industry, regulators, and public health officials [45,46]. Those plans called for multiple points of action, including increased regulatory guidance over the manufacture of ready-to-eat foods. Also in 2003, following a large outbreak linked to deli turkey meat, the USDA issued new regulations aimed at further reducing L. monocytogenes contamination of ready-to-eat meat and poultry products [39]. Published dietary recommendations for consumers may also have contributed to the decreased disease incidence [29,44,137]. Noncommercial sources of food, such as raw milk cheese, continue to be sources of listeriosis, especially among Latin American women [33,146].

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For persons who are at increased risk for listeriosis, including those who are pregnant or immunocompromised, specific dietary measures can be taken to decrease risk [23]. Such persons should avoid high-risk foods such as hot dogs; deli meats or luncheon meats (particularly deli sliced), unless they are reheated until steaming hot; soft cheeses (such as feta, Brie, and Camembert, blue-veined cheeses, or Mexican-style cheeses such as queso blanco, queso fresco, and Panela), unless they have labels that clearly state they are made from pasteurized milk; unpasteurized (raw) milk; refrigerated p aˆ tés or meat spreads (canned or shelf-stable p aˆ tés and meat spreads may be eaten). In addition, they should avoid getting fluid from hot dog or other deli meat packages on other foods or food preparation surfaces and wash hands after handling hot dogs and deli meats. Dietary and food preparation measures have been recommended to the general public; these should decrease the risk not only of listeriosis but also of other common foodborne diseases, such as salmonellosis and campylobacteriosis. These measures include thorough cooking of raw food from animal sources; washing raw vegetables thoroughly before eating; keeping uncooked meats separate from vegetables, cooked foods, and ready-to-eat foods; avoiding raw (unpasteurized) milk or foods made from raw milk; and washing hands, knives, and cutting boards after handling uncooked foods [29]. In addition to individual advice for consumers, control of listeriosis requires action from public health agencies and the food industry. Important control strategies from public health agencies include developing and maintaining timely and effective disease surveillance programs, promptly investigating clusters of listeriosis cases, and enforcing current regulations designed to minimize L. monocytogenes in foods consumed without further cooking. A survey in the United States found that 1.8% of ready-to-eat foods were contaminated with L. monocytogenes [57]. For foods such as ready-to-eat salad vegetables, only rare servings may be contaminated but the level of contamination may be high [127]. It is unlikely that such contamination would be found during routine product testing. It is imperative therefore that the food industry develop an understanding of how contamination occurs and then implement hazard analysis critical control point (HACCP) programs to minimize the presence of L. monocytogenes at important points in the processing, distribution, and marketing of processed foods [2].

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150. Watson, G. W., T. J. Fuller, J. Elms, and R. M. Kluge. 1978. Listeria cerebritis: Relapse of infection in renal transplant patients. Arch. Intern. Med. 138:83–87. 151. Weis, J. 1975. The incidence of L. monocytogenes on plants and in soil. In Problems of listeriosis, ed. M. Woodbine. Leicester, U.K.: Leicester University Press, pp. 61–65. 152. Welshimer, H. J. 1960. Survival of Listeria monocytogenes in soil. J. Bacteriol. 80:316–320. 153. Welshimer, H. J. 1968. Isolation of L. monocytogenes from vegetation. J. Bacteriol. 95:300–303. 154. Wenger, J. D., A. W. Hightower, R. R. Facklam, S. Gaventa, and C. V. Broome. 1990. Bacterial meningitis in the United States, 1986: Report of a multistate surveillance study. The Bacterial Meningitis Study Group. J. Infect. Dis. 162:1316–1323. 155. Wenger, J. D., B. Swaminathan, P. S. Hayes, S. S. Green, M. Pratt, R. W. Pinner, A. Schuchat, and C. V. Broome. 1990. Listeria monocytogenes contamination of turkey franks: Evaluation of a production facility. J. Food Prot. 53:1015–1019.

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Virulence 5 Molecular Determinants of Listeria monocytogenes Michael Kuhn and Werner Goebel CONTENTS Introduction ....................................................................................................................................111 Molecular Aspects of the Invasion of Mammalian Cells..............................................................113 Invasion of Nonprofessional Phagocytic Cells ....................................................................114 Cellular Adhesion .................................................................................................................118 Uptake by Macrophages and Dendritic Cells......................................................................118 Escape from the Phagocytic Vacuole.............................................................................................120 Growth in the Host Cell Cytoplasm ..............................................................................................125 Intracellular Motility and Cell-to-Cell Spread ..............................................................................126 Bile Salt Hydrolase—A Novel Virulence Factor ..........................................................................129 Accessory Virulence Factors..........................................................................................................130 p60, Product of the iap Gene...............................................................................................130 Superoxide Dismutase and Catalase ....................................................................................131 Stress Response Mediators...................................................................................................131 Iron Uptake Systems ............................................................................................................132 PrfA and Regulation of Virulence Gene Expression in L. monocytogenes ..................................132 The Positive Regulatory Factor A (PrfA) ............................................................................132 PrfA-Dependent Promoters, Transcripts, and Mechanism of Temperature-Dependent Virulence Gene Expression......................................................134 Environmental Signals Affecting Virulence Gene Expression ............................................136 Two-Component Systems and Regulation of Virulence Gene Expression .........................137 Lessons Learned from Genome Sequence of L. monocytogenes .................................................137 Evolutionary Aspects .....................................................................................................................138 Open Questions ..............................................................................................................................139 Acknowledgments ..........................................................................................................................139 References ......................................................................................................................................139

INTRODUCTION Studies that aimed to unravel the mechanisms of Listeria monocytogenes pathogenicity and its interaction with hosts on the cellular, molecular, and genetic levels were initiated about two decades ago. The early studies used transposon mutagenesis and infection of primary and established cell lines to obtain insights into the interaction of L. monocytogenes with eucaryotic host cells. The recent sequencing of the genomes of L. monocytogenes and Listeria innocua together with development of genetic tools now allows manipulation of L. monocytogenes, which has, together with cell culture 111

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and transgenic animal models, greatly broadened our understanding of the molecular and cell biology of L. monocytogenes infections. Most of the early studies on the cell biology of L. monocytogenes infections used epithelialike and macrophage-like cell lines [103,229,315]. Macrophages actively ingest L. monocytogenes, but internalization of the bacterium by normally nonphagocytic cells is triggered by L. monocytogenes -specific products. Aside from the internalization step, the intracellular life cycle of the bacteria in phagocytes or normally nonphagocytic mammalian cells is, however, very similar. The pathogen first appears in a vacuole, which is subsequently lysed by some of the ingested bacteria allowing L. monocytogenes to escape into the cytoplasm. Whereas most of the bacteria begin to replicate in the cytoplasm, those remaining in the phagosome are killed and digested. Concomitant with the onset of intracellular replication, L. monocytogenes induces nucleation of host actin filaments arranged to a polar tail. Formation of a tail at one pole of the bacterial cell produces a propulsive force that moves the bacteria through the cytoplasm. Bacteria that reach the surface of the infected host cell induce the formation of pseudopode-like structures with the bacterium at the tip and the actin tail behind it. These pseudopods are taken up by neighboring cells. The bacteria thus entering the neighboring cells are within a vacuole surrounded by a double membrane, which is subsequently lysed to release the bacteria into the cytoplasm of the newly infected host cell. The line drawing shown in Figure 5.1 summarizes this intracellular life cycle. The different steps and the listerial virulence factors involved in this life cycle are discussed in detail later.

FIGURE 5.1 Schematic drawing of the intracellular life cycle of Listeria monocytogenes (A) and representative electron micrographs showing adhesion (B), entry (C), bacteria inside vacuoles (D), bacteria free in the cytoplasm (E), moving bacteria in protrusions (F and G), and bacteria in a double membrane vacuole formed during cell-to-cell spread (H). (Reprinted with permission from Vazquez-Boland, J. A. et al. 2001. Clin. Microbiol. Rev. 14:584–640.)

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FIGURE 5.2 Organization of the central virulence gene cluster of L. monocytogenes and structure of the locus in other Listeria species. Genes belonging to the virulence gene cluster are in gray, and the flanking loci are in black. The virulence gene cluster is inserted in a chromosomal region delimited by the prs and ldh genes. In the plcB-ldh intergenic region, two ORFs, orfA and orfB, are found in all Listeria species, indicating that the insertion point of the cluster is between the prs and orfB loci. In the plcB–orfB intergenic region of L. monocytogenes, there are two small ORFs, orfX and orfZ (stippled), which delimit the putative excision point of the cluster in L. innocua. In the plcB–orfB intergenic region of L. ivanovii, two small ORFs, orfX (encoding a homologue of the orfX product from L. monocytogenes) and orfL (stippled), are also present, which, like those in L. monocytogenes, delimit the deletion point of the cluster in the nonpathogenic species L. welshimeri. The additional ORFs found in the L. seeligeri virulence gene cluster are hatched. (Reprinted with permission from Vazquez-Boland, J. A. et al. 2001. Clin. Microbiol. Rev. 14:584–640.)

Some of the known virulence genes whose products are involved in the intracellular life cycle of L. monocytogenes are clustered on the chromosome in the so-called PrfA-dependent virulence gene cluster. The cluster comprises six well-characterized genes, prfA, plcA, hly, mpl, actA, and plcB (Figure 5.2) along with four small open reading frames (ORFs) of unknown functions downstream of plcB, called orf X, Z, B, and A. The ends of the gene cluster are defined by genes coding for housekeeping enzymes. Distal from prfA, defining the “left” border of the gene cluster, the prs gene is located, encoding a phosphoribosyl-pyrophosphate synthetase [117,127,185]. The ldh gene coding for lactate dehydrogenase together with the orfs A and B [45,117,127,319] mark the “right” border of the gene cluster downstream from plcB and the small orfs X and Z. The products of these virulence genes are listeriolysin (LLO, encoded by hly), a phosphatidylinositolspecific phospholipase C (PI-PLC, encoded by plcA), a phosphatidylcholine-specific phospholipase C (PC-PLC, encoded by plcB), a metalloprotease (Mpl, encoded by mpl), ActA, a protein involved in actin polymerization (encoded by actA), and the positive regulatory factor PrfA (encoded by prfA). The well-studied internalins internalin A (InlA) and InlB are encoded by the inlAB operon [102]. Many other internalin-like genes are found dispersed around the L. monocytogenes chromosome [38,117]. Several other genes suggested to play a role in virulence are located outside the virulence gene cluster [320]. Some of them are, however, connected to the virulence cluster genes because they are also regulated by the transcriptional activator PrfA (discussed later).

MOLECULAR ASPECTS OF THE INVASION OF MAMMALIAN CELLS Uptake of L. monocytogenes by macrophages of different origin is well documented [183,208,253]. Invasion of L. monocytogenes into different, normally nonphagocytic mammalian cell types, including murine and human fibroblasts [87,142,183,253], murine and human epithelial cells

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[8,87,103,253], murine hepatocytes [76,332], human endothelial cells [82,130,131,293,294], and mouse and human dendritic cells [135,173,174,247], has also been described.

INVASION

OF

NONPROFESSIONAL PHAGOCYTIC CELLS

Transposon mutagenesis and an appropriate in vitro invasion assay using Caco-2 epithelial cells resulted in the identification of internalin (InlA), a surface protein of L. monocytogenes, to mediate bacterial invasion into epithelial cells [102]. The mutants identified exhibit a lower invasive capacity than the wild-type strain when tested on different cells. Transposon insertions occur in a chromosomal region, which represents an operon consisting of the inlA and inlB genes. Expression of inlA in L. innocua, a noninvasive Listeria species closely related to L. monocytogenes, renders this species invasive (at least to some extent). This experiment shows that the inlA gene product is necessary and sufficient to mediate invasion. A large number of internalin homologues have since been identified in L. monocytogenes [38,78,89,117,257]. Common to all internalins is an element of several leucine-rich repeats with leucine residues at a fixed position in a typical 22 amino acid (aa) unit. Internalin is an acidic protein of 800 amino acids [77,102] that possesses two extended repeat domains. Domain A consists of 15 leucine-rich repeats, whereas domain B consists of 2.5 repeats of about 70 amino acids (aa) each. The InlA protein has a typical N-terminal transport signal sequence and a cell wall anchor in the C-terminal part comprising the sorting motif LPXTG followed by a hydrophobic membrane-spanning region of 20 aa and a few positively charged aa (Figure 5.3) [77]. This distal LPXTG motif, like similar motifs in other surface proteins covalently linked to the peptidoglycan in Gram-positive bacteria, is responsible for the attachment of InlA to the bacterial cell envelope in a process mediated by the enzyme sortase [21,68,109]. InlB, a 630-amino acid protein also carries an N-terminal transport signal sequence, eight leucine-rich repeats and three C-terminal GW modules; however, in contrast to InlA, it has no LPXTG motif and no cell wall-spanning region (Figure 5.3) [77]. Nevertheless, InlB is a listerial surface protein targeted to the bacterial surface via the interaction of the GW modules with lipoteichoic acid in the listerial cell wall. This type of association appears to be relatively weak because significant amounts of InlB are found in the supernatant fluid [27,158]. To get more insight into the molecular details of InlA- and InlB-mediated cellular invasion, three-dimensional structures of important parts of both proteins were solved at the atomic level [211,284,285]. In InlA and InlB (and also InlH [284]), three N-terminal parts in each protein are combined to form a contiguous internalin domain. In this internalin domain, a central LRR region is flanked contiguously by a truncated EF-hand-like cap and an immunoglobulin-like fold (Figure 5.3). The extended beta-sheet, resulting from the distinctive fusion of the LRR and the immunoglobulin-like folds, constitutes an adaptable concave interaction surface proposed to interact with the respective mammalian receptor molecules during infection. In the case of InlB it was shown that four surface-exposed aromatic amino acids along its concave face are essential for host cell invasion and binding to its receptor Met (discussed later) [206]. Four eucaryotic receptors for internalin and InlB were recently identified [28,159,219,290]. Human E-cadherin was identified as the internalin receptor by a biochemical approach using matrixbound purified InlA to isolate the internalin ligand from epithelial membrane proteins [219]. A member of the cadherin family, E-cadherin is mainly expressed at the basolateral site of enterocytes and is a major constituent of adherence junctions, where it connects adjacent cells by homophilic interactions of its extracellular domains [232]. It binds internalin directly and its location on the basolateral membrane of epithelial cells is in line with previous observations suggesting the basolateral membrane as the entry site for L. monocytogenes [311]. Antibodies directed against the leucine-rich repeat region of internalin block entry of L. monocytogenes into cells expressing E-cadherin, thereby underlining the importance of the repeat regions of internalin for its function as an invasin [219].

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(A)

(B)

FIGURE 5.3 A: Structure of the members of the internalin multigene family present in L. monocytogenes strain EGD-e. S: signal peptide; B: B-repeats; C: Csa domain repeats C-repeat; D: D-repeats. See text for details. (Reprinted with permission from Vazquez-Boland, J. A. et al. 2001. Clin. Microbiol. Rev. 14:584–640.) B: Crystal structure of the N-terminal part of InlA and the distal domain of its receptor E-cadherin. (Reprinted with permission from Heesemann, J. et al. 2003. Biospektrum 9:486–489.)

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The first InlB receptor to be isolated also by a biochemical approach was the complement receptor for the globular part of the C1q-fragment (gC1q-R). Direct interaction of the ubiquitously expressed gC1q-R and InlB was demonstrated and it could be shown that soluble C1q or antibodies against gC1q-R block InlB-mediated entry of L. monocytogenes [28]. The receptor tyrosine kinase Met was recently identified as a second InlB receptor required for InlB-dependent entry of L. monocytogenes into various cells [290]. Treatment of mammalian cells with InlB protein or infection with L. monocytogenes induces rapid tyrosine phosphorylation of Met, a receptor tyrosine kinase for which the only other known ligand is hepatocyte growth factor (HGF). Like HGF, InlB binds to the extracellular domain of Met and induces “scattering” of epithelial cells. Finally, glucosaminoglycans (GAGs) were identified as a third type of InlB ligand [159]. GAGs are present on the surface of mammalian cells, where they decorate the proteoglycans; they promote the oligomerization of growth factors such as HGF. InlB binds to GAGs through its C-terminal GW repeats, which anchor the protein to the bacterial cell surface. GAGs are hence believed to detach InlB from the bacterial surface, thus allowing its interaction with the Met receptor at the contact site of L. monocytogenes on the host cell surface [210]. L. monocytogenes uptake by macrophages and other mammalian cells depends on functional actin microfilaments because invasion requires membrane extensions formed by rearrangement of actin filaments and is hence blocked by treatment with actin depolymerizing drugs such as cytochalasins [103,183]. L. monocytogenes is taken up in a way described as a zipper mechanism, which means that the host cell membrane is in close contact with the bacterium without surface changes in the vicinity. Entry can also be blocked by tyrosine kinase inhibitors such as genistein [272,310,322] and the tyrosin phosphatase inhibitor vanadate [180], which shows that signal transduction events involving various cellular kinases and phosphatases are needed. Epithelial cell invasion by L. monocytogenes is initiated by binding of InlA to its receptor E-cadherin. The structural basis for this highly specific interaction became obvious recently, when the crystal structure of the functional InlA domain, bound to the N-terminal domain of its receptor E-cadherin, was solved [285]. The concave interaction domain formed mainly by the LRR surrounds and specifically recognizes human E-cadherin. Individual amino acid residues in InlA were probed for their role in the InlA-E-cadherin interaction. These studies explained the tremendous specificity of InlA for human E-cadherin [191] because the aa proline at position 16 in human E-cadherin (which is changed to glutamic acid in the mouse homologue) represents a very exposed aa in the molecule responsible for the intimate contact with InlA [285]. The cytoplasmic domain of Ecadherin directly interacts with β-catenin, which in turn recruits α-catenin [193]. α-Catenin directly binds to actin filaments and hence completes the direct link of the listerial receptor to the host cell cytoskeleton. Other proteins normally present in the adherence junction complex are also recruited to the site of L. monocytogenes entry [54], but their precise role in the uptake process, and the rearrangement of the actin network particularly, is unknown. Originally proposed to be a specific factor for hepatocyte invasion [76], InlB was recently shown to be the only relevant internalin for endothelial cell invasion [17,129,130,244]. InlB-specific invasion of human brain microvascular endothelial cells is very sensitive to the presence of adult human serum, which regularly harbors anti-Listeria antibodies [145]. InlB is also involved in epithelial cell invasion [17,203], but InlA and InlB play no role in fibroblast invasion [203]. As mentioned before, three mammalian receptors for InlB have been characterized and a complex series of intracellular signal transduction events are triggered by the interaction of InlB with Met (reviewed in detail by Bierne and Cossart [19] and Cossart et al. [54]), the only one of the three receptors that has a cytoplasmic domain. The PI-3 kinase is in the center of the signal transduction pathway leading to actin rearrangement [150] and is most likely activated upon interaction with adaptor proteins recruited to the phosphorylated cytoplasmic domain of Met [151,290]. The generation of PIP3 by the PI-3 kinase [150] then leads to an activation of the downstream kinases Rac, PAK, and the LIM-kinase [20]. It is currently believed that these events finally lead to the activation of the Arp2/3 complex (see later discussion),

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which is necessary to induce the actin polymerization involved in rearrangement of the actin cytoskeleton during the uptake process [54]. Additionally, the InlB-Met interaction triggers signals leading to activation of the Ras-mitogen-activated protein kinase pathway [52], the activation of phospholipase Cγ, protein kinase C, and Akt, which in turn activate the central transcription factor NF-κB [19]. For a long time, the significance of the listerial surface proteins InlA and InlB in in vivo host cell invasion was less clear. An inlAB mutant is only slightly impaired in virulence in the mouse model upon intravenous infection and the mutant was only transiently impaired in persistence in the liver and behaved like the wild-type in spleens and lymph nodes of infected mice [76]. In a different study inlAB mutants were only rarely found inside hepatocytes compared with the wildtype strain, indicating a role for the inlAB locus in hepatocyte invasion in vivo [105]. Most surprisingly, penetration of the intestinal barrier in orally infected mice was very inefficient and totally independent of the presence of InlA or InlB [191]. This finding is in sharp contrast to the unequivocally proven role of InlA in epithelial cell invasion in vitro. The identification of a single nucleotide exchange in mouse E-cadherin compared to human E-cadherin (Pro16Glu), which renders the mouse homologue unable to bind InlA [191], gave a first hint to clarify the unexpected findings in the mouse model of listeriosis. A recent publication by Lecuit et al. [194] elegantly used a transgenic mouse model to demonstrate convincingly the role of internalin in the penetration of the intestinal barrier. They constructed transgenic mice expressing human E-cadherin in their enterocytes and showed that, in these mice, L. monocytogenes mutants lacking InlA were significantly less able to cross the intestinal barrier because they were severely impaired in promoting their uptake by the enterocytes forming the intestinal barrier [192,194]. A large number of other internalins are present in L. monocytogenes as deduced from the genome sequence [38,117]; some, including InlC, InlE, InlF, InlG, and InlH [78,89,256], were already described in the pregenomic era. None of these proteins seems to be able to induce phagocytosis in mammalian cells and their roles in the infection process are largely unknown. By the construction of various combinations of in-frame deletions in most of the respective internalin genes it could recently be shown that—in contrast to InlB—InlA by itself triggers uptake into Caco-2 epithelial cells poorly and needs the help of other internalins [17]. This additional trigger function can be supplied by InlC alone or by InlC together with InlG, InlH, and InlE, or by InlB. Because no cellular receptors for internalins other than InlA and InlB are known, it is presently unclear whether these internalins trigger host cell function via the known InlB receptors or via other mammalian surface molecules. Several other L. monocytogenes surface structures have been described as involved in invasion. LpeA (Lipoprotein promoting entry) [262], a listerial lipoprotein with homology to a Streptococcus pneumoniae adherence factor, was implicated in the invasion of hepatocytes (and to a lesser extent of epithelial cells) because the respective mutant shows clearly diminished capacity of cellular invasion, but not of adhesion or intracellular growth. LpeA is the first listerial lipoprotein involved in invasion to be identified. The major extracellular protein p60 of L. monocytogenes [182,249] was initially postulated to be involved in fibroblast invasion, but its role in mediating cellular uptake is still under debate (see later discussion). The listerial surface protein ActA, a major virulence factor allowing actin-based intracellular motility [74,168] (see following), also was suggested recently to play a role in internalin-independent uptake of L. monocytogenes by epithelial cells [7]. Analysis of the invasive capacity of strains lacking or overexpressing ActA suggests that ActA may function as an invasion-mediating protein—at least when overexpressed [307]. Such an ActA-promoted attachment and invasion of CHO epithelia-like cells as well as IC-21 murine macrophages was shown to be mediated by interaction of the listerial surface protein ActA with a heparan-sulfate proteoglycan receptor [7]. It is supposed that electrostatic interactions between heparan sulfate and positively charged residues in the N-terminal part of ActA could lead to low-stringency binding to the cell surface proteoglycan

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receptors widely distributed in mammalian cells [7]. Whether the proposed low-stringency binding of L. monocytogenes to heparan sulfate proteoglycan receptors directly triggers uptake or results in adequate presentation of other bacterial factors to the host cell membrane that ultimately lead to phagocytosis remains to be clarified.

CELLULAR ADHESION The mechanisms allowing L. monocytogenes adhesion to various mammalian cells without following uptake are not well understood. InlA can clearly mediate binding to the surface of epithelial cells because mutants lacking the inlA gene bind less well to Caco-2 epithelial cells [17,262]. In contrast, InlB, which by itself triggers high-efficiency invasion of endothelial cells, is obviously not involved in cell adhesion. Mutants lacking the inlB gene adhere to human brain microvascular endothelial cells essentially as the wild-type strain [17,129]. Because even the nonpathogenic species L. innocua (which lacks the internalins of L. monocytogenes [117]), binds to endothelial cells, one must assume that common cell wall structures mediate the interaction of L. monocytogenes and L. innocua with the endothelial cells [129]. One of the listerial surface structures that may mediate internalin-independent binding to mammalian cells are the lipoteichoic acids found in listerial cells walls [91]. These polymers are known to interact with mammalian pattern recognition receptors of the scavenger- and toll-receptor families [85]. It was shown that purified lipoteichoic acid from L. monocytogenes can trigger host cell responses in macrophages [141] and such interactions may also confer cell binding. A recent paper by Abachin et al. [1] has now shown that an L. monocytogenes mutant with an altered lipoteichoic acid lacking D-alanine is severely impaired in binding to epithelial cells, hepatocytes, and even macrophages. It is believed that the mutation increases the electronegativity of the bacterial surface, which then interferes with cell binding even in the presence of internalin and InlB. Various extracellular bacterial pathogens exploit the extracellular matrix protein fibronectin as a bridging receptor for cell binding [92]. Five fibronectin binding proteins were identified in L. monocytogenes, one of which was a 55-kDa cell surface protein [114]. Additionally, the gene of one fibronectin binding protein of 25 kDa was cloned and sequenced [115]. However, the role of these listerial proteins in virulence in general and specifically in cell binding is not known. As mentioned before, the L. monocytogenes surface protein ActA can act as an invasion—at least when overexpressed. However, it is possible that in the situations tested, ActA actually acts as an adhesin allowing more efficient contact of the bacteria to the cell surface followed by interaction of the invasion-promoting molecules with the host cell [7]. Another surface protein, called p104, probably involved in listerial adhesion to Caco-2 epithelial cells was recently identified by transposon mutagenesis [243]. The mutants that lack p104 expression are reduced about tenfold in adhesion to the Caco-2 cells and antibodies against the protein also inhibit adhesion. Up to now, the only listerial protein clearly shown to mediate L. monocytogenes adhesion without promoting uptake is the surface molecule Ami [224,225]. Ami is a 102-kD autolysin with the catalytic activity in the N-terminal part of the molecule. The C-terminal cell wall anchor region is made up of repeated modules containing a GW dipeptide as also found in InlB [27]. Ami confers cell binding to epithelial cells and hepatocytes in a ∆inlAB background. It was shown by complementation that the C-terminal GW modules are responsible for mediating cell adhesion and the purified C-terminal part binds to epithelial cells in vitro [224].

UPTAKE

BY

MACROPHAGES

AND

DENDRITIC CELLS

Macrophages of different origin were used in in vitro studies to analyze the mechanisms of L. monocytogenes uptake by professional phagocytes, which are generally assumed to take up L. monocytogenes by conventional phagocytosis involving actin-polymerization. If present, complement factors C1q and C3 are deposited on the bacterial surface and stimulate L. monocytogenes uptake by binding the bacteria to the respective receptors [4,59,80].

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Macrophages ingest L. monocytogenes very rapidly and intracellular killing starts shortly after phagocytosis and leads to destruction of most of the ingested bacteria [64,66,259,325]. In a single macrophage, killed bacteria inside acidified phagosomes and phagolysosomes and growing bacteria that have escaped into the cytoplasm can be detected. These findings suggest competition between phagosome–lysosome fusion followed by killing of the bacteria and their escape from the acidified phagosome before phagosome–lysosome fusion occurs. The result of this competition is a population of cytoplasmic bacteria able to grow inside the macrophage. The route of uptake by the macrophages may also be important for the fate of the invading bacteria. As demonstrated by Drevets et al. [81], the mode of uptake is critical for subsequent survival because L. monocytogenes taken up in the presence of complement C3 leads to enhanced killing of the bacterium. Whether the surface protein InlA substantially contributes to the triggering of phagocytosis of L. monocytogenes by macrophages is still under debate. Using bone marrow-derived macrophages, InlA had only a slight effect on invasion because an inlAB mutant still showed more than 60% invasion when compared with the wild-type strain [146]. Uptake of L. monocytogenes by the mouse macrophage -like cell line J774A.1 was inhibited by at least 50% by the pretreatment of L. monocytogenes with anti-InlA antibodies and that recombinant InlA specifically bound to the macrophages [281]. It was suggested recently that the listerial protein p60 might enhance phagocytosis by macrophages because a Salmonella typhimurium strain that expresses and secretes p60 seems to be more invasive in phagocytic cells but not in enterocytes [146]. In line with this assumption is that pretreatment of L. monocytogenes with a polyclonal anti-p60 antiserum inhibits uptake of the bacteria by a macrophagelike cell line [146]. Another factor that could be involved in attachment and invasion of L. monocytogenes in macrophages is the listerial cell wall polymer lipoteichoic acid. L. monocytogenes binds to the macrophage scavenger receptor most likely via lipoteichoic acid [85,128]. This interaction may also trigger conventional receptor-mediated phagocytosis of L. monocytogenes. The mechanisms specifically underlying L. monocytogenes uptake by macrophages are not well understood. As expected, a functional actin cytoskeleton is necessary as deduced from inhibitor studies with cytochalasins [103,183]. In addition, microtubules also seem to be involved because the drugs nocodazole and cholchicin, which depolymerize microtubules, inhibit L. monocytogenes uptake by different macrophages [181]. The early signaling events associated with L. monocytogenes uptake by J774 macrophages were analyzed by Goldfine and coworkers (summarized in Goldfine and Wadsworth [125]). They demonstrated that very rapidly and before attachment and invasion, the addition of the bacteria to the macrophages results in three peaks of calcium mobilization within 10 min [323]. Whereas the first two peaks result from calcium influx, the third results from the mobilization of intracellular calcium stores. Linked to the calcium peaks is the mobilization of several protein kinase C isoforms that are translocated to the cell membrane [324]. Calcium signaling and protein kinase C activation depend on the listerial virulence factors LLO and PI-PLC (see later discussion) and modulate the uptake of L. monocytogenes into the macrophages. It appears that calcium mobilization and protein kinase C activation negatively influence the speed of the uptake process because mutants lacking LLO and PI-PLC are taken up more rapidly as the wild-type strain and specific inhibitors of PKC also induce the speed of uptake [323]. Another class of professional phagocytic cells important in the innate immune response against L. monocytogenes is the dendritic cells. They can be found in different tissues where they take up foreign material and present it to T-cells to stimulate adaptive responses [264]. Dendritic cells of mouse and human origin efficiently take up L. monocytogenes [135,173,174,247]. Invasion of dendritic cells seems to be independent of internalin and InlB [135,173], but requires a functional cellular cytoskeleton. Furthermore, the uptake is significantly improved in the presence of human serum and specifically enhanced by antibodies against the listerial protein p60, which act as opsonins [174]. At least some of the intracellular bacteria are released into the cytoplasm, but intracellular replication seems to be very low in dendritic cells. Because dendritic cells are able to migrate over long distances toward lymphoid tissue, they might also be important vectors during the early dissemination of the bacteria [255].

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ESCAPE FROM THE PHAGOCYTIC VACUOLE Hemolytic activity detected around colonies of L. monocytogenes growing on blood-agar plates was long supposed to represent a major virulence determinant because all clinical isolates of L. monocytogenes show this hemolytic phenotype. The hemolytic activity is due to the action of a cytolysin, called listeriolysin O (LLO). In experimental infections all virulent strains were found to be hemolytic, whereas nonhemolytic strains were avirulent. Nonhemolytic mutants obtained after transposon mutagenesis using the conjugative transposons Tn1545 or Tn916 [104,163,253] always proved to be avirulent in the mouse model. Virulence is restored in hemolytic revertants that have lost the transposon insertion or by the introduction of the cloned hly gene into a nonhemolytic L. monocytogenes transposon mutant [55]. Listeriolysin O, a secreted protein of 58 to 60 kDa, belongs to a family of thiol-activated, cholesterol-dependent, pore-forming toxins (CDTX) for which streptolysin O is the prototype [242]. All members of this family are inhibited by low concentrations of cholesterol and oxygen and activated by reducing agents like DTT. Cholesterol is the receptor for these cytolysins because only membranes containing cholesterol are attacked and this component inhibits pore formation and toxicity [242]. Upon addition to erythrocytes, toxin monomers oligomerize in the target cell membrane to form stable pores that can be visualized by electron microscopy (Figure 5.4) [246]. Listeriolysin O has been purified to homogeneity and its toxicity, determined by intraperitoneal injection into the mouse, shows LD50 of 1.7 µg per mouse. Optimal hemolytic activity is found at pH 5.5, a pH value much lower than that determined for the other CDTX [111], a property in agreement with the function of LLO in the acidified phagosome (see later discussion). The gene encoding LLO, hly, was cloned from strains of different serovars of L. monocytogenes and sequenced [71,216,220]. The deduced amino acid sequence for LLO yielded 529 amino acids including an N-terminal signal sequence of 25 amino acids. As expected, the sequence shows extended homologies with the protein sequences of other CDTX. The highest homology is observed in the C-terminal part and includes a highly conserved undecapeptide containing the unique cysteine thought to be essential for cytolytic activity. Site-directed mutagenesis revealed, however, that the cysteine is not essential for hemolytic activity. In contrast, a tryptophan residue, in close vicinity to the cysteine, appears to be required for hemolytic activity and virulence [222] as it was also shown for an alanine residue also located in that motif [152]. The three-dimensional structure of LLO is not known. However, the structure of the closely related toxin perfringolysin O (PFO) was solved. This toxin—and hence most likely also LLO—is composed of four domains that form an L-shaped molecule (Figure 5.4) [273]. The expression of domain 4 alone or of domains 1 to 3 of LLO showed that domain 4 is responsible for membrane binding and oligomerization, whereas the first three domains are necessary for full hemolytic activity [83,172]. Strikingly, when expressed simultaneously, the two secreted domains LLO-d123 and LLO-d4 reassembled into a hemolytically active form [83]. The role of LLO in virulence was determined by injection, intravenous and intraperitoneal, of wild-type and nonhemolytic mutants of L. monocytogenes into mice and by following the fate of the bacteria in liver and spleen. In contrast to the wild-type strain, the nonhemolytic mutants are eliminated from these organs within a few hours without eliciting protective immunity [55,104,163,253]. The role of LLO in the intracellular survival was determined using different mouse and human cell lines. In the human enterocyte-like cell line Caco-2 [103], mouse 3T6 fibroblasts [183], and mouse CL.7 fibroblasts [253], nonhemolytic L. monocytogenes mutants are as invasive as the isogenic wild-type strains. The nonhemolytic mutants are, however, incapable of survival and intracellular growth within these host cells and also in mouse peritoneal macrophages [183], mouse bone marrow-derived macrophages [253], and the mouse macrophage-like cell line J774 [253]. Electron microscopy of infected macrophages and epithelial cells revealed that nonhemolytic L. monocytogenes mutants found inside the cells are unable to open the phagosome and hence unable to escape into the

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A

B FIGURE 5.4 A: Pore-forming activity of LLO. Sheep erythrocyte ghost after treatment with purified LLO, showing the ring-shaped oligomeric structures of the toxin attached to the membrane (bar, 100 nm). (Reprinted with permission from Jacobs, T. et al. 1998. Mol. Microbiol. 28:1081–1089.) B: Crystal structure of PFO, a close relative of LLO. (Reprinted with permission from Rossjohn, J. et al. 1997. Cell 89:685–692.)

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cytoplasm of the host cells [103,315]. Additional evidence that LLO is essential for lysis of the phagosomal membrane was obtained by infection of macrophages with a Bacillus subtilis strain expressing LLO [18]. This engineered strain escapes from the phagosome into the cytoplasm, whereas the nonhemolytic B. subtilis parental strain stays in the phagosome as do the nonhemolytic L. monocytogenes mutants. The low pH optimum of LLO is in agreement with its function in the acidified phagosome. Bafilomycin or ammonium chloride treatment inhibits vacuolar acidification and inhibits the escape of L. monocytogenes from phagosomes of infected epithelial cells or macrophages. These findings sustain the importance of the low pH activity optimum of LLO for its role as a vacuole opener [15,51]. Recently, Portnoy and coworkers [156,157] analyzed the role of LLO (and especially the role of its low pH optimum) by constructing L. monocytogenes strains that secrete the closely related extracellular cytolysin perfringolysin instead of LLO. Such a strain escaped from the vacuole, but damaged the host cell. An elegant selection procedure was used to isolate mutants that do not damage the host cell upon perfringolysin expression in the cytoplasm. The mutated perfringolysins were reduced in activity at neutral pH, had a generally reduced hemolytic activity, or showed a shorter half-life in the cytoplasm. These results show that the low activity of LLO at neutral pH values and its short half-life in the cytoplasm are critical parameters for its suitability as a phagosome opener without concomitant cytotoxicity. The strains expressing mutated perfringolysins allowing intracellular growth without cell damage are totally avirulent, however [157]. Further study [118] now showed that a single amino acid exchange in LLO (Leu461Thr) results in an LLO variant with ten times higher hemolytic activity at neutral pH and an L. monocytogenes strain expressing this LLO variant is about 100-fold less virulent. The reduction in virulence is most likely associated with a higher toxicity of the molecule to the host cells, which are killed by membrane damage resulting from activity of LLO [118,119]. The importance of restriction of LLO activity to the phagosomal compartment is further supported by other lines of evidence. A so-called PEST sequence was recently identified in the N-terminal region of LLO [65,202]. PEST sequences target proteins to the proteasome degradation pathway [260], hence dramatically reducing their cytoplasmic half-life. LLO variants lacking the PEST sequence demonstrate normal hemolytic activity and allow vacuolar escape, but are toxic to their host cells. Their expression results in a decrease in virulence in the mouse model [65]. A recent publication by Lety et al. [201], however, showed that rather than the actual PEST sequence, some amino acids immediately downstream of PEST could be responsible for the correct function of LLO, thus questioning the results mentioned earlier. The expression of LLO in the infected host cell is tightly controlled; expression is induced when the bacteria are taken up by mammalian cells and seems to be highest in the phagosome [36], although it also continues in the cytoplasm [227]. A recent paper by Dancz et al. [61] finally showed that induction of LLO expression (with an IPTG-inducible expression system) at different time points after infection allows bacteria trapped in the cytosol to escape into the cytoplasm of macrophages and then grow intracellularly, again demonstrating the crucial role of LLO in phagosomal escape. Fusion of L. monocytogenes-containing phagosomes with endosomes has been observed in electron microscopy studies [315]. However, it is not known whether such an event is necessary for L. monocytogenes to progress through its intracellular life cycle [325]. The recent description of Rab5-regulated fusion of L. monocytogenes-containing phagosomes with endosomes and results that indicate that Rab5a controls early phagosome–endosome interactions and governs the maturation of the early phagosome leading to phagosome–lysosome fusion [6] show that phagosome maturation events take place upon ingestion of L. monocytogenes into macrophages [3]. On the other hand, live L. monocytogenes delays phagosome maturation and subsequent degradation by yet unknown mechanisms. It is believed that this allows the bacteria to prolong their survival inside the phagosome/endosome, assuring their viability as a prelude to escape into the cytoplasm [5]. Prolonged intraphagosomal survival of L. monocytogenes in macrophages was

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recently demonstrated [61]; this could be nicely explained by the data on an L. monocytogenesinduced delay in phagosome maturation. Listeriolysin O-independent escape of L. monocytogenes from primary vacuoles in human epithelial cells [253] is mediated by two listerial phospholipases and a metalloprotease [213] that also contribute to vacuole escape in other cells like bone marrow-derived macrophages [42]. Phospholipase activity of L. monocytogenes was first discovered as a zone of opacity surrounding colonies on egg yolk agar [100]. Transposon mutants of L. monocytogenes lacking phospholipase activity were identified by formation of small plaques on fibroblast cell monolayers [309] and by reduced hemolysis on blood-agar plates [164], indicating a participation of phospholipase activity in hemolysis. The gene encoding a phosphatidylinositol-specific phospholipase C (PI-PLC), called plcA [41,195,215], encodes a protein of 34 kDa that exhibits high homology to several Grampositive phospholipases and contains a typical transport signal sequence. This enzyme was purified from culture supernatant fluid of an overexpressing L. monocytogenes strain [124] and was highly specific for phosphatidylinositol with no detectable activity on phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. It also does not cleave phosphatidylinositol-4-phosphate or phosphatidylinositol-4,5-bisphosphate, but is active, albeit with low specific activity, on glycosylated phosphatidylinositol-anchored proteins [108]. The crystal structure of PI-PLC was recently determined (Figure 5.5) [228]. The enzyme consists of a single (βα)8-barrel domain with the active site located at the C-terminal side of the β-barrel. Unlike other

FIGURE 5.5 Ribbon diagram of the structure of PI-PLC viewing toward the active site pocket where the bound myoinositol molecule (labeled Ins in yellow) is shown. α-Helices (A to G) are colored in red, β-strands (I—VIII) in blue, loops in green. β-Strands are labeled with roman numerals. The N-terminus is labeled with N′, the C terminus with C. (Reprinted with permission from Moser, J. et al. 1997. J. Mol. Biol. 273:269–282.)

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(βα)8-barrels, the barrel in PI-PLC is open. Interaction between the substrate and the active site pocket is made by specific hydrogen bonds with a number of charged amino acid side-chains. Two histidine residues (His38 and His86) not present in the active site are, however, important for the activity of the enzyme because their mutagenesis results in PI-PLC variants with activity reduced 100-fold [11]. L. monocytogenes strains expressing these variants behave like plcA-deletion mutants in cell culture assays. In addition to the highly specific PI-PLC, L. monocytogenes produces a second phospholipase C, which hydrolyzes phosphatidylcholine (lecithin), and is thus a phosphatidylcholine-specific phospholipase C (PC-PLC) or lecithinase [112], also called broad-spectrum phospholipase C. A 32-kDa protein was detected in the supernatant liquid of an L. monocytogenes culture that showed phospholipase activity on egg yolk overlays [164]. The protein was purified to homogeneity [112,123] and is a zinc-dependent phospholipase C of 29 kDa. The pH optimum of the enzyme is between pH 6 and 7 and its activity is stimulated by 0.5 M NaCl and 0.05 mM ZnSO4. In addition to phosphatidylcholine, it also hydrolyzes phosphatidylethanolamine, phosphatidylserine, and, with lower efficiency, sphingomyelin. Phosphatidylinositol is not a suitable substrate. The purified protein exhibits weak hemolytic activity but is not toxic to mice [112]. The gene plcB, encoding PC-PLC, is part of the lecithinase operon, which consists of mpl, actA, plcB, and the small orfs X and Z [319]. PC-PLC is a protein of 289 amino acids with a 25-amino-acid N-terminal transport signal and a putative propeptide of 26 amino acids. Maturation of the 32-kDa precursor of PC-PLC occurs after secretion because both forms of the protein can be found in the supernatant liquid; it is obviously accomplished by the metalloprotease of L. monocytogenes [254,258]. Use of in-frame deletions in the plcA gene enabled clear demonstration that PI-PLC is required for efficient escape of L. monocytogenes from the phagosome of mouse bone marrow-derived macrophages. However, the mutation in plcA has only a slight effect on virulence [42]. It is assumed that PI-PLC acts in concert with listeriolysin inside the acidified phagosomal vacuole of the host cell to mediate lysis of the vacuolar membrane. The broad pH optimum of PI-PLC, ranging from pH 5.5 to 7.0, is consistent with its postulated function in the acidified phagocytic vacuole of infected cells. To further assess the role of PI-PLC, the plcA gene was expressed in L. innocua, which lacks the prfA-dependent virulence gene cluster and is therefore unable to escape from the host cell vacuole. The PI-PLC expressing L. innocua strain cannot escape from the phagosome of J774 macrophages, but shows limited intracellular growth inside the vacuoles, which appear to be structurally intact [286]. The role of PC-PLC in escaping from the primary vacuole is not clear and differs from cell type to cell type. In mouse bone marrow-derived macrophages, PC-PLC has no role in lysis of the vacuole [299]. However, in the human Henle 407, HEP-2, and HeLa epithelia-like cell lines, where escape of L. monocytogenes occurs at low efficiency independently from LLO [133,213,253], PC-PLC is required for lysis of the phagocytic vacuole together with the metalloprotease. PI-PLC is not required in this system, but the efficiency of escape was reduced in a hly, plcA double mutant [213]. The metalloprotease Mpl of L. monocytogenes indirectly contributes to pathogenicity and intracellular replication of the bacteria. Transposon mutants with insertions in the mpl gene are less virulent but grow normally inside mammalian cell lines [258]. The reduced virulence was attributed to lack of proteolytic processing and hence activation of the 32-kDa PC-PLC proform [254,305]. This also seems the way in which Mpl contributes to lysis of the vacuole in Henle 407 cells, where vacuolar escape is independent of LLO but depends on PC-PLC and Mpl [258]. Located immediately downstream of the hly gene, the mpl gene [72,218] encoding Mpl is the first gene of the lecithinase operon [72,218,319]. The deduced amino acid sequence of this protease shows high homology to several zinc-dependent metalloproteases from Bacillus species and yields 510 amino acids with a typical N-terminal signal sequence and a putative internal cleavage site. Like other metalloproteases, the enzyme is activated by proteolytic maturation resulting in a mature 35-kDa protein [72,218]. A 60-kDa protein is detected with an antiserum raised against Bacillus stearothermophilus thermolysin, which probably represents the proform of the metalloprotease.

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Only small amounts of the mature 35-kDa form of the protein were detected in the supernatant liquid of an L. monocytogenes culture [72]. Recently, the protein Mpl was purified and biochemically characterized and shown to be an enzyme active at a wide range of temperatures and pH values. The protein exhibits a high thermal stability and shows a narrow substrate specificity cleaving, besides the pro-PC-PLC, also casein and actin [50]. The translocation through and release of PC-PLC from the bacterial cell wall occurs most efficiently upon a decrease in pH as regularly encountered in primary and secondary phagosomes. The release coincides with the proteolytic activation of PC-PLC by Mpl, which both colocalize at the cell wall–membrane interface [302]. Listeria monocytogenes encodes a large number of putative lipoproteins [38,117] with largely unknown functions. By deleting the gene encoding a putative lipoprotein-specific signal peptidase, Reglier-Poupet et al. [261] recently got first insights into the role of at least some members of this protein family. The deletion mutant failed to process several lipoproteins and showed reduced virulence in the mouse model. Expression of the signal peptidase is strongly induced while the bacteria reside in the phagosome and mutant bacteria are clearly impaired in phagosomal escape. The mechanism of how listerial lipoproteins contribute to lysis of the phagosomal membrane is, however, still unknown.

GROWTH IN THE HOST CELL CYTOPLASM As mentioned earlier, phagosomal escape is a prerequisite for L. monocytogenes to replicate intracellularly and is hence a critical virulence mechanism of this species. In principle, intracellular bacteria have two possibilities for intracellular multiplication: They replicate in a membrane-bound vacuole as exemplified by many pathogens, including Salmonella Typhimurium, Legionella pneumophila, Mycobacterium tuberculosis, and Coxiella burnetii or they escape into the host cell cytoplasm. This second intracellular niche is only chosen by L. monocytogenes, L. ivanovii, Shigella flexneri, and Rickettsia spp. [92]. Because the vacuole is a potentially hostile environment, bacteria that stay there have evolved many different ways to interfere with phagosome maturation that allow them to live in an intracellular compartment fulfilling their needs [138]. L. monocytogenes starts intracellular multiplication shortly after escape from the vacuole with intracellular generation times of 40 to 60 min—close to the approximately 40-min generation time observed in rich broth culture [253]. The host cell cytoplasm hence allows listerial growth with high efficiency. However, the cytosol is poorly characterized as a substrate supporting bacterial growth and the relative abundance of nutrients is unknown. Whereas various auxotrophic mutants of L. monocytogenes are able to grow intracellularly [212], expression of several metabolic genes is increased intracellularly [166], indicating that at least some metabolites may be limiting in the cytosol, but there is no general upregulation of expression of stress proteins in intracellularly growing L. monocytogenes [139]. Early studies addressing the question whether even nonpathogenic bacteria not adapted to an intracytoplasmic lifestyle can grow in the host cell cytoplasm used B. subtilis [18] or L. innocua [90] strains engineered to express listeriolysin to allow phagosomal escape after uptake. Results of these studies showed that nonpathogenic bacteria were able to multiply—at least to some extent—in the host cell cytoplasm and hence implied that this compartment may be favorable for bacterial growth. A recent study by Goetz et al. [122] addressed this question differently by directly microinjecting L. monocytogenes and other bacteria into cytoplasm of Caco-2 epithelial cells and J774 macrophages. Intracellular multiplication of the bacteria was followed microscopically because they were constructed to express the green fluorescent protein. In contrast to the studies just mentioned that used this method, only bacteria naturally capable of intracytoplasmic growth (L. monocytogenes, S. flexneri, and enteroinvasive Escherichia coli) grew in the cells; others such as B. subtilis, L. innocua, and S. Typhimurium did not [122]. Furthermore, an L. monocytogenes mutant lacking the central virulence regulator PrfA (discussed later) did not

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multiply upon microinjection, clearly pointing to the need of specific virulence determinants necessary for efficient intracytoplasmic multiplication. The obvious discrepancies in the studies concerning the ability of bacteria to use host cell cytosol for growth have been discussed in detail elsewhere [120,240,318], but further investigation is needed before a clear decision can be made. Probably some cytosolic compartments permit bacterial growth and others do not, depending on conditions of the infected cell and infecting bacterium. In search of specific bacterial factors allowing intracellular growth, a bacterial homologue of the mammalian glucose-6-phosphate translocase (Hpt) [48] and a lipoate protein ligase (LplA1) [239] were identified that are necessary for efficient intracellular proliferation of L. monocytogenes. Expression of the Hpt permease is tightly controlled by the central virulence regulator PrfA, which induces a set of virulence factors required for listerial intracellular parasitism upon entry into host cells. Loss of Hpt resulted in impaired listerial intracytosolic proliferation and attenuated virulence in mice but did not affect bacterial growth in a rich medium like BHI broth [48]. However, Hpt alone is not sufficient for cytoplasmic growth because L. innocua expressing Hpt together with LLO cannot grow intracytoplasmically upon infection of macrophages [298]. Lack of LplA1 results in bacteria that cease intracellular replication after about five rounds of replication and are clearly defective in mouse virulence assays. A major target for LplA is the E2 subunit of the pyruvate dehydrogenase enzyme (PDH) complex; in intracellularly grown lplA1 mutants, PDH is no longer lipolyated. Studies of lipoic acid metabolism have shown that little free lipoic acid exists in mammalian cytosol [241]. Thus, LplA1 may not be important in replication of L. monocytogenes when free lipoic acid is available, but it is required in the host cell wherein lipoic acid may be scavenged by host molecules [239].

INTRACELLULAR MOTILITY AND CELL-TO-CELL SPREAD Intracellular movement of L. monocytogenes inside the host cell cytoplasm as well as intercellular spread mediated by actin polymerization were initially described by Mounier et al. [229] and Tilney and Portnoy [315]. Their studies were followed by a series of analyses describing the cell biology of the process. It was shown that L. monocytogenes moves rapidly through the cytoplasm with the help of the formed actin tails with a speed of up to 1.5 µm/sec. Quite surprisingly, L. monocytogenes rotates around its long axis as it is propelled by actin polymerization [271]. The rate of actin assembly, which occurs at the barbed ends of the actin filaments near the bacterial surface, equals the rate of actin-based motility; actin polymerization provides the propulsive force for intracellular movement, which can also take place in cytoplasmic extracts from Xenopus oocytes [60,280,312,313,314]. Mutants defective in intracellular motility were obtained by transposon mutagenesis. These mutants have lost the ability to initiate actin polymerization [309] because of insertion into a gene called actA or still induce actin polymerization but are unable to rearrange actin filaments to actin tails [184]. The gene actA is located downstream of mpl in the lecithinase operon and codes for a proline-rich protein (ActA) of 639 amino acids (Figure 5.6). Its apparent molecular weight determined by SDS-PAGE is 92 kDa [74,319]. ActA is a surface protein consisting of three domains: the N-terminal domain with the transport signal sequence, the central proline-rich repeat region, and the C-terminal part, which includes a membrane anchor [74,168,319]. Mutations in the actA gene result in loss of virulence in mice [74], lack of intracellular actin polymerization around bacteria, and inability of intracellular movement [74,168]. Inside the host cells, actA mutants form microcolonies located near the nucleus [74]. Several assays were used to prove that ActA alone is sufficient to stimulate F-actin assembly and promote intracellular movement: First, the nonmotile species L. innocua was engineered to express ActA; the recombinant bacteria induce formation of actin tails and move in cytoplasmic extracts as the wild-type L. monocytogenes strain [170]. Second, actA was transfected into mammalian cells [99,250,251] where the ActA protein (including the membrane anchor) was targeted

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FIGURE 5.6 Diagram of the molecular components required for actin-based motility of L. monocytogenes. A: Interactions between host-cell proteins and ActA at the bacterial surface. Two domains of ActA are required for normal motility. The amino-terminal domain activates actin filament nucleation through Arp2/3. The central proline-rich domain binds VASP and profilin interacts with VASP, enhancing filament elongation. B: Host protein functions throughout the comet tail. In addition to the factors that act at the bacterial surface, capping protein binds to the barbed end of actin filaments to prevent elongation of older filaments, α-actinin crosslinks filaments to stabilize the tail structure, and ADF/cofilin disassembles old filaments. (Reprinted with permission from Cameron, L. A. et al. 2000. Nat. Rev. Mol. Cell Biol. 1:110–119.)

to mitochondria, which subsequently assemble F-actin on their surface. Third, polystyrene beads were coated with purified ActA that induced F-actin polymerization in in vitro systems that also support listerial movement [39]. The ActA protein is distributed asymmetrically on the surface of L. monocytogenes. After cell division, it is concentrated at the old pole [169,311] and absent or present only at low concentrations at the new bacterial pole. This asymmetric distribution of the ActA protein is required and sufficient to direct actin-based motility by coating streptococci asymmetrically with genetically engineered ActA protein [300]. In a cell-free system, these streptococci, but not uniformly coated ones, moved efficiently in cytoplasmic extracts [300]. Additionally, only polystyrene beads coated with ActA asymmetrically induce true F-actin tails and move in extracts [39]. ActA seems to be an elongated protein [49] that thus far resists crystallization efforts. It was postulated that ActA is present on the bacterial surface as a dimer [230], but these data were questioned recently by demonstration that functional ActA is a monomeric protein [207]. Elucidation of the precise mechanisms by which ActA allows actin recruitment and intracellular movement by stimulating F-actin polymerization is in the center of research interests of several laboratories and has been reviewed in detail elsewhere [14,40,53]. Expression of mutated forms of ActA in mammalian cells [99, 250] or in L. monocytogenes [49,186–188,252,297,301,306] made it possible to define regions of the ActA protein with specific functions in recruitment of cellular proteins and hence in actin polymerization and movement. Deletion of the whole N-terminal domain of ActA was followed by total abolishment of actin polymerization and intracellular movement in both systems, showing the absolute necessity of this domain in ActA function [186]. Within the N-terminal part of ActA, two smaller regions were identified that are required for filament elongation (aa 117 to 121) or for continuity of the actin tail (aa 21 to 97); their deletion led to discontinuous actin tail formation [187,188,252]. In contrast, deletion of the C-terminal domain did not inhibit actin assembly [186]. The actin tails produced for L. monocytogenes strains expressing ActA without the central proline-rich repeats were significantly shorter and movement speed was drastically reduced [301]. The protein composition of the F-actin tails was analyzed using different methods and several actin binding proteins and proteins regulating the actin dynamics were colocalized with tails, including α-actinin, tropomyosin, vinculin, talin, fimbrin, villin, ezrin/radixin, cofilin, coronin, Rac,

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capZ, profilin, VASP, Mena, and the Arp2/3 complex [44,60,63,70,113,167,311,313,328]. From these proteins only profilin, Mena, the Arp2/3 complex, and the vasodilator-stimulated phosphoprotein (VASP) are associated with the surface of moving bacteria and colocalize with ActA [44,113,313]; only VASP, Mena, and the Arp2/3 complex directly bind to ActA [44,113,252,334]. Another cellular protein, called LaXp180, directly binds to ActA-expressing intracellular L. monocytogenes. This resulted in recruitment of the cellular phosphoprotein stathmin [13,248]. However, the role of these proteins in intracellular motility, if any, is unknown. The Arp2/3 complex, named after two of its members (Actin related proteins 2 and 3), consists of seven host cell proteins and was initially characterized as a profilin-binding protein complex [205]. Arp2/3 initiates F-actin polymerization because of its nucleation activity [231], which is activated significantly by the presence of ActA [328,329]. Studies with ActA mutants clearly demonstrated that the N-terminal region of ActA is sufficient for this activation [297]. The regions of the ActA protein that directly interact with proteins from the Arp2/3 complex during activation were mapped to two regions spanning the acidic aa 41 to 46 and the basic aa 146 to 150 [26,252,334]. In this interaction, ActA is thought to mimic the activity of proteins of the WASP family, which are natural ligands and activators of Arp2/3 [26]. Arp2/3 can also bind to the side of pre-existing actin filaments and initiate a new filament at this point, creating branched structures found throughout the comet tails associated with moving L. monocytogenes [327]. In summary, current data point to a central role of the Arp2/3 complex in initiating actin-based listerial intracellular movement. However, a recent study showed that ActA can also generate new actin structures in an Arp2/3independent but VASP-dependent manner [94]. This clearly shows that a complex protein like ActA can interfere with host cell actin polymerization machinery in multiple ways. The phosphoprotein VASP binds directly to the proline-rich repeats of ActA [44] via its Ena/VASP-homology domain [238]. On the other hand, VASP is a natural ligand of profilin [263] and could stimulate actin assembly by binding to ActA and enhancing profilin concentration in the vicinity of the bacterium. In this respect it is important to note that ActA can interact simultaneously with four Ena/VASP homology domains [207] and thus recruit at least four profilins to the bacterium. Mena, which is closely related to VASP, also binds ActA and profilin directly and might function in concert with VASP to recruit profilin–actin complexes to the site of actin polymerization [113]. However, profilin is dispensable, at least in vitro, because profilin-depleted cytoplasmic extracts still supported actin assembly and bacterial movement [209]. ActA can bind G-actin with a region in its N-terminal part, but deletion of this region does not interfere with actin tail formation in infected cells [297]. It is believed that, inside cells, the VASP-mediated profilin/G-actin recruitment can bypass defects in actin binding of ActA [296]. Another function recently attributed to Ena/VASP proteins is control of temporal and spatial persistence of bacterial actin-based motility [9]. Furthermore, purified VASP binds to F-actin [189] and enhances actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing frequency of actin branch formation. The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility [279,296]. The 92-kDa ActA protein on the bacterial surface is cleaved by the listerial metalloprotease Mpl, resulting in a major 72-kDa degradation product and, depending on the Listeria strains tested, additional smaller degradation products [121,237]. These products are found on the bacterial surface or in the supernatant liquid as 65- and 30-kDa fragments [237]. ActA is also degraded inside the cytoplasm of the infected host cell [226]. However, this type of degradation seems to be mediated by the proteasome of the host cell because it can be blocked by proteasome inhibitors. Additionally, ActA becomes phosphorylated inside the host cell as shown by the presence of three distinct forms of the protein with slightly different motilities in SDS-PAGE found in infected cells [33]. A genetically engineered ActA variant, which is fully functional but lacks the C-terminal region, is no longer phosphorylated inside host cells, suggesting that phosphorylation may not be necessary for movement [186]. The roles of Mpl- or proteasome-mediated ActA degradation as well as the

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phosphorylation inside the host cell are currently not understood, but one could imagine that different variants of ActA could interact differently with host cell proteins regulating the dynamics of the F-actin cytoskeleton. Expression of ActA is controlled by PrfA (discussed later) as are the other genes of the virulence gene cluster. However, different virulence factors are required at different steps of the intracellular life cycle and in different quantities. These necessities are reflected by complex regulatory networks governing expression of the virulence genes; this is far from being completely understood. ActA expression is maximally induced when bacteria have reached host cell cytoplasm and the expression level is increased more than 200-fold in cytosolic bacteria in comparison to broth-grown cultures [291]. ActA expression in intracellularly located bacteria was analyzed by reporter gene fusions (β-galactosidase, gfp, or β-glucuronidase) to actA [36,95,217,291] or by direct measurement of actA-specific transcripts [36]. In all these studies, the principle finding was identical, showing a marked induction of ActA expression in intracytoplasmically located L. monocytogenes. L. monocytogenes can spread from cell to cell without leaving the cytoplasm by forming microvilli-like protrusions on the host cell surface that are phagocytosed by neighboring cells [229,315]. The mechanisms of microvilli formation and induction of phagocytosis by the neighboring cell are largely unknown. Listeria randomly move through the cytoplasm; bacteria are finally propelled into the host’s distended plasma membrane and long protrusions are formed (Figure 5.1). In a monolayer of cells, the protrusions enter neighboring cells; they are subsequently taken up and a secondary vacuole with a double membrane is formed. This secondary vacuole is disrupted, which requires the action of LLO and the listerial phospholipase PC-PLC. Electron micrographs of plcB mutants inside mammalian cells [319] show numerous bacteria followed by actin tails and trapped in vacuoles surrounded by double membranes, indicating that plcB mutants are unable to lyse the double membrane of the vacuole. Careful examination of the plaque formation capacity of different mutants, which is thought to be a good correlate for intercellular spread, revealed that in addition to the broad spectrum phospholipase PC-PLC, PI-PLC and the metalloprotease contribute to plaque formation, most likely by supporting lysis of the double membrane vacuole [214,299]. The listerial metalloprotease Mpl probably supports this lysis by proteolytic activation of PC-PLC, but host cell proteases may also cleave and hence activate PC-PLC in the absence of Mpl [214]. The pivotal role of LLO in escape from the secondary vacuole was shown in an elegant study using an L. monocytogenes hly mutant coated with recombinant LLO, which allowed bacteria to escape from the primary vacuole and spread into neighboring cells once. However, dilution of the recombinant LLO through bacterial replication inhibited further spread of bacteria [110]. The predominant role of LLO in lysis of the secondary vacuole and hence in cell-to-cell spread was confirmed by microinjection of hly mutants into the cytosol [122] and use of strains allowing temporal control of LLO expression [61]. Listerial factors other than ActA, LLO, Mpl, and PCPLC are not known to contribute to intercellular spread. The timeline and mechanics of cell-tocell spread were analyzed in some detail by Robbins et al. [270], who presented a model for this process. Upon membrane contact, bacteria continue to move and form the protrusion. The fitful bacterial movement stops after several minutes during which the protrusion is formed. After about 20 min, the protrusion suddenly collapses and the double membrane vacuole is formed and rapidly acidifies to activate LLO and PC-PLC. Upon lysis of the membrane, motility recovers after one to two bacterial generations.

BILE SALT HYDROLASE—A NOVEL VIRULENCE FACTOR Comparison of L. monocytogenes and L. innocua genomes [117] has revealed the presence of an L. monocytogenes-specific putative gene, termed bsh, encoding a bile salt hydrolase (BSH) [86]. Bile salts are end products of cholesterol metabolism in the liver; they are stored in the gall bladder and released into the duodenum, helping fat digestion. In addition, bile salts are known to have

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antimicrobiol activity because they are amphipathic molecules that can attack and degrade lipid membranes. Some intestinal microorganisms have hence evolved mechanisms to resist the detergent action of bile, including synthesis of porins, efflux pumps, and transport proteins [134]. Others produce bile salt hydrolases that transform and inactivate bile salts. Deletion of the bsh gene from the L. monocytogenes chromosome results in increased bile sensitivity, reduced virulence, and reduced liver colonization after infection of mice. This demonstrates that BSH is a novel L. monocytogenes virulence factor involved in the intestinal and hepatic phases of listeriosis. In addition, the bsh gene is positively regulated by the central listerial virulence regulator PrfA (see later discussion), further demonstrating that it is a true virulence factor.

ACCESSORY VIRULENCE FACTORS The listerial proteins reviewed so far are true virulence factors because their sole function is specifically connected to colonization of the vertebrate host; they are found exclusively in pathogenic Listeria species. Other listerial factors involved in virulence have been identified that also contribute to successful colonization of the host. They have functions outside the pathogenic lifestyle and are present in nonpathogenic Listeria species. Such factors are sometimes called accessory virulence factors. P60,

PRODUCT

OF THE IAP

GENE

Protein p60 is a major protein secreted by all L. monocytogenes isolates [34,182]. It is also found on the cell surface of the bacteria [278]. Because it possesses murein hydrolase activity that appears to be involved in a late step of cell division, p60 is an important enzyme for cell metabolism of L. monocytogenes cells [333]. The gene for this protein, initially called iap (invasion associated protein), codes for an extremely basic protein of 484 amino acids, with a 27-amino-acid signal sequence and an extended repeat domain consisting of 19 threonine-asparagine units separated by a proline–serine–lysine motif. A single cysteine found in the C-terminal part of p60 is probably essential for its enzymatic activity [171,333] and amino acids in the N-terminal region of the protein define its intracellular stability [295]. p60 is a member of a protein family in L. monocytogenes with three members: p45, which is a peptidoglycan lytic protein [283] encoded by the spl gene; the putative protein encoded by lmo394 [117]; and p60. At least one type of rough mutant of L. monocytogenes characterized by expression of reduced amounts of p60 shows significantly reduced uptake by 3T6 fibroblast cells [182]. These mutants form long cell chains that possess double septa between the individual cells. Treatment of L. monocytogenes rough mutants with partially purified p60 protein disaggregates the cell chains to normally sized single bacteria, which again become invasive for fibroblasts. Ultrasonication leads to physical disruption of the cell chains, producing similar single cells that are noninvasive. The reduced invasiveness of the p60 mutants is only observed with certain mammalian host cells. Cell chains of p60 mutants adhere normally to Caco-2 epithelial cells and are perfectly invasive for these cells upon disruption of bacterial cell chains by ultrasonication without addition of p60 [34]. Other rough mutants of L. monocytogenes that show normal or even increased levels of p60 expression have been isolated [277]. These mutants are adherent and invasive like WT bacteria despite formation of long filaments. The genetic basics of these phenotypes are unknown. p60 was long regarded as an essential protein [333]. However, viable mutants with in-frame deletions in the iap gene recently demonstrated that p60 is not an essential protein for L. monocytogenes [199,249]. Detailed characterization of one of the deletion mutants showed that the mutant forms cell chains but is nearly as invasive as the WT strain. However, the mutant grows in microcolonies inside host cells and does not form F-actin tails; it only induces actin clouds around the bacteria. A defect in polar ActA distribution caused by impaired cell division is the reason for lack of intracellular motility in this p60-lacking strain. According to these findings, p60, renamed

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CwhA for cell wall hydrolase A, seems not to be linked directly to cell invasion; rather, it indirectly modifies bacterial behavior via its impact on cell division [249]. Another L. monocytogenes peptidoglycan hydrolase, called MurA or p66, was recently identified [43]. Deletion of the gene encoding MurA, which shows homology with p60 in its C-terminal domain, also results in the formation of long cell chains. It is currently believed that p60 and p66 may act in concert to control proper cell separation during the last step of bacterial division [43]. Two recent papers by Portnoy and coworkers [199,200] also shed light on genetic and biochemical bases of reduced p60 expression and the rough phenotype. They described an additional secA gene in L. monocytogenes, called secA2, located immediately upstream of the iap gene and involved in the smooth–rough transition. They found that rough mutants with reduced p60 expression have a deletion in the secA2 gene or express truncated SecA2 proteins. Furthermore, deletion of the secA2 gene resulted in reduced p60 expression and conversion to the rough phenotype. Rough mutants displaying normal p60 levels are not affected in their secA2 gene. A number of additional proteins, the secretion of which is secA2 dependent, have been identified [199]; it has been shown that the hydrolytic activity of p60 is crucial for L. monocytogenes virulence through generation of glucosaminylmuramyl dipeptide, which modifies host inflammatory responses.

SUPEROXIDE DISMUTASE

AND

CATALASE

Possible roles of catalase and superoxide dismutase in the virulence of L. monocytogenes have recently been reviewed [137,320]. Both enzymes act in concert to detoxify potentially harmful superoxide radicals. Generated by the oxidative burst in a phagocytic cell, these radicals are converted into hydrogen peroxide by action of superoxide dismutase, which is then cleaved by catalase into water and molecular oxygen. Bacterial catalases and superoxide dismutases have long been suspected to be important virulence factors of intracellular bacteria, but no correlation of superoxide dismutase expression with virulence was found in L. monocytogenes [326]. The gene for superoxide dismutase from L. monocytogenes [29], called lmsod, reveals an ORF coding for a protein of 202 amino acids with high homology to manganese-containing superoxide dismutases from other organisms. Catalase mutants obtained by transposon mutagenesis show wild-type virulence in infected mice [190]. Whereas catalase-negative L. monocytogenes mutants are killed by mouse resident macrophages already at low serum concentrations, killing wild-type bacteria requires high serum concentrations, suggesting that resistance to fully activated macrophages is partially mediated by catalase activity [316]. Isolation of catalase-negative L. monocytogenes strains from listeriosis patients supports the notion that catalase does not seem to be necessary for intracellular growth of L. monocytogenes [35,88]. Also in line with these data is the finding that all species of Listeria produce the same type of SOD, which is constitutively expressed and not regulated by environmental factors [317], as true virulence factors are.

STRESS RESPONSE MEDIATORS Bacterial survival under stress conditions requires an adaptive response mediated by a set of conserved proteins that are upregulated upon exposure to many different stress conditions and when bacterial growth is restricted. However, in contrast to other facultative intracellular bacteria, L. monocytogenes does not induce expression of general stress proteins during intracellular proliferation in macrophages [139]. Nevertheless, stressful conditions are likely to be encountered by L. monocytogenes during transient residence in the phagosome upon uptake by macrophages. In that stage, expression of the hly gene encoding LLO is induced as it is under other stress conditions like heat treatment [303–305]. A group of virulence-associated stress mediators involved in phagosomal escape and intracellular multiplication were identified in L. monocytogenes. The first was ClpC, an ATPase belonging

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to the heat-shock protein 1000 family [282]. The gene encoding the ClpC ATPase, called clpC, was identified by Tn917 mutagenesis and a selection for mutants depending on iron [274]. The clpC mutants are highly susceptible to stress, including iron limitation, elevated temperatures, and high osmolarity. Virulence of these mutants is severely impaired in the mouse with restricted capacity to grow in bone marrow-derived macrophages [275,276]. Electron microscopy of infected macrophages showed that clpC mutants remained inside the phagocytic vacuole, indicating that ClpC is involved in phagosomal lysis or helps the bacteria to survive in the harsh conditions of this environment to allow production of sufficient amounts of LLO and PI-PLC to destroy the vacuolar membrane [275]. Expression of ClpC is not directly controlled by PrfA and no PrfAboxes (discussed later) are found in the promoter region of the clpC gene. In contrast, overexpression of the PrfA regulon leads to a loss of ClpC expression, showing that expressional cross-talk between PrfA and ClpC is most likely mediated by a negative regulator activated by PrfA [269]. Recent evidence also points to a role for ClpC in expression of internalin, InlB and ActA, because ClpC is required for efficient uptake into several cell lines [235]. ClpE is closely related to ClpC and is also involved in virulence of L. monocytogenes [234]. L. monocytogenes mutants with deletions in the clpC and clpE genes are totally avirulent in mice; it is believed that both proteins have redundant functions in stress tolerance because the absence of one is compensated by the transcriptional upregulation of the other [234]. ClpP is a stress-induced L. monocytogenes serine protease of 22 kDa belonging to a highly conserved protein family. ClpP is required for growth under stress conditions and for survival in macrophages and infected animals [107]. ClpP seems to be involved in stress-induced upregulation of LLO expression because clpP-mutants secrete only small amounts of functional LLO [106]. Expression of ClpC, ClpE, and ClpP is negatively controlled by ctsR, the first gene of the operon containing ClpC [233]. L. monocytogenes CtsR is homologous to the B. subtilis CtsR repressor of stress response genes. Consistent with the function of CtsR as a repressor is the fact that a ctsR deletion mutant showed enhanced survival under stress conditions and normal virulence, whereas overproduction of CtsR resulted in a significantly attenuated virulence in the mouse model of infection [233].

IRON UPTAKE SYSTEMS Iron is a key element for all bacteria, serving as a cofactor in many proteins involved in electron transport processes. Inside the host organism, however, iron is not freely available because it is tightly bound to transferrin or ferritin. Pathogenic bacteria therefore had to evolve specific mechanisms to capture iron from host tissues and these uptake mechanisms play an important role in virulence. In L. monocytogenes, different iron-uptake mechanisms have been described [32]: The first uses the direct transport of ferric citrate [2] and the second involves an extracellular ferriciron reductase that uses iron-loaded catecholamines as a substrate [12,57,67]; the third system may use a surface-located transferrin-binding protein [140]. Unlike many other pathogenic bacteria, L. monocytogenes does not secrete siderophores to capture extracellular iron [58]. The genetic basis of different iron uptake systems has not been studied and there is no experimental evidence whether these systems directly contribute to pathogenesis of L. monocytogenes infection.

PRFA AND REGULATION OF VIRULENCE GENE EXPRESSION IN L. MONOCYTOGENES THE POSITIVE REGULATORY FACTOR A (PRFA) The first indications for coordinate regulation of virulence genes in L. monocytogenes by a trans acting factor were obtained from analysis of spontaneously occurring nonhemolytic mutants of L. monocytogenes, which carry deletions in a region upstream of the hly gene [126,197]. Cloning and

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sequencing of the locus affected by the deletion led to identification of the prfA (positive regulatory factor A) gene. Its product, PrfA, a cytoplasmic protein of 27 kDa [198,217], regulates all virulence genes of the virulence gene cluster. The prfA deletion mutants can be complemented in trans by introduction of the cloned prfA gene to yield a wild-type phenotype again [198]. Site-specific mutations or transposon insertions in the prfA promoter or the prfA coding region block transcription of the entire gene cluster, i.e., plcA, hly, mpl, actA, and plcB [47,217]. This indicates that the prfA gene encodes a transcriptional activator required for expression of the L. monocytogenes virulence gene cluster. Additional evidence for this presumptive role of PrfA was provided by transcriptional activation of the cloned hly gene by PrfA in B. subtilis [98]. Also present in the closely related species L. ivanovii and L. seeligeri are prfA-like genes with high sequence similarity to prfA from L. monocytogenes [178,185]. In L. seeligeri the prfA gene is silenced [178] and activity of the protein is not known; however, PrfA from L. ivanovii also controls sets of virulence genes similar to those of L. monocytogenes [185]. PrfA is a member of the Crp/Fnr family of transcriptional activators, which have been identified so far mainly in Gramnegative bacteria. Like all members of this family, PrfA contains a conserved helix–turn–helix motif in its C-terminal part. In addition, adjacent to this motif, PrfA carries a sequence with features of a leucine zipper and a second helix–turn–helix motif at its N-terminus [30,185,288] (Figure 5.7). In hyperhemolytic strains of L. monocytogenes, an altered PrfA, termed PrfA*, has been identified and characterized and it was shown that all these PrfA variants carry a Gly145Ser

FIGURE 5.7 Structure of PrfA (A) and its target DNA sequences in PrfA-regulated promoters (B). In PrfA, amino acid coordinates correspond to the peptide sequence deduced from the prfA gene (position 1 corresponds to the Met residue of the first triplet), whereas in Crp they correspond to the actual amino acid position in the primary structure of the native protein, which lacks the residue encoded by the first triplet. Therefore, the amino acid numbering of Crp is shifted one position with respect to that of PrfA and hence position 144 of Crp, where the Crp* mutation Ala144Thr lies, aligns exactly with position 145 of PrfA, where the Gly145Ser substitution leading to the PrfA* mutant phenotype is located. In Crp, A to F designate the α-helical stretches of the protein and AR1 is activating region 1 (two other activating regions [AR2 and AR3] are embedded within the β-roll structure). In PrfA, structures specific for this protein are in light gray. (B) N indicates any nucleotide. (Reprinted with permission from Vazquez-Boland, J. A. et al. 2001. Clin. Microbiol. Rev. 14:584–640.)

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substitution [23,267]. Furthermore, all genes of the virulence gene cluster are highly expressed in these strains and do not respond to conditions under which expression of virulence genes is induced in wild-type strains [267]. Interestingly, Crp mutants are known that carry a mutation at similar positions and lead to a transcriptionally active conformation without the need of the cofactor cAMP (Figure 5.7). This suggests that wild-type PrfA also needs a conformational change for activity and that the Gly145Ser substitution results in a constitutively active form of PrfA [321]. Three other mutations in PrfA were recently reported to increase PrfA function: a Gly155Ser mutation that appears to be similar in nature to Gly145Ser and a Glu77Lys substitution located in the β-roll structure in the N-terminal part of the protein [292]. A Ser183Ala substitution located in the second HTH motif that also resulted in increased PrfA activity was described by Sheehan et al. [288]. On the other hand, it was shown that inactive PrfA can be present in large quantities in L. monocytogenes [266]. In addition, PrfA mutants were selected with single point mutations rendering the protein partially or totally inactive [144]. These mutants were unable to bind to DNA and harbored mutations in the leucin zipper or the HTH motif or they still bound to the target DNA but were unable to form a stable complex with RNA polymerase and were located in the β-roll structure. Other studies suggested that PrfA-mediated activation of gene expression requires presence of a coactivator protein [22, 69] the nature of which is still unknown.

PRFA-DEPENDENT PROMOTERS, TRANSCRIPTS, AND MECHANISM OF TEMPERATURE-DEPENDENT VIRULENCE GENE EXPRESSION A 14-bp palindromic sequence first identified in the promoter region of the hly gene [221] was found to be present in promoters of all PrfA-dependent genes, located about 40 bp upstream of the transcriptional start site. However, the 14-bp palindrome, called PrfA-box, is not perfectly conserved in all promoters and the differences could contribute to differential regulation of the adjacent genes by PrfA (Figure 5.7) [30,176,289,331]. Meanwhile, it was shown that PrfA directly binds to these palindromic sequences [22,24,69,288], thereby activating the virulence genes. Purified PrfA alone is able to bind specifically to the target sequence as shown by gel motility shift assays. However, addition of PrfA-free cell extracts results in formation of additional PrfA-containing DNA-binding complexes that also contain RNA polymerase [24,69]. Whether PrfA first binds to the PrfA-box and enhances binding activity for RNA polymerase or whether the PrfA/RNA polymerase complex may already form before interaction with DNA is still unknown. The observation that PrfA can exist in an inactive form and the striking similarity of the cAMP-independent mutant Crp* and the constitutively active PrfA* strongly suggest involvement of a cofactor in regulation of PrfA activity. Despite extensive investigations, such a cofactor remains unknown [178]. The transcriptional organization of most PrfA-regulated genes—especially the virulence gene cluster—is complex. Three transcripts of the prfA gene have been identified: a long (2.1 kb) transcript cotranscribed with the plcA gene and autoregulated by PrfA, and two shorter ones (0.8 and 0.9 kb) transcribed from three distinct promoters located in front of the prfA gene [97]. The listeriolysin gene, hly, is the only gene in the virulence gene cluster transcribed in a monocistronic mRNA from two PrfA-dependent promoters, P1 and P2, located in the intragenic region between hly and plcA [221]. A third hly promoter, P3, downstream from P1 and P2, was recently identified and shown to be PrfA independent; it results in low-level transcription of the hly gene [73]. The three genes of the lecithinase operon are transcribed from at least two PrfA-regulated promoters: One, located in front of the mpl gene, yields a 5.4- to 5.7-kb transcript comprising mpl, actA, and plcB, as well as an additional mRNA of 1.8 kb comprising mpl alone [24]. A second promoter, located directly in front of the actA gene, leads to a 3.6-kb bicistronic transcript comprising actA and plcB [24]. The inlAB operon is transcribed from multiple PrfA-dependent and -independent start sites upstream of inlA. In different studies, three or four start sites were mapped in the promoter region upstream of inlA that harbors a degenerated PrfA-box with two mismatches

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(Figure 5.7) [79,203]. In addition, an inlB monocistron is detectable and a putative transcription terminator is located between inlA and inlB. Overall, transcriptional control of inlAB expression is complex and not fully understood. Full expression of PrfA-dependent virulence genes requires synthesis of monocistronic prfA transcripts and the bicistronic plcA-prfA transcript [96,42]. In an initial step of the infectious process, it is believed that transcription of prfA via the prfA promoters results in synthesis of a limited amount of PrfA sufficient to activate the high-affinity PrfA-dependent hly and plcA promoters. This, in turn, would result in synthesis of the plcA-prfA transcript, which leads to enhanced PrfA synthesis. The higher cellular level of PrfA activates the mpl and actA promoters, which seem to have a lower affinity for PrfA because of base mismatches in their palindromic PrfA boxes [53]. As in many other instances [149,175], PrfA-dependent virulence gene expression in L. monocytogenes is thermoregulated and the shift from 30 to 37°C results in a dramatic increase in expression of virulence genes [196,217]. Detailed recent analysis of this phenomenon culminated in identification of an RNA thermosensor controlling temperature-dependent gene expression [155]. Low expression of virulence genes at temperatures below 30°C coincides with absence of PrfA protein. However, quite surprisingly, the prfA gene is still transcribed under these conditions from its own promoter, resulting in a monocistronic prfA transcript [196,266]. At 37°C, prfA is transcribed from the prfA promoter and the PrfA-dependent plcA promoter, resulting in monocistronic and bicistronic plcA-prfA messengers [196,217,266]. Johannson et al. [155] could now demonstrate that, at 30°C, the monocistronic prfA messenger is not translated because the upstream untranslated mRNA (UTR) preceding prfA forms a secondary structure that masks the ribosome-binding region. This secondary structure is thermosensitive and hence unstable at higher temperatures (37°C), which then allow efficient translation resulting in an approximately fivefold increase in PrfA levels. As mentioned earlier, in the presence of PrfA, the bicistronic plcA-prfA messenger is transcribed from the PrfA-dependent promoter upstream of plcA, further increasing the amount of PrfA in the bacterial cell and finally allowing virulence gene expression from low-affinity promoters. L. monocytogenes has hence evolved a sophisticated twostep mechanism to control PrfA-dependent virulence gene expression (reviewed in Johansson and Cossart [154] and Newman and Weiner [236]). Before completion of the genomic sequence of L. monocytogenes [117], knowledge of the PrfA regulon was very limited. At that time, the only known genes that did not belong to the virulence gene cluster but were also regulated by PrfA were inlAB, [79] and inlC [75,89]. The availability to sequence the complete genome of L. monocytogenes allowed in silico screening of the sequence for genes preceded by putative PrfA-boxes. This screen identified four additional previously unknown genes harboring PrfA-boxes. One of the genes, later called hpt, encodes for a putative hexose permease, expression of which was shown to depend strictly on PrfA [48]. The other three genes preceded by PrfA-boxes are genes of unknown function. A recent systematic approach to elucidate the complete PrfA regulon used a whole genome macroarray based on the complete genome sequence of L. monocytogenes [223]. With this macroarray, expression profiles of the wild-type strain and a prfA-deletion mutant were compared upon growing the bacteria in standard BHI medium or in media known to induce or to repress virulence gene expression. With this approach, three groups of differently regulated genes were identified. In addition to the 10 already known PrfA-regulated genes, Group I comprises 2 new genes, positively regulated and preceded by a PrfA box. Group II comprises eight negatively regulated genes: One is preceded by a PrfA box and the others form an operon. Group III comprises 53 genes of which only 2 are preceded by a PrfA box and that are activated or repressed under different conditions; most of the genes in this group are transcribed from sigma B-dependent promoters. Taken together, the results suggest that PrfA positively regulates a core set of 12 genes preceded by a PrfA box that is probably expressed from a sigma A-dependent promoter and negatively regulates 8 genes. A second set of PrfA-regulated genes lacks PrfA boxes and is expressed from sigma B-dependent promoters. The data presented reveal that PrfA can act as an activator or a repressor and suggest

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that PrfA may directly or indirectly activate sets of genes in association with different sigma factors [223]. The important role of sigma B in expression of L. monocytogenes virulence genes was also confirmed by recent studies [165,308] showing that sigma B regulates not only stress response genes but also several well-known virulence genes like those encoding the internalins and the bile salt hydrolase.

ENVIRONMENTAL SIGNALS AFFECTING VIRULENCE GENE EXPRESSION Pathogenic bacteria that can also live in the free environment are forced to sense their surroundings in order to regulate expression of genes needed for living inside or outside their host. Facultative intracellular pathogens additionally should be able to know whether they are inside or outside their individual mammalian host cell. An increasing number of signals have been shown to affect virulence gene expression in L. monocytogenes (reviewed in Brehm et al. [30] and Kreft and Vazquez-Boland [178]). The signals can be classified into physicochemical signals (temperature, iron, glucose, cellobiose, salt, pH, activated charcoal) or stress conditions (heat shock, oxidative stress, nutritional stress, growth inside host cells). The mechanisms of altered gene expression under such conditions are unknown or only poorly understood. However, in all systems analyzed, PrfA plays a role in regulation of environmentally modulated gene expression. At temperatures below 30°C, the PrfA-dependent genes are not transcribed because of a lack of translation of the monocistronic prfA transcript (see earlier discussion) [155]. A shift in temperature to 37°C results in onset of prfA expression followed by transcription of the virulence cluster genes [79,196]. Treatment of culture medium with activated charcoal probably depletes a signal molecule from the medium; this would result in increased transcription of prfA and the PrfA-dependent genes [268]. Carbohydrates modulate virulence gene expression in a complex and poorly understood manner. Glucose directly influences prfA gene expression and thereby interferes with PrfA regulation. Addition of larger amounts of glucose to the medium results in acidification, which reduces LLO expression by unknown mechanisms [62,97]. The disaccharide cellobiose results in inhibition of hly and plcA expression without reduction in monocistronic prfA mRNA levels and with only slightly decreased amounts of PrfA protein. This indicates that the inhibitory mechanism exerted by cellobiose leads to a change in PrfA activity [16,166,245]. Two genomic loci have been identified [31,148] that contribute to cellobiose-mediated virulence gene repression; one of these, the bvrABC system, most likely encodes for a sensor system for extracellular cellobiose [31]. The mechanisms of stress-mediated altered gene expression are even less understood. Heat-shock conditions increase hly, plcA, and actA expression. P60 expression is inhibited by heat shock and also by oxidative stress (H2O2) [303–305]. Shift of L. monocytogenes from a rich medium into a minimal essential medium (MEM) induces expression of the virulence cluster genes as well as other surface-associated proteins [268]. Phagocytosis and intracellular localization are supposed to be natural stress factors. It has been shown that expression of numerous proteins is selectively induced during phagocytosis of L. monocytogenes by macrophages [139]. An insertional mutagenesis study [166] and two IVET studies [84,101] tried to identify genes preferentially expressed inside mammalian cells using the hly gene as a reporter. The first study resulted in identification of genes involved in nucleotide biosynthesis, an arginine transporter, and plcA [166]; the IVET studies only identified some of the known virulence genes and some unknown genes whose function is unknown in the intracellular life cycle. Experiments directly measuring bacterial mRNA levels inside host cells by RT-PCR or gfp-reporter gene assays revealed that the genes hly, actA, and inlC are heavily expressed inside the mammalian cell [24,36,89,95,291]. It was also shown that PrfA is upregulated during interaction of L. monocytogenes with host cells [265]. The complexity of regulation of gene expression inside the host cell is, despite all progress, far from understood.

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AND

REGULATION

OF

137

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Signal transduction mechanisms allowing bacteria to modulate gene expression in response to diverse stimuli often involve two-component systems (TCSs) composed of a sensor kinase and a response regulator [132]. The sensor kinase is often localized in the cytoplasmic membrane to sense outside signals and contains a highly conserved cytoplasmic kinase domain which, upon activation, phosphorylates the cognate response regulator. The phosphorylated response regulator then binds to specific DNA sequences and activates transcription of its target genes. TCSs have not been studied intensively in L. monocytogenes; lisR/K [52], cheY/A [93], agrA/C [10], and cesRK [161] were characterized in some detail and shown to contribute to virulence of L. monocytogenes. Kallipolitis et al. [160] identified a total of seven putative TCSs in a PCR-based approach including the already known lisR/K. Availability of the complete genomic sequence of L. monocytogenes strain EGD-e allowed the in silico identification of 16 putative two-component systems in addition to a large array of other putative regulatory systems belonging to different families. The largest of these are GntR-like regulators and BglG-like antiterminators, many of which are associated with PTS. Surprisingly, only 1 of 16 putative TCSs identified had no counterpart in the closely related but totally apathogenic species L. innocua. This points to a minor role of TCSs for virulence gene regulation in L. monocytogenes [117]. To study the role of the listerial TCS for in vitro and in vivo survival and growth of L. monocytogenes more systematically, in-frame deletion mutants in 15 of 16 TCSs were constructed by introducing large deletions in the individual response regulator genes, removing most of the open reading frames [330]. The mutants are currently characterized by testing them in various in vitro growth assays and several cell culture assays and by assessing their virulence potential in experimentally infected mice. At least one of the previously undescribed response regulators (degU) (lmo2515) also contributes to virulence because the L. monocytogenes degU mutant is severely impaired in colonizing experimentally infected mice [330].

LESSONS LEARNED FROM GENOME SEQUENCE OF L. MONOCYTOGENES Availability of complete genomic sequences of L. monocytogenes and its close nonpathogenic relative L. innocua [117] opens new possibilities for investigation of listerial virulence. Sequencing of the 2,944,528 base pairs of the circular chromosome of L. monocytogenes revealed the presence of 2,853 protein-coding genes, from which about 35% are without a predicted function. Comparison of this sequence with that of L. innocua and the closely related species B. subtilis revealed some special features of L. monocytogenes [37,117]. The first surprise was the presence of many putative surface proteins belonging to six families (19 internalins, 21 other LPXTG proteins, 9 GW module-containing proteins, 11 hydrophobic-tail proteins, 4 p60-like proteins, and up to 68 putative lipoproteins). The high number of related members in each family seems to be—at least in part—because of extensive gene duplications. Of particular interest are surface proteins characterized by the common LPXTG motif in their C-terminus necessary for covalent linkage of the proteins to the cell wall peptidoglycan. L. monocytogenes harbors a total of 41 genes encoding for LPXTG proteins (more than any other bacterial species analyzed). These can be further grouped into the family of internalins and internalin-like proteins (characterized by the leucine-rich repeat motif) and the LPXTG proteins lacking this leucine-rich repeat. The function of the vast majority of these genes is unknown, but one may speculate that internalins and internalin-like proteins confer host-species and cell-type specificity during infection. They may also mediate attachment of bacteria to other surfaces during their life outside the mammalian host [38,117]. Another surprising feature of L. monocytogenes revealed by analysis of the genome was the high number of transport proteins (331 in total), of which 88 are devoted to carbohydrate transport by phosphoenolpyruvate-dependent phosphotransferase systems (PTS). Hence, L. monocytogenes

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has nearly three times as many PTS systems as B. subtilis, which probably gives L. monocytogenes the ability to take up, and therefore grow on, a large number of carbohydrates. As mentioned earlier, one specific hexose-phosphate transporter is necessary for intracellular multiplication of L. monocytogenes and further studies will most likely reveal the importance of other uptake systems for adaptation to different environmental conditions. As anticipated, because of the wide range of different conditions faced by L. monocytogenes during extracellular and intracellular growth, a high number of transcriptional regulators (209 in total) have also been identified. This number is second only to that of Pseudomonas aeruginosa, another ubiquitous opportunistic pathogen [117].

EVOLUTIONARY ASPECTS Based on in vitro data, all Listeria species are regarded as normally noncompetent [25]. It was therefore totally unexpected to find genes in L. monocytogenes and L. innocua coding for putative DNA uptake systems homologous to B. subtilis competence genes [25,117]. The uptake apparatus may no longer be functional, or its regulation or signals that induce competence may differ from those of B. subtilis and correct conditions to induce competence have not yet been found during laboratory culture. Nevertheless, the possibility of gene transfer by transformation could well explain genomic differences between sequences of the two Listeria species characterized on a genomic level to date. These differences are mainly found in blocks dispersed around the chromosome, resulting in a mosaic genome structure. Furthermore, the collinearity identified for L. monocytogenes and L. innocua chromosomes (see Figure 1.1) also extends to numerous regions of the B. subtilis chromosome. The hundreds of insertions found in the three chromosomes are best explained by multiple independent transformation events followed by DNA integration at various sites in the chromosomes. The origin of the known virulence genes in Listeria is still unclear. The large family of internalins seems to have evolved in Listeria after initial combination of the LPXTG membrane anchor motif with a leucine-rich repeat motif (both of unknown origin) to form a proto-internalin. This then (probably) duplicated several times and evolved further by recombinations and by point mutations. The virulence gene cluster was obviously acquired a long time ago because traces of the transfer event are barely detectable, or it evolved in Listeria. Of the individual genes present in the virulence gene cluster, homologues of the listeriolysin gene [242], the metalloprotease gene [72,218], and the two phospholipase genes [195, 215, 319] are present in many related species but never found in a cluster as in L. monocytogenes. The origin of the actA gene is unknown because no bacterial protein with significant homology has yet been isolated. It has been speculated, however, that the actA gene may be of eukaryotic origin because parts of the protein show some homology to eukaryotic cytoskeletal proteins [45]. At present, fully functional virulence clusters are found in L. monocytogenes and in the animal pathogen L. ivanovii; a similar cluster with additional genes is present in the nonpathogenic species L. seeligeri (Figure 5.2) [45,127]. Interestingly, one of the ORFs with unknown function at the right border of the cluster shows some weak homology to genes of Listeria phages [45,204]. This might point to phage transduction events involved in early evolution of this gene cluster. Analysis of the sequences flanking the virulence gene cluster in different Listeria species indicates that it is ancestral to the genus Listeria because it is present in all isolates exactly at the same chromosomal position [45,46,179]. Most likely, the virulence gene cluster was lost by two independent events from the nonpathogenic species L. innocua and L. welshimeri as indicated by the presence of short DNA sequences believed to belong originally to the virulence gene cluster [179]. The situation in L. grayi is still unknown, but current sequencing efforts [116] will soon resolve the situation in this more distantly related species. Availability in the near future of genome sequences of L. ivanovii, L. welshimeri, L. grayi, and L. seeligeri—in addition to those of L. monocytogenes and L. innocua (thus, the complete genome sequences of the whole Listeria genus) [46,116]—and their comparison will certainly greatly enlarge understanding of the evolution of the genus Listeria.

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OPEN QUESTIONS The last years have seen an enormous increase in understanding of the molecular basis of infectious diseases. Knowledge of the genes determining virulence of L. monocytogenes and the role played by virulence gene products in the infectious process is rapidly expanding. However, many problems concerning the virulence of L. monocytogenes remain unsolved. For instance, L. ivanovii, a species largely apathogenic for humans [287], resembles L. monocytogenes in its intracellular life cycle [162]. Genes homologous to most of the known virulence genes of L. monocytogenes are also detectable in this species [127,136,177] and the complete PrfAregulated gene cluster identified in L. monocytogenes is also present in L. ivanovii [127]. However, L. ivanovii is virulent for animals and avirulent for humans; an experimental infection in mice yielded a different outcome than that of L. monocytogenes [147]. What is the molecular explanation for this obvious difference in the pathogenic potential of these two Listeria species? Is it the result of a different mechanism in regulation of known virulence genes inside the infected cells or differences in specific activity of the known virulence gene products? What is the role of the many small internalins present in L. ivanovii but absent in L. monocytogenes? The availability of the genomic sequence of L. ivanovii in the near future [116] will probably help to answer at least some of these questions. Expression of L. monocytogenes virulence genes inside infected mammalian host cells and tissues is another important but unsolved problem. The expression pattern of known L. monocytogenes virulence determinants is complex under in vitro growth conditions and regulated by PrfAdependent and PrfA-independent mechanisms. Very little is known of how PrfA and other putative regulatory factors control these genes while the bacteria reside inside host cells and tissues. Application of available high-throughput methods like macro- and microarrays to measure gene expression on the mRNA level [223] or two-dimensional gel electrophoresis on the protein level [257] of many genes in parallel under different conditions will certainly dramatically increase understanding of gene expression regulation of L. monocytogenes. These data, together with others, will greatly add to understanding of how this fascinating human pathogen thrives under very different conditions inside and outside the human host.

ACKNOWLEDGMENTS We thank T. Williams and J. Kreft for carefully and critically reading this manuscript. We apologize to all those who contributed to our current knowledge on the biology of L. monocytogenes but were not mentioned in this chapter. Work from this group was supported by the Deutsche Forschungsgemeinschaft through the grants SFB 479-B1 (WG) and SFB 479-B5 (MK), the European Union through the grants BMH4-CT96-0659, BIO4-CT98-0036, and QLG2-CT1999-00932 (WG), and the Fonds der Chemischen Industrie (WG).

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of Listeria 6 Characteristics monocytogenes Important to Food Processors Beatrice H. Lado and Ahmed E. Yousef CONTENTS Introduction ....................................................................................................................................158 Temperature....................................................................................................................................159 Growth Temperature.............................................................................................................159 Range and Optimum ................................................................................................159 Growth Kinetics........................................................................................................160 Cold Tolerance..........................................................................................................161 Stress Adaptation at Elevated Sublethal Temperatures............................................163 Variations in Virulence with Temperature ...............................................................163 Freezing ................................................................................................................................163 Lethal Temperature...............................................................................................................164 Mechanisms of Thermal Inactivation.......................................................................164 Kinetics of Thermal Inactivation..............................................................................165 Extrinsic Factors in Resistance to Heat ...................................................................167 Intrinsic Factors in Resistance to Heat ....................................................................167 Surrogate Microorganisms for Thermal Studies..................................................................168 Acidity ............................................................................................................................................169 Survival at Low pH ..............................................................................................................169 Inactivation at Low pH.............................................................................................169 Factors Influencing Acid Tolerance .....................................................................................170 Mechanisms of Acid Damage and Tolerance ......................................................................170 Consequences of Enhanced Acid Tolerance ........................................................................171 Water Activity ................................................................................................................................171 Water Activity and Growth or Survival ...............................................................................171 Osmotolerance Factors .........................................................................................................172 Antimicrobial Components in Food ..............................................................................................174 Salt ........................................................................................................................................174 Survival at Extreme Salt Concentrations .................................................................174 Physiology at High Salt Concentrations ..................................................................174 Organic Acids and Their Salts .............................................................................................175 Lactate.......................................................................................................................176 Sodium Diacetate......................................................................................................176 Sodium Propionate ...................................................................................................177 Potassium Sorbate ....................................................................................................177

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Sodium Benzoate......................................................................................................178 Parabens and Other Benzoic Acid Derivatives ........................................................178 Fatty Acids and Related Compounds...................................................................................178 Free Fatty Acids .......................................................................................................178 Fatty Acid Monoesters .............................................................................................179 Sodium Nitrite ......................................................................................................................180 Antioxidants..........................................................................................................................180 Smoke ...................................................................................................................................181 Spices, Herbs, and Plant Extracts ........................................................................................181 Lysozyme..............................................................................................................................182 Hydrogen Peroxide...............................................................................................................183 Lactoperoxidase System.......................................................................................................183 Lactoferrin ............................................................................................................................184 Biocontrol.......................................................................................................................................185 Live Fermentate....................................................................................................................185 Bacteriocins ..........................................................................................................................185 Modified Atmosphere.....................................................................................................................186 Alternative Processing Technologies .............................................................................................187 Irradiation .............................................................................................................................187 Ionizing Radiations...................................................................................................187 Ultraviolet Radiation and High-Intensity Pulsed Light...........................................188 High-Pressure Processing.....................................................................................................189 Pulsed Electric Field Processing..........................................................................................189 Attachment and Biofilm Formation...............................................................................................190 Sanitizers ........................................................................................................................................192 Chlorine and Chlorinated Compounds.................................................................................193 Quaternary Ammonium Compounds ...................................................................................194 Acid Sanitizers......................................................................................................................194 Ozone....................................................................................................................................194 Miscellaneous Sanitizing Agents .........................................................................................195 Active Packaging............................................................................................................................196 Multiple Antimicrobial Treatments................................................................................................197 References ......................................................................................................................................198

INTRODUCTION The goal of food processing is to produce a safe, wholesome product that has a suitable shelf life and is acceptable to the consumer. Food manufacturers rely on a variety of processing and preservation methods to reach this goal. These methods inactivate or inhibit growth of spoilage and pathogenic microorganisms, suppress undesirable chemical and biochemical changes and hence ensure food’s safety, and maintain its desirable physical and sensory properties. Methods currently used in food preservation involve physical, chemical, or biological factors. Physical preservation factors include heating, cooling, freezing, radiation, high-pressure processing, and packaging. Chemical treatments include addition of antimicrobial agents (e.g., benzoate, propionate, and sorbate), acidifying agents (e.g., acetic and lactic acids) or curing agents (e.g., sodium chloride and sodium nitrite). Preservation by biological means (biopreservation) includes fermentations that control spoilage and pathogenic microorganisms through competition for substrate, gradual lowering of pH, and release of antimicrobial metabolites. Success of a preservation technology depends on meeting the processing goal described earlier. Heat is the most reliable and commonly used preservation factor, but thermal processing alters

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quality of food and decreases availability of nutrients. Alternative technologies, such as radiation and high-pressure processing, may maintain the critical balance between food’s safety and its quality. Similarly, treatment combinations are used in food processing and the value of using multiple preservation factors is best expressed as the hurdle concept. Growth, inhibition, or inactivation of Listeria monocytogenes in response to food-processing and preservation techniques will be detailed in this chapter. To eliminate any ambiguities, some basic concepts will be defined, and every effort will be made to use these terms uniformly in this chapter. The expression “log” or “log count” refers to the microbial count in “log10 CFU/mL or g.” When measurable increase in count is encountered, it is described as such, unless multiplication of the microorganism is reported; in this instance, the increase will be described as “growth.” Growth of microorganisms is enhanced when the lag phase decreases, generation time decreases (i.e., maximum specific growth rate increases), or the gain in count attained after a given growth period increases. Conversely, “growth inhibition” or simply “inhibition” is the condition when opposite change in the growth parameters just described occurs. Growth inhibitors may also be described as bacteriostatic and, for inhibition of Listeria, as listeriostatic agents; such agents do not usually cause measurable inactivation. “Inactivation” refers to a decrease in cell population; it will be expressed as a decrease in log count. An agent that causes microbial inactivation is described as bactericidal, but may also be reported as listericidal when it is active against Listeria. The D-value is the time of exposure to a lethal factor (e.g., heat or radiation) required to inactivate 90% (i.e., one log) of the population of a given microorganism at a given dose of the deleterious factor. D-value is a measure of resistance of the microorganism to the deleterious (lethal) factor; the larger the D-value is, the greater is the resistance. When the count does not change appreciably, the status of the microorganism is best described as survival. The word “survival” also refers to ability of the microorganism to maintain its viability during the treatment. Some generalizations and conclusions in this chapter should be viewed with caution. These are based on published research using bacterial strains available to researchers at the time of the study. Recent reports show that strains of L. monocytogenes vary considerably in resistance to processing and new processing-resistant strains are occasionally discovered [190,310]. If highly resistant pathogenic strains are discovered in the future, the conclusions in this chapter will need to be modified accordingly.

TEMPERATURE The temperature to which food is exposed may have growth-conducive, preserving, or lethal effects on microorganisms in the product. At ~0 to 45°C, L. monocytogenes grows to various extents when present in a suitable medium. Temperatures below 0°C freeze the culture or food and preserve or moderately inactivate the pathogen. Temperatures greater than 50°C are lethal to the pathogen. These three ranges of temperature will be addressed separately.

GROWTH TEMPERATURE Range and Optimum The temperature range that permits growth of L. monocytogenes is of particular interest to food processors because this pathogen is a psychrotrophic bacterium. Listeria monocytogenes was reported to grow at temperatures between –1.5 and 45°C [148,261]. In contrast, L. innocua (19 strains), L. murrayi (1 strain), and L. grayi (1 strain) failed to grow at temperatures below 1.7 (± 0.4), 2.8, and 3.0°C, respectively [161]. Difficulty in determining experimentally the minimum temperature for growth of L. monocytogenes led Tienungoon et al. [316] to use mathematical modeling to estimate this value. According to these investigators, the estimated minimum growth temperature, under optimum pH and aw, was –1.6 and 0.41°C for L. monocytogenes Scott A and L5, respectively. Limits of growth at refrigeration temperature depended strongly on medium pH [316].

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Microorganisms grown at optimum incubation conditions exhibit a short lag phase, short generation time during the exponential growth phase, and high cell count or density at the stationary phase. Interestingly, incubation conditions producing the shortest generation time do not always result in the shortest lag phase or the largest cell density. Therefore, reported “optimum” growth temperatures are only estimated determinations. Optimum temperature for growth of L. monocytogenes, as frequently reported in publications, occurs between 30 and 37°C. Growth Kinetics This section addresses the growth rate of L. monocytogenes, related parameters, and influence of temperature on parameters. Specific growth rate (µ), a commonly used kinetic parameter, reaches a maximum at the midexponential phase of growth. Specific growth rate and generation time (or doubling time) are inversely related as follows: Generation time = 0.693/µ Bacteria at the lag phase show no detectable growth; however, duration of this phase is a useful indicator of subsequent growth. Researchers from the U.S. Department of Agriculture, Eastern Regional Research Center (USDAERRC) modeled growth of L. monocytogenes in broth culture at different incubation conditions [88]. According to these models, lag phase and generation time were smallest (1.7 and 0.3 h, respectively) when the bacterium was incubated aerobically at 37°C, and the medium has 0.997 water activity (aw) and pH 7.0 initially. Decreasing incubation temperature progressively increased these two growth parameters. Robinson et al. [277] used a different model to estimate growth parameters of L. monocytogenes at different incubation temperatures. These authors observed a shorter lag phase at 15°C than at 20 or 25°C. Zaika and Fanelli [351] reported minor variation in generation time of L. monocytogenes when the bacterium was grown in brain–heart infusion at temperatures ranging from ~30 to 42°C; the corresponding value was ~0.6 h. Determination of L. monocytogenes growth kinetics in food produced different results. The generation time of L. monocytogenes decreases substantially as temperature increases from –1.5 to 30°C. The pathogen grew at –1.5°C in vacuum-packaged sliced roast beef with a calculated generation time of 100 h [148]. Generation times of 62 to 131 h in chicken broth and pasteurized milk were observed during extended incubation at –0.1 to –0.4°C [330]. Generation time was 28.5 to 46 h, 1.8 h, and 0.7 h when the pathogen was incubated at 4, 21, or 35°C in dairy products, respectively [280]. Therefore, food storage at refrigeration temperature retards but does not prevent growth of Listeria contaminants. In the absence of antimicrobials, holding food at 20 to 43°C supports rapid growth of the pathogen. A relationship between incubation temperature and generation time of Listeria would be useful in risk assessment studies, but large variations have been observed at a given temperature. Analysis of some published data (e.g., [280]) shows a linear relationship between the square root of the maximum specific growth rate ( µmax ) and incubation temperature (Figure 6.1). Other studies produced a nonlinear relationship between these two variables, with ( µmax ) showing a plateau after the optimum growth temperature is reached [277,351]. Medium composition contributes to the large variations in generation time at a given temperature. High salt (4.5 to 7.5%) or high EDTA (0.1 to 0.3 mM) concentration, for instance, may mask the effect of temperature on growth by increasing generation time, therefore contributing to deviation from linearity [351]. The listeriostatic activity of 7.5% NaCl or 0.3 mM EDTA was higher at 37 to 42°C than at 19°C [351]. Strains of L. monocytogenes vary considerably in growth characteristics. Lag phase durations for 39 strains varied from 70 to 270 h at 4°C and from 36.5 to 70 h at 10°C in trypticase soy broth–yeast extract [15]. Scott A, a strain extensively used in Listeria-related research, had the longest (209 h) and the second longest (62.8 h) average lag phase at 4 and 10°C, respectively.

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1

Square root of max

0.8

0.6

0.4

0.2

0

0

10

20

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Incubation Temperature (°C) FIGURE 6.1 Linear relationship between incubation temperature and the square root of the maximum specific growth rate ( µmax ) of L. monocytogenes in various foods. Note that µmax values were calculated from minimum generation times (h) as follows: µmax = 0.693/generation time. (From Hudson, J. et al. 1994. J. Food Prot. 57:204–208; Iturriaga, M. H. et al. 2002. J. Food Prot. 65:1745–1749; and Rosenow, E. M. and E. H. Marth. 1987. J. Food Prot. 50:452–457.)

However, correlation between growth parameters and strains’ serotype was not established [15]. Variability among strains shows the importance of selecting a target strain for use in challenge studies. Selecting a fast growing and process-resistant target strain would be particularly valuable for food processing optimization research. Such a strain could be used to illustrate the worst-case scenario for lethality of a process, as well as survivability or growth during subsequent storage. Cold Tolerance The ability of L. monocytogenes to grow at refrigeration temperature is problematic to food processors. Mechanistic studies on bacterial cell membrane and osmoprotectants help explain how this pathogen maintains its physiological functions in a cold environment. Membrane phospholipids must remain in a liquid–crystalline state to maintain membrane fluidity and therefore growth at low temperature. The fatty acid composition determines whether membrane phospholipids are in the liquid–crystalline state. Membranes of L. monocytogenes contain >95% branched-chain fatty acids [12]. When grown at 37°C, major fatty acids are anteiso-C15:0 (41 to 52%), anteiso-C17:0 (24 to 51%), and iso-C15:0 (2 to 18%) (Figure 6.2). When grown at 5°C, the anteiso-C15:0 form becomes a strongly predominant group, representing 65 to 85% of total membrane fatty acids [12]. This reduction in the proportion of long aliphatic chains (C17:0) and the increase in asymmetric branching reduce van der Waals

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O

CH3 C

OH

H3C Iso-C 15:0

O C OH H3C

CH 3 Anteiso-C 15:0

O C OH H3C

CH 3 Anteiso-C 17:0

FIGURE 6.2 Structure of the major membrane fatty acids in L. monocytogenes. (From Annous, B. A. et al. 1997. Appl. Environ. Microbiol. 63:3887–3894.)

bonds among membrane constituents. Tight packing of membrane phospholipids at low temperature is therefore reduced; this helps maintain the pathogen’s membrane fluidity. Food-grade agents interfering with the biosynthesis of anteiso-C15:0 would be useful in controlling growth of L. monocytogenes in refrigerated foods. Highest anteiso-C15:0 fatty acid proportions in the membrane were found when the pathogen was grown in the presence of glycine betaine [12]. Growth of L. monocytogenes at low temperature is stimulated by presence of glycine betaine and carnitine [10,226]. When L. monocytogenes was grown at 4°C, addition of 130 µM glycine betaine nearly doubled the specific growth rate [181]. Listeria does not synthesize glycine betaine and carnitine, but imports these compounds from the environment. Plants are rich in betaine, and meat is rich in choline, a precursor of betaine [26]. Processed meat contains approximately 340 to 480 nmol/g glycine betaine [297]. Abundant levels of carnitine are found in foods of animal origin; processed meats (sausages and ham) and skim milk contain 230 to 950 and 120 to 140 nmol/g free carnitine, respectively [11,297]. The ATP-dependent glycine betaine porter II (Gbu) and, to a lesser extent, the glycine betaine-Na+ symporter (BetL) allow accumulation of glycine betaine into the cytoplasm at refrigeration temperature, even when the osmolyte concentration is very low in the growth medium [226]. Uptake is higher in the late exponential phase than in the stationary phase [11], but this uptake does not appear to be under σB control [180]. Carnitine was transported into the pathogen cytoplasm via an ATP-dependent transporter (OpuC) [120]. Prior treatment of a microorganism affects its subsequent physiology and growth kinetics. Temperature downshift, from 25 to 4°C, increased σB transcription [17]; expression of this alternative sigma factor is known to contribute to resistance of L. monocytogenes to heat, carbon starvation, acid, and osmotic and oxidative stresses [17,109]. Another study showed that growth of L. monocytogenes at 10°C, compared to 37°C, upregulated genes involved in cold-adaptive response (flaA and flp), regulatory adaptive response (rpoN, lhkA, yycJ, bglG, adaB, and psr), general

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microbial stress responses (groEL, clpP, clpB,flp, and trxB), amino acid metabolism (hisJ, trpG, cysS, and aroA), cell surface alteration (fbp, psr, flaA), and degradative metabolism (eutB, celD, and mleA) [201]. An oligopeptide permease (OppA) and a high-affinity potassium uptake system (Kdp) were required for growth at low temperature [29,37]. Stress Adaptation at Elevated Sublethal Temperatures Physiological changes caused by exposure to elevated, yet sublethal, temperature has been extensively studied for its consequences on stress resistance, or heat-shock response. Heat-shock response of L. monocytogenes has been triggered at temperatures ranging from 43 to 52°C, i.e., ~8 to 17°C above the pathogen’s optimal growth temperature [105,124,296]. Temperature upshift, from 25 to 48°C, increased transcription of the σB general stress regulon [17]. Expression of major molecular chaperones DnaK, DnaJ, GrpE, GroEL, and GroES also increased [124,136]. These molecular chaperones are referred to as heat-shock proteins because they fold newly synthesized proteins, repair misfolded proteins, and prevent protein aggregation upon heat shock [124]. In general, heatadapted L. monocytogenes cells are more resistant to stress than cells grown at optimal temperature. Holding food at temperatures in the heat-shock range should be avoided because the pathogen may gain resistance to subsequent processing treatments. Variations in Virulence with Temperature Virulence of Listeria increases when the pathogen is grown at refrigeration rather than optimum temperature. Durst [84] reported that 7 of 36 weakly virulent L. monocytogenes strains became markedly virulent to mice by intraperitoneal injection after maintaining the cultures on agar slants for 6 months at 4°C. Similarly, Wood and Woodbine [340] found a strain of L. monocytogenes that was more virulent to chick embryos when grown at 4°C, instead of 37°C. Cold storage, therefore, may enhance virulence of some L. monocytogenes strains. In contrast, activity of listeriolysin O appeared lost after several weeks of storage at 4°C [47]. The decrease in activity of listeriolysin O was more pronounced at pH 7.0 than at pH 5.5. However, pathogenicity was recovered in ≤ 24 h by incubating these refrigerated cells at 37°C [47]. Listeriolysin and other PrfA-regulated virulence genes are thermoregulated and expressed only in Listeria cultures grown above 30°C [192]. Heat shock and other environmental stresses affect the virulence of L. monocytogenes. When L. monocytogenes was heat shocked at 48°C for 2 h, listeriolysin O was almost totally lost; however, subsequent growth of the heat-shocked cells at 37°C resulted in production of listeriolysin 40 times greater than that present immediately after heat shock [174]. Restoration of virulence after heat shocking reinforces the importance of eliminating L. monocytogenes from minimally processed, ready-to-eat food.

FREEZING Although L. monocytogenes does not grow below –1.5°C, this pathogen can readily survive at much lower temperatures. The Listeria population decreased < 1 log over 3 months’ storage at –18 to –20°C in inoculated samples of fish, shrimp, ground beef, ground turkey, frankfurters, corn, and ice-cream mix [140,250]. Populations of the pathogen in tomato soup (pH 4.7) decreased ~3 logs of L. monocytogenes under similar inoculation and frozen storage conditions [250]. The high inactivation rate for Listeria in tomato soup was attributed to freeze–thaw injury [250]. Freezing–thawing ruptured the cell wall, altered plasma membrane integrity, and caused leakage of cytoplasmic contents [92]. Survival and injury of L. monocytogenes during storage at frozen temperature vary with the temperature, freezing rate, and freezing menstruum [91,92,94]. A low freezing temperature and rapid freezing rate were most favorable to bacterial survival [94]. No evidence of cell death was observed when L. monocytogenes was frozen and stored at –198°C in liquid nitrogen. Freezing and storage at –18°C inactivated 1 to 2 logs and injured >50% of the pathogen population.

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Similar ranges of inactivation were observed in five foods (spinach, cheese, fish, chicken, and beef) during initial quick freezing to –50°C in 57 min and subsequent storage at –18°C for up to 300 days [128]. The quick freezing and subsequent storage only inactivated 0.1 to 1.6 and 0.0 to 1.0 logs, respectively. Injury of the surviving population ranged from a nondetectable level to 90%. Multiple freeze–thaw cycles are more detrimental to survival of Listeria than is a single cycle [94]. Such treatment is also far more damaging to the pathogen when frozen at –18°C than at –198°C. Four freeze–thaw cycles in phosphate buffer inactivated > 2 logs L. monocytogenes and caused no detectable injury when the freezing temperature was –18°C. In phosphate buffer lethality was only 34% and injury was 15% when the pathogen was subjected to four freeze–thaw cycles and the freezing temperature was –198°C [94]. Freezing and subsequent storage cause limited inactivation of L. monocytogenes, so contamination of frozen food should be prevented through good manufacturing practices and, whenever possible, by subjecting the food or ingredients to listericidal processes (e.g., pasteurization) before freezing. Some food ingredients may protect Listeria against freeze–thaw injury. Addition of 2 to 4% glycerol or 2% milk to phosphate buffer markedly decreased the extent of cell death and injury during freezing at –18°C [91]. Survival of Listeria over 5 months of frozen storage is higher in the presence of 2% glycerol than in 2% milk [91]. Casein, lactose, and fat were identified as the main milk fractions protecting Listeria against freeze injury. Cryoprotection by lactose and milk fat was observed during the first month of storage at –18°C. Cryoprotection by glycerol and casein exceeded 5 months under similar storage conditions and was, therefore, longer lasting than that of lactose or milk fat [91]. Simulated milk ultrafiltrate, compared to phosphate buffer, caused almost no change in death rate, but decreased cell injury during the first 24 h of frozen storage at –18°C [91]. Adaptation of L. monocytogenes to sublethal levels of environmental stress from acid, ethanol, sodium chloride, heat, or starvation increases survival of the pathogen during freezing, frozen storage, and freeze–thaw cycles [204]. Although freezing and frozen storage may cause a limited decrease in viability of L. monocytogenes, such treatments can cause injury and thus sensitize L. monocytogenes cells to listeriostatic or listericidal agents. After frozen storage, viability of the pathogen decreased in the presence of acid [23,250], lysozyme, or lipase [93]. Cross-contamination of meat during freezing by immersion in nitrogen was reduced, although not fully prevented, by 2% lactic acid wash before freezing [23].

LETHAL TEMPERATURE Thermal processing, such as pasteurization, is the most widely used method to preserve food. These processes target microorganisms of concern in a given food, thus rendering the product safe for human consumption. Listeria monocytogenes is ubiquitous in the environment, and it has been increasingly associated with foodborne diseases and food recalls [312]. The pathogen appears relatively resistant to processing compared to other nonsporing bacteria; processors consider L. monocytogenes to be the primary pathogen of concern in minimally processed and ready-to-eat products. Researchers, therefore, have been actively pursuing new intervention strategies and evaluating efficacy of conventional preservation processes, particularly those involving heat. Mechanisms of Thermal Inactivation Elevated temperature causes multiple irreversible cellular damage in Listeria that results in cell death. Heating L. monocytogenes at temperatures above 56°C causes ribosomal damage, protein unfolding and denaturation, and, consequently, enzyme inactivation [8,49]. Upon heating, ribosomes lose Mg2+, which results in dissociation of the 30S and 50S ribosomal subunits [233,342]. The 30S is more heat labile than the 50S subunit, which is in turn more heat labile than the entire 70S ribosome [79]. Ribosomal damage, particularly denaturation of the 30S ribosomal subunit, has been

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associated with thermal inactivation of vegetative bacteria and is believed to be the main cause of bacterial death [233]. Superoxide dismutase and catalase activities in L. monocytogenes decreased at temperatures higher than 45 to 50°C and 55 to 60°C, respectively. Higher processing temperatures may completely inactivate these enzymes [72], sensitizing the pathogen to aerobic storage conditions [177,254]. However, no apparent correlation between these enzymes and the pathogen’s heat resistance was found [72]. Heating probably releases Mg2+ from teichoic acids in the cell wall of Gram-positive bacteria [8]. Mild heat (56°C), however, did not damage the membrane permeability of Listeria [49]. Recovery of thermally injured Listeria requires synthesis of mRNA and repair proteins [49]. Kinetics of Thermal Inactivation Measurement Measuring thermal inactivation kinetics involves heating an inoculated medium uniformly at the desired temperature and monitoring survivors during the course of the treatment. Small thickness of samples maximizes the rate of heat transfer, minimizes temperature come-up and cooling down times, and ensures treatment homogeneity [18,99]. Researchers used capillary tubes, sealed bags, sealed tubes, or open tubes to measure Listeria thermal inactivation kinetics [18,78,99], but variations among these methods led to conflicting results [78]. For fluid products, capillary tubes are generally recommended to establish reliable survivor plots [18,99]. Data are analyzed and inactivation rates or thermal death times are calculated. Before implementation in food processing, it is recommended that the kinetic results be confirmed in pilot-scale pasteurizers, using inoculated food; operating parameters, pasteurizer design, and treated matrix may affect the efficacy of thermal processes. D-Value and z-Value The relationship between thermal treatment time and the log count of survivors is commonly referred to as a “survivor plot.” If this relationship is linear, thermal resistance parameters can be readily calculated. Time required to inactivate one log of the microbial population at a given temperature (i.e., D-value) is a popular expression of its thermal resistance. If the survivor plot is nonlinear, the D-value cannot be determined accurately. Implications of the nonlinearity of inactivation data have been addressed in a recent publication [142]. Pooled data from multiple sources (411 data points) show a log-linear relationship between treatment temperature and D-values of L. monocytogenes (Figure 6.3). Thermal inactivation rates at any given temperature varied considerably among studies and when the pathogen was heated in different media. Using raw and reconstituted nonfat dry milk, for example, produced D63°C and D71.7°C values of ~0.33 and 0.015 min, respectively [32,99]. In contrast, the pathogen appeared more resistant to heat when present in ice-cream mix; D-values at 68.3, 73.9, and 79.4°C were 3.9, 0.53, and 0.043 min, respectively [33]. Although Figure 6.3 shows large variability in thermal resistance of L. monocytogenes in different studies, these data were pooled and used to estimate the temperature change necessary to vary the D-value of L. monocytogenes by one log (i.e., z-value); this calculation resulted in a z-value of 7.6°C. Recently, a large number of published inactivation studies were reviewed and D- and z-values of L. monocytogenes were compared for various laboratory media, and dairy, meat, egg, seafood, and vegetable products [79]. The authors reported average and median z-values of 7.1 and 6.4°C, respectively (minimum: 4.3°C; maximum: 29.3°C). Heat treatment of low-acid refrigerated food should be sufficient to decrease the target pathogen ≥ 5 logs. Manufacturers of fruit juice and other acid foods also adopt this principle. Presence of L. monocytogenes in ready-to-eat food is not permitted in the United States. This zero-tolerance policy, in addition to the pathogen’s ubiquity and resistance to adverse conditions and mild processing, makes L. monocytogenes a suitable target for thermal processing.

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100

D-value (min)

10

1

0.1

0.01 50

54

58

62

66

70

74

Temperature (°C)

FIGURE 6.3 Decrease of L. monocytogenes D-values with heating temperature of inoculated meat products, dairy products, seafood, fruits, juices, and vegetables. (From Chhabra, A. T. et al. 1999. J. Food Prot. 62:1143–1149; Doyle, M. E. et al. 2001. J. Food Prot. 64:410–429; Juneja, V. K. and B. S. Eblen. 1999. J. Food Prot. 62:986–993; Mazzotta, A. S. 2001. J. Food Prot. 64:315–320; and Murphy, R. Y. et al. 2002. J. Food Prot. 65:53–60.)

The following are examples of thermal treatments that successfully inactivated L. monocytogenes in various foods. Cooking ham and brined salmon to internal temperatures of 65 and 82.8°C, respectively, eradicates Listeria from these foods [79]. Pasteurization of apple cider at 71.1°C for 11 sec inactivates > 5 logs L. monocytogenes. When processed at 68.1°C for 14 sec, low numbers of injured Listeria were recovered from artificially contaminated apple cider with relatively high pH (4.1) and low sugar content (11°Brix); however, survivors died when the processed cider was stored at 4°C for 24 h [216]. Minimum temperature-time pasteurization processes required by the U.S. Food and Drug Administration (FDA) for dairy products are sufficient to eliminate ≥ 105 CFU/g L. monocytogenes [13,51,99,341]. Processing food at a different temperature should achieve equivalent microbial inactivation [114]. The hardiness of L. monocytogenes to mild thermal processes has frequently been observed. At temperatures < 60°C, Listeria spp. had a substantially higher D-value than Salmonella spp. in meat products, especially in chicken [239]. Equal or slightly lower D-values for Listeria were observed at 70°C, indicating that the z-value for Listeria was probably lower than that for Salmonella in these products. In fruit juices processed at 56 to 60°C, Listeria was more heat resistant than Salmonella, but

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less heat resistant than Escherichia coli O157:H7 [220]. The z-value of the latter pathogen, however, was smaller than that of Listeria [220]. Populations of L. monocytogenes decreased only 1.6 and 0.38 logs during heat treating egg white (56.7°C for 3.5 min) and salted/sweetened egg yolk (63.3°C for 3.5 min), respectively [230]. Because low populations of the pathogen are typically found in contaminated egg products, current pasteurization protocols for whole egg and egg yolk may be sufficient to eliminate the organism, even though these thermal processes do not provide a wide safety margin [230]. Extrinsic Factors in Resistance to Heat Food Composition Some food components may protect L. monocytogenes against heat. Listeria monocytogenes was more resistant in presterilized or repasteurized milk than in raw milk, but the reason for this increased resistance has not been identified [34]. Resistance of L. monocytogenes to mild heat increases with the food’s pH [160], fat content [210], salt concentration [160], freedom from antimicrobials [215], highfructose corn syrup solids concentration [146], and presence of stabilizers such as guar gum and carrageenan [266]. However, acid, salt, and phosphate in beef gravy caused only a minor increase, if any, in D-values at temperatures higher than 62.5°C [160]. High salt concentration increased the denaturation temperature of Listeria’s 30S ribosomal subunit, which contributes to heat tolerance of the pathogen [302]. The fat fraction from sheep milk protected L. monocytogenes against heat [210]. Increased viscosity in the presence of gum decreased the rate of heat transfer in the food; this may have been sufficient to induce heat adaptation [266]. Variations in heat resistance because of food composition may be associated with availability of nutrients that support growth of Listeria. Starvation of Listeria can trigger a stress-adaptive response and thereby increase the pathogen’s tolerance to heat [205]. Listeria monocytogenes Engulfment Most in vitro inactivation studies processed freely suspended cells of L. monocytogenes. Animals suffering from listeric mastitis produce milk in which L. monocytogenes is usually entrapped within phagocytic leukocytes. Bunning et al. [52] reported average D71.7°C values of 5.0 and 3.1 sec for intracellular (zintracellular = 8.0°C) and freely suspended (zsuspension = 7.3°C) L. monocytogenes, respectively. The higher thermal resistance of intracellular compared to freely suspended Listeria was also observed by Doyle et al. [80], except when contaminated milk was stored ≥ 4 days at refrigeration temperature. During cold storage of milk, heat resistance of Listeria decreased in parallel to leukocyte breakdown; therefore, the researchers speculated that leukocytes protected the engulfed pathogen against thermal inactivation [80]. Homogenization of raw milk disrupts phagocytes; this frees the pathogen, which loses thermal protection during the subsequent thermal process. As infected cows develop fever, L. monocytogenes grown at these elevated temperatures may become adapted to heat [80]. Consistent with this hypothesis, heat shocking (48°C, 15 min) Listeria in milk increased the D71.7°C value from 3.0 to 4.6 sec [51]. The latter value is comparable to that measured for intracellular L. monocytogenes [52]. Intrinsic Factors in Resistance to Heat Strain Listeria monocytogenes strains vary in resistance to heat [99]. Strains from serovar 4b tend to be slightly more heat resistant than those from serovar 1/2a [46]. This trend, however, is not always confirmed. One of the most commonly studied strains, Scott A (serovar 4b), has low heat resistance, compared to V7 (serovar 1a) [99]. Sporadic survival of hardy strains may be encountered if process parameters are based on inactivation studies using heat-sensitive strains, such as Scott A. Process validation, therefore, should aim at destruction of a target Listeria strain selected for its high resistance to heat. Some studies have assessed thermal resistance using mixtures of

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strains (“cocktails”). Presence of at least one problematic (target) strain and strains from various serovars is recommended in these cocktails to minimize the risk of underestimating the pathogen’s thermal resistance. Physiological State As frequently observed in bacteria, Listeria is more heat resistant in the stationary than in the exponential phase of growth. When L. monocytogenes is exposed to sublethal stress, it may develop an adaptive response to subsequent thermal treatments. Environmental and processing stresses that may cause this phenomenon include sublethal heat, acids, oxidants, starvation, and high osmolarity [158,205]. Prewarming, slow heating, or cooking food; hot water washing; underprocessing; and holding food in warm trays (as may happen in food service establishments) are examples of sublethal heat shock. Holding L. monocytogenes at sublethal temperatures (e.g., 45 to 48°C, for 15 min to 1 h) induces adaptive thermotolerance [124,206]. The D52 to 71.1°C-values of heat-adapted cells were 1.5 to 4 times greater than that observed in cells incubated at optimal growth temperature (30 to 38°C) before the thermal process [51,175,199,296]. A nearly twofold increase in D62°C was observed when the pathogen was heated in ground pork at 1.3°C/min compared to 8°C/min [175]. The z-value of heat-adapted L. monocytogenes, however, remained similar to that of unadapted cells [199]. The average D56°C of L. monocytogenes suspended in a phosphate buffer (pH 7.0) was 4.1, 8.8, and 2.9 min, when cells had been previously exposed to pH 4.5, 4 to 8% (v/v) ethanol, and 500 ppm hydrogen peroxide, respectively. For comparison, the corresponding nonadapted L. monocytogenes strain had a D56°C of 1.0 min [205]. Starvation at 30°C for up to 163 h in phosphate buffer increased the D56°C value of the remaining viable cells to 13.6 min [205]. The pathogen’s thermotolerance at 60°C increased 1.3- to 8-fold in the presence of 0.5 to 1.5 mol/L NaCl, compared to cells exposed to 0.09 mol/L NaCl [211]. Adaptive thermotolerance is a transient, nonheritable property [157,158]. Acquired thermotolerance of L. monocytogenes lasted at least 24 h at 4°C in a sausage mix [105], 365 days in cheddar cheese (pH 5.1) stored at 13 and 6°C, respectively [282]. The effect of pH on cell viability, however, depends strongly on other environmental factors and on the physiological state of the microorganism. Inactivation at Low pH Absence of growth and decrease in cell viability may be observed at pH ≤ 5.5, when other environmental conditions (e.g., temperature) are not optimal for survival of L. monocytogenes. The Pathogen Modeling Program from the USDA estimated the decrease in count in nutrient broth at pH 3.2 to 4.4 [88]. Within this pH range, the predicted D-value decreased with pH in a log-linear manner (Figure 6.4). This decrease in D-value was also more marked as the temperature increased from 4 to 35°C.

1000

10

15

20 0

2 25

30

35 5

pH

2

5

va

0 4. 3.

10

lue

4 4.

100

6 3.

3 x D-value (hours)

10000

Temperature (°C) FIGURE 6.4 Exposure time, under lethal acidic conditions, needed to decrease L. monocytogenes populations 3 logs (i.e., 3 × D-values) at different storage temperatures, as determined by the USDA Pathogen Modeling Program. (From Eastern Regional Research Center. 2003. USDA. Pathogen Modeling Program [version 7.0]. http://ars.usda.gov/services/docs.htm?docid=6786.)

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During prolonged storage in orange serum at pH ≤ 4.8, counts of L. monocytogenes were first constant (~40- and 20-day lag periods at pH 4.8 and 4.0, respectively), then decreased at a rate of approximately 1 log/5 to 8 days [252]. When pH of the orange serum decreased, the lag period decreased and the subsequent inactivation rate increased [252]. At 5°C, the pathogen population decreased < 2 logs after 49 days and >4 logs after 12% NaCl decreases with increasing salt concentration or incubation temperature. Presence of 16% NaCl was listeriostatic for at least 33 days at ≤4°C. Presence of 26% NaCl decreased Listeria populations 2 and 3.5 logs when the pathogen was incubated at 0 and 4°C, respectively, for a similar storage period [147]. Incubation of L. monocytogenes in the presence of 14% NaCl for 36 days at 10 and 25°C decreased populations ~2 and ≥ 6 logs, respectively [299]. Physiology at High Salt Concentration Listeria formed colonies with a rough surface and irregular borders on agar media containing ≥6% NaCl [152]. Cell morphology changed from short rod to filamentous and deformed shapes [22,351], with a strongly hydrophilic surface [22]. Morphological changes were less pronounced when incubating the cells at refrigeration temperature [38]. Interestingly, elongated cells were also observed when L. monocytogenes was grown in media adjusted to pH 5.0 to 6.0 with citric acid adjusted to pH > 9.0 with NaOH containing ≥ 1.75 mM H2O2 containing ≥ 0.3 mM EDTA This suggests that filamentous morphology contributes to adaptation to adverse conditions [152,351]. Changes in cell morphology, in response to medium salinity, altered the adhesion properties of the pathogen [22]. Whether these changes facilitate persistence of the pathogen in foodprocessing environments or on food and equipment surfaces has not been reported. Viability of cells requires maintenance of an electrochemical potential across the membrane as well as a low cytoplasmic Na+ concentration regardless of the extracellular NaCl concentration. Regulation of potassium uptake (kdp), Na+ efflux (gbu and ykpA), and multidrug efflux (mdrL) were overexpressed under high salinities, presumably to expel excess Na+ ions from cytoplasm [37,126]. An increase in anteiso-C15:0 and decrease in anteiso-C17:0 fatty acid levels in the Listeria membrane have been observed upon exposure to high salinities [61]. A similar membrane fatty acid profile has been observed when the pathogen is stored at refrigeration temperature. Reasons for this change in membrane fatty acid profile have not been determined and may be linked to increased osmoprotectant (e.g., glycine betaine) concentration in the cell cytoplasm. Activation of the osmotic stress response was discussed in the previous section.

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Up to 6% sodium chloride protected L. monocytogenes against thermal inactivation in beef gravy (pH 4.0) [160]. This thermoprotective effect was particularly evident at treatment temperatures ≤ 60°C. The highest D55°C-value was observed in gravy containing 1.5 to 4.5% NaCl. When heating the gravy at 55°C, increasing the salt concentration to 4.5% decreased the rate of thermal inactivation of L. monocytogenes. Furthermore, thermotolerance of the pathogen increased slightly with NaCl concentration when sodium pyrophosphate was included in the gravy formulation. As explained earlier, high salt concentration increases the temperature of denaturation of the pathogen’s ribosomes and hence increases its thermotolerance [302].

ORGANIC ACIDS

AND

THEIR SALTS

Organic acids and their salts (Table 6.1) are frequently incorporated into foods as acidulants (e.g., acetic acid) or preservatives (e.g., sodium diacetate). Acid sprays and acid dips are used to inactivate

TABLE 6.1 Organic Acids Commonly Used as Acidulants or Antimicrobial Agents in Food Name

Molecular Weight (g/mol)

pKa

Acetic acid

60.05

4.74

Sodium diacetate

142.09

Citric acid

192.1

3.13 4.76 6.40

Benzoic acid

122.12

4.19

Lactic acid

90.08

3.79

Parabens: e.g., methylparaben

152.14

8.47

137.13

4.65 4.80

Propionic acid

74.08

4.87

Sorbic acid

112.12

4.76

Para-aminobenzoic acid

Source: Budavari, S. 1989. The Merck index. Rahway, NJ: Merck & Co., Inc.

Structure

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L. monocytogenes on food surfaces. According to the USDA Pathogen Modeling Program [88], bactericidal activity of acid increases with temperature. When listeriostatic doses of organic acids are used, storage at refrigeration temperature is essential to prevent further growth of the pathogen [151]. Under listericidal conditions, however, refrigeration diminishes acid lethality (Figure 6.5). The growth rate of L. monocytogenes in the presence of organic acids varies markedly with type and concentration of acid and medium pH. Acetic and lactic acids (50 mM) inhibited growth of the pathogen at 37°C when the medium pH was 4.7, but not when it was 6.0 [344]. Growth of Listeria on turkey frankfurters was retarded when frankfurters were dipped into 15 to 25% sodium diacetate or sodium benzoate and to a lesser extent when they were dipped into 15 to 25% potassium sorbate or sodium propionate [151]. At equal pH and equimolar total acid, growth inhibition of L. monocytogenes was in the following order: acetic > lactic > citric [344]. However, at equal pH and equimolar undissociated acid concentration, listeriostatic activity was in the following order: citric > lactic > acetic. These organic acids were more potent bacteriostatic agents than was HCl [344]. Although relatively high concentrations of citric acid (>0.5 M ) have listeriostatic activity, smaller concentrations promoted growth of L. monocytogenes; these data suggest that citric acid contributes to the pathogen’s metabolism [41,344]. When multiple organic acids are applied simultaneously, these acids may act additively or synergistically against targeted microorganisms. A combination of 1.5 to 2.5% sodium lactate and 0.15% sodium diacetate successfully prevented growth of L. monocytogenes in cured meat products (wieners, ham, light bologna, and Cotto salami) at 4°C during 18 weeks [292]. It is widely accepted that antimicrobial activity of weak organic acids, such as acetic acid, is associated with the acid’s undissociated form. The undissociated acid freely permeates the cytoplasmic membrane and dissociates inside the cytoplasm, thus accumulating protons and anions within the cell. This decreases the proton motive force and interferes with microbial metabolism [304,344]. Concentration of the undissociated form of a weak organic acid at a given pH can be calculated according to the Henderson–Hasselbalch equation: pH = pKa + log ([dissociated form]/[undissociated form]) Concentration of the undissociated (uncharged) organic acid form increases as the pH decreases. Contrary to the weak-acid theory, some researchers believe that hydrophobicity of sorbic acid is the cause of its antimicrobial properties [304]. According to these researchers, weak organic acids are membrane-active agents that act to disrupt cytoplasmic membrane function, producing cell death. Lactate Salts of lactic acid (Table 6.1) are used at 1 to 4% as additives in baked goods, meat, and poultry products. When added to food, lactates do not change pH of the products [59]. Sodium or potassium lactate (4%) is listeriostatic at refrigeration temperature [59]. Sodium, potassium, and calcium lactates were equally effective in inhibiting growth of L. monocytogenes in cooked strained beef stored at 20°C [59]. Calcium lactate, however, was most antilisterial in pork-liver sausage [335]. Application of NaCl (2 to 3%), nitrate (125 ppm), or low temperature enhanced the listeriostatic effect of lactate against L. monocytogenes in meat and smoked salmon [259,335]. A combination of lactate (4%) and nisin (400 IU/mL) is listericidal at pH 5.5 and 4°C, and the inactivation rate increases with addition of polyphosphate (0.5%) [48]. Lactate–nisin and lactate–nisin–polyphosphate combinations caused ~2.3- and 4.2-log reductions in 28 and 20 days, respectively; nisin alone only resulted in an initial 1.1-log decrease and did not prevent subsequent growth of L. monocytogenes [48]. Addition of 0.3% sorbate did not improve antilisterial activity of lactate (4%) [48]. Sodium Diacetate Sodium diacetate (Table 6.1) is a GRAS additive [115]. The additive is used as an acidulant, flavoring compound, and antimicrobial agent in foods. When sodium diacetate was added to brain

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heat infusion (BHI) broth at the 18- to 35-mM level (mixture pH of 6.3 to 5.25), a small inoculum of L. monocytogenes (103 CFU/mL) was inhibited in a concentration-dependant manner [293]. A low incubation temperature (5°C, compared to 35°C) enhanced inhibitory action of the diacetate. Minimum inhibitory concentrations of diacetate in the broth were 35, 32, and 28 mM at 35, 20, and 5°C, respectively. Based on equal levels of undissociated acetic acid at different pH values, sodium diacetate was more effective and had lower minimum inhibitory concentrations at 35°C than did acetic acid; minimum inhibitory concentrations were 5, 20, 30, 40, and >100 mM for sodium diacetate and 5, 20, >50, >100, and >150 mM for acetic acid, at pH 4.7, 5.0, 5.5, 6.0, and 6.5, respectively. Dipping turkey frankfurters into 15 to 25% sodium diacetate (pH 4.6) at 22°C for 1 min decreased L. monocytogenes populations 1.7 to 2.0 logs [151]. Sodium acetate on the surface of these frankfurters inhibited Listeria growth ≥14 days when the treated product was stored at 4 to 13°C. The pathogen population increased 2.5 logs over the same time period when frankfurters were stored at 22°C [151]. Sodium diacetate also reportedly enhanced listericidal activity of gamma irradiation [298] and 5,000 AU/mL of pediocin [289]. Sodium Propionate Propionic acid (Table 6.1) and its salts are useful antimycotic agents, and their potential role as antilisterial preservatives has been investigated. Broth media containing >2,000 ppm sodium propionate inhibited growth of L. monocytogenes at pH 5 [96]. Generation times for L. monocytogenes in tryptose broth at pH 5.6 and without sodium propionate decreased from 68 to 49 min as the incubation temperature increased from 4 to 35°C. When 3,000 ppm sodium propionate was added to the medium, generation times decreased from 3.0 days to 4.5 h as the incubation temperature increased from 4 to 35°C [96]. Sodium propionate (3,000 ppm) at pH 5.0 was listeriostatic at 4°C and listericidal at 35°C in tryptose broth incubated for 67 days. Combinations of propionate and acetic acid in growth media produced strong antilisterial action [98]. Lowering the incubation temperature from 35 to 13°C not only diminished the rate of growth of L. monocytogenes, but also decreased maximum populations of the bacterium in the presence of propionate and other organic acids [98]. Sodium propionate (3,000 ppm) was less effective than sorbic acid in eliminating L. monocytogenes from cold-pack cheese at pH 5.2 to 5.5 [285]. Growth of the pathogen on the surface of frankfurters dipped into 15 to 25% sodium propionate (pH ~9.0) was inhibited at 4°C for ≥14 days [151]. Refrigerated storage is essential for long-term listeriostatic activity of sodium propionate at pH >5.5. When contaminated frankfurters were stored at 13 and 22°C, growth was observed after ~7 and 3 days, respectively [151]. Potassium Sorbate Sorbic acid (Table 6.1) is primarily active against yeasts and molds, but this antimicrobial agent also inhibits a wide range of bacteria, particularly aerobic, catalase-positive organisms. Hence, several investigators have assessed the ability of potassium sorbate and sorbic acid to inhibit L. monocytogenes in laboratory media and various foods. Sorbate had a minimum inhibitory concentration of 400 to 600 and >5,000 mg/L at pH 5.0 and 6.0, respectively, when L. monocytogenes was cultured in BHI broth at 35°C [234]. Antilisterial activity of sorbate was enhanced by other organic acids, with acetic and tartaric acids being most effective [97]. The lower the storage temperature and pH of the medium were, the greater was the effectiveness of sorbate against L. monocytogenes. Strong listericidal effects were observed when 0.3% sorbate was used in combination with 125 ppm sodium nitrite, 0.5% polyphosphate, or 400 IU/mL nisin [48]. Potassium sorbate is widely used to extend the shelf life of many foods, including butter, cheese, meat, cereals, and bakery items. Listeria did not grow on turkey frankfurters (pH ~ 9.0) dipped for 1 min in 15 to 25% potassium sorbate when the frankfurters were subsequently stored at 4°C for

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≥14 days [151], with any decrease in viability being minimal (24 h [251]. These active compounds (e.g., thymol) damage the cell membrane and may interfere with peptidoglycan synthesis [271]. Because of the potential link between consumption of chocolate milk and listeriosis, Pearson and Marth [258] studied the effect of caffeine and theobromine, two methylxanthine compounds in cocoa, against L. monocytogenes (103 CFU/mL, initially) in skim milk at 30°C. Limited antilisterial activity was observed with 2.5% theobromine; however, the authors found that 0.5% caffeine exhibited antilisterial activity in skim milk through extending the lag phase ~3 to 6 h, increasing the generation time 1 h, and decreasing the final maximum population ~1.4 log. The combination of 2.5% theobromine and 0.5% caffeine slightly increased antilisterial activity when compared with 0.5% caffeine alone.

LYSOZYME Lysozyme is an antimicrobial enzyme naturally present in foods of animal origin, including hen’s eggs and milk. The enzyme is active against many Gram-positive bacteria [267] and has been proposed for use in cheese [113]. Lysozyme is particularly attractive as a food preservative because this enzyme is active between 4 and 95°C and over the pH range generally encountered in food

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[155]. The mechanism of inactivation of Listeria by lysozyme is unclear, but appears to be nonlytic; cell leakage but no peptidoglycan hydrolysis was observed in lysozyme-treated L. innocua [218]. Inhibition of L. monocytogenes by lysozyme in tryptic soy broth increased as temperature and pH decreased [155]. The presence of lysozyme in food, on food surfaces, or on packaging surfaces contributes to control of L. monocytogenes [31,130,169]. Listeriostatic or listericidal activity has been reported in vegetables, fresh meat, processed meat products (e.g., ham), fish fillets, mayonnaise, milk, and cheese [101,149,150,169]. Lysozyme activity in food, however, is diminished in the presence of minerals and meat [150,169]. Addition of EDTA (1 to 5 mM) or lipase (12.5 IU/mL) enhances lysozyme (0.02 to 0.1 mg/g) activity against the pathogen [150,198]. Lysozyme sensitized the pathogen to mild heat [267]. Combinations including 0.05% potassium sorbate, 5 mM sodium acetate, 0.95% ethanol, or 10 mM ascorbic acid did not substantially increase antilisterial activity of lysozyme [149].

HYDROGEN PEROXIDE The United States permits the use of hydrogen peroxide solution (< 35% H2O2) to sterilize multilayer packaging materials in aseptic processing systems. According to FDA regulations, residual hydrogen peroxide must be < 0.5 ppm [117] immediately after packaging. Hydrogen peroxide is used as a preservative, particularly with raw milk, in several parts of the world. Use of this agent as a direct food additive in the United States is very limited. The FDA permits adding 0.05% (w/w) hydrogen peroxide to raw milk for making cheese [115]. Addition of 0.02% hydrogen peroxide to cheese brine inactivates L. monocytogenes, when the brine is held at 12°C; lower levels were sufficient as the storage temperature decreased [191]. Hydrogen peroxide (0.05%) was relatively ineffective in decreasing the Listeria count in raw milk or milk artificially contaminated with equal numbers of S. aureus and S. faecalis [77]. It was listericidal when combined with mild heat [327], lactic acid [327], or peroxyacetic acid [179]. The population of L. monocytogenes decreased >5 logs when inoculated produce was sprayed or dipped in a solution containing a mixture of hydrogen peroxide and lactic acid, at 1.5% each [327]. Alternatively, Listeria may be controlled when lactic acid bacteria that produce hydrogen peroxide are immobilized on the surface of produce [138]. Hydrogen peroxide causes oxidative damage in bacterial DNA, RNA, protein, and lipids [271]. Catalase from L. monocytogenes detoxifies low levels of hydrogen peroxide. A high concentration of this antimicrobial agent, however, overwhelms a cell’s catalase and hence overcomes the natural resistance of this pathogen. Resistance of L. monocytogenes to 0.1% hydrogen peroxide was observed after the bacterium was adapted to pH 4.5 to 5.0, 500 ppm H2O2, 5% ethanol, 7% NaCl, or 45°C heat for 1 h [206]. Adaptation of L. monocytogenes to H2O2 increased the D56°C value of this pathogen 2.9-fold [205].

LACTOPEROXIDASE SYSTEM The lactoperoxidase system is a naturally occurring antimicrobial mechanism in milk. Activation of this system has been proposed to extend the shelf life of raw milk in countries with inadequate refrigeration. A functional lactoperoxidase system in raw milk may inactivate or inhibit growth of Listeria. The lactoperoxidase system extended the lag phase of L. monocytogenes in the presence of 35 mg/L thiocyanate, 0.2 g/L glucose, and 1 mg/L glucose oxidase [30,295]. Listericidal activity of the lactoperoxidase system was reported in ultrahigh temperature-treated milk supplemented with potassium thiocyanate (84 mg/L), glucose (10 g/L), and glucose oxidase (2 mg/L) [87]. The lactoperoxidase system enhanced destruction of Listeria during thermal processing [166], high-pressure processing [125], and nisin treatment [30]. However, processors should consider

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the biphasic inactivation patterns of L. monocytogenes when the lactoperoxidase system is activated [166]. Lactoperoxidase catalyzes oxidation of thiocyanate (SCN–) by hydrogen peroxide to hypothiocyanate (OSCN–) and hypothiocyanous acid (HOSCN) [187] as follows: 2SCN − + H 2O 2 + 2H + Lactoperoxidase →(SCN)2 + 2H 2O (SCN)2 + H 2O → HOSCN + H + + SCN − HOSCN
OSCN − + H +

( pKa = 5.3)

The hypothiocyanate and hypothiocyanous acid oxidize sulfhydryl residues (R-SH) in membrane proteins of bacteria, generating sulphenic acid derivatives (R-SOH). When the lactoperoxidase system is activated, leakage of potassium ions, amino acids, and peptides was first observed. Uptake of glucose, amino acids, purines, and pyrimidines was subsequently inhibited. Consequently, bacterial replication and protein synthesis decreased [187]. Efficacy of the lactoperoxidase system depends on sufficient quantities of lactoperoxidase and reactants in milk. Lactoperoxidase represents 1% of whey proteins [274], which is adequate for a functional lactoperoxidase system. Bovine milk naturally contains 1 to 7 ppm thiocyanate. Supplementation of milk with thiocyanate (>35 mg/L, final concentration) is necessary for satisfactory listeriostatic activity of the lactoperoxidase system [87]. Hydrogen peroxide must also be added exogenously or generated by exogenous enzymes such as glucose oxidase [75]. This enzyme oxidizes glucose to gluconic acid and hydrogen peroxide. Production of gluconic acid decreases the pH; this may increase the bactericidal effect of the lactoperoxidase system beyond what would have been observed in similar model systems with pH values near neutrality [75]. Listeria monocytogenes was inhibited and inactivated in milk containing ≥0.75% gluconic acid during extended incubation at 13 and 35°C, respectively [95]. The lactoperoxidase system in TSBYE was more effective at 5 to 10°C than at 20 to 30°C; corresponding increases in lag time were 32 to 98 h and 2.8 to 8.9 h [295].

LACTOFERRIN Lactoferrin is an iron-binding glycoprotein found in mammalian milk. At 23 to 46 mg/mL, lactoferrin was slightly listeriostatic in milk stored at 35°C for 18 h [255]. Its listericidal action is localized in the N-terminus domain of the protein and not at its iron-binding site [20,76]. Dialysis reduces the degree of lactoferrin saturation with iron, and the resulting product is known as apo-lactoferrin. At 15 and 30 mg/mL, apo-lactoferrin was listeriostatic and listericidal in milk stored at 35°C for 18 h, respectively [255]. Lactoferricin B is a small antimicrobial peptide (25 amino-acid residues) resulting from hydrolysis of the N-terminal region of bovine lactoferrin by gastric pepsin [76]. At 1 to 3 µg/mL and ≥15 µg/mL, respectively, lactoferricin B was listeriostatic and listericidal in peptone glucose yeast extract broth at 30°C [19]. Storage of milk at refrigeration temperature was recommended for optimal lactoferrin hydrolysate activity [237]. Activity of lactoferrin and related compounds strongly depends on composition of the medium. Lactoferricin maintained its antilisterial activity over a pH range of 5.5 to 7.5. The listericidal activity of lactoferrin or lactoferricin decreased as cation concentrations in the medium increased [19]. Listeria monocytogenes grew in milk supplemented with ferric ammonium citrate (0.125 M) and apo-lactoferrin (30 mg/mL) after incubation at 35°C for 18 h. In this study, Listeria counts decreased 1.5 logs in the absence of ferric ammonium citrate [255]. A combination of 15 mg/mL apo-lactoferrin and 150 µg/mL lysozyme retarded growth of L. monocytogenes in milk at 36°C [256].

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BIOCONTROL The terms “biopreservation” and “biocontrol” refer to use of microorganisms or their metabolites to inhibit or inactivate undesirable microorganisms in food. Biocontrol may be attributed to the starter microorganisms, their metabolites, and fermentation end products. Biocontrol of L. monocytogenes in food is achieved by adding a bacteriocin-producing microorganism, bacteriocin-containing fermentate, bacteriocin crude extract, or purified bacteriocin [238]. Bacteria that produce antilisterial bacteriocins include strains of Lactococcus lactis, Lactobacillus bavaricus, Lb. reuteri, Lb. acidophilus, Lb. curvatus, Lb. saké, Lb. plantarum, Leuconostoc carnosum, Leuconostoc mesenteroides, Carnobacterium piscicola, Pediococcus acidilactici, Propionibacterium thoenii, and Enterococcus spp. [186,209,238,347]. Numerous studies listed potential applications of identified bacteriocins in food. This section gives an overview of the most potent bacteriocins against Listeria and regulatory issues regarding applications of bacteriocins in food. To list all bacteriocins with known efficacy against Listeria is beyond the scope of this review. Bacteria that have been used traditionally in food fermentation (i.e., most lactic acid bacteria) include bacteriocin-producing strains. Starter cultures, including these bacteriocin-producing strains, may be used to ferment food or food ingredients. Legal issues arise, however, when purified bacteriocins are applied to foods. Approval of U.S. regulatory agencies, mainly the FDA, is required before commercial use of purified bacteriocins [110]. A company can self-affirm whether a bacteriocin of interest is GRAS; however, the company is required to justify application of the bacteriocin if requested by the FDA [110]. Among purified bacteriocins, FDA has approved only nisin, for use in pasteurized cheese spreads [115].

LIVE FERMENTATE Live fermentate is a preparation with high microbial biomass that is generally the product of fermentation. The microorganism of interest is used as starter culture, but the exact composition of the live fermentate is unknown because it contains microbial metabolites and the unused portion of substrates. Live fermentate used for antimicrobial purposes may be incorporated in the food, distributed over the food surface, or immobilized on the packaging surface in contact with the food [138,238,303]. Lactic acid bacteria are most suitable for biocontrol purposes because these bacteria are used in many traditional fermented foods and generally recognized as safe. These bacteria compete with other microorganisms for nutrients or produce antimicrobial compounds, such as weak acids, hydrogen peroxide, and bacteriocins [303]. Biocontrol of L. monocytogenes in refrigerated storage requires the use of suitable antagonistic psychrotrophic bacteria. Nisin-producing lactococci are poor biocontrol microorganisms because they do not grow well at refrigeration temperature or in meat products. Pediocin-producing pediococci were not suitable meat biopreservatives because they were effective against L. monocytogenes only at abuse temperatures [347]. The psychrotrophs C. piscicola L103 [290], Leuconostoc carnosum 4010 [45], and several strains of Lb. bavaricus [339] were proposed as alternative bacteriocin producers to prevent growth of Listeria at refrigeration temperature. Growth of the pathogen may also be retarded in ready-to-eat, fresh-cut vegetables inoculated with Lb. delbrueckii [138].

BACTERIOCINS Bacteriocins are polypeptides synthesized by bacteria; these agents are generally active against strains related to the producer [238]. Bacteriocins are suitable as processing aids, complementing other preservation methods. Heat, freezing–thawing, acid [238], high hydrostatic pressure [163],

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and pulsed electric field [163] were more listericidal in the presence of bacteriocins than they were in their absence. Nisin and pediocin are the most investigated bacteriocins against L. monocytogenes. Nisin is produced by a limited number of Lc. lactis subsp. lactis strains [67]. This bacteriocin is useful in preventing outgrowth of Clostridium spp., including C. botulinum [64]. Pediocins are produced by strains of Pediococcus spp. [74,197]. Dipping meat [104,352] or salmon [306] in a nisin solution (1.3 × 103 to 5.0 × 103 IU/mL) inhibited growth of L. monocytogenes during refrigerated storage. Listeria populations decreased ~1.5 logs on packaged cheese stored at 4°C for 84 days when nisin (61.4 mg/cm2) was immobilized on the package [288]. Nisin activity, however, varies with environmental conditions. Listeriostatic activity of nisin increased with decreasing temperature [306] and pH [139]. Addition of 2% sodium chloride enhanced the listericidal activity of nisin in laboratory media, particularly at levels < 10 µg nisin/mL [139]. Stearic acid (≥10%) decreased the antimicrobial efficacy of nisin (1.0 × 105 IU/mL) [67] and carbon dioxide sensitized the pathogen to nisin [241]. After 10 days, L. monocytogenes decreased ~4 logs in the presence of 2.5 µg/mL nisin in phosphate buffer (pH 6.2) with 100% carbon dioxide atmosphere at 4°C [241]. Maximum counts were reached after 35 days’ incubation; in the absence of nisin or when aerobic incubation was used, maximal counts were reached after 10 to 15 days [241]. Listeria monocytogenes strains vary in their sensitivity to nisin [21]. Listeriostatic activity of pediocin has been demonstrated in numerous foods, including meat and dairy products [74,134,197]. Conflicting results were reported on pediocin activity in milk and liquid eggs, likely because compounds in the crude extract offset pediocin activity [197]. Antilisterial activity of this bacteriocin in food slurries was improved when pediocin was encapsulated in phosphatidylcholine-based liposomes or when 0.1% Tween 80 was added to the product [74]. Unlike nisin, pediocin is not in the FDA’s list of food-grade GRAS substances. Food supplementation with purified pediocin, therefore, requires regulatory approval in the United States. Pediocin bound to heat-killed producer cells [134] or in dehydrated fermented whey permeate [197] was recommended for food applications. Bacteriocins such as nisin and pediocin disrupt the bacterial membrane, thereby depleting the proton motive force of the membrane [134]. Acid adaptation or starvation increased resistance of L. monocytogenes to nisin and pediocin [204]. Resistant Listeria subpopulations were not detected when nisin was immobilized on edible cellulose-based packaging material [288]. Relatively stable populations of bacteriocin-resistant mutants have been isolated from bacterial suspensions exposed to a bacteriocin for extended periods [202]. Application of bacteriocin cocktails and rotation of bacteriocins could ensure their efficacy against Listeria over extended processing times. Incorporation of bacteriocins in food, however, should not preclude use of proper sanitary practices.

MODIFIED ATMOSPHERE “Modified atmosphere” refers to an altered gaseous environment that aims to extend shelf life of a food. Proper packaging maintains the food under this modified atmosphere. A defined mixture of nitrogen (N2), carbon dioxide (CO2), or oxygen (O2) commonly replaces air in modifiedatmosphere packaging [65]. A vacuum replaces air in vacuum-packaged food. Sous vide-processed foods are vacuum packaged, then cooked, chilled, and finally stored at refrigeration temperature. Modified-atmosphere packaging is frequently used to maintain the quality of fresh meat, vegetables, and fruits at refrigeration temperatures [65]. Listeria monocytogenes grows well under aerobic and anaerobic conditions and at refrigeration temperatures. These properties make L. monocytogenes a potential threat to the safety of foods packaged under vacuum or modified atmospheres [65]. Growth of L. monocytogenes is not inhibited in food that has been packaged under vacuum [148] or superatmospheric oxygen (>5 kPa O2) [4]. Presence of ≥80% carbon dioxide or 100% nitrogen gas increased the lag phase of L. monocytogenes in Stilton cheese stored at 4°C by 2 to 3 weeks [338]. The presence of 10% oxygen in a nitrogen-rich (e.g., 80% nitrogen) environment did not satisfactorily inhibit growth of the pathogen [338]. An increase in the amount of carbon dioxide

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increases the length of the lag phase and the generation time [106,338]. The presence of > 80% carbon dioxide is recommended to retard growth of Listeria [106,184,338]. Growth inhibition by carbon dioxide (≥ 50%) was more pronounced at 4°C than at 10°C, and at pH 5.5 than at pH 6.5 [106]. Incorporation of organic acids or bacteriocins increases the safety of modified-atmosphere packaged foods. Inhibition of L. monocytogenes was increased by incorporating 0.5% sodium acetate, 2% sodium lactate, or 0.26% potassium sorbate into vacuum-packaged bologna stored at 4°C [336]. The presence of nisin in vacuum or modified-atmosphere packaging (gaseous mixture not described by the authors) was slightly listericidal during storage of cheese and cooked ham at 4°C [288].

ALTERNATIVE PROCESSING TECHNOLOGIES Heat is most commonly used for food preservation, but alternative methods are gradually being implemented in food processing. Processors hope these alternative technologies will preserve nutritional quality and fresh taste and texture while decreasing the risk of disease transmission. Some alternative technologies may be applied to packaged food to overcome accidental postprocess contamination of the product with L. monocytogenes. Physical alternative processes include radiation, high hydrostatic pressure, pulsed electric field, and high-intensity pulsed light [176,189,281]. These alternative technologies are gradually gaining acceptance from regulatory agencies as well as from consumers. For example, the United States has approved irradiation for control of foodborne pathogens in fresh or frozen meat, dehydrated spices, seeds for sprouting, and whole eggs [118]. Alternative technologies are perceived as nonthermal, although some heat may be generated during their application [82,190]. However, processing conditions are generally selected so that heat is minimal and quickly dissipated [189]. Control of pathogens is therefore accomplished mainly by nonthermal means.

IRRADIATION Microwaves, radio frequencies, high-intensity pulsed light, ultraviolet, electron beam, and gamma are the forms of radiation of interest to food manufacturers. These radiations differ in wavelength and penetrating power [27]. Gamma radiation and electron beams are the most effective and applicable forms for decontamination of food [66,309]. Ultraviolet radiation has poor penetrating power and is therefore limited to decontamination of surfaces, water, and juices [118]. Pulsed light treatment controls microorganisms on food surfaces only [118]. Microwave penetrates food, but treatment uniformity is questionable: Listeria spp. survived on chickens cooked in microwave ovens [107]. Because microwave lethality is caused by heating, it will not be addressed in this section. Ionizing Radiations Food irradiation, i.e., processing food with ionizing radiation, generally refers to treatment with gamma radiation or electron beam. Cobalt-60 or cesium-137 emits gamma rays for food treatment. Accelerators ( 3 logs when exposed to white light (200 to 900 nm, with peak emission at 500 to 650 nm) for 100 pulses (100 nsec/pulse) at a frequency

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TABLE 6.5 High-Pressure Processing Treatments Decreasing L. monocytogenes 4 Logs in Selected Foods Food

Pressure (MPa)

Temperature (°C)

Time (min)

Ref.

250 300 300 414 414 300

30 20 5 2 25 25

5.0 5.0 15 5.1 8.7 6.7

5 269 127 7 7 207

Fruit juices Jams Ovine milk (6% fat) Fresh pork loin Fresh pork loin Frankfurters

of 1 Hz [281]. In this study, no inactivation was observed when cells were treated with white light that was low in ultraviolet rays (350 to 900 nm), which suggests that pulsed light inactivation results from the bactericidal action of ultraviolet radiation [281].

HIGH-PRESSURE PROCESSING Treating food with high hydrostatic pressure may improve a product’s safety without damaging its quality. Pressures ≥ 300 MPa are commonly used and food pressure processors able to deliver up to 900 MPa are currently available. The pressure is transferred instantaneously and homogeneously throughout the treated product, thus making this technique suitable for surface and in-depth decontamination of food in flexible packages [350]. Adiabatic heating is generated as pressure increases [270]. The treatment temperature refers to the average temperature during the pressure-holding time. Listeria is particularly susceptible to inactivation at pressures ≥250 MPa (Table 6.5). Inactivation increases with pressure [207]. D-values are difficult to estimate because survivor plots have a sigmoid shape with considerable tailing during prolonged processing time [222,310]. With respect to pressure resistance, heterogeneity among cells within a bacterial population may explain the tailing [324]. The efficacy of high pressure against L. monocytogenes varies with treatment conditions, medium composition, and different strains of the pathogen. High-pressure inactivation of Listeria follows a second-order relationship with processing temperature [7,127]. Inactivation of Listeria in milk was minimal at around 20°C, intermediate at ~5°C, and highest at 50°C [127]. Food with high levels of proteins or glucose favored survival of the pathogen during the pressure treatment [294]. The level of fat in food did not consistently affect survival of Listeria [127,294]. Food formulations containing bacteriocins [162], the lactoperoxidase system [125] or carvacrol [167] synergistically enhanced the action of high-pressure processing against Listeria. Sensitivity to high pressure varied among Listeria strains [125,310]. Interestingly, strains resistant to high pressure [310] also expressed superior resistance to pulsed electric field and heat [188,190]. Pressure ≥ 200 MPa causes irreversible protein denaturation, rupture of cytoplasmic membrane, and leakage of cell contents [189]. This range of pressure also induces autolysis of Listeria [218]. Expression of cold-shock proteins is induced in survivors of high-pressure processing [218]. During storage, expression of cold-shock proteins also enhances the cell’s resistance to high pressure [337].

PULSED ELECTRIC FIELD PROCESSING Pulsed electric field (PEF) treatment involves exposing food to pulses ≥20 kV/cm for a total treatment time of microseconds to milliseconds [82]. The continuous PEF process is well adapted to decontamination of liquids. The PurePulse system received a no-objection letter from the FDA and can be used for acid and low-acid foods. Other systems are currently being developed for industrial-scale processing of acid foods.

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TABLE 6.6 Pulsed Electric Field Processing Parameters for Decreasing Listeria spp. 2 Logs in Selected Foods Food Whole milk 50% Acid whey (pH 4.2) Liquid whole egg

Electric Field (kV/cm)

Treatment Time (µs)

Ref.

35 25 40

300 ≤48 64

273 190 53

Pulsed electric field rapidly inactivates Listeria in fluid products (Table 6.6). Numerous processing parameters influence the bactericidal efficacy of the process. Inactivation of Listeria spp. with PEF increases with the electric field intensity, pulse length, pulse frequency, gap between electrodes, number of product passages between electrodes, and process temperature [82]. Process efficacy decreases as a product’s flow rate increases [82]. The efficacy also varies with food composition. Inactivation of Listeria spp. increases with decreasing pH and conductivity [82]. The population of L. monocytogenes Scott A decreased ~0.2 and 3.4 logs in sweet whey (pH 6.8; 0.46 S/m) and sweet whey acidified with lactic acid (pH 4.2; 0.46 S/m), respectively, when processed with a PEF at 25 kV/cm and 23°C for 48 µsec [188]. Cell injury was detected when treated cells were grown on acidified agar [343]. Inactivation of L. monocytogenes by PEF varies among strains. Listeria monocytogenes OSY8578, a meat isolate with superior resistance to the process compared to other strains, is a potential target strain for PEF process optimization [190]. Lactobacillus plantarum ATCC 8014 has greater resistance to the process than does L. monocytogenes OSY-8578 in acid whey; therefore, it is a potential surrogate microorganism for PEF process validation in this medium [188]. The electric field permeabilizes the bacterial membrane, resulting in leakage of intracellular contents [54]. Expression of molecular chaperones (DnaJ, GroEL, and GroES) decreases ~5 to 20 min after the PEF process [188]. Cell permeabilization [342] and chaperone decrease [188] are more pronounced in process-sensitive than process-resistant strains. When the input energy and maximum treatment temperature exceeded ~80 J/mL and 60°C, respectively, damage of Listeria cells resulted from the combined effect of heat and electric field [82]. Nonthermal treatment with mild electric pulses strongly sensitized L. monocytogenes to mild heat [188]. The population of L. monocytogenes decreased 3.3 to 6.1 logs when treated with heat (55°C for 10 min) 10 min after PEF treatment (27.5 kV/cm for 144 µsec). Treatments with PEF or heat killed ~0.5 log only. Heat sensitization subsequent to PEF treatment paralleled the decrease in expression of molecular chaperones [188].

ATTACHMENT AND BIOFILM FORMATION Listeria monocytogenes attaches to numerous surfaces, including stainless steel, glass, wood, porcelain, iron, plastic, polyester, propylene, rubber, waxed cardboard, and paper [85,185, 212,236,301]. Consequently, equipment surfaces, conveyor belts, floor sealants, and drains are potential reservoirs for Listeria spp. in food-processing plants and may lead to secondary food contamination. Floor drains should therefore not be located next to filling and packaging equipment, and using high-pressure hoses to clean these drains should be avoided to prevent formation of an L. monocytogenes aerosol [300]. The ability of Listeria to attach to objects with different surface properties suggests that packaging material is a potential source of contamination of food with this pathogen. Different materials, however, were not equivalent in hosting the pathogen. Attachment was stronger on polyvinyl chloride and polyurethane than on stainless steel [231]. Stainless steel was more supportive of surface colonization by Listeria at refrigeration to ambient temperatures than was polytetrafluoroethylene [58].

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1. Planktonic cells

2. Cell deposition on surface • Hydrophilic interactions • Flagella

3. Cell adhesion to surface • Hydrophilic interactions • ±Fibrils • Synthesis of exopolymers

4. Surface colonization • Cells monolayer

5. Biofilm formation • Layers with variable cell density • Homogeneous cell distribution horizontally

6. Biofilm development • Biofilm growth • Presence of capillary water channels (3-D structure)

FIGURE 6.7 Formation of L. monocytogenes biofilms under static conditions, on a flat nonporous surface. Deviations from this representation may be observed among strains and with the properties of attachment surface. (From Chae, M. S. and H. Schraft. 2000. Int. J. Food Microbiol. 62:103–111; Chavant, P. et al. 2002. Appl. Environ. Microbiol. 68:728–737; Kalmokoff, M. L. et al. 2001. J. Appl. Microbiol. 91:725–734; Mafu, A. A. et al. 1990. J. Food Prot. 53:742–746; Midelet, G. and B. Carpentier. 2002. Appl. Environ. Microbiol. 68:4015–4024; and Vatanyoopaisarn, S. et al. 2000. Appl. Environ. Microbiol. 66:860–863.)

Attachment and biofilm formation of L. monocytogenes on solid surfaces progress in the following sequence: cell deposition on the surface, adhesion to the surface, surface colonization, biofilm formation, and biofilm development (Figure 6.7) [56,58,164,212,231,326]. Adhesion of Listeria to surfaces has been attributed to hydrophilic interactions, presence of flagella, fibrils, and synthesis of exopolysaccharides. Listeria monocytogenes is a hydrophilic microorganism with surface free energy ~66 mJ/m2 [213]. Therefore, cell attachment and surface colonization are rapid (≤ 2 h at 20 and 37°C) on hydrophilic substrata [58]. Because of its flagella, Listeria movement overcomes electrostatic repulsion forces of the surface thereby facilitating attachment of the pathogen [58,326]. Strongest negative electrophoretic mobility of L. monocytogenes (3.95 µm/s) was observed at pH 6.0 and 20°C [229]. Listeria produces flagella at 20°C but not at 37°C; these are rich in negatively charged groups (COO–) [229]. The net charge of the pathogen was neutral at pH 2.0 to 3.5 [213,229]. Listeria flagella also act as adhesive structures during early stages of cell attachment [326]. Highly adherent strains synthesize fibrils [164]. Fibril formation was observed at ≤ 21°C and pH 6.0 to 9.0, after 9-h incubation on an iron surface [300].

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Exopolysaccharide fiber secretion, after a 1-h incubation at 20°C, seemed to aid attachment of the pathogen to polypropylene and glass surfaces [212]. Attachment to stainless steel, polyvinyl chloride, or polyurethane at 25°C was greater for Listeria, compared to Staphylococcus sciuri, Pseudomonas putida, and Comamonas sp. [231]. Attachment of L. monocytogenes to surfaces varies in the presence of other bacteria. The adherence of L. monocytogenes was enhanced in the presence of Flavobacterium spp. [35], but decreased when the pathogen was cocultured with S. sciuri [195] or with a nisin-producing strain of Lc. lactis [196]. The three-dimensional structure of L. monocytogenes biofilms changes with time, and it is influenced by incubation conditions (i.e., static vs. constant agitation). After 2 to 3 days’ incubation at 37°C, dense bacterial mats (i.e., biofilm) were formed on the colonized surface [56,58]. The bottom layer of the biofilm had a higher cell density than the upper layer, after 72-h incubation at 37°C on a glass slide [56]. Cell distribution horizontally appeared homogeneous [56]. Water channels are clearly observed with scanning electron microscopy as the biofilm develops [58,217]. Water channels are important to circulate nutrients and dissolved gases inside the biofilm, as well as to eliminate metabolic waste products [58]. The structure and physiology of adherent cells are different from those of planktonic (i.e., suspended) cells. Listeria monocytogenes in the stationary phase in a biofilm changed from a rod to a coccoid shape as the population aged [319]. The pathogen grew more slowly when immobilized than in a planktonic mode. Strains of L. monocytogenes varied in their ability to adhere and form biofilms, even when exhibiting a growth pattern similar to that of planktonic cell populations [56,164]. The ability to mount a stringent response, via induction of the relA gene, and physiological adaptation to nutrient deprivation were essential for growth of adhered Listeria [311]. Microscopic analysis shows an increase in cell density within the Listeria biofilm over time, while enumeration on agar indicates a constant colony count [319]. This difference in count by these two enumeration methods may have been caused by an increase in noncultivable or dead L. monocytogenes as the biofilm aged [319]. During biofilm development, synthesis of the following proteins was upregulated: pyruvate dehydrogenase, 6-phosphofructokinase, the 30S ribosomal proteins YvyD and rpsB, superoxide dismutase, a sensing protein CysK, a DNA repair protein RecO, and a cell division initiation protein DivIVA. These proteins are associated with carbohydrate metabolism, stress response, quorum sensing regulation, DNA repair, and cell multiplication [319]. Synthesis of flagellin, a flagellar protein, decreased during biofilm growth [319]. This decrease was expected because flagella help during early stages of cell attachment [326], but they may impair formation of a structured biofilm. Listeria in a biofilm is harder to remove and inactivate than when it is present as freely suspended (planktonic) cells. Planktonic Listeria is less resistant to inimical treatments than is adherent Listeria. Resistance to antimicrobial agents increased with the initial inoculum size [196], age of the biofilm [247], and when the environmental temperature decreased [249]. Food soils protected biofilms against removal by cleaning [143]. Proteins enhanced attachment to silica surfaces in the following decreasing order: bovine serum albumen < β-casein and α-lactoglobulin < β-lactoglobulin [3]. Active packaging is proposed as a means to prevent growth and attachment of the pathogen to surfaces in direct contact with food. Coating surfaces with nisin, an antagonistic starter culture (e.g., bacteriocin producer), egg white lysozyme, or bacteriophages was proposed to prevent bacterial attachment and colonization of surfaces [31,196]. Surfactants also decrease adhesion of the pathogen to surfaces [229].

SANITIZERS Cleaning the food-processing environment is generally a multistep process [308]. A first rinse removes loose soils. Detergents, followed by a second rinsing step, are then applied to eliminate residual soils. The washed surface is then sanitized to inactivate residual microbial contaminants.

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A final rinsing step with potable water removes the sanitizer and microbial residues. Sanitization is a critical step that decreases pathogenic and spoilage microorganisms in food-processing facilities and therefore prevents cross-contamination of food. Water may also disseminate microorganisms, unless a suitable sanitizer is added to the processing water. To be deemed effective, sanitizing agents must reduce at least 5 logs of a given test organism population within 30 sec of exposure at ambient temperatures [171]. Most sanitizing agents active against Listeria spp. belong to one of five categories: chlorine-containing compounds quaternary ammonium compounds acid sanitizers ozone iodophors Efficacy of sanitizers decreases with temperature [214], increasing surface porosity [214], and as an L. monocytogenes biofilm develops [247]. Plasmid-mediated resistance to multiple sanitizers is a problematic phenomenon because it can be transferred at high frequency (8 × 10–6 to 1 × 10–3 transconjugant CFU per one donor CFU) between Listeria spp. and S. aureus [194].

CHLORINE

AND

CHLORINATED COMPOUNDS

Chlorine and its derivatives are used extensively in food, water, and surface decontamination. Chlorinated compounds include hypochlorite solution, chloramines, and chlorine dioxide (gaseous or aqueous). According to the Grade-A Pasteurized Milk Ordinance, utensils and equipment are preferably sanitized with ≥ 50 ppm available chlorine at 24°C for ≥ 1 min [86]. A survivor plot of Listeria exposed to 1 ppm available chlorine follows a biphasic pattern. The pathogen population decreased rapidly during the first 2 min of exposure, whereas a subpopulation survived for > 60 min [90]. Listericidal efficacy of active chlorine is strongly impaired by organic loads, phosphate, temperatures close to 25°C, and pH > 6 [89,90,307]. Although a solution containing 100 ppm available chlorine was effective in broth [203], 200 ppm decreased populations only 0.6 log CFU on tomatoes after 3 h [25]; 1,000 ppm inactivated 2 logs when the pathogen formed a biofilm [243]. Sensitivity of L. monocytogenes to chlorine varies among strains [90] and with the physiological state of the Listeria population [89]. High chlorine concentrations may alter the taste of food and increase the risk of chlorine gas release into the processing environment, thus increasing safety hazards for workers, and necessitating the removal of chlorine residuals from wastewater. After considering chlorine’s advantages and adverse effects, 120 to 200 ppm active chlorine is recommended for treatment of fresh fruits and vegetables [25,307]. Addition of 10 to 100 ppm sodium hypochlorite to cheese brine will also inactivate Listeria in this product [191]. Addition of chlorine or hypochlorite to water generates hypochlorous acid (HOCl) and hypochlorite ion (OCl–) [86]: Cl2 + H2O → HOCl + H+ + Cl− Ca(OCl)2 + H2O → Ca2+ + H2O + 2 OCl– Ca(OCl)2 + 2 H2O → Ca(OH)2 + 2 HOCl HOCL
H + + OCl − The bactericidal activity of chlorine increases with the concentration of undissociated hypochlorous acid [86]; therefore, chlorine activity increases with decreasing pH. Its dissociated form (OCl–)

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has weak bactericidal activity [86]. Undissociated hypochlorous acid diffuses into the bacterial cell, where this acid induces formation of toxic oxidative species and combines with proteins. This leads to inhibition of mRNA, protein synthesis, and oxidative phosphorylation [86,271].

QUATERNARY AMMONIUM COMPOUNDS Quaternary ammonium compounds are noncorrosive cationic surfactants frequently used to sanitize equipment surfaces [208,318]. Application of 100 to 200 ppm active compounds on surfaces is listericidal [203]. Listeria monocytogenes is slightly more resistant to quaternary ammonium compounds than to other sanitizers [214]. However, when the pathogen was exposed to cleaning compounds (4°C for 30 min) before treatment with quaternary ammonium compounds (25°C for 30 sec), >7 logs were inactivated [308]. Quaternary ammonium compounds react with bacterial carboxylic groups. Consequently, the cytoplasmic membrane is permeabilized and the cytoplasmic content may coagulate [318]. Enhanced resistance (≤10-fold) of L. monocytogenes to sanitizers has been reported after 2-h exposure to sublethal concentrations of quaternary ammonium compounds [208]. Adaptation to quaternary ammonium compounds involves a decrease in efflux pump activity [318] and modifications in cell wall structure [228]. Cells adapted to benzalkonium chloride were more elongated and filamentous than unadapted cells [318]. Adapted cells doubled their contents of C17:0 (iso and anteiso) fatty acids in the membrane and increased activity of the efflux pump system. The multidrug efflux pump, MdrL, which prevents accumulation of chemicals in bacterial cells, was present in L. monocytogenes adapted to sanitizers [278]. The gene encoding for this pumping system in L. monocytogenes can be chromosomal and plasmid encoded [278]. The contribution of MdrL to adaptation to sanitizers, however, is unclear [228,278]. Adaptation to multiple sanitizers may contribute to persistence of the pathogen in food-processing environments. However, the minimal inhibitory concentration of sanitizers against L. monocytogenes adapted to quaternary ammonium compounds is below the concentration of sanitizers generally used in food-plant sanitation [208].

ACID SANITIZERS Acid sanitizers are nonvolatile agents that retain bactericidal activity at 5 logs in 25 min [247]. At 80 ppm, peracid and peroctanoic acids were more potent than chlorine against a Listeria biofilm in the presence of milk residues on stainless steel [108]. When fruits were inoculated with L. monocytogenes and washed with lactic acid (1.5%) and hydrogen peroxide (1.5%) at 40°C for 15 min, pathogen populations decreased ≥ 6 logs [327]. Mixtures of peroxyacetic acid and hydrogen peroxide (120 ppm) were proposed as alternatives to a chlorine wash for lettuce [307]. Inclusion of sodium lactate and sodium acetate in wiener or bratwurst formulations inhibited growth of L. monocytogenes during extended storage at refrigeration temperature [133]. Dipping these sausages in lactate–diacetate solution was less effective than including this mixture in product formulations [133].

OZONE Ozone (O3) has been used in European countries for decades and has recently been approved in the United States by the FDA for treatment, storage, and processing of foods, including meat and poultry, unless use is precluded by a standard of identity [119]. Aqueous ozone reduces the microbial load on surfaces of food, packaging material, and equipment [172]. Gaseous ozone minimized

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TABLE 6.7 Inactivation of L. monocytogenes in Suspensions or on Food Surfaces after Exposure to Ozonated Water Medium Water (pH 6.1) Soluble starch solutionb BSA solutionb,c Phosphate buffer (pH 5.9) Alfalfa sprouts Beef

Temperature (°C)

Ozone (ppm)

Time (min)

Inactivationa (log CFU/mL)

Ref.

20 20 20 25 4 4

0.2 0.2 0.2 1.8 23 3

2 2 2 0.5 2 5

5.0 5.0 3.7 7.0 0.9 1.1

275 275 275 173 329 244

a

Decrease in log count compared to control.

b

20 ppm, pH 6.0.

c

Bovine serum albumin.

growth of microbial contaminants during storage of fresh produce [172]. In pure cell suspensions, L. monocytogenes is inactivated rapidly by small concentrations of ozone. The pathogen population decreased ~7 logs in 50 mM phosphate buffer (pH 5.9) after 30-sec exposure to 1.8 ppm ozone at 25°C [173]. Inactivation of L. monocytogenes increases with ozone concentration [111]. Listericidal activity of ozone varies with temperature, medium composition, strain, and physiological status. At an equivalent ozone concentration and exposure time (0.25 ppm; 2-min exposure), the inactivation rate of the pathogen almost doubled as temperature decreased from 37 to 4°C [111]. Processing at refrigeration temperature is therefore desirable to optimize the listericidal activity of ozone. Organic compounds such as proteins react with ozone [275], thus reducing the concentration of active ozone available for microbial inactivation in food. Consequently, efficacy of ozone decreases when L. monocytogenes is present in food, compared to deionized water or phosphate buffer (Table 6.7). Listeria is more sensitive to ozone than are other bacteria [173,275]. Sensitivity to ozone varied among strains of L. monocytogenes [111]. Resistance to ozone increased as L. monocytogenes entered the stationary phase. Ozone decomposes very rapidly in an aqueous solution; this generates numerous free radical species, such as hydroxyl (⋅OH) and superoxide (⋅O2–) [172]. These reactive species have high oxidizing power and initiate the oxidation of organic compounds. As expected from these findings, ozone causes oxidative damage in bacteria. Cell wall, membrane, intracellular enzymes, and DNA are possible sites of damage by ozone. Catalase and superoxide dismutase may protect L. monocytogenes against ozone; superoxide dismutase is more important than catalase for this protection [111]. Sublethal treatment with ozone did not sensitize L. monocytogenes to mild heat, alkali, or 6% NaCl [244].

MISCELLANEOUS SANITIZING AGENTS Iodophors, such as octylphenoxy-polyethoxy ethanol iodine complexes, are used to sanitize surfaces in contact with food. Iodophors are polymeric organic molecules that complex iodine species (I–, I2, and I3–) [135]. Listericidal activity of 25 ppm available iodophor is equivalent to that of 200 ppm chlorine [203]. Listeria monocytogenes is ~10 times more resistant to iodophors when the pathogen is attached to the surface of polypropylene or rubber than to stainless steel or glass [214]. The activity of these sanitizers against L. monocytogenes attached to stainless steel was 2 to 13 times higher at 20°C than at 4°C [212]. No inactivation of Listeria, however, was detected when an iodophor (80 ppm I2) was used in the presence of milk or human serum [24].

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Iodophors cause multiple damage in bacterial cells. The bacterial cell wall is permeable to iodine, which interacts with double bonds of membrane phospholipids; oxidizes free sulfhydryl groups; and combines with tyrosine, histidine, cytosine, and uracil [135]. Therefore, death of cells treated with iodine was attributed to loss of membrane integrity, protein inactivation, and DNA denaturation [135]. The population of L. monocytogenes decreased >7 logs CFU on tomatoes when surfaceinoculated fruit was sprayed with a 0.5% calcinated calcium solution (HYCEA-S, Kaiho Ltd., Tokyo, Japan) [16]. Addition of 100 ppm hydrogen peroxide inactivated the pathogen in contaminated cheese brines [191]. Suspended in peptone water, the population of L. monocytogenes decreased >6 logs when incubated at 37°C for 8 h in the presence of a packaging material coated with nisin (188 µg/mL) and lauric acid (200 mg/mL) [145]. This type of active packaging successfully decreased L. monocytogenes by 1 log on the surface of contaminated turkey bologna, and no growth was reported for >21 days at 21°C [73]. Compounds that have profound listericidal efficacy on nonfood surfaces include sodium dichloroisocyanurate [24], glutaraldehyde [24], chitosan [179], carvacrol [179], monolaurin [325], and listeriophages [144]. Listeria monocytogenes is ubiquitous in nature and prevention of cross-contamination during handling of ready-to-eat food is essential. Regular use of listericidal hand sanitizers can contribute to prevention of cross-contamination with Listeria during food handling. Kerr et al. [168] estimated that 12 and 7% of food workers carried Listeria spp. and L. monocytogenes, respectively. Thimothe et al. [314] detected Listeria spp. on 5.1% of employee-contact surfaces (gloves and apron) in seafood-processing plants. Thorough hand rubbing for 30 sec with nonmedicated soap reduced the aerobic mesophilic flora 6 logs L. monocytogenes [71]. Similar listericidal activity was reported

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when this zeolite was wet-process or powder coated [71]. When coextruded in plastic film, silverion zeolite showed strong antimicrobial action, even though it was not tested specifically against Listeria [36]. Bactericidal activity of silver ion zeolite was associated with transfer of silver ions to the cell, which led to formation of reactive oxygen species [219].

MULTIPLE ANTIMICROBIAL TREATMENTS Controlling L. monocytogenes in food may require application of multiple hurdles. Thermal pasteurization alone is generally considered adequate for eliminating this pathogen. This heat treatment, however, may damage heat-sensitive nutrients and flavor compounds. Thermal treatments less than pasteurization require additional hurdles (i.e., treatment combinations) to achieve the desired lethality [193]. Treatments that are commonly combined include mild heating (normally, 55 to 75°C), addition of antimicrobial agents (e.g., nitrites), acidification, and nonthermal alternative processing (e.g., ultrahigh pressure). These treatments may be applied simultaneously or sequentially to maximize antimicrobial efficacy and retention of product quality. When milk and liquid egg were experimentally treated with mild heat, limited inactivation of Listeria spp. was observed. Combining heat with nisin resulted in greater listericidal action (Table 6.8) compared to heat alone. Hurdles with lethal or growth-inhibitory action against foodborne microorganisms may be combined, depending on food and processing conditions. Compatibility between hurdles is essential and a synergistic or additive antimicrobial action is expected. Synergistic action is most desirable; this is noticeable in combinations producing inhibition or lethality greater than the additive actions of individual hurdles [53]. Satisfactory hurdle treatments cause multiple cell injury, such as physical cell damage, disruption of cell homeostasis, or metabolic exhaustion [193]. Multiple hurdles are particularly useful in eliminating L. monocytogenes in ready-to-eat meat and poultry products [323]. Listeriosis is increasingly associated with consumption of these products;

TABLE 6.8 Inactivation of Listeria by Heat, Pulsed Electric Field (PEF), and High-Pressure Processing (HPP) in Combination with Nisin Food

Processing Parameters

Nisin (µg/mL)

Milk

Heat: 60°C, 2 min Heat: 60°C, 2 min PEF: 40 kV/cm, 64 µs, 28°C PEF: 40 kV/cm, 64 µs, 28°C No treatment Heat: 54°C, 16 min Heat: 54°C, 16 min HPP: 300 MPa, 20°C, 10 min + 0 day storage at 4°C + 0 day storage at 4°C + 18 days storage at 4°C + 18 days storage at 4°C PEF: 40 kV/cm, 64 µs, 31°C PEF: 40 kV/cm, 64 µs, 31°C No treatment + 0 day storage at 4°C + 18 days storage at 4°C

0 7 0 37 ≤ 37 0 10

0.5–0.7 2.2–4.0 2.0 3.0 0 1.9 3.0

215 215 54 54 54 178 178

0 5 0 5 0 100

0.8 0.7 0 3.5 2.1 4.7

268 268 268 268 53 53

100 100

0 0

291 291

Liquid whole egg

Log CFU/mL Decreased

Ref.

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recent recalls of contaminated batches have caused major economic losses to the industry [312]. Ready-to-eat meat and poultry products may be formulated to contain antilisterial agents (e.g., diacetate), are thermally treated, and occasionally may be stored in contact with antimicrobial packaging materials [36,73]. Increasing awareness of possible postprocessing contamination of ready-to-eat meat prompted processors to apply lethal treatments at late stages of production. These are referred to as “post-lethality treatments” because they are applied after the traditional critical control point, e.g., the main heating step during production of frankfurters [323]. In-package thermal pasteurization and high-pressure processing are examples of post-lethality treatments that target primarily environmental food-surface contaminants. Careful integration of hurdles is crucial to the success of the combined treatment. Consideration of hurdle–hurdle interactions is essential to avoid antagonism. Inactivation of L. monocytogenes increased when high-pressure treatment increased from 300 to 700 MPa [310]. Using pressures much lower in magnitude than those just described, Pagan et al. [248] observed some antagonism between pressure and ultrasonic treatments. When ultrasonic waves of 117 µm and pressurization were applied simultaneously, D40°C of L. monocytogenes decreased to a greater extent when pressure increased from 0 to 100 kPa than from 300 to 400 kPa. It is presumed that ultrasonic treatments inactivate bacteria through intracellular cavitation and high pressure counteracts this mechanism. Improper application of hurdles may have a negative impact on the safety of treated food. Treating L. monocytogenes with commonly used hurdles, such as mild heat and acid, induced stressadaptive responses and protected the pathogen against lethal preservation factors [17,206,345]. Simultaneous exposure to multiple hurdles, therefore, may be necessary to minimize the risk of stress hardening of foodborne pathogens. Unlike conventional preservation technologies, emerging alternative methods, such as high-pressure processing, target a limited number of loci in a microbial cell [189]. These technologies cause limited microbial lethality and considerable stress and injury. Adaptation of pathogens to stresses caused by application of these technologies may protect pathogens from subsequent lethal treatments and thus compromise the safety of food. Alternative processing technologies may benefit from the hurdle concept. Addition of bacteriocins, for example, improved the lethality of high-pressure processing against L. monocytogenes [268]. Treating L. monocytogenes with PEF decreased levels of molecular chaperones for a short period after treatment [188]. When heat was applied to PEF-treated cells to coincide with maximum depression in the level of chaperones, great lethality was observed. Applying heat before PEF processing resulted only in a limited additive lethal effect. Careful choice of treatment combinations, therefore, makes alternative technologies feasible in today’s food applications.

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Methods 7 Conventional to Detect and Isolate Listeria monocytogenes Catherine W. Donnelly and David G. Nyachuba CONTENTS Introduction ....................................................................................................................................216 Cold Enrichment ............................................................................................................................217 Selective Enrichment and Plating at 30 to 37°C...........................................................................219 Selective Agents ...................................................................................................................219 Potassium Tellurite ...................................................................................................219 Lithium Chloride/Phenylethanol ..............................................................................220 Nalidixic Acid...........................................................................................................221 Trypaflavine/Acriflavine ...........................................................................................221 Potassium Thiocyanate .............................................................................................222 Thallous Acetate .......................................................................................................222 Polymyxin B.............................................................................................................222 Moxalactam ..............................................................................................................223 Ceftazidime...............................................................................................................223 Selective Media for Enrichment and Isolation of Listeria............................................................223 Selective Enrichment Media ................................................................................................223 UVM Broth...............................................................................................................225 Fraser Broth ..............................................................................................................226 Isolation Media.....................................................................................................................227 McBride Listeria Agar (MLA).................................................................................227 LPM Agar .................................................................................................................228 Oxford Agar (OXA) and Modified Oxford Agar (MOX)........................................228 PALCAM Agar .........................................................................................................228 Other Selective Plating Media .................................................................................229 Comparative Evaluation of Direct Plating Media for Recovery of Listeria from Foods .............................................................................................230 Incubation Conditions ..............................................................................................232 Oblique Illumination ............................................................................................................232 β-Hemolysis ............................................................................................................233 Official Methods for Isolating L. monocytogenes from Food .............................................234 FDA Method.............................................................................................................234 International Dairy Federation Method ...................................................................238 USDA–FSIS Method................................................................................................239 The Netherlands Government Food Inspection Service..........................................243 Research Advances.........................................................................................................................244 215

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Considerations for Recovery of Injured Listeria ..........................................................................244 Conclusions ....................................................................................................................................248 References ......................................................................................................................................249

Die Methode ist alles. Ralovich, German proverb [122]

INTRODUCTION Listeria monocytogenes is a nonfastidious organism that can be subcultured on most common bacteriological media such as tryptose agar, nutrient agar, and blood agar. However, attempted isolation or re-isolation of Listeria from inoculated or naturally contaminated food and clinical specimens by use of nonselective media is often challenging. Difficulties encountered in isolating L. monocytogenes date back to initial characterization of this pathogen in 1926 when Murray and his coworkers [127] stated, “The isolation of the infecting organism is not easy and we found this to remain true even after we had established the cause of the disease.” Although efforts to isolate L. monocytogenes from blood and cerebrospinal fluid of infected patients have met with considerable success (mainly because of the presence of Listeria in pure culture), obvious difficulties arise when food and clinical specimens (tissue biopsies and autopsy specimens) contain small populations of L. monocytogenes in combination with large numbers of other organisms. The first isolation methods were generally based on direct inoculation of samples on simple agar media. Isolation was problematic in cases of low numbers of viable Listeria cells, and inoculation into test animals (for example, embryonated eggs) was recommended. Upon conducting experiments with guinea pigs and mice, Larsen concluded that this biological method could be of value for detecting small numbers of Listeria in samples without competitive microflora [104]. However, in the presence of other microorganisms, especially Gram-negative mixed flora, test animals died of septicemia. Direct plating, cold enrichment, selective enrichment, and several rapid methods can be used in various combinations to detect L. monocytogenes in food, clinical, and environmental samples. In 1948, Gray et al. [76] introduced the cold enrichment procedure as an alternative method to isolate L. monocytogenes from highly contaminated samples. Although this method has contributed much to present-day knowledge concerning the epidemiology of listeriosis, the prolonged incubation period necessary to obtain positive results is a serious disadvantage. Major improvements in selective enrichment and plating media have since decreased analysis times from several months to less than 1 week. Outbreaks of foodborne listeriosis coupled with the high mortality rates associated with sporadic cases of illness and the advent of mandatory hazard analysis critical control point (HACCP) programs have underscored the need for faster and more efficient methods to detect small numbers of Listeria in a wide range of foods. The purpose of this chapter is to review and update development of various enrichment broths, as well as plating media and methods, used to isolate Listeria spp. (including L. monocytogenes) from food, environmental, and clinical samples. Numerous enrichment broth and plating media formulations have been developed and used during the past five decades for selective cultivation of Listeria. Detection and isolation of Listeria remain complicated tasks. Unfortunately, researchers have not yet been able to identify a single procedure sensitive enough to detect L. monocytogenes in all types of foods within a reasonable time. Furthermore, many selective enrichment broths and plating media fail to allow repair or growth of sublethally injured Listeria frequently present in semiprocessed and processed foods [32] or food-processing environments.

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Despite these inherent shortcomings, research efforts in response to foodborne listeriosis outbreaks have led to development of numerous regulatory procedures, including the U.S. Department of Agriculture Food Safety and Inspection Service (USDA–FSIS) and the U.S. Food and Drug Administration (FDA) procedures [86,88,91,178]. Both of these methods have been adopted in the United States as “standard methods” to isolate L. monocytogenes from a wide variety of foods and food-processing environments. However, in an effort to detect more rapidly and reliably healthy and sublethally injured Listeria in the wide range of foods currently examined, these methods and others have proved to be somewhat insensitive and do undoubtedly require further modifications and refinement.

COLD ENRICHMENT Difficulties in detecting and isolating L. monocytogenes typically arise when small numbers of Listeria are present in environmental and clinical or food samples containing large numbers of indigenous microorganisms. Hence, numbers of Listeria must be increased significantly, relative to that of the background flora, before the bacterium can be detected. Thirteen years after the first description of L. monocytogenes by Murray et al. [127], Biester and Schwarte [17] observed that Listerella (Listeria) could be frequently isolated from naturally infected sheep organs that were held refrigerated in 50% glycerol for several months. Although the organism was only rarely isolated after initial plating of diluted specimens, Biester and Schwarte failed to comment on the significance of cold storage. Following similar chance observations, a young graduate student, M. L. Gray, recognized the benefits of low-temperature incubation for recovering L. monocytogenes from clinical specimens. In 1948, Gray et al. [76] reported that in three of five bovine listeriosis cases, L. monocytogenes was only isolated after brain tissue was diluted in tryptose broth, stored for 5 to 13 weeks at 4°C and then plated on tryptose agar. Although a few Listeria colonies were observed after directly plating the remaining two brain tissue samples on tryptose agar, the bacterium was more readily isolated following cold enrichment. These results clearly demonstrated the ability of L. monocytogenes to multiply to detectable levels in the presence of other microbial contaminants during extended storage at refrigeration temperature (4°C). Gray’s cold enrichment method, in which samples homogenized in tryptose broth were incubated at 4°C and plated weekly or biweekly on tryptose agar during 3 months of storage, was soon adopted as the standard procedure for recovering L. monocytogenes. Normally only a few weeks of cold enrichment are required before Listeria can be detected; however, in one instance [74], 6 months of refrigerated storage was necessary before L. monocytogenes could be isolated from calf brains. Although the cold enrichment procedure is clearly slow and laborious, this method greatly enhances the likelihood of isolating Listeria (if present) from a variety of specimens, including food. In 13 studies summarized by Bojsen-Møller [21], Listeria was identified in 995 tissue and organ specimens from naturally and experimentally infected domestic animals. Using direct plating and cold enrichment procedures, Listeria was isolated from 684 of 995 (68.7%) specimens, whereas 307 of 995 (30.8%) specimens required cold enrichment before the bacterium could be detected. Furthermore, cold enrichment failed to detect Listeria in only 4 of 684 (0.6%) samples that were previously positive by direct plating. A study by Ryser et al. [156] stressed the importance of cold enrichment for recovery of L. monocytogenes from cottage cheese manufactured from milk inoculated with this pathogen. Using direct plating, L. monocytogenes was recovered from 43 of 112 (38.4%) cottage cheese samples stored at 3°C for up to 28 days, whereas cold enrichment of the same samples in tryptose broth for up to 8 weeks yielded Listeria in 59 of 112 (52.7%) samples. Thus, cold enrichment was necessary to detect this pathogen in 16 of 112 (14.3%) cheese samples. Ryser and Marth also found cold enrichment to be of great value in detecting low levels of L. monocytogenes in cheddar [157], Camembert [158], and brick cheese [160] manufactured from pasteurized milk inoculated with the bacterium.

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Lewis and Corry [109] compared cold enrichment and the FDA method [86] for isolating L. monocytogenes and Listeria spp. from ready-to-eat (RTE) foods at retail in the United Kingdom. Of the 57 food samples examined using cold enrichment, 5 yielded L. monocytogenes, and 2 L. innocua while the FDA method yielded 3 samples positive for L. monocytogenes only. Despite the proven success of cold enrichment, the mechanism by which numbers of L. monocytogenes are enhanced during prolonged incubation at 4°C is not fully understood. Although cold enrichment exploits the psychrotrophic nature of L. monocytogenes and simultaneously suppresses growth of indigenous nonpsychrotrophic organisms, Gray and Killinger [74] indicated that, at times, growth of Listeria was too rapid to attribute enhanced growth of this pathogen to mere multiplication. When this procedure was first described in 1948, Gray et al. [76] suggested possible involvement of an inhibitory factor in bovine brain tissue that suppressed growth of competing organisms. However, this theory has since been dispelled by subsequent studies that demonstrated enhanced growth of Listeria during cold enrichment of such diverse samples as mouse liver [144], oat silage [73], feces [139], sewage [56], cabbage [78], raw milk [170], and cheese [156–160]. A more plausible explanation is that, in many clinical specimens, Listeria may exist within monocytes, macrophages, or other phagocytic cells, with cold storage facilitating release of the intracellular organism. More recent research on the role of cold-shock proteins, cold-acclimating proteins, and other mechanisms that enable psychrotrophic growth of L. monocytogenes may help further explain the preferential growth of Listeria during cold enrichment [11,98]. For instance, anteiso-C15:0 fatty acid reportedly plays a critical role in adaptation of L. monocytogenes to cold temperatures [4]; mutants deficient in this fatty acid have been shown to be cold sensitive. As previously reported by Ryser and Marth [161], over 20 media formulations have been successfully used to cold enrich a diverse group of samples naturally or artificially contaminated with L. monocytogenes. Incubation at 4°C is partially selective for growth of L. monocytogenes, so nonselective broths such as tryptose broth and Oxoid nutrient broth no. 2 (ONB2) rapidly emerged as media of choice; tryptose broth was generally recognized as superior. In earlier studies, cold enrichment was used as the sole enrichment procedure and was followed by plating a portion of the enriched sample on tryptose agar at intervals during 2 to 12 months [164]. Following incubation, plates were examined under oblique lighting for typical bluish green, Listeria-like colonies. Cassiday and Brackett also have briefly reviewed the methods and media used by various researchers [22a,35,36]. Although growth of L. monocytogenes is favored at 4°C, other organisms, including Proteus, Hafnia, Pseudomonas, enterococci, and certain lactic acid bacteria are also able to multiply in nonselective media at refrigeration temperatures [2], thus making detection of Listeria more difficult. To prevent overgrowth by non-Listeria organisms, investigators began adding selective agents to various nonselective cold enrichment broths in an effort to inhibit non-Listeria microbes. In 1972, Bojsen-Møller [21] recognized that supplementing tryptose phosphate broth with polymyxin B substantially reduced populations of Gram-negative rods (i.e., Escherichia coli, Pseudomonas aeruginosa, and Proteus spp.) and enterococci while at the same time allowing rapid growth of L. monocytogenes. Unfortunately, certain species of lactic acid bacteria resistant to polymyxin B can ferment lactose to lactic acid and reduce the pH to the point where L. monocytogenes fails to grow at 4°C. Attempts at maintaining a pH of 7.2 by adding 0.1 M MOPS (3-N-morpholino propane sulfonic acid) to cold-enriched raw milk samples were unsuccessful [80]. Recovery of L. monocytogenes is enhanced when cold enrichment is used as a secondary enrichment following selective primary enrichment at 30 to 37°C. Bannerman and Bille [10] subjected numerous cheese and cheese factory environmental samples to secondary cold enrichment in the FDA Listeria enrichment broth (LEB) following primary enrichment at 30°C for 48 h. After plating enrichments on two selective agars, 34 and 62 of 96 isolates were obtained using warm and cold enrichment, respectively. Thus, cold enrichment for 28 days resulted in a 29.2% (28 of 96) increase in recovery of L. monocytogenes from cheese and cheese factory samples. However, with the advent of improved selective media and methods, most investigators have concluded that

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cold enrichment offers no advantages over selective enrichment [82]. In addition, the lengthy incubation period necessary for cold enrichment makes this procedure impractical for routine regulatory analysis of foods that most often require quick reporting of results.

SELECTIVE ENRICHMENT AND PLATING AT 30 TO 37 °C The principle of enrichment at elevated temperatures (30 to 37°C) is based on selective inhibition of indigenous microflora through addition of inhibitory agents while at the same time allowing unhindered growth of Listeria. Given the many months required for cold enrichment, the scientific community soon became aware of the need for a shorter incubation period. In 1950, Gray et al. [75] isolated L. monocytogenes from contaminated material that was inoculated into nutrient broth containing 0.05% potassium tellurite and incubated at 37°C for 6 to 8 h before being plated on tryptose agar with or without 0.05% potassium tellurite. Even though subsequent studies showed both potassium tellurite-containing media to be partially inhibitory to Listeria [96,108,133,145], Gray and his colleagues can still be credited with introducing the first cold enrichment procedure and the first warm enrichment media for selective isolation of L. monocytogenes. Since 1950, various combinations of selective agents have been added to basal media (i.e., tryptose broth, ONB2, and tryptose phosphate broth) to obtain media suitable for selective enrichment of Listeria at 30 to 37°C. Mavrothalassitis [120] reported an optimum incubation temperature of 30°C for enrichment of L. monocytogenes from heavily contaminated samples. Results from at least two additional studies [41,130] also showed that laboratory cultures of L. monocytogenes, L. seeligeri, or L. ivanovii were more susceptible to commonly used Listeria selective agents (i.e., ceftazidime, cefotetan, laxamoxef, and fosfomycin) when incubated at 37 rather than 30°C. Hence, most Listeria enrichments are done at 30°C. Ryser and Marth [161] previously reviewed the wide range of media formulations developed for selective enrichment of L. monocytogenes from environmental, clinical, and food specimens.

SELECTIVE AGENTS Modest, nonspecific nutritional requirements of L. monocytogenes have led to difficulties in formulating media that enhance growth of this pathogen. Consequently, efforts have primarily focused on inhibition of the indigenous bacterial flora by taking advantage of the resistance of L. monocytogenes to various selective agents, including chemicals, antimicrobials, and dyes. The major advances that have contributed to present-day ability to isolate Listeria from heavily contaminated environments are shown in Table 7.1. Although many inhibitory agents have proven to be at least somewhat useful for selective isolation of L. monocytogenes from naturally and artificially contaminated biological specimens, others have demonstrated very little value when added to basal media, as previously reviewed by Ryser and Marth [161]. Throughout the following discussion of selective agents, one must keep in mind that formulating media that are selective and now differential for L. monocytogenes is not a straightforward process; many selective agents may partially or completely inhibit growth of this pathogen, particularly when the organism is sublethally injured [32,46,131,167]. Potassium Tellurite Many selective media, including the early formulation by Gray et al. [75], contain inhibitory substances that are now of questionable value. As previously described, in 1950, Gray et al. [75] examined the potential usefulness of potassium tellurite and sodium azide in Listeria-selective media. Sodium azide prevented growth of L. monocytogenes in tryptose broth, whereas potassium tellurite was quite selective for the pathogen. However, shortly after these findings were published, Olson et al. [133] observed that potassium tellurite prevented growth of numerous L. monocytogenes

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TABLE 7.1 Recognition of Selective Agents Useful in Isolation of Listeria Year

Compound

1950

Potassium tellurite

1960

Lithium chloride/phenylethanol

1966

Nalidixic acid

1971

Acriflavin(e)/trypaflavin(e)

1971

Polymyxin B

1986

Moxalactam

1988

Ceftazidime

Role in Selective Media Selective/differential for Listeria that reduces tellurite to tellurium, producing black colonies Amplification of Listeria in the presence of Gramnegative bacteria Inhibitory to Gram-negative bacteria through interference with DNA gyrase Inhibitory to Gram-positive cocci Prevents growth of Gramnegative rods and streptococci Broad spectrum; inhibitory to many Gram-positive and Gram-negative contaminants, including Staphylococcus, Proteus, and Pseudomonas Broad-spectrum cephalosporin antibiotic

Ref. 24,75,96,100,108,121,133,145,171

47,59,77,78,80,106,113,121,157,158,170

1,20,52,55,71,92,96,134,135,147,166

3,19,44,51,52,57,77,89,93,94,134,135, 145,146,148,149,152 21,43,47,113,134,152,168

86,106,124,134

10,114,117,130

strains. Other investigators [96,100,108,121,145] have substantiated these findings and discouraged the use of potassium tellurite as a selective agent. The advantage of adding potassium tellurite to selective media is that the resulting Listeria colonies appear black from reduction of potassium tellurite to tellurium. Unlike the typical black-yellowish and gray colonies produced by Gram-positive cocci, the marginal zone of Listeria colonies appears green when the organism is grown on media containing potassium tellurite and viewed with oblique illumination [164]. A modification of Vogel Johnson agar (MVJA) was evaluated by Buchanan et al. [24] for isolating Listeria from foods. Selective agents, including moxalactam, nalidixic acid, bacitracin, and potassium tellurite, permitted growth of Listeria while suppressing background contaminants. Furthermore, the ability to distinguish colonies readily was not predicated on the need for obliquely transmitted light. Buchanan et al. [27] also found that lithium chloride-phenylethanol-moxalactam agar (LPM) and MVJA generally gave comparable recovery of Listeria from naturally contaminated samples of fresh meat, cured meat, poultry, fish, and shellfish. Adding tellurite and mannitol to MVJA greatly aided in differentiating Listeria colonies from those formed by naturally occurring contaminants, including various species of enterococci and staphylococci. However, Smith and Archer [171] reported that potassium tellurite prevented repair of heat-injured L. monocytogenes. Lithium Chloride/Phenylethanol Using the combination of phenylethanol and lithium chloride, McBride and Girard [121] succeeded in amplifying numbers of L. monocytogenes in the presence of Gram-negative bacteria. The usefulness of phenylethanol and lithium chloride as Listeria-selective agents has since been

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confirmed by other investigators, resulting in the earlier widespread use and acceptance of McBride Listeria agar (MLA) as a plating medium for L. monocytogenes [38,59,77,78,80,106, 113,121,157,158,170]. A modification of MLA (omission of sheep blood and addition of cycloheximide as an antifungal agent) was once recommended by the FDA for analyzing food samples suspected of harboring Listeria [113,114]. Ryser and Marth [159,160] and Yousef and Marth [186] reported that increasing the lithium chloride concentration to 0.5% (0.05% lithium chloride in the original formulation [121]) increased selectivity of the medium without appreciably decreasing recovery of healthy Listeria [159,160,183]. Nalidixic Acid Beerens and Tahon-Castel [14] were first to report the usefulness of nalidixic acid in isolating L. monocytogenes from heavily contaminated pathological specimens. Increased isolation of Listeria using media containing nalidixic acid primarily resulted from inhibition of indigenous Gram-negative bacteria [71]. The benefits of adding nalidixic acid to otherwise noninhibitory media were soon confirmed in many laboratories [20,52,92,96,134,135,166]. After discovering the benefits of adding nalidixic acid to enrichment broth [14], Ralovich et al. [147] effectively used serum agar containing nalidixic acid to isolate L. monocytogenes from feces, organs, and other clinical specimens. Although the microbial background flora was largely inhibited on this medium, streptococci and other nalidixic acid-resistant organisms occasionally persisted. Nalidixic acid was eventually recognized as one of the most important selective agents, and it is now used alone or more commonly in combination with other selective agents for isolating L. monocytogenes from food and clinical specimens. Farber et al. [55] developed an improved Listeria-selective plating medium by combining the positive attributes of McBride Listeria agar and LPM agar. In their formula for Farber Listeria agar, oxolinic acid was substituted for nalidixic acid. Both agents function by interfering with the activity of DNA gyrase, an enzyme needed to maintain proper DNA structure and resealing of chromosomal nicks [71]. Trypaflavine/Acriflavine Despite successful use of nalidixic acid, Ralovich et al. [148,149] found that growth of certain Gram-positive cocci and Gram-negative rods in the presence of this selective agent complicated the isolation of Listeria. Such difficulties led to inclusion of trypaflavine, a known inhibitor of Gram-positive cocci, in media containing nalidixic acid. This medium soon became known as trypaflavine nalidixic acid serum agar (TNSA). The end result was the selective inhibition of virtually all other bacteria; growth of L. monocytogenes was only slightly decreased [19,133]. Following successful use of this medium in many European studies [19,93,134,135,148], Ralovich et al. [145] endorsed TNSA as the plating medium of choice for isolating L. monocytogenes from contaminated materials. Additional work revealed that contaminating organisms, predominantly streptococci, grew infrequently on clear media containing both antibiotics and were generally discernible from L. monocytogenes with the naked eye. In 1972, Seeliger [165] reported that combined use of acriflavine and nalidixic acid greatly suppressed Gram-negative organisms and fecal streptococci without apparently affecting recovery of L. monocytogenes. These findings were subsequently confirmed by Bockemühl et al. [20], who reported easy recovery of L. monocytogenes from enriched fecal samples using an agar medium that contained nalidixic acid and acridine dye. Confirmation of these findings in other European laboratories [52,57,77,94] led to widespread use of trypaflavine/nalidixic acid as Listeria-selective agents. In 1974, Hofer [89] proposed using a medium prepared from tryptose agar containing nalidixic acid, trypaflavine, and thallous acetate. Trypaflavine can be replaced by other acridine dyes, including xanthacridine, acriflavine, or proflavinehemisulfate [146]. According to Gregorio et al. [77], use of nalidixic acid together with

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acriflavine or trypaflavine gave rise to media that were equally inhibitory to background microflora, suggesting that similar results can be obtained by substituting acriflavine for trypaflavine. Based on results from European laboratories [44,51,93,146], a serum agar- or blood agar-based medium containing trypaflavine, acriflavine, and nalidixic acid appeared to be satisfactory for selective isolation of L. monocytogenes from samples containing a mixed microbial flora. In 1984, Rodriguez et al. [152] developed a blood agar medium containing acriflavine and nalidixic acid (Rodriguez isolation medium [RIM]) that was far superior to the earlier formulations of Ralovich et al. [147,149]. During the last decade, numerous media containing acriflavine and nalidixic acid with or without other antibiotics have been developed for selective isolation/enrichment of Listeria from food and environmental samples, including Merck Listeria agar [22,79], which is commercially available in Europe. Potassium Thiocyanate In 1961, Fuzi and Pillis [65] proposed a medium containing 0.35% potassium thiocyanate for selective enrichment of L. monocytogenes. Although it was reported useful by some researchers [52,107,166], others found that potassium thiocyanate inhibited L. monocytogenes [100,108,148]. Despite these reports, several studies demonstrated that an enrichment broth containing this selective agent in combination with nalidixic acid was useful in isolating L. monocytogenes from cabbage [78], milk [80,170], and other dairy products [102]. In 1972, Ralovich et al. [148] endorsed Levinthal’s broth and Holman's medium, which contain nalidixic acid and trypaflavine, for selective enrichment of Listeria. Results obtained by Slade and Collins-Thompson [170] demonstrated that growth of L. monocytogenes in ONB2 containing nalidixic acid and potassium thiocyanate can be improved by adding acriflavine. Thallous Acetate During the early 1950s, thallous acetate was employed as a selective agent for lactic acid bacteria; however, it was not until 1969 that Kramer and Jones [100] recommended the combined use of thallous acetate and nalidixic acid in Listeria-selective media. Three years later, Khan et al. [96] found that, unlike potassium tellurite, thallous acetate used alone or together with nalidixic acid did not adversely affect recovery of L. monocytogenes from biological specimens and silage samples. In 1979, Leighton [108] demonstrated that the combined use of thallous acetate and nalidixic acid completely suppressed growth of E. coli strains previously resistant to nalidixic acid. Greater inhibition of Gram-positive bacteria also occurred when both selective agents were used together rather than separately. Although Leighton [108] recommended a medium composed of tryptose phosphate broth, thallous acetate, and nalidixic acid for recovery of L. monocytogenes from mixed bacterial populations, thallous acetate (as well as potassium thiocyanate, potassium tellurite, and lithium chloride) altered the colonial morphology of L. monocytogenes from the smooth to rough form. In view of this experience, most currently used formulations of Listeria-selective media omit thallous acetate. Polymyxin B In 1971, Despierres [43] reported that the combination of polymyxin B and nalidixic acid was useful for recovering L. monocytogenes from feces and that these antibiotics prevented growth of many background organisms, including Enterococcus faecalis. That same year, Ortel [134] proposed another medium containing polymyxin B and bacitracin to isolate L. monocytogenes from stool samples. According to Bojsen-Møller [21], Gram-negative rods and enterococci failed to grow in tryptose phosphate broth containing polymyxin B, but growth of L. monocytogenes was relatively unaffected. After examining six different enrichment and isolation media, Rodriguez et al. [152] concluded that little if any benefit was gained by adding polymyxin B to media already containing nalidixic acid and acriflavine.

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Doyle and Schoeni [47] successfully isolated L. monocytogenes from milk and clinical and fecal samples after enrichment in a selective broth containing polymyxin B, acriflavine, and nalidixic acid that resembled isolation medium II developed by Rodriguez et al. [152]. Although the selective enrichment broth developed by Doyle and Schoeni gained some attention [113], the necessity for polymyxin B in this medium remains somewhat questionable. Siragusa and Johnson [168] successfully isolated L. monocytogenes from yogurt using a medium containing polymyxin B, nalidixic acid, and acriflavine. Their medium reportedly prevented growth of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, thus making it particularly suitable for isolating L. monocytogenes from certain fermented dairy products. Moxalactam Results from antibiotic susceptibility tests [134] led Lee and McClain [106] to add moxalactam (a broad-spectrum antibiotic that is inhibitory to many Gram-positive and Gram-negative bacteria, including Staphylococcus, Proteus, and Pseudomonas) to MLA containing 0.25% phenylethanol and 0.5% lithium chloride. The result was a highly selective medium for recovery of L. monocytogenes from raw beef and many other foods. This medium, LPM agar, is recommended by the USDA–FSIS for isolating L. monocytogenes from raw meat and poultry [124] and also has been incorporated into the current FDA procedure as a second selective plating medium [86]. Ceftazidime Bannerman and Bille [10] used Columbia agar base in combination with acriflavine and ceftazidime (AC agar), a broad-spectrum cephalosporin antibiotic, to isolate L. monocytogenes from cheese samples. AC agar was superior to FDA-modified McBride Listeria agar (MMLA) [114,117]; it recovered approximately 50% more L. monocytogenes isolates from soft cheese and cheese manufacturing environments than did FDA-MMLA. Except for a few enterococci, the combination of acriflavine and ceftazidime inhibited all other non-Listeria organisms, including yeasts and molds. However, van Netten et al. [130] reported that PALCAM agar, which contains polymyxin B and lithium chloride along with half or less the concentration of acriflavine and ceftazidime found in AC agar, was superior to the latter medium. After comparing 13 different plating media, these authors also concluded that media containing ceftazidime and 1.5% lithium chloride afforded more selectivity than did phenylethanol alone. However, increased selectivity results in decreased recovery of stressed or sublethally injured cells that are frequently present in foods.

SELECTIVE MEDIA FOR ENRICHMENT AND ISOLATION OF LISTERIA SELECTIVE ENRICHMENT MEDIA Frequent outbreaks of foodborne illness caused by L. monocytogenes in the recent past and the high mortality rate associated with listeriosis have highlighted the need for more sensitive, reliable, and rapid detection methods for the pathogen. The logical approach was to use some of the previously described enrichment broths containing selective agents and to incubate samples at an elevated temperature, generally 30°C. In response to numerous requests from the food industry, several enrichment schemes have been developed that include primary or secondary selective enrichments. An outbreak of listeriosis epidemiologically linked to consumption of pasteurized milk [63] led Hayes et al. [80] to develop a two-stage enrichment procedure for isolating L. monocytogenes from raw milk. Primary cold enrichment in ONB2 followed by secondary enrichment at 35°C in ONB2 containing potassium thiocyanate (KSCN) and nalidixic acid and plating on GBNA yielded the highest number of positive milk samples. No statistically significant difference in recovery of Listeria was observed using Stuart transport medium or selective enrichment broth containing

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potassium thiocyanate and nalidixic acid. Although 15 milk samples were positive when plated on GBNA medium as compared with 11 on MLA2 without blood, the difference was not statistically significant. The authors concluded that primary cold enrichment in ONB2 followed by secondary selective enrichment at 35°C and plating on GBNA medium were the most useful for identifying positive raw milk samples. Slade and Collins-Thompson [170] developed a somewhat shorter two-stage enrichment procedure to isolate Listeria from foods. Their method was tested using raw milk inoculated to contain approximately 100 L. monocytogenes CFU/mL. Results showed that tryptose broth was superior to ONB2 as a primary cold enrichment medium. In addition, diluting milk samples 1:10, rather than 1:5, increased the number of Listeria isolations on selective media. The more dilute samples probably maintained a higher pH (≥6) during cold enrichment as a result of fewer lactic acid bacteria and little lactose being present; this in turn led to faster growth and increased detection of Listeria on solid media. Original MLA without blood was the only medium tested that proved to be useful for plating primary cold enrichments because tryptose agar and trypaflavine nalidixic acid agar were typically overgrown by competing microflora. Favorable results were, however, obtained using tryptose agar after secondary enrichment at 37°C. Addition of acriflavine to thiocyanate nalidixic acid broth proved beneficial for recovery of L. monocytogenes. Thus, following 7 to 14 days of cold enrichment in tryptose broth, L. monocytogenes was most frequently isolated after plating samples enriched in thiocyanate nalidixic acid broth on MLA with blood or tryptose agar. A “shortened” enrichment procedure and a two-stage cold/selective enrichment procedure were developed in Canada by Farber et al. [54] for isolating Listeria spp. from raw milk. In the shortened enrichment procedure, milk samples underwent primary and secondary enrichment at 30°C as well as primary cold enrichment in two selective media (FDA enrichment broth and University of Vermont medium [UVM]). Although no single step within the procedure was completely satisfactory for isolating Listeria from raw milk, the two steps that were most helpful involved surface plating the primary FDA enrichment broth culture on MLA2 with blood after 1 day of incubation at 30°C, and surface plating the 30-day-old cold enriched FDA enrichment broth culture (initially incubated 7 days at 30°C) on MLA2 with blood. Collectively, these steps detected Listeria spp. in 31 of 51 (60.8%) positive raw milk samples. Although 11 isolations were made after 1 day but not 7 days of primary selective enrichment at 30°C, 6 isolations were only possible after 7 days of primary selective enrichment. Thus, incubating the primary selective enrichment at 30°C for 7 days before plating on MLA2 with blood markedly enhanced recovery of Listeria from raw milk. The two-stage cold/warm enrichment method, which was the second of two procedures developed by Farber et al. [54], also detected Listeria spp. in raw milk samples. Using this procedure, Listeria spp. were isolated from 12 samples that tested negative using the shortened enrichment procedure. Similarly, 10 samples that tested positive for Listeria spp. using the shortened enrichment procedure were negative with the two-stage cold/warm enrichment method. Thus, when used alone, neither procedure detected Listeria in all positive samples. Following cold enrichment, similar numbers of samples were positive for Listeria spp. after enrichment in FDA enrichment broth and UVM. However, eight raw milk samples were only positive after 2 weeks of cold enrichment as compared with three samples in which Listeria was only detected after 4 weeks of cold enrichment. These results are similar to those of Doyle and Schoeni [47], who also observed that Listeria spp. could be more readily isolated from raw milk and soft, surface-ripened cheese [48] during the first 2 weeks of cold enrichment. Food-associated outbreaks of listeriosis along with discovery of L. monocytogenes in many European varieties of soft- and smear-ripened cheese prompted two Swiss investigators, Bannerman and Bille [10], to develop a two-stage selective/cold enrichment procedure to recover Listeria spp. from cheese and dairy plant surfaces. Their isolation method is similar to the shortened enrichment

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procedure just described [54] with the exception that the secondary selective enrichment step has been eliminated and AC agar has been included as an additional selective plating medium. Using this method, Listeria spp. were isolated from 157 of 1,099 (14.3%) cheese and environmental samples. A total of 99 samples were positive for Listeria using both plating media. Following selective enrichment, 56 of 99 (57%) and 35 of 99 (35%) samples were positive after surfaceplating enrichment cultures on AC agar and FDA-MMLA, respectively. Increased selectivity of AC agar was presumably responsible for detection of approximately 50% more Listeria isolates as compared with FDA-MMLA. Important information concerning presence of Listeria spp. in food and environmental samples can be gained using the three procedures just described as well as procedures developed by Hayes et al. [80] and Slade and Collins-Thompson [170]; however, the need for cold enrichment in these procedures increased the length of analysis to 30 to 40 days. Hence, the time constraints of this method negate its use in any isolation procedure that is to be adopted by the food industry as a “standard” enrichment method. Rodriguez et al. [154] developed a complicated scheme to isolate Listeria from raw milk that more importantly paved the way for subsequent development of several widely used enrichment media, including UVM enrichment broth [33,45]. Their protocol included three noninhibitory collection (primary enrichment) media, three selective (secondary) enrichment media, and one selective plating medium, RIM III, all of which were previously described by Rodriguez et al. [152]. The three selective enrichment media used in this protocol contained nalidixic acid and trypan blue with or without polymyxin B; nalidixic acid and acriflavine were used as selective agents in the plating medium. Milk was added to all three collection media, with collection medium B streaked onto RIM III after 7 and 15 days of storage at 4°C. Collection medium A was incubated at 4°C for 24 h, subcultured in all three secondary enrichment media, which were incubated at 22°C until a color change occurred, and then samples were streaked onto plates of RIM II. A portion of collection medium A also was diluted in collection medium C, which was streaked on to RIM III following 7 and 15 days at 4°C. According to these authors, 11 L. monocytogenes isolates were obtained after primary cold enrichment, with collection medium C accounting for 9 of 11 isolations. Although results for collection medium C appear impressive, the increased number of isolations using this medium may have resulted from a more dilute sample: approximately 1:40 as compared with approximately 1:8 in collection media A and B. Under these conditions, collection medium C should have maintained a higher pH during cold enrichment because fewer lactic acid bacteria and less lactose were likely present, thereby enhancing the growth environment for L. monocytogenes. In contrast to cold enrichment, 49 L. monocytogenes isolates were obtained following secondary enrichment at 22°C with 16, 32, and 1 colonies originating from Rodriguez enrichment media 1, 2, and 3, respectively. Recovery of only one Listeria isolate using Rodriguez enrichment medium 3 is not surprising considering that collection medium A was diluted approximately 1:68 in collection medium C after only 24 h of enrichment at 4°C. Transfer of the culture after 24 h of cold enrichment provides little opportunity for appreciable growth of L. monocytogenes, so the organism was likely diluted out of the sample. Overall, primary cold enrichment of milk samples diluted approximately 1:8 followed by secondary enrichment in Rodriguez enrichment media 1 and 2 at 22°C and plating on an isolation medium containing nalidixic acid and acriflavine provided the best opportunity for detecting L. monocytogenes in raw milk. UVM Broth Selective media originally recommended by the FDA [114,117] and USDA–FSIS [123,124] for enrichment of food samples containing L. monocytogenes were modifications of media proposed by Ralovich et al. [149] and Rodriguez et al. [152] as modified by Donnelly and Baigent [45], respectively.

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Donnelly and Baigent [45] explored the use of several selective enrichment media to inhibit growth of raw milk contaminants and select for L. monocytogenes. The most successful medium for this application was a modification of Rodriguez enrichment medium III [152]. This medium, designated LEB by Donnelly and Baigent [45], consisted of proteose peptone (5.0 g/L), tryptone (5.0 g/L), Lab-Lemco powder (5.0 g/L), yeast extract (5.0 g/L), sodium chloride (20.0 g/L), disodium phosphate2-hydrate (12.0 g/L), potassium phosphate monobasic (1.35 g/L), esculin (1.0 g/L), nalidixic acid (40 mg/L), and acriflavine HCl (12 mg/L). McClain and Lee [123] modified this formula to contain 20 mg/L nalidixic acid, and this formulation was known as USDA LEB I. These authors further modified LEB I to contain 25 mg/L acriflavine and used this medium, LEB II, for secondary enrichment of meat and poultry samples. USDA–FSIS currently recommends use of UVM broth (LEB I) for primary enrichment of meat, poultry, egg, and environmental samples [33,91,178]. Fraser Broth Fraser broth [64] is a modification of USDA LEB II which contains lithium chloride (3.0 g/L) and ferric ammonium citrate (0.5 g/L). This medium reportedly was advantageous for detecting Listeria spp. in enriched food and environmental samples. Because Listeria will turn Fraser broth black from esculin hydrolysis within 48 h of incubation [23], this broth has now replaced USDA LEB II in the USDA protocol as the preferred secondary enrichment medium for meat, poultry, and environmental samples [91,178]. In 1986, Doyle and Schoeni [47] used the microaerophilic nature of L. monocytogenes in developing a shortened one-step enrichment procedure to isolate this organism from milk as well as fecal and biological specimens. In their protocol, the sample was placed inside an Erlenmeyer flask equipped with a side arm and then diluted 1:5 in Doyle and Schoeni selective enrichment broth (DSSEB). Following 24 h of incubation at 37°C in an atmosphere of 5% O2:10% CO2:85% N2, a portion of the sample was streaked onto plates of MLA (original formulation with blood), which were similarly incubated under microaerobic conditions. Using DSSEB, L. monocytogenes was consistently isolated from raw milk samples inoculated to contain 10 L. monocytogenes CFU/mL. In addition, about two and five times as many L. monocytogenes isolates were recovered from fecal and biological specimens, respectively, using DSSEB rather than cold enrichment and direct plating. Another enrichment procedure, which is partially based on microaerobic incubation, was developed by Skovgaard and Morgen [169] to isolate Listeria spp. from heavily contaminated samples, including feces, silage, minced meat, and poultry. In this two-step enrichment procedure, microaerobic incubation (24 h/30°C/95% air: 5% CO2) of the sample in USDA LEB I is followed by aerobic secondary selective enrichment in USDA LEB II, after which untreated and KOH-treated samples are surface plated on LPM agar. Using this isolation scheme, which, with the exception of microaerobic incubation, closely resembles the original USDA procedure, numerous fecal, silage, minced beef, and poultry samples were positive for Listeria spp., including L. monocytogenes. Based on these results, the authors concluded that their method was suitable for detecting Listeria in heavily contaminated materials, including samples of raw ground beef and poultry. Although both procedures just described decrease the Listeria detection time to approximately 3 days, incubating enrichment cultures under microaerobic conditions is particularly awkward and not feasible for large-scale testing programs. A large listeriosis outbreak in which coleslaw was implicated as the vehicle of infection prompted Hao et al. [78] to compare various media and methods to detect L. monocytogenes in cabbage. Preliminary results clearly demonstrated a need for some type of enrichment procedure before L. monocytogenes could be isolated from inoculated samples. After comparing results from various plating and enrichment media, these investigators proposed a two-step enrichment procedure for isolating L. monocytogenes from cabbage. A cold enrichment period of 14 or 30 days at 5°C in ONB2 or brain heart infusion broth (BHI) led to increased recovery of Listeria from cabbage

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following secondary enrichment (30°C/48 h) in FDA enrichment broth or ONB2 containing potassium thiocyanate and nalidixic acid. A comparison of nine selective plating media, with and without an additional 5 mg of Fe3+/L, led to the recommendation of modified Doyle/Schoeni selective agar II and MLA with glycine anhydride rather than glycine (MLA2) for isolating L. monocytogenes from cabbage. Both media contained 5% sheep blood, which was beneficial for picking Listeria-like colonies. As was true for the cold enrichment broths, several popular plating media, including FDA-MMLA and LPM agar, were not examined in this study. However, their efficacy in isolating L. monocytogenes from cabbage and other vegetables needs to be determined before recommending this procedure for use in routine analysis of such products. Despite repeated efforts toward developing an effective enrichment medium for recovery of L. monocytogenes, no one single selective enrichment broth has proven to be totally reliable for analysis of food products containing Listeria. Nevertheless, several enrichment broths have moved to the forefront, including the FDA enrichment broth [86], UVM broth [91,178], and Fraser broth [91], all of which are commercially available from BBL, Difco Laboratories, and other manufacturers. Truscott and McNab [174] developed a selective enrichment medium called Listeria test broth (LTB) as an alternative to UVM broth for detecting L. monocytogenes in meat products. After primary and/or secondary enrichment of 50 frozen ground beef samples in both enrichment broths, L. monocytogenes was detected in 19 of 50 (38%) and 16 of 50 (32%) samples using UVM and LTB, respectively. Although Listeria recovery rates for these two broths are not appreciably different, neither medium alone was able to detect the pathogen in all 29 samples that were positive. In addition, LPALCAMY broth, which was developed by van Netten et al. [130], has shown superior results to USDA LEBs I and II as well as the tryptose broth-based antibiotic medium of Beckers et al. [13] for detecting L. monocytogenes in naturally contaminated cheese, minced meat, fermented sausage, raw chicken, and mushrooms. However, given wide variations in the type and the number of naturally occurring microbial contaminants in the food supply, development of a single enrichment broth for truly optimal recovery of Listeria from all types of food appears improbable.

ISOLATION MEDIA McBride Listeria Agar (MLA) MLA was the first widely used plating medium for selective isolation of L. monocytogenes. This medium, introduced by McBride and Girard [121] in 1960, is prepared from phenylethanol agar to which lithium chloride, glycine, and sheep blood are added. At least seven subsequent changes in the original formulation of MLA have led to considerable confusion as to the exact composition of this medium. Ironically, the first reported modification of MLA by Bearns and Girard [12] dates back to 1959, nearly 1 year before the original formulation appeared in the literature [121]. This medium, named modified McBride medium (MLA2) by the authors and known today as one of several modified MLAs, is similar to the original formulation except that sheep blood is omitted and glycine anhydride is substituted for glycine [106]. In most instances, MLA2 was more Listeria selective than nalidixic acid agar [59,134], acriflavine nalidixic acid agar [170], or acridine nalidixic acid agar [59]. The selectivity of MLA2 can be further improved, without affecting recovery of Listeria, by increasing the lithium chloride content to 0.5%. With the further addition of sheep blood, this medium became partially differential and hence was better suited than MLA2 for recovering L. monocytogenes from brick [160], feta [137], and blue cheese [138], as well as cold-pack cheese food [159]. Following an earlier report in which glycine was found to partially inhibit L. monocytogenes [106], many individuals began to replace glycine with glycine anhydride, which is far less inhibitory to Listeria. Nevertheless, two widely used formulations of the original MLA containing

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glycine have been commercially available since 1985 from Difco Laboratories and BBL (now Becton Dickinson). Although addition of blood provides one means of identifying possible L. monocytogenes colonies (virtually all are at least somewhat β-hemolytic) and enhances growth of the pathogen in certain B vitamin- and/or amino acid-deficient media, many workers preferred to omit blood from the various formulations of MLA and examine the plates under oblique illumination for blue to bluish green Listeria-like colonies. In 1987, Lovett et al. [117] added cycloheximide to blood-free MLA2 and named this particularly useful medium FDA-modified McBride Listeria agar (FDA-MMLA). Although one earlier study claimed that TNSA was superior to MLA2, subsequent data indicated that FDA-MMLA [113,114,115,117] and MLA2 [69,78,80,117, 149,170], which contain glycine anhydride, were the MLA formulations of choice for isolating Listeria spp. from foods, particularly dairy, vegetable, and seafood products. The FDA formulation previously served as one of two plating media (the other is LPM agar) in the FDA procedure [116]. LPM Agar In 1986, Lee and McClain [106] added 4.5 g of lithium chloride and 20 mg of moxalactam to MLA2 and named their new medium lithium chloride-phenylethanol-moxalactam (LPM) agar. Although this selective medium (commercially available from Becton Dickinson is particularly well suited for isolating Listeria from raw meat and poultry—as evidenced by its inclusion as the medium of choice in an earlier version of the USDA procedure—LPM agar has since been replaced by modified Oxford agar [33], which produces black Listeria colonies, each with a black halo following 24 h of incubation. However, LPM plus esculin and ferric iron is still used as one of the selective isolation agars in the FDA procedure [88]. Oxford Agar (OXA) and Modified Oxford Agar (MOX) In 1989, Curtis et al. [42] developed an agar medium that eliminated the need for oblique illumination. Their medium, Oxford agar (OXA), was prepared from Columbia agar base to which several selective agents, including colistin sulfate (20 mg/L), fosfomycin (10 mg/L), cefotetan (2 mg/L), cycloheximide (400 mg/L), lithium chloride (15 g/L), and acriflavine (5 mg/L), were added. Esculin and ferric ammonium citrate also were added as differential agents to produce black Listeria colonies from esculin hydrolysis. This medium was slightly modified by McClain and Lee by incorporating moxalactam; this new medium was designated modified Oxford agar (MOX) [33]. In May 1989, the USDA–FSIS procedure was changed to incorporate MOX as the recommended plating medium. Late in 1990, the FDA modified its procedure by replacing FDAMMLA with Oxford agar (OXA). In the present version of the FDA method [88], one of the following selective media must now be used: PALCAM, OXA, MOX, or LPM fortified with esculin and Fe3+. PALCAM Agar In 1988, van Netten et al. [129] reported that RAPAMY agar, a modification of TNSA developed by Ralovich et al. [149] that includes acriflavine, phenylethanol, esculin, mannitol, and egg yolk emulsion, was suitable for enumerating Listeria spp. Virtually identical populations were observed when overnight broth cultures of L. monocytogenes, L. seeligeri, and L. ivanovii were surfaceplated on RAPAMY and nonselective agar; growth of all non-Listeria organisms tested, except Enterococcus faecalis and Enterococcus faecium, was completely inhibited on the selective medium. Like OXA [42], RAPAMY agar also produced distinctive black Listeria colonies surrounded by a dense black halo from esculin hydrolysis. Although such characteristic colonies were present

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against a deep red background (inability to utilize mannitol) on RAPAMY agar, E. faecalis and E. faecium generally produced colonies with blue-green halos. Although attempts to eliminate growth of these two species of enterococci by adding cefoxitin (moxalactam) to this medium failed, results suggested that RAPAMY agar could be used to quantify Listeria spp. in thermally processed and dried foods with total aerobic plate counts of ≤106 CFU/g and Enterococcus counts of ≤102 CFU/g. However, as might be expected, high populations of enterococci severely hampered detection of Listeria spp. in chicken, minced meat, and mold-ripened cheese. Further attempts by van Netten et al. [128] to eliminate growth of enterococci by adding fosfomycin (20 mg/L) to RAPAMY agar met with only limited success. Addition of lithium chloride (1.5%) to RAPAMY agar inhibited many Listeria spp.; however, an improved selective and differential medium was obtained by adding lithium chloride to RAPAMY agar and omitting nalidixic acid. The resultant medium was named ALPAMY agar because it contains acriflavine, lithium chloride, phenylethanol, esculin, mannitol, and egg yolk emulsion agar. In a study with pure cultures, ALPAMY agar allowed uninhibited growth of all 10 L. monocytogenes strains tested but completely prevented growth of single strains of L. seeligeri and L. ivanovii. Selectivity tests showed that ALPAMY agar supported growth of only 2 of 41 non-Listeria organisms—one strain each of Staphylococcus aureus and Micrococcus spp., both of which were readily differentiated from Listeria colonies. Subsequent studies indicate that ALPAMY agar is far superior to RAPAMY agar for detecting Listeria in raw milk and soft cheeses manufactured from raw milk, as well as in raw vegetables and chicken. This medium is the forerunner to PALCAM agar [130], which contains polymyxin B and lithium chloride along with half or less the concentration of acriflavine and ceftazidime found in AC agar. It is recommended that PALCAM agar plates be incubated for 40 to 48 h at 30°C under microaerobic conditions (5% oxygen, 7.5% carbon dioxide, 7.5% hydrogen, and 80% nitrogen). This medium, along with L-PALCAMY enrichment broth, is the basis for the Netherlands Government Food Inspection Service (NGFIS) method for Listeria detection and isolation. Other Selective Plating Media Interest in foodborne listeriosis during the 1980s led to development of many additional Listeriaselective media for examining milk and dairy products. In 1984, Martin et al. [119] developed gum base nalidixic acid medium (GBNA), a synthetic agar-free solid medium superior to the MMLA of Bearns and Girard [12] for isolating L. monocytogenes from raw milk [80]. Bailey et al. [8] also found that a modified version of this medium containing lithium chloride and moxalactam was suitable for isolating L. monocytogenes from raw chicken. A selective agar medium [78] based on the enrichment broth of Doyle and Schoeni [47], from which acriflavine was omitted and Fe3+ was added, compared favorably with the original formulation of MLA [121]. Supplementation of selective [78] and nonselective [38] media with Fe3+ enhances growth of L. monocytogenes and may be beneficial for isolating sublethally injured cells from food samples containing a mixed microbial flora. Attempts to isolate L. monocytogenes from food products have focused on enhancing the selectivity of currently available blood-free plating media, as well as development of media that incorporate differential agents other than blood to aid microbiologists in differentiating Listeria and L. monocytogenes colonies in mixed cultures. In 1987, Buchanan et al. [24] found the combination of moxalactam, nalidixic acid, and bacitracin to be effective in allowing growth of Listeria spp. while preventing growth of most other foodborne organisms, including micrococci and streptococci. These selective agents were used to formulate MVJ on which L. monocytogenes colonies appear entirely black (reduction of tellurite) on a red background (due to the microbe’s inability to use mannitol). Thus, suspect Listeria colonies could be readily identified on MVJ without using oblique illumination. Adding the same three selective agents to the MMLA of Bearns and Girard [12] resulted in Agricultural Research Service-modified McBride Listeria agar (ARS-MMLA) which could be used in conjunction with oblique lighting to quantitate Listeria in a wide range of dairy and meat products.

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In a subsequent study, Buchanan et al. [26] found that MVJ was slightly superior to ARS-MMLA for recovery of L. monocytogenes from inoculated samples of milk, dairy products, meat, and coleslaw. Although ARS-MMLA was more selective than MVJ, the black Listeria-like colonies that appeared on MVJ were more readily discernible. Initial comparisons of ARS-MMLA and MVJ with LPM agar indicated that both of the new media functioned well. In a follow-up study, Buchanan et al. [25] assessed the ability of MVJ and LPM Agar to detect Listeria in retail samples of raw meat, fish, and shellfish. Listeria populations were generally too low to be detected by direct plating on either medium. However, using USDA Listeria enrichment broth I (USDA LEB I) in a three-tube/24-h most probable number (MPN) method, comparable isolation rates were obtained for MVJ and LPM agar. The differential capability of MVJ was again extremely useful in selecting presumptive Listeria colonies. Comparative Evaluation of Direct Plating Media for Recovery of Listeria from Foods The need for reliable media in routine food analysis precipitated several studies to identify the most suitable direct plating media. Rijpens and Herman [151] conducted a study to compare selective and nonselective primary enrichments for detection of L. monocytogenes in cheese. A completely selective enrichment procedure was compared with two partially nonselective protocols. After enrichment for approximately 48 h, the enrichment media were streaked on selective agars and presumptive Listeria colonies were confirmed using PCR. In some instances, PCR was also done directly on the enrichment broth. The conventional, completely selective enrichment procedure was not always the best choice to detect stressed L. monocytogenes in cheeses. The methods that incorporated a nonselective enrichment step gave better results than the completely selective method. However, for mold-ripened soft cheeses, best results were obtained using the completely selective enrichment procedure. In another study by Johansson et al. [90], enrichment in half-Fraser broth for 24 h at 30°C, followed by plating onto L. monocytogenes blood agar (LMBA) and PALCAM medium combined with additional streaking proved to be the most rapid and specific method to detect indigenous L. monocytogenes populations from soft mold-ripened cheese in comparison with standard methods. With a high sensitivity (93%) and a low detection limit (1 to 10 CFU/25 g–1), this procedure provided negative and presumptive positive results within 2 to 3 days. Differences among LMBA, PALCAM, and Oxford medium were highly significant (at 99% significance level). Overall, plating on LMBA after standard enrichment protocols gave the best results. An improvement in detection was also obtained by modifying the confirmation procedure. A loopful of culture (an additional streak) from PALCAM or Oxford medium was streaked on a nonselective medium in addition to streaking only separate colonies as specified in the standards. A year-long survey of two Northern Ireland milk-processing plants for L. monocytogenes was carried out by Kells and Gilmour [95]. Sample sites included the milk-processing environment (walls, floors, drains, and steps), processing equipment, and raw and pasteurized milk. The FDA Listeria-selective enrichment procedure was used to process samples and an additional agar medium, LMBA, was utilized as part of the isolation procedure to compare its performance to that of the recommended Oxford and PALCAM agars. LMBA was able to isolate L. monocytogenes from 94.1% of sites compared to isolation rates of 76.5 and 79.4% using Oxford and PALCAM agars, respectively. Duarte et al. [50] examined four secondary enrichment protocols (conventional methods: UVM II, Fraser 24 h and Fraser 48 h; and an impedimetric method: Listeria electrical detection medium) for their ability to detect Listeria spp. and L. monocytogenes in fish and environmental samples collected along the processing chain of cold-smoked fish. From all methods, Listeria spp. and L. monocytogenes were present respectively in 56 and 34 of the 315 samples analyzed. Fraser broth incubated for 48 h gave the fewest false-negative Listeria spp. results (4/56; [7.1%]), but concurrently

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only 15/34 (44.1%) samples were correctly identified as containing L. monocytogenes. Listeria electrical detection (LED) medium detected only 36/56 (64.3%) Listeria spp.-positive samples. Despite this lower isolation rate, LED identified 20/34 (58.8%) L. monocytogenes-positive samples correctly and gave fewer false positive results. The overall conclusion of these authors, similar to those of many others, was that more than one isolation method is needed to estimate L. monocytogenes contamination rates accurately. Golden et al. [68], Hao et al. [78], and Cassiday et al. [34] collectively compared 20 selective plating media for their ability to recover uninjured cells of L. monocytogenes from samples of pasteurized milk, Brie cheese, ice cream mix, raw cabbage, dry-cured/country-cured ham, and/or raw oysters inoculated to contain approximately 102, 104, and 106 L. monocytogenes colony-forming units per gram or milliliter. Gum base nalidixic acid tryptose soya medium (GBNTSM), MLA2, FDA-MMLA, and modified Despierres agar (MDA) were consistently superior to nine other media used by Golden et al. [68] for enumerating all three inoculum levels of Listeria in samples of pasteurized milk and ice cream mix. Ability to recover low levels of Listeria from both products was facilitated by the lack of significant levels of non-Listeria contaminants. Five of fourteen plating media used in this study failed to recover L. monocytogenes from inoculated samples of pasteurized milk as well as Brie cheese and were therefore omitted for analysis of ice cream and raw cabbage. Examination of Brie cheese containing approximately 102 and 104 L. monocytogenes CFU/g indicated that none of the nine remaining direct plating media was sufficiently selective to prevent overgrowth of Listeria by molds, yeasts, and Gram-positive cocci. Despite these inherent difficulties in detecting small numbers of Listeria, modified Rodriguez isolation medium III (MRIM III), MLA2, FDA-MMLA, and MDA were judged to be satisfactory when Brie cheese contained ≥106 Listeria CFU/g. However, subsequent results from the same laboratory [35] indicate that LPM agar was superior to these four media for isolating L. monocytogenes from Brie cheese. With raw cabbage, enumeration of Listeria was a problem only at the lowest inoculum level where large populations of microbial contaminants (i.e., Gram-positive and Gram-negative rods as well as Gram-positive cocci) typically interfered with recovery. At the two higher inoculum levels, L. monocytogenes was readily quantitated by direct plating on MDA, GBNTSM, and MLA2. However, this same investigative team [35] later obtained even better results using LPM agar. One year earlier, Hao et al. [78] successfully recovered L. monocytogenes from inoculated samples of cabbage using GBNA, Doyle and Schoeni selective enrichment agar (DSSEA), DSSEA + ferric citrate, DSSEA + acriflavine + ferric citrate, thiocyanate nalidixic acid agar (TNAA) + glucose + ferric citrate, and MLA2, but concluded that DSSEA + acriflavine + ferric citrate and MLA2 outperformed the other media tested. When results from the previous three studies are combined, LPM agar, GBNTSM, MLA2, FDA-MMLA, and MDA generally emerged as the plating media of choice for detecting uninjured Listeria in dairy and vegetable products. Overall, these findings agree with those of at least four other studies [66,84,102,110] in which LPM agar outperformed other popular plating media, including FDA-MMLA, RIM III, and/or MVJ for recovery of L. monocytogenes from raw milk, ice cream, yogurt, soft cheese, and/or vegetables inoculated with the pathogen. In addition, Rodriguez et al. [153] found that RIM III containing 6 rather than 12 g of acriflavine hydrochloride was superior to the original formulation of MLA for isolating L. monocytogenes from artificially contaminated raw milk and hard cheese. Although the best media for recovering Listeria from dairy products and vegetables remain to be defined, OXA, MOX, LPM, and PALCAM agar appear to be the present plating media of choice in the United States for selective isolation of Listeria from such products as evidenced by their inclusion in the FDA and USDA procedures [83,86,91,114,115]. Given the inherent differences that exist between the natural microflora found in various foods, one can easily surmise that Listeria-selective plating media best suited for dairy products and vegetables might be somewhat less than ideal for analysis of meat, poultry, and seafood. Consequently, Cassiday et al. [34] evaluated 10 selective plating media for their ability to enumerate

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L. monocytogenes in artificially contaminated dry- and country-cured ham as well as raw oysters. According to their results, MDA, FDA-MMLA, and LPM agar recovered approximately equal numbers of uninjured Listeria from dry-cured ham. However, ease in differentiating L. monocytogenes colonies from those formed by background contaminants led these authors to recommend LPM agar for analysis of dry-cured ham. Not surprisingly, LPM agar also was equal or superior to three other plating media (i.e., MRIM III, MVJ, and UVM) deemed acceptable for isolating Listeria from country-cured ham. Unlike both types of ham, high populations of indigenous microflora in raw oysters greatly complicated detection of Listeria on virtually all 10 plating media. Although MRIM III and MVJ supported less growth of Listeria than other marginally acceptable plating media (including MLA2, FDAMMLA, and GBNTSM), MRIM III and MVJ were somewhat more reliable for differentiating L. monocytogenes from background contaminants. Therefore, these authors hesitantly recommended MRIM III and MVJ for examination of raw oysters. Several less extensive studies also have dealt with the ability of various plating media to recover Listeria from meat, poultry, and seafood. According to a 1988 report by Loessner et al. [110], recognition of L. monocytogenes in inoculated samples of raw ground beef and scallops was only possible using LPM agar. Among the three other plating media tested, RIM III and the original formulation of MLA proved to be insufficiently selective, whereas MVJ was inhibitory to the L. monocytogenes strain tested. Unlike these findings, Garayzabel and Genigeorgis [66] indicated that LPM agar and RIM III were acceptable for detecting Listeria in raw meat and both media were superior to FDA-MMLA. Bailey et al. [8] found that LPM agar and GBNA fortified with lithium chloride and moxalactam were superior to unfortified GBNA and MLA for recovering L. monocytogenes as well as other Listeria spp. from naturally contaminated raw poultry. Incubation Conditions Most plating media used to isolate Listeria are normally incubated aerobically at 30 to 37°C. Plates containing popular selective media such as LPM agar or MOX agar normally are incubated for 48 h; plates containing pure or near-pure cultures of Listeria on nonselective media can generally be examined after 24 h. Because growth of L. monocytogenes is reportedly enhanced under conditions of reduced oxygen [164], inoculated plates [47,128,129,156–158,160] as well as selective enrichment broths [38] have been incubated under microaerobic conditions (5% O2:10% CO2:85% N2). Microaerobic conditions are especially recommended when using PALCAM agar.

OBLIQUE ILLUMINATION Except for plating media that contain esculin, xylose, mannitol, or other differential agents, most formulations of Listeria-selective plating media can be classified into one of two categories based on presence or absence of blood. Recognition of Listeria-like colonies on blood-free media such as MMLA, TNSA, and GBNA is greatly facilitated when colonies are observed under oblique illumination with a binocular scanning microscope. When the Henry technique [85], in which plates are examined under obliquely transmitted white light at an angle of 45° (Figure 7.1), is used, Listeria colonies are small, round, finely textured, bluish green to bluish gray with an entire margin. In 1984, Martin et al. [119] compared the appearance of L. monocytogenes on nalidixic acid agar and tryptone soya gum base nalidixic acid medium and found that the uniformly transparent nature of the gum-base medium greatly enhanced the bluish-green color of Listeria colonies when observed under oblique illumination, as described by Henry [85]. Noting that the angle of transmission in the Henry method is 135°, Lachica [101] found that the bluish-green hue of Listeria colonies was more easily observed if plates were viewed from the backside at an angle of 45° with a 5× magnification hand lens while colonies were directly illuminated with a high-intensity beam of light that traveled perpendicular to the bench surface (Figure 7.2). This latter method has

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FIGURE 7.1 Oblique illumination technique developed by Henry. Angles of reflected light (β) and transillumination (α) equal 45 and 135°, respectively. (From Henry, B. S. 1933. J. Infect. Dis. 52:374–402.)

eliminated many of the problems (i.e., reproducibility and convenience) associated with the classical technique developed by Henry [85] more than 70 years ago. Given enough experience, either of these two lighting techniques can be used easily to differentiate probable Listeria colonies from background organisms, even on heavily contaminated plates. However, these procedures are time consuming and not readily adaptable for routine use in large testing laboratories. β-Hemolysis Addition of blood to solid media also can be used to differentiate Listeria, including L. monocytogenes, from other microorganisms. When grown on media containing blood, such as MLA, L. monocytogenes colonies are typically surrounded by a narrow zone of β-hemolysis. In some instances, β-hemolytic activity is so weak that the clearing zone cannot be observed until the colony is gently removed from the agar surface.

FIGURE 7.2 Modified Henry technique developed by Lachica [101]. Angle of transillumination (α) equals 135°. (From Lachica, R. V. 1989. Annu. Meeting, Soc. Ind. Microbiol., Seattle, Washington, August 13–18, Abstr. P-44.)

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In 1989, Blanco et al. [18] proposed overlaying previously inoculated plates of blood-free Listeria selective agar with a thin layer of blood agar so that the β-hemolytic activity associated with pathogenic Listeria could be directly observed after reincubation. According to these authors, hemolysis was more readily observed using this procedure than when blood was incorporated into plating media before incubation. However, further work using highly contaminated samples such as raw milk showed that the success of this procedure primarily depended on selectivity of the initial plating medium, with highly selective media yielding the best results.

OFFICIAL METHODS

FOR ISOLATING

L.

MONOCYTOGENES FROM

FOOD

Heightened worldwide interest in foodborne listeriosis coupled with the advent of mandatory HACCP programs for meat and seafood products in the United States has led to development of more reliable commercial screening methods for Listeria. Two protocols developed in the United States by the FDA and USDA–FSIS have emerged as “standard methods” to isolate L. monocytogenes from dairy foods, seafoods, vegetables and meat, poultry, and egg products. Despite widespread use of these methods in the United States, Canada, and Western Europe, both procedures are still plagued with difficulties that include the inability to isolate Listeria from all positive samples as well as difficulties in recovering sublethally injured cells. In response to these concerns, the USDA–FSIS and FDA protocols have been modified to enhance their ability to recover injured Listeria. Working in cooperation with the International Dairy Federation (IDF), other official European agencies have developed somewhat similar protocols partially based on current FDA methodology. In this section, positive and negative aspects of the most widely used Listeria testing protocols will be discussed, along with identification of some of the most critical steps involved in isolating L. monocytogenes from different foods. FDA Method The FDA method, originally developed by Lovett et al. [114,117], is the most frequently used procedure in the United States for detecting and enumerating L. monocytogenes in milk, milk products (particularly ice cream and cheese), seafood, and vegetables. The original protocol [114] has been modified several times since it was developed [86,88,114,117,177]. The most current standard FDA methodology [88] and permitted alternative rapid methodologies recommended to detect and isolate L. monocytogenes from foods are as follows. Presumptive contaminated food lots are sampled. Generally, subsamples are composited, if required, according to FDA field laboratory instructions. Analytical portions (25 g) are preenriched for Listeria species at 30°C for 4 h in buffered Listeria enrichment broth (BLEB M52), equivalent [87] to AOAC/IDF dairy products enrichment broth [5,175] base containing sodium pyruvate. Four hours after nonselective incubation, the selective agents (acriflavin, 10 mg/L; sodium nalidixate, 40 mg/L; optional antifungal, e.g., cycloheximide 50 mg/L) are added. Incubation for selective enrichment is continued at 30°C for a total of 48 h. The enrichment culture is streaked at 24 and 48 h on one of the prescribed differential selective agars to isolate Listeria species or L. monocytogenes. Surveillance enumeration of L. monocytogenes levels in contaminated food is now required for regulatory samples that test positive for the pathogen [88]. Detection may be done first and if contamination is detected, a reserve sample portion can be tested for numbers. This is probably the preferable method because, generally, only a small percentage of samples can be expected to be positive and most often at low levels approaching 1 CFU/25 g. However, the option of combining regulatory detection and enumeration is permitted. Enumeration of L. monocytogenes in positive samples is performed on reserve samples by colony count on L. monocytogenes differential selective agar in conjunction with MPN enumeration using selective enrichment in BLEB with subsequent plating on ALOA (a diagnostic, chromogenic

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isolation medium) [179a] or BCM [50,167] differential selective agar. Most samples are likely to be negative for Listeria and most positive samples will only contain a few colony-forming units per 25 g. Hence, it is efficient to delay enumeration of reserve samples until the Listeria detection stage is completed. Nevertheless, it may sometimes be more convenient to detect and enumerate simultaneously. To accomplish this, the enrichment homogenate is prepared as described earlier and 0.1 mL is immediately spread on ALOA, BCM, or an equivalent L. monocytogenes selective agar. Plates are incubated at 35°C for 24 to 48 h. The combined minimal method will allow the cell number of presumptive L. monocytogenes to be categorized as 25,000 CFU/g. More replica plates and more decimal dilutions in trypticase soy broth with 0.6% yeast extract (TSBye M157) are optional to obtain more precise enumeration. Five representative colonies are tested for ability to ferment L-rhamnose by the conventional fermentation method, by the BCM rhamnose confirmatory agar, or by a rapid L. monocytogenes identification kit to rule out definitively the uncommon occurrence of L. ivanovii in foods. Alternatively, prescribed rapid detection kits with their respective enrichment media may be conditionally used to screen for presence of Listeria contaminants. Putative Listeria isolates on selective agars from standard or screen-positive enrichments are purified on nonselective agar and confirmed by conventional identification tests or by a battery of such tests in kit form. Isolates may be rapidly confirmed as L. monocytogenes (or not) by using specific test kits. Subtyping of L. monocytogenes isolates is optional except for FDA isolates, which must be typed serologically, and strain typed by pulsed-field gel electrophoresis (PFGE) and ribotyping. Nonobligatory pathogenicity testing of L. monocytogenes isolates is described. The major changes in the revised FDA methodology (Figure 7.3) include: Certain prescribed rapid detection kits and their enrichments are now authorized screening alternatives to the standard selective enrichment. It is now necessary to use only one instead of two of the several prescribed selective isolation agars (Oxford agar, PALCAM, LPM plus esculin and ferric iron, MOX). Oxford agar is still the preferred standard selective isolation medium. MOX has been added to the list of prescribed selective agars and LPM without added esculin and ferric iron has been removed. Use of the new chromogenic differential selective agars, like BCM, ALOA, CHROMagar Listeria, and Rapid L’mono, is encouraged as long as it is in parallel with one of the prescribed selective agars. The new agar media differentiate L. monocytogenes and L. ivanovii colonies from those of other Listeria spp. and will greatly facilitate choosing L. monocytogenes colonies when colonies of more than one species are present on a plate. The Henry illumination technique is de-emphasized because only differential selective isolation agars are prescribed. The current enrichment medium, which resembles the step-1 enrichment of the internationally harmonized method proposed by Asperger et al. [6], is basically unchanged. However, pimaricin (natamycin), a much less toxic compound than cycloheximide, is introduced as the alternative antifungal compound in the Listeria enrichment medium. If L. monocytogenes is detected in a food sample, enumeration of the level of contamination in the food is required. The isolation procedure involves streaking BLEB culture onto one of the following esculincontaining selective isolation agars: OXA or PALCAM or MOX or LPM fortified with esculin and Fe3+. These esculin-containing media are listed in order of preferred use, subject to their availability. OXA, PALCAM, or MOX plates are incubated at 35°C for 24 to 48 h and fortified LPM plates at 30°C for 24 to 48 h. It is strongly recommended that one of the L. monocytogenes–L. ivanovii differential selective agars, such as BCM, ALOA, RapidL’mono, or CHROMagar Listeria be streaked at 48 h (optionally at 24 h) in addition to the chosen esculin-containing selective agar. This will reduce the problem of masking of L. monocytogenes by L. innocua.

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Add 25 g or 25 mL sample to 225 mL BLEB

Stomach or blend

Screening with rapid detection kits

Incubate 24–48 h at 35°C

Spread 0.1 mL on ALOA M10a, BCM M17a or an equivalent L. monocytogenes selective agar, and if enumeration is required, more replica plates and more decimal dilutions in TSBye

Streak onto

OXA/MOX and LPM (with or w/o Esculin & Fe3+) or PALCAM

OXA/MOX 35° C 24–48 h

LPM 30° C 24–48 h

PALCAM 35° C 24–48 h

Examine Listeria-like colonies

FIGURE 7.3 FDA procedure for isolating L. monocytogenes from foods. (From Hitchins, A. D. 2003. In Food and Drug Administration bacteriological analytical manual, 8th ed. AOAC International, pp. 10.01–10.13.)

BCM has been collaboratively validated by FDA [87]. An ISO TC34 SC9 comparative validation showed that all the media (and a selective blood agar—LMBA, Sifin, Germany) inhibited Listeria competitors more or less equally well. ALOA was preferred only because its formulation is public. Another differential selective medium, chromogenic Listeria agar (M40b) is now available. Listeria colonies are black with a black halo on esculin-containing media. Certain other bacteria can form weakly brownish-black colonies, but color development takes longer than 2 days. Five or more typical colonies are transferred from OXA and PALCAM or modified LPM or MOX to TSAye, streaking for purity and typical isolated colonies. If BCM plates are streaked as recommended previously and blue colonies are observed, they are presumptive L. monocytogenes colonies because L. ivanovii is not often reported in foods. L. monocytogenes and L. ivanovii colonies on ALOA are blue and have a zone of lipolysis around them. Purification on TSAye is a mandatory step in the conventional analysis because isolated colonies on selective agar media may still be in contact with an invisible weak background of partially inhibited competitors. At least five isolates are necessary because more than one species of Listeria may be isolated from the same sample. BCM and ALOA plates are used to help in reducing the number of colonies that need to be picked. L. monocytogenes and L. ivanovii can be distinguished using a commercial confirmatory medium (Biosynth International, Inc.) or by conventional

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TABLE 7.2 Serotypes of Listeria Species L. monocytogenes L. L. L. L. a

ivanovii innocua welshimeri seeligeri

1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b 4c, 4d, 4e, “7” 5 4ab, 6a, 6b, Una 6a, 6b 1/2b, 4c, 4d, 6b, Un

Un = undefined.

Source: von Koenig, C. H. et al. 1983. Infect. Immun. 40:1170–1177.

rhamnose/xylose fermentation broths or agars. TSAye plates are incubated at 30°C for 24 to 48 h. The plates may be incubated at 35°C if colonies will not be used for wet-mount motility observations (one of the many classical identification tests). For the approved rapid methods [88], the selective isolation agar recommended by the manufacturer must be used but auxiliary use of the new L. monocytogenes–L. ivanovii differential agars is also recommended. Isolates should be typed serologically and genetically. For serological typing, commercial sera are used to characterize isolates as type 1 or 4 or not type 1 or 4 (types 3, 5, 6, etc.) [88]. Table 7.2 exhibits the serological relationships of Listeria spp. Most L. monocytogenes isolates obtained from patients and the environment are type 1 or 4. More than 90% of L. monocytogenes isolates can be serotyped with commercially available sera. All nonpathogenic species, except L. welshimeri, share one or more somatic antigens with L. monocytogenes. Serotyping alone without thorough characterization, therefore, is not adequate for identification of L. monocytogenes. The BCM L. monocytogenes detection system (LMDS), mentioned earlier [50,167], consists of a selective pre-enrichment broth (LMPEB), selective enrichment broth (LMSEB), selective/ differential plating medium (LMPM), and identification on a confirmatory plating medium (LMCM). Restaino et al. [150] explored the efficacy of the BCM LMDS using pure cultures and naturally and artificially contaminated environmental sponges. The BCM LMPEB allowed growth of Listeria and resuscitation of heat-injured L. monocytogenes. The BCM LMSEB, which contains the fluorogenic substrate 4-methylumbelliferyl-myo-inositol-1-phosphate and detects phosphatidylinositol phospholipase C (PI-PLC) activity, provided a presumptive positive test for the presence of pathogenic Listeria (L. monocytogenes and L. ivanovii) after 24 h at 35°C. An initial inoculum of 10 to 100 CFU/mL of L. monocytogenes in BCM LMSEB yielded a fluorogenic response after 24 h. On BCM LMPM, L. monocytogenes and L. ivanovii were the two Listeria species forming turquoise convex colonies (1.0 to 2.5 mm in diameter) from PI-PLC activity on the chromogenic substrate, 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate. L. monocytogenes was distinguished from L. ivanovii by its fluorescence on BCM LMCM or acid production from rhamnose. False-positive organisms (Bacillus cereus, Staphylococcus aureus, Bacillus thuringiensis, and yeasts) were eliminated by at least one of the media in the BCM LMDS. Using a pure culture system, the BCM LMDS detected one to two L. monocytogenes cells from a sponge rehydrated in 10 mL of DE neutralizing broth. In an analysis of 162 environmental sponges from facilities inspected by the USDA, the values for identification of L. monocytogenes by BCM LMDS and the USDA method were 30 and 14 sites, respectively, with sensitivity and specificity values of 85.7 and 100.0% with BCM LMDS vs. 40.0 and 66.1% with the USDA method. No false-positive organisms were isolated by BCM LMDS, whereas 26.5% of the sponges tested by the USDA method produced false-positive results.

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Serology is useful when epidemiological considerations are crucial. A TSBye culture is used to inoculate tryptose broth. This is incubated for 24 h at 35°C, the temperature at which flagella (H) antigen expression is reduced. The culture is transferred to tryptose agar slants and incubated for 24 h at 35°C. Both slants are washed in a total of 3 mL Difco fluorescent antibody (FA) buffer and transferred to a sterile 16- × 125-mm screw-cap tube. These slants are heated in a water bath at 80°C for 1 h. Cells are sedimented by centrifugation at 1600 g for 30 min, 2.2 to 2.3 mL of supernatant fluid are removed, and the pellet is resuspended in the remaining buffer. The manufacturers usually provide recommendations for sera dilution and agglutination procedures to be followed. Genetic subtyping involves submission of data from pulsed-field gel electrophoresis (PFGE) of DNA restriction fragments of FDA isolates to PulseNet (CDC, Atlanta, Georgia). Isolates should also be ribotyped or sent to a ribotyping laboratory [184,185]. Present versions of the FDA procedure have greatly shortened and simplified isolation of Listeria spp. from many foods compared with earlier methods developed to detect the pathogen in clinical specimens; revised procedures have afforded many improvements over the original FDA protocol [114,117]. In 1987, Doyle and Schoeni [48] compared the original FDA classic cold enrichment and shortened enrichment procedures for their ability to recover L. monocytogenes from 90 samples of commercially produced, soft, surface-ripened cheese that was previously identified as likely to contain L. monocytogenes. Although L. monocytogenes was isolated from 41 of 90 (46%) cheeses, no single procedure detected the pathogen in all positive samples. A total of 21 samples were positive after cold enrichment as compared with only 16 and 13 samples that were positive using the FDA and shortened enrichment procedures, respectively. Thus, the latter two protocols failed to recover L. monocytogenes from 5 of 21 (23.8%) and 8 of 21 (38.1%) samples that were positive following cold enrichment. Furthermore, because Listeria was never isolated from the same positive sample by all three protocols, it appears that the original FDA method was inferior to cold enrichment. Similar results were obtained by Doyle et al. [49] when these same three enrichment procedures were used to isolate L. monocytogenes from milk samples after HTST pasteurization. Researchers in Canada [55] and England [141] found negligible differences between numbers of Listeria recovered from naturally contaminated samples of raw milk and soft cheeses analyzed by the FDA and cold enrichment procedures, although both methods again failed to detect Listeria in all positive samples. These variable findings for the original FDA and cold enrichment procedures have been attributed to nonuniform distribution of Listeria within samples. However, Doyle and Schoeni [48,49] found cold enrichment superior to the FDA method for analysis of soft, surface-ripened cheese, when nonuniform distribution of Listeria is expected, as well as in pasteurized milk. Hence, variations in the ability of the FDA and cold enrichment procedures to detect Listeria in dairy products probably result from inherent differences between the two methods (media, incubation conditions) and/or presence of microbial competitors rather than nonuniform distribution of Listeria in the product. Although these results indicate that cold enrichment was generally superior to the original FDA protocol, the time-consuming nature of cold enrichment makes this procedure unacceptable as a commercial screening method for L. monocytogenes. International Dairy Federation Method Using the original FDA method as a starting point, the IDF initiated development of a “reference” method in 1988 [173] to recover L. monocytogenes from dairy products. Development of the IDF method essentially followed that of the FDA protocol as previously reviewed by Ryser and Marth [161] with the eventual elimination of pre-enrichment (for detecting sublethally injured Listeria) and the KOH treatment of the enrichment broths before plating on Listeria-selective media. The present IDF method [7] received AOAC approval in 1993 based on results from an AOAC

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Add 25 g or 25 mL into 225 mL IDF Selective Enrichment Medium

Blend or stomach for about 2 min

If necessary, adjust pH 48 h/30°C Streak onto Oxford Agar 48 h/37°C

Pick 5 presumptive Listeria colonies (black with black halos) for confirmation

FIGURE 7.4 IDF procedure for isolating L. monocytogenes from milk and dairy products. (From Association of Official Analytical Chemists. 1996. In Official methods of analysis of the association of official analytical chemists. Gaithersburg, Md.: AOAC International, 17.10.01.)

collaborative study [175] assessing the ability of this method to recover L. monocytogenes from inoculated samples of raw milk, ice cream, Camembert cheese, Limburger cheese, and skim milk powder. The AOAC-approved IDF method (Figure 7.4) closely resembles the FDA protocol (see Figure 7.3); the sample is enriched in IDF enrichment broth that contains the same concentrations of selective agents found in LEB [88]. Following 48 h of incubation at 30°C, enrichments are plated on Oxford agar as opposed to the FDA procedure, which calls for Oxford agar and LPM without esculin/Fe3+ or PALCAM. This method requires a minimum of 4 days to obtain presumptive results and continues to be popular in Europe for detecting Listeria in dairy products. USDA–FSIS Method The USDA–FSIS devised a method for detecting L. monocytogenes in meat and poultry products (Figure 7.5) [91,176,178]. The original USDA protocol developed in 1986 by Lee and McClain [106,124] differs from the original and revised FDA procedures in that primary and secondary enrichment steps are included for detecting Listeria. The original USDA procedure enabled Listeria detection within 3 days compared with 9 to 11 or 5 to 6 days using the original and revised FDA methods, respectively. The original USDA–FSIS procedure was revised in May 1989 [33] and differed from the original method in that LEB II was replaced by Fraser broth [64] as the secondary enrichment medium; LPM agar was replaced by MOX; and the regulatory sample size was increased to 25 g. Fraser broth and modified Oxford agar will blacken during incubation because Listeria spp. and other contaminants can hydrolyze esculin; colonies of Listeria will exhibit black halos on modified Oxford agar following 24 to 48 h of incubation. However, MOX is more selective than LPM or Oxford agar [42], and staphylococci and streptococci are generally unable to grow on it.

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Add 25 g meat sample to 225 mL UVM broth, stomach 2 min

Incubate at 30°C

0.1 mL + 10 mL Fraser broth

Incubate 26 ±2 h 35° C

Incubate 48 h 35°C

Streak onto MOX

Examine for black colonies (presumptive Listeria)

If negative

Streak onto MOX

Incubate 35°C, 48 h

Examine for black colonies

FIGURE 7.5 USDA procedure for isolating L. monocytogenes from red meat, poultry, egg products, and environmental samples. (From USDA/FSIS. 2002. In Microbiology laboratory guidebook, 3rd ed., revision 3, chapter 8.)

Reported inadequacies in the previous [33] USDA–FSIS procedure were related to use of Fraser broth for secondary enrichment. False-negative results caused by reliance on Fraser broth darkening and a 24-h secondary enrichment have been reported by several laboratories [9,99]. Kornacki et al. [99] compared recovery of L. monocytogenes from Fraser broth incubated for 26 vs. 48 h. L. monocytogenes was isolated from 60 of 1,088 meat product and environmental swab samples from meat and dairy plants. False-negative rates as high as 6.7% were attributed to the inability of L. monocytogenes to be detected in Fraser broth at 26 h but not at 48 h, and to the failure of Fraser broth to blacken. Furthermore, investigators failed to detect L. monocytogenes in eight Fraser broth enrichments that were positive by primary enrichment. These findings clearly stress the importance of incubating Fraser broth enrichments for 48 h. The USDA–FSIS has recommended several modifications to its original procedure. The latest USDA–FSIS method to isolate and identify L. monocytogenes from red meat, poultry, egg, and environmental samples has been in effect since April 2002 [178]. This revised method includes use of rapid screening tests and has a sensitivity of 1000 CFU/g) than samples produced by other manufacturers (4% of 781 samples). Further, serotype 4b phage type 6,7 and serotype 4bX accounted for 96% of isolates from manufacturer Y’s pâté samples, compared to 19% of samples from other producers. Patients infected with the two predominant subtypes were significantly more likely to have consumed pâté during the 3 weeks before illness, compared to patients infected by other subtypes (87% of 15 vs. 35% of 17, χ2 test P = 0.0095).

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350 Total Cases Non-pregnancy Pregnancy

300

Number of Cases

250

200

150

100

50

0 1983 84 85 86 87 88 89 90 91 92 93 94 Year

FIGURE 10.9 Listeriosis in England, Wales, and Northern Ireland, 1983–1994. (Adapted by E.T. Ryser from McLauchlin, J. and L. Newton 1995. Human listeriosis in England, Wales and Northern Ireland: a changing pattern of infection. Proc. XIIth International Symposium on Problems of Listeriosis, Perth, Western Australia, October 2–6, pp. 177–181.)

The chance finding of L. monocytogenes in pâté obtained from a patient’s refrigerator also prompted a government health warning (May–June 1989) to at-risk consumers regarding pâté consumption. Following this warning and suspension of sales of pâté from manufacturer Y, the incidence of listeriosis declined dramatically (Figure 10.9). Further, a concurrent decline in infection by the two predominant subtypes was also observed. Last, in a 1990 survey of 626 pâté samples, the frequency of L. monocytogenes serotype 4b phage type 6,7 and 4b X-positive samples declined to 4%. In 1999, a small outbreak of invasive listeriosis in three U.S. states (New York, Connecticut, and Maryland) was attributed to consumption of pâté [11]. Eleven cases caused by an unusual subtype of serotype 4b were identified. Two patients had consumed the same brand of pâté, and a third had consumed an unknown brand of pâté. Based upon USDA–FSIS investigation findings, the company recalled approximately 400 pounds of pâté and other similar products produced from poultry meat [17]. The recall was subsequently expanded to encompass approximately 80,000 pounds of product [17]. One of the largest documented outbreaks of invasive listeriosis occurred in France in 1992 [72a,73,89,130] and was associated with consumption of pork tongue in aspic (jelly). Detection of this outbreak was facilitated by two National Reference Centers (NRC), components of a surveillance system for listeriosis established in 1987. Clinical isolate serotyping at the Nantes facility, followed by phage typing at the Pasteur Institute, revealed that 279 of 758 captured cases (37%) were caused by a specific subtype of serotype 4b (Figure 10.10), compared to only 6 to 27 cases during previous years [89]. Among the 279 outbreak-associated cases, 182 (65%) outbreak-associated cases occurred in nonpregnant adults, 5 occurred in children (2%), and 92 (33%) in pregnant women.

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120 Epidemic

Non-epidemic

Number of Cases

100 80 60 40 20 0

J

F

M

A

M

J

J

A

S

O

N

D

Month

FIGURE 10.10 Listeriosis in France, 1992. (Adapted by E.T. Ryser from Jacquet, C., B. Catimel, R. Brosch, C. Buchrieser, P. Dehaumont, V. Goulet, A. Lepoutre, P. Veit, and J. Rocourt 1995. Investigations related to the epidemic strain involved in the French listeriosis outbreak in 1992. Appl Environ Microbiol 61: 2242–2246.)

This outbreak resulted in 63 deaths in nonpregnancy-associated cases, 22 fetal deaths, and 7 neonate deaths, for an overall case fatality rate of 33% [72a]. Health officials launched epidemiologic and laboratory-based investigations to identify risk factors for infection and potential control measures [72a, 73]. The first case–control study, conducted among 144 case-patients and 288 matched controls, did not identify risk factors for infection. However, a second case-control study among women with perinatal infection found that casepatients were more likely than controls to have consumed pork tongue in aspic (60% of casepatients vs. 6.1% of controls, OR = 9.2) and, to a lesser extent, other delicatessen items. A third study implicated a specific brand of pork tongue in aspic (OR = 14.8). In the contemporaneous laboratory-based based investigation, the clinical isolates were further characterized by PFGE and ribotyping along with a subset of >14,000 nonhuman isolates submitted to the NRC and isolates from samples collected in conjunction with this investigation [89]. A total of 247 (89%) human clinical isolates shared the same 3-enzyme PFGE profile, designated as the epidemic subtype sensu stricto. Notably, this subtype is closely related to that implicated in several U.S. and European outbreaks described in this chapter (California, 1985; Switzerland, 1983–1987; Denmark, 1989–1990). The sensu stricto subtype of L. monocytogenes was recovered from 154 food items collected during various stages of production and distribution, including abattoirs, processing facilities, retailers, and patient refrigerators. Among these food items were pork tongue in aspic from delicatessens (n = 112), other meat products (n = 19), cheeses (n = 12), and other food items with lower frequency. This subtype was further isolated from opened and unopened packages of the brand of pork tongue in aspic cited by patients, from environmental samples collected at the facility that produced that brand, and from retail stands. Thus, results of the laboratory-based investigation strongly supported the epidemiologic investigation findings. In another report describing the investigation of six processors suspected to have produced contaminated product, L. monocytogenes was isolated from 35% of 270 samples collected throughout the processing area [130]. Among these, 33% were from cooked product contact surfaces, correlating well with environmental investigation findings from other ready-to-eat meat and poultry products. The epidemicassociated phage type was isolated from raw brine in a facility producing pork tongue in aspic. In follow-up investigations in two plants after cleaning and disinfection, L. monocytogenes was isolated

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from 17% of raw product surfaces and 7% of finished product contact surfaces, most of which remained visibly soiled or covered by a biofilm; these results highlight the difficulty of eliminating this organism from the food processing environment (see Chapter 19 for a detailed discussion). Two invasive listeriosis outbreaks in France were linked to consumption of contaminated rillettes, a ready-to-eat pâté-like product produced by cooking ham meat in grease [52,74]. In July 1993, the National Reference Center notified Ministry of Health officials of a cluster of 10 listeriosis cases caused by an unusual phage type [73]. This serotype 4b subtype had accounted for 100 food items was administered to case-patients and controls matched by gestational age or age and underlying condition as appropriate. Within 6 days, preliminary analyses implicated rillettes

16

Sporadic cases Epidemic cases (Materno-neonatal)

14

Epidemic cases (Non-pregnant)

12

Cases

10

8

6

4

2

0 17 24 31

May

7

14 21 28

June

5

12 19 26

July

2

9

August

16 23 30

6

13 20 27

September

4

11 18 25

October

1

8

November

FIGURE 10.11 Listeriosis in France, 1993. (From Goulet, V., J. Rocourt, I. Rebiere, C. Jacquet, C. Moyse, P. Dehaumont, G. Salvat, and P. Veit 1998. Listeriosis outbreak associated with the consumption of rillettes in France in 1993. J Infect Dis 177: 155–160. With permission.)

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purchased at a specific retail chain as the most likely vehicle of transmission (OR = 18.0, CI = 2.2–208.0). The retail chain was supplied with a specific brand (designated “Brand A”) of meat products, including rillettes, produced at a single plant. Based upon these results and isolation of the epidemic phage type from a raw product sample from the implicated establishment, the producer recalled the product and ceased production. Further analysis of case–control study data resulted in the implication of additional Brand A meat products, including pâtés and sausages. Two Brand A products remained significantly associated with illness in a multivariate statistical model, and included rillettes (OR = 10.9, CI = 2.1–54.4) and country pâté (OR = 5.0, CI = 1.0 –24.1). Each of 35 case-patients interviewed reported exposure to Brand A products; 30 had consumed rillettes, 4 had consumed other meat products, and 1 had a family member who had consumed rillettes, suggestive of the potential for crosscontamination of other items during storage. Further interview of case-patients and controls who had consumed rillettes revealed that case-patients were more likely than controls to have stored the rillettes longer (> 6 days, 48% of case patients vs. 28% of controls, P = 0.12) and to have eaten them over more meals (> 4 meals, 48% of case patients vs. 23% of controls, P = 0.05). Results of the environmental investigation correlated well with the epidemiologic investigation findings. The epidemic-associated subtype was isolated from 15 of 508 containers of Brand A rillettes sampled prior to their sell-by dates, 20 containers removed from retail shelves, 11 containers returned to retailers in response to the recall, and 2 containers sampled from patients’ refrigerators and from several other Brand A meat products. Sell-by dates indicated contamination of at least 14 batches. The majority of unopened containers contained L. monocytogenes concentrations of 10,000 CFU/g, demonstrative of the growth potential during home storage. An intensive environmental investigation at the processing facility revealed several potential sources of contamination. Records indicated isolation of coliforms from the rillettes production line. Production of rillettes had increased during that time, and time of disinfection had been shortened to the apparent detriment of product safety. Further, the epidemic-associated subtype of L. monocytogenes was isolated from smoked pork breast, the hood over the frankfurter processing line, and, two months after the outbreak ended, from one of the filling and packaging machines for rillettes. These results support both raw materials and the processing environment as potential sources of product contamination. As a result of these findings, raw materials and cooked product areas were better separated, a Hazard Analysis Critical Control Point (HACCP) plan was developed and implemented, bacteriological monitoring throughout the processing chain was reinforced, and the processor initiated postpackaging pasteurization of rillettes. Analysis of data from hospital surveys and two primary surveillance systems for invasive listeriosis in France indicated a dramatic estimated 68% decline in the incidence of listeriosis from 1987 to 1997 [71]. Data from a network of general and regional hospitals (EPIBAC) indicated a decline from 12.3 bacteremia and 3.4 meningitis cases per million population to 3.5 and 0.9 cases per million, respectively, whereas data from the National Reference Center showed a decline from 6.3 to 4.1 cases of listeriosis per million population. Contemporaneous declines among retail-level food product samples evidenced the contribution of aggressive prevention efforts implemented during that decade, including enhanced surveillance, intensified microbiological monitoring of food products throughout the production and distribution chains, improved hygiene at the retail level (particularly at delicatessen counters), and targeted health education messages for high-risk populations. Due to the severity of disease and high case-fatality rate, invasive listeriosis remained a public health priority. In 1998, health officials mandated notification of all laboratory-confirmed listeriosis cases, along with standardized collection of food exposure history (complete with purchase information). Detection and investigation of the two coincidental outbreaks described below were greatly facilitated by these initiatives, in addition to the molecular surveillance system already in place, which also enabled their differentiation.

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Between October 1999 and February 2000, health officials identified 10 cases of invasive listeriosis throughout France caused by the same serotype 4b subtype; clinical isolates were of the same phage type, and matched by two-enzyme PFGE analysis [52]. Six cases occurred in nonpregnant adults with underlying conditions known to be associated with increased risk of listeriosis, three cases were perinatal, and one occurred in a previously healthy adult. Two adults with underlying conditions and one infant died. A food history review for the first five recognized cases revealed that all patients had reported consumption of rillettes during the 2 months prior to illness, with four of five having purchased the products from the same market chain. In a review of nonclinical isolates, 21 matched the epidemic-associated subtype; 7 of those had been isolated from rillettes produced by the manufacturer that supplied the market named by the patients. In-plant records indicated isolation of L. monocytogenes from finished product stock during the outbreak period, and isolates from rillettes were later shown to match the epidemic-associated subtype. The manufacturer recalled its products (rillettes, pork tongue in aspic) in January, and the outbreak ended several weeks later. In response to this outbreak, French manufacturers reduced the shelf life for rillettes to 28 days to help reduce the potential growth of inadvertent contaminants to high levels during storage. A second, simultaneous outbreak occurred from November 1999 to February 2000 [52]. This outbreak involved a total of 32 cases and was caused by a subtype of serotype 4b with a different PFGE pattern than that associated with the first outbreak. Nine perinatal cases, 11 cases in nonpregnant adults with underlying conditions, and 12 cases in previously healthy nonpregnant adults (all male, median age 52.5 years, all with central nervous system listeriosis) were distributed throughout France. This outbreak had an overall case fatality rate of 31% and included death in five adults with underlying conditions, four premature neonates, and one fetus. A case–control study, in which patients infected with nonoutbreak-associated L. monocytogenes subtypes served as controls, found that case-patients were significantly more likely to have consumed pork tongue in aspic (OR = 28.0, 95% CI = 3.2–1,222.1), along with several ready-to-eat items including cervelas sausage, cooked ham, and pâté de campagne. Having consumed at least one meat product purchased from a delicatessen counter was also significantly associated with illness. On multivariate analysis, consumption of pork tongue in aspic (OR = 75.5, 95% CI 4.7–1,216.0) and pâté de campagne (OR = 8.9, 95% CI = 1.7–46.1) remained significantly associated with illness. It is likely that more than one vehicle of transmission was associated with this outbreak, as consumption of pork tongue in aspic explained 14 of 29 cases (48%) and all other case patients reported consumption of pâté and/or ham. Patients reported a variety of purchase sites. A review of distribution records allowed identification of two processors that had supplied the majority of purchase sites named by patients. Available records, extensive product sampling, and in-plant environmental investigations did not, however, enable investigators to implicate a single brand of product or processor. At the time of this outbreak, L. monocytogenes was permissible in ready-to-eat meat products manipulated between the microbial inactivation and packaging steps if studies were available to show that the concentration of L. monocytogenes would not exceed 100 CFU/g at the end of the product shelf life. In response to this outbreak, the food safety agency advised a production level zero-tolerance policy for these products. In 2000, investigators used PCR-based detection methods and automated ribotyping to quickly link a cluster of three cases of invasive listeriosis (serotype 1/2) in Western Australia, one of which resulted in fetal death, to contaminated cooked chicken products [86]. The efficient turn-around time (8 hours for ribotyping) resulted in data that guided generation of hypotheses for illness and allowed the investigators to make efficient recommendations for intervention. The products were recalled, and isolation of high levels of L. monocytogenes from unopened packages of cooked chicken pieces prompted implementation of a more rigorous cleaning and sanitation program and additional heat treatment at the end of production. Other isolated listeriosis cases linked to consumption of meat or poultry products have been reported and include (1) isolation of >106 CFU/g of L. monocytogenes serogroup 4 from a homemade sausage consumed by a man prior to

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development of listeriosis caused by the same serogroup [44]; (2) in the context of a study to identify risk factors for listeriosis, isolation of the same electrophoretic enzyme type from two patients and retail packages of pork sausage and ground beef [133]; and (3) isolation of the same phage type from cooked-chilled chicken and from fetal tissue obtained from a patient who had consumed the chicken prior to development of listeriosis [93].

REDUCTION

OF

MEAT

AND

POULTRY PRODUCT–ASSOCIATED INVASIVE LISTERIOSIS

Findings from the investigation of outbreaks associated with meat and poultry products strongly support the USDA–FSIS/FDA joint risk assessment, in which analysis of per serving and per annum relative risk rankings placed deli meats and frankfurters (not reheated) in the highest risk category (very high risk) and pâté and meat spreads in the second highest category (high risk) [16]. The categorization of unreheated frankfurters and deli meats reflects several factors, including (1) a relatively high rate of contamination, (2) rapid growth of L. monocytogenes under refrigerated storage temperatures, (3) likely storage for extended periods, and (4) frequent consumption by members of the U.S. population. Supportive data indicated a relatively low annual consumption rate for pâté and meat spreads (6 to 10-day range) contributed to the high risk ranking. Thus, these products warranted particular attention with respect to reducing the incidence of listeriosis. Outbreak investigation findings, in conjunction with risk assessment results, have led to an aggressive USDA–FSIS regulatory policy. Following the 2002 turkey deli meat-associated outbreak in the United States, USDA–FSIS issued a final rule mandating that establishments producing ready-to-eat meat and poultry products have in their HACCP plans or Sanitation Standard Operating Procedures (SSOPs) controls to prevent product contamination by L. monocytogenes from the processing environment [8]. These controls must be scientifically validated, and are stratified in accordance with the number of control steps taken (i.e., a post-lethality step treatment and/or a growth inhibitor). Controls could include a postpackaging thermal step or the addition of growth inhibiting compounds. Irradiation would also be an effective measure; however, FDA has not yet approved it for use with these products. The final rule also states that processors relying solely upon sanitation for prevention of contamination would be subject to intensified testing by USDA–FSIS. Establishments would also be mandated to share in-plant control measure verification data. Last, this final rule clarified that isolation of L. monocytogenes from processing equipment would provide sufficient justification for product recall, regardless of whether finished product yielded this organism or not. In addition to efforts to reduce the frequency and level of L. monocytogenes contamination, another critical prevention strategy for reduction of listeriosis associated with ready-to-eat meat and poultry products lies in effective consumer education programs.

SEAFOOD PRODUCTS Several small outbreaks of listeriosis have been linked to seafood items, including imitation crab meat, smoked mussels, gravad, and cold-smoked fish. Two 1996 cases in Canada were suspected to have been caused by consumption of imitation crab meat produced from the Alaskan Pollock fish [61]. A healthy adult female was hospitalized with severe gastrointestinal symptoms including explosive diarrhea and projectile vomiting. L. monocytogenes serotype 1/2b was isolated from her blood and stool. Her husband experienced similar but less severe symptoms; L. monocytogenes serotype 1/2b was isolated from his stool. Both reported consuming imitation crab meat during the 18 hours before onset of illness. Samples of imitation crab meat and four other food items obtained from the patients’ refrigerator yielded L. monocytogenes isolates that matched the patient isolates by serotyping, PFGE, and randomly amplified polymorphic DNA (RAPD) analysis. The imitation crab meat, along with two other items, was contaminated at a high level (2 × 109 CFU/g). Retail samples

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of imitation crab meat also yielded matching isolates, although at low levels, suggestive of growth during subsequent home storage. Based upon these findings, along with information indicating that two well meal companions had not consumed this item, investigators concluded that the imitation crab meat was the most likely vehicle of infection. Although one case was invasive, the predominance of gastrointestinal symptoms following consumption of a high inoculum correlates well with reports of noninvasive L. monocytogenes-associated gastroenteritis [120]. This outbreak also highlights the importance of consumer education regarding safe food storage, as food sampling results demonstrated the likelihood of cross-contamination of several other food items.

SMOKED MUSSELS Investigators in New Zealand linked three cases that occurred from October to December 1992 to consumption of contaminated smoked mussels [37]. Initially, two perinatal cases caused by serotype 1/2a were detected. A food exposure history indicating consumption of the same brand of mussels prior to development of listeriosis prompted investigators to analyze all 1991 and 1992 serotype 1/2a clinical isolates by PFGE. Two additional patient isolates, along with L. monocytogenes isolates from an unopened package of mussels obtained from a patient’s refrigerator, were indistinguishable by PFGE. One patient with illness in October 1992 reported consumption of mussels, but the fourth patient, with illness in May 1991, did not. Isolates from three patients reporting mussel consumption and the mussel isolates were indistinguishable by phage typing, sensitivity to arsenic and cadmium, and restriction fragment length polymorphism (RFLP). The fourth patient’s isolate was untypable via phage typing. This is an oft-encountered caveat of this subtyping method, and likely means that the isolate was of a type not represented in the phage set [105]. These laboratory findings highlight the higher discriminatory power offered by characterization with more than one typing method when feasible. Confirmation of “Brand X” mussels as the vehicle of transmission prompted officials to screen additional Brand X products, along with samples collected from the processing environment. Of 27 isolates from Brand X products, 26 matched the human clinical isolates, along with 4 of 7 from environmental samples taken from the processing facility. Notably, this subtype had been isolated from the processing environment 3 years earlier. These data are supportive of long-term colonization of the processing environment, as has been observed in other studies focused upon the smoked seafood industry [23,24,58,64,83,118,126], which can then serve as an ongoing source of contamination. Another small outbreak with four cases of L. monocytogenes serotype 1/2b associated gastroenteritis, which occurred in Tasmania in 1991, was also microbiologically confirmed to have been caused by mussels produced in New Zealand [112,113]. A high concentration of L. monocytogenes (1.6 × 107 CFU/g) was isolated from both open and unopened packages of mussels. Traceback investigations identified three batches for which the shelf life had been overestimated by at least 3 months, prompting a recall. The isolates were indistinguishable by RFLP and phage typing (J. McLauchlin, unpublished data) but did not match those isolated from Brand X products or the respective processing environment.

COLD-SMOKED FISH PRODUCTS Despite frequent isolation of L. monocytogenes in cold-smoked fish products [29,57,92,118], few listeriosis outbreaks have been attributed to these food items. In a 1995 report from Australia, smoked salmon was implicated as the vehicle of transmission for two cases of perinatal listeriosis which resulted in fetal death [20]. Another outbreak, associated with cold-smoked rainbow trout and gravad (made by curing a raw fillet with sugar, salt, pepper, and dill), occurred in the province of Värmland, Sweden from August 1994 to June 1995 [58,144]. Of nine cases due to serotype 4b, three were perinatal infections and six cases were in elderly or immune-compromised nonpregnant adults.

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Two patients, including one neonate, died. All patients reported consumption of gravad or smoked rainbow trout or salmon, five of whom recalled definitely or possibly consuming the same brand. Four subtypes were identified among patient isolates using restriction enzyme analysis (REA) with three enzymes and phage typing (designated clonal types A–D); six isolates were clonal type B, seen only once previously in Sweden. Investigators launched an environmental investigation, in which samples from the producer processing area along with gravad and smoked fish collected from patient refrigerators, retailers, and the producer were screened. Samples from the processing area and from fish yielded L. monocytogenes. Molecular subtyping results linked environmental and food isolates to eight of nine cases. Type B was recovered from samples of gravad fish from the home of one patient and from the producer, along with residue found in the packing machine, providing laboratory confirmation of the vehicle and identifying the likely environmental source of product contamination. Similar to several other investigations, the same subtype had been isolated from a product from this producer 6 months earlier, indicating that this subtype was likely among the resident environmental microflora. In addition, L. monocytogenes types C and D were isolated from product sampled at the processing plant, explaining two additional cases. One sample yielded two subtypes, highlighting the utility of characterizing more than one L. monocytogenes colony from a given sample during investigations. In the 2003 USDA–FSIS/FDA joint risk assessment, smoked fish was categorized as a high risk food on a per serving basis [16]. The high risk classification resulted largely from several characteristics: (1) a high frequency of contamination (12.9%), as indicated by data compiled from 30 studies evaluating prevalence; (2) a high estimated contamination level at retail (>0.6% of servings containing 103–106 CFU); (3) a moderate growth rate during storage; and (4) a typical storage duration of 3 to 5 days. Given the risk ranking, the infrequency of association of these products with listeriosis outbreaks is intriguing. Differences in pathogenic potential among L. monocytogenes subtypes common to these products do not provide an explanation, as several studies have reported the isolation of human epidemic- and sporadic-case–associated subtypes from smoked fish and smoked fish processing environments [23,29,118,144]. Potential contributors to this phenomenon could include the relatively low frequency of consumption by a large segment of the population or an inherent feature of the food matrix or production process that affects the organisms’ ability to cause disease once they are consumed. It is also possible that outbreaks have occurred but have gone undetected. Control of L. monocytogenes in the cold-smoked fish-processing environment and products presents a unique and significant challenge to the industry. This organism is ubiquitous in the environment, thus easily introduced into production facilities. Further, persistence of L. monocytogenes in smoked fish processing facilities, thereby establishing an environmental reservoir that is difficult to eliminate, has been well documented [23,24,58,64,83,118,126]. The production process, which typically involves brining via rubbing with salt, cure injection, or soaking in 3 to 5% aqueous phase NaCl followed by smoking at 25 to 30°C [144], does not include a step that would inactivate L. monocytogenes or prevent growth during refrigerated storage. As a result of these factors, sporadic contamination with low levels of viable L. monocytogenes may be difficult to control. Strategies to minimize L. monocytogenes contamination should include a continued focus on rigorous in-plant cleaning and sanitation programs, verified and modified in accordance with data obtained from routine environmental screening, and exploration of measures effective against established microflora. Options to inhibit growth of sporadic contaminants include exploration into process modification to employ potential inhibitory properties of lactic acid bacteria, salt and smoke, post-packaging pasteurization (i.e., irradiation), and frozen storage [5].

VEGETABLES Compared to other food categories, few invasive listeriosis outbreaks have been linked to consumption of contaminated vegetables or vegetable products. The large coleslaw-associated outbreak that occurred in the Canadian Maritime Provinces, discussed earlier in this chapter, was among the first

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to provide convincing evidence for transmission of L. monocytogenes by food. Although reported after this outbreak, an outbreak likely associated with raw vegetables occurred among hospitalized patients in Massachusetts a few years earlier in 1979 [82]. Twenty-three patients hospitalized during September and October had invasive listeriosis. Of these cases, 20 (87%) were caused by serotype 4b (identified as the epidemic serotype), compared to 33% of cases over the previous 26 months. Fifteen of these 20 patients acquired their infection while hospitalized. All cases occurred among nonpregnant adults, half of whom had underlying conditions contributing to immune suppression. Five patients died, although two deaths were attributed to other causes. Epidemiologic studies among patients and controls matched by age, gender, and hospitalization date did not result in identification of specific dietary risk factors. Case-patients were more likely than controls to have consumed tuna fish, chicken salad, and hard cheese; the common factor was the inclusion of these items in salads that also contained lettuce, celery, and/or tomatoes. This investigation also revealed intriguing clinical findings. Outbreak-associated case patients were more likely than patients with sporadic listeriosis to have had gastrointestinal symptoms at the time of fever onset (85% of outbreak-associated case-patients, compared to 22% of patients with sporadic listeriosis), a phenomenon similar to that observed in the 1987 outbreak that occurred in Philadelphia [137]. They were also more likely to have received antacids or histamine-blocking drugs prior to the onset of listeriosis (60% compared to 17%). These results were corroborated by case–control study findings; 60% of case-patients took antacids or histamine blockers before onset of listeriosis, compared to 25% of controls, and were also more likely to have undergone gastrointestinal procedures. Decreased stomach acidity may have resulted in improved survival of L. monocytogenes during passage through the stomach, as has been observed for other enteric pathogens [66]. In addition, underlying gastrointestinal lesions, for which these medications may have been used, may have resulted in an increased risk for invasive disease. A small outbreak of serotype 4b infections that occurred among patients subsequently admitted to a hospital on the Texas–Mexico border was epidemiologically associated with frozen vegetables [140]. There were five cases (four perinatal, one in an immunocompromised nonpregnant adult) detected over a 5-week period, with two additional cases identified over the following 2 months. A case–control study implicated frozen broccoli and cauliflower, and L. monocytogenes serotype 4b was later isolated from opened and unopened packages of these frozen vegetables. Human clinical and food isolates were indistinguishable by PFGE subtyping. In the recent USDA–FSIS/FDA risk assessment, vegetables were classified in the moderate risk category, due largely to the high number of annual servings and moderate frequency of contamination by L. monocytogenes (about 2 to 5% of samples). However, vegetables remain infrequently associated with outbreaks of listeriosis. Potential differences in pathogenic potential among different subtypes of L. monocytogenes do not offer a conclusive explanation for this phenomenon, as both outbreak and sporadic-case–associated subtypes have been isolated from vegetables (see Chapter 16 for a detailed discussion). The overall low growth rate during storage [16], along with the relatively short shelf life of unpreserved vegetable products may contribute to the infrequent association with invasive disease, compared to other food categories. Continued emphasis on processing facility hygiene, along with careful handling of raw vegetables and ready-to-eat vegetable products by individuals in high-risk populations, will help to further reduce the risk of listeriosis from consumption of vegetables.

L. MONOCYTOGENES–ASSOCIATED FEBRILE GASTROENTERITIS A second, increasingly well-described disease state resulting from infection by L. monocytogenes is febrile gastroenteritis (for a recent review with a focus on clinical practice, see Ooi and Lorber [120]). As shown in Table 10.2, at least eight outbreaks of foodborne gastroenteritis have been attributed to this organism. These outbreaks have largely been recognized as point-source outbreaks among otherwise healthy members of a cohort and are characterized by consumption of a high-level

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TABLE 10.2 Listeria monocytogenes-Associated Febrile Gastroenteritis Outbreaks Year

Location

Serotype

No. Cases

Implicated Vehicle

Reference

1993 1994 1997 1998 2000 2001 2001 2001

Northern Italy Illinois Northern Italy Finland New Zealand Los Angeles Sweden Japan

1/2b 1/2b 4b 1/2a 1/2 1/2a 1/2a 1/2b

18 45 1,566 5 31 16 48 38

Rice salad Chocolate milk Cold corn and tuna salad Cold-smoked rainbow trout Ready-to-eat meat products Turkey delicatessen meat Fresh, raw-milk cheese Cheese

[129] [50] [22] [110a] [139] [65] [46,51] [101]

inoculum followed by onset of fever, gastrointestinal symptoms including watery diarrhea and nausea, headaches, and joint and muscle pain within 24 hours. Symptoms typically resolve within a few days. Here, we discuss several well-characterized outbreaks.

POTENTIAL ASSOCIATION WITH A MILD CLINICAL SYNDROME: SHRIMP, 1989, UNITED STATES The suggestion of milder illness in otherwise healthy adults was reported by Riedo et al. following an investigation that grew from follow-up of two perinatal cases of L. monocytogenes bacteremia [125]. After learning that the patients’ only common exposure was attendance at a party, public health officials launched a retrospective cohort investigation among 36 attendees to evaluate the potential for mild illness and determine risk factors for illness. In this type of study, food and/or other exposures are collected from all or a representative sample of persons in a well-defined population among whom disease occurred (e.g., attendees of an event after which illness occurred among some attendees), allowing the calculation of attack rates of illness in those who did or did not report a certain exposure [53]. The attack rates can then be compared to determine the food item or other exposure associated with the greatest increase in risk. For this investigation, based upon the body of knowledge regarding foodborne listeriosis at that time, a case of illness was defined as isolation of L. monocytogenes from blood or stool or illness with any two concurrent gastrointestinal or musculoskeletal symptoms in a party attendee within 6 weeks after the party. Illness in 10 patients met the case definition, 9 met the symptom criteria, and L. monocytogenes was isolated from 1 of 25 stool samples collected from attendees. Symptom onset and/or isolation of L. monocytogenes from a clinical specimen occurred throughout the 6 weeks after the event. The 3 clinical isolates (2 from the blood of the women with perinatal infection and the stool isolate) were serotype 4b and matched by multilocus enzyme electrophoresis. Among 24 food items served at the party, persons who consumed greater amounts of shrimp (boiled and chilled, served with a sauce) and, to a lesser extent, nonalcoholic beverages, Camembert cheese, and cauliflower were at significantly increased risk for illness (relative risk [RR] for shrimp consumption 7.0, 95% CI = 2.3–21.5). After controlling for consumption of other foods, only consumption of shrimp and cauliflower remained significantly associated with illness. Only three ill persons, however, recalled consumption of cauliflower. No leftover foods were available for screening, and foods purchased from suppliers approximately 6 weeks after the party did not yield L. monocytogenes. Although a matching L. monocytogenes subtype was isolated from a person with noninvasive illness, the longer incubation period (19 to 23 days for index cases) is not representative of other L. monocytogenes-associated outbreaks of foodborne gastroenteritis. It is possible that another organism was responsible for the mild gastrointestinal illness observed in the patients who subsequently

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developed invasive listeriosis and that the syndrome observed in others was not associated with food consumed at the party. However, this report highlighted the potential for a milder illness caused by L. monocytogenes in otherwise healthy adults and raised awareness of this hypothesis for consideration in subsequent investigations.

RICE SALAD, 1993, NORTHERN ITALY In 1993, an outbreak of febrile gastroenteritis occurred among 39 attendees of a supper [129]. Four attendees were hospitalized during the 24 hours following the event with high fever (average 39.6 °C), diarrhea, nausea, abdominal pain, arthromyalgia, headache, and sore throat. Several days later, blood cultures yielded L. monocytogenes serotype 1/2b, whereas stool cultures were negative for enteric pathogens including Salmonella and Shigella. Routine public health follow-up revealed that the outbreak occurred among nonpregnant, otherwise healthy persons. Based upon these unusual findings, health officials launched epidemiologic and laboratory investigations to better characterize the outbreak. Investigators defined a case as onset of fever plus diarrhea, nausea, vomiting, and/or arthromyalgia within 3 days of attending the supper. Among the 39 attendees, 18 met the case definition, for an attack rate of 46%. Ill persons ranged in age from 17 to 54 years; their median age was significantly higher than that of well attendees (36 years compared to 22 years, P < 0.001). Their clinical syndrome largely mirrored that of hospitalized attendees, with the exception of a flu-like syndrome (arthromyalgia, sore throat, headache) in four persons in the absence of gastrointestinal symptoms. Among patients with gastrointestinal symptoms, onset occurred a median of 18 hours following the supper. None of the stool specimens collected from all attendees approximately 1 month after the event yielded L. monocytogenes. Among convalescent serum tested from 18 ill attendees, however, 39% showed αsomatic antigen type 1, compared to 1 of 4 well persons. None had α-type 4b antibodies, and none of 11 community members who served as a comparison group had detectable antibody titers against either. Consumption of rice salad was significantly associated with illness. Eighteen of 20 persons (90%) who consumed rice salad developed illness, compared to none who did not consume that item (RR undefined, P < 0.001). A food preparation review revealed that, due to limited refrigeration space, the rice salad had been held at ambient temperature during the 24 hours before the supper. Samples of rice salad and other available food items did not yield pathogens including pathogenic Escherichia coli, Salmonella, Bacillus cereus, or Staphylococcus aureus. Although there was no rice salad left for further screening, samples of several other food items, along with environmental samples collected from the blender and freezer in the home of the cook, yielded L. monocytogenes. The clinical, food, and freezer isolates all matched by MEE and phage typing. Although it is possible that the gastrointestinal syndrome characterizing this outbreak may have been caused by an organism not detected in clinical specimens, which were collected approximately 1 month after the event, several factors support L. monocytogenes as the etiologic agent: (1) isolation of the same subtype from the blood of two patients, food, and environmental samples; (2) several ill persons with high titers of α-Listeria antibodies, compared to a low percentage among well attendees and nondetectable titers in a comparison population; and (3) a median incubation period longer than that typically observed for toxin-mediated illness, yet shorter than that typical for viral etiologies. This outbreak highlights the importance of proper food handling and storage in noncommercial settings, as L. monocytogenes was isolated at >103 CFU/g from two food items. Further, investigation findings are supportive of considering L. monocytogenes as a potential etiology in outbreaks of febrile gastroenteritis.

CHOCOLATE MILK, 1994, UNITED STATES The investigation of a 1994 outbreak linked to contaminated chocolate milk provided overwhelming evidence for the association of L. monocytogenes with febrile gastroenteritis [50]. Again recognized

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as a point-source outbreak, illness occurred among approximately 92 persons who attended a picnic at a Holstein cow show in Illinois. Of 82 persons interviewed, 45 (55%) met the case definition of onset of symptoms from two to four symptom complexes including (1) fever; (2) diarrhea, nausea, or vomiting; (3) myalgia or arthralgia; and (4) headache within a week of attending the picnic or consuming food from it. As observed in the previous outbreak, the median age of ill persons was higher than that of well persons (31 years vs. 24 years). None of the attendees reported underlying conditions; one pregnant woman at 40 weeks gestation had diarrhea the day after the picnic and delivered a healthy baby several days later. Common symptoms among ill persons included diarrhea, fatigue, fever, chills, headache, myalgia, and abdominal cramps. The median incubation period was 20 hours (range 9 to 32 hours), and diarrhea lasted a median of 42 hours (range 3 to 50 hours). Four patients were hospitalized as a result of their illness, and others reported loss of work time due to the severity of their symptoms. L. monocytogenes was isolated from 11 stool specimens. All positive specimens were from persons with illness meeting the case definition. Analysis of convalescent serum collected from 48 attendees and a control group with nonoutbreak-associated enteric infections showed a significantly higher median α-Listeriolysin O antibody titer (143 ELISA units) in persons with illness meeting the case definition than in controls (63 ELISA units, P < 0.001). Lower titers were also reported among attendees who were not ill and persons with mild illness. The strong association of higher α-Listeriolysin O antibody titers with illness highlights the diagnostic utility of this serologic assay. Among 60 persons who consumed chocolate milk, 45 (75%) became ill, compared to none of 22 persons who did not consume this item (RR undefined, 95% CI = 13.6 – ∞). Consumption of Swiss cheese was also significantly associated with illness; however, fewer attendees had consumed the cheese, and all ill persons who consumed cheese had also consumed chocolate milk. Culture of a carton of milk leftover from the picnic and a carton collected from the dairy yielded L. monocytogenes at 1.2 × 109 and 8.8 × 108 CFU/mL, respectively. An 8-ounce carton of milk would, therefore, potentially provide an inoculum of 2.9 × 1011 CFU. These and all but one clinical isolate were indistinguishable by MEE, ribotyping, and PFGE analysis. The PFGE pattern of one clinical isolate differed by one band. Inspection of the dairy and review of handling during transport to the picnic provided a clear explanation for the presence of high levels of L. monocytogenes in the chocolate milk. After flavor addition and milk pasteurization, the chocolate milk was held in a jacketed tank for 2 hours before being pumped to a filler line over a 7-hour period. The refrigerated jacket had been in disrepair, thus unrefrigerated, for 3 years. Inspection revealed a breach in the tank lining, which allowed milk to leak into the insulation jacket and pool there. When milk was pumped out, the pooled milk could leak back into the tank. Sanitizer spray nozzles were clogged, thus inhibiting the flow of sanitizer into the tank lining. A total of 180 cartons of chocolate milk for the picnic were in transport for over 2 hours without refrigeration. Upon delivery, they were held in a noncommercial refrigerator overnight. They were placed in an unrefrigerated cooler on the morning of the picnic and were available throughout the day. Based upon these data, it is likely that postpasteurization contamination of the milk occurred due to poor facility hygiene practices, with subsequent temperature abuse allowing rapid growth of L. monocytogenes. Records further indicated that chocolate milk had been distributed to three additional states. Consumption of chocolate milk from the implicated dairy was associated with similar illness in a family of five traveling through a neighboring state. A review of clinical isolates submitted to health officials in Illinois, Michigan, and Wisconsin resulted in identification of three additional cases of invasive listeriosis with some association with chocolate milk from the implicated dairy. Two patients had consumed chocolate milk from the implicated dairy, and the third had consumed chocolate milk purchased at a store that sold milk from the implicated dairy. Further, each clinical isolate was indistinguishable from the outbreak-associated subtype. Recommendations resulting from this investigation included diagnostic screening for L. monocytogenes in outbreaks with similar clinical features, enhanced monitoring of food products for pathogens including L. monocytogenes, and rigorous cleaning and sanitation programs.

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AND

TUNA SALAD, 1997, NORTHERN ITALY

A massive outbreak of L. monocytogenes-associated febrile gastroenteritis occurred in northern Italy in 1997 [22]. On a single day, local health units in three different towns received unusually high reports of febrile illness and gastroenteritis among students and staff of two primary schools and students of a university. All had dined at cafeterias supplied by the same local caterer, which prepared approximately 8,000 meals daily. Health officials immediately launched epidemiologic and laboratory-based investigations to investigate the scope, magnitude, and potential source of illness. Investigators determined that a cohort of 2,930 persons had consumed cafeteria meals on the day before the illnesses began. Of 2,189 primary school students and teachers enrolled in a cohort study, 93 adults and 1,473 students reported having at least one flu-like or gastrointestinal symptom, for an overall attack rate of 72%. Headache (88% in adults, 86% in children), abdominal pain (72% in both groups), and fever (68% in adults, 86% in children) were most commonly reported, and the median incubation period was 24 hours. Fever and vomiting occurred with significantly more frequency in children, whereas diarrhea, arthralgia, and myalgia were significantly more frequent in adults. A total of 292 persons (19%) were hospitalized as a result of their illness for a median of 3 days, underscoring the severity of associated symptoms. Of 292 stool specimens screened by 9 hospital laboratories, 290 were negative for bacterial and viral enteric pathogens and two yielded different Salmonella serotypes. L. monocytogenes was isolated from the blood of one patient. Among 141 stool specimens subsequently screened for L. monocytogenes, 123 (87%) were positive. All isolates were serotype 4b, and were indistinguishable by PFGE and RAPD analysis. Persons who consumed a salad made from corn and tuna were over six times more likely to become ill than those who did not (84% vs. 14%, RR = 6.2, 95% CI = 4.8–8.0, P < 0.001). No other food items were significantly associated with illness. Investigators conducted an environmental investigation at the catering facility and tested food samples obtained there. Although unopened cans of corn and tuna did not yield microorganisms, a sample of the leftover corn and tuna salad yielded L. monocytogenes at >106 CFU/g. These, along with L. monocytogenes isolates from facility sink drains and a meal preparation surface, were indistinguishable from the clinical isolates by PFGE and RAPD analysis. In a food preparation review, caterers reported opening the cans of tuna and corn during early morning on the day of preparation and allowing them to drain at ambient temperature. They were then mixed (no additional ingredients were added), portioned, and transported to the schools for lunch service. A corn salad without tuna was prepared later that day for dinner service at the university that had reported gastrointestinal illness among students. Studies with inoculated corn showed the potential for growth of L. monocytogenes to high concentrations (>106 CFU/g) during storage at 25°C. Based upon the environmental and laboratory-based investigation results, salad ingredients or the prepared salad were likely cross-contaminated with L. monocytogenes at the catering facility. Storage at ambient temperatures for several hours prior to serving could then allow the contaminants to grow to high levels. This outbreak highlights the importance of rigorous cleaning and sanitation programs in food production areas and proper food handling practices in reducing illness caused by L. monocytogenes. The high hospitalization rate underscores the potential severity of febrile gastroenteritis.

COLD-SMOKED RAINBOW TROUT, FINLAND A small outbreak in Finland was linked to consumption of cold-smoked rainbow trout [110a]. Four adults and one child developed a gastrointestinal illness with nausea, abdominal cramps, and diarrhea within 27 hours of sharing a meal together. Fever was reported by three adults, and other symptoms included vomiting, headache, fatigue, and arthralgia. The child was hospitalized overnight as a result of the illness. Information from patient interviews identified cold-smoked rainbow

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trout as the most likely vehicle of infection. No leftover fish was available; however an unopened package from the same lot obtained from the retailer yielded 1.9 × 105 L. monocytogenes CFU/g. Retail inspection findings indicated that the storage temperature of the cold-smoked trout was higher than that recommended by the producer. Stool samples collected from the patients did not yield enteric pathogens. However, subsequent stool swabs obtained from two ill persons yielded L. monocytogenes. Clinical and smoked trout isolates were both serotype 1/2a, and were indistinguishable by two-enzyme PFGE analysis.

READY-TO-EAT MEAT AND POULTRY PRODUCTS, NEW ZEALAND AND UNITED STATES In 2000, investigators in New Zealand linked several independent foodborne illness reports to consumption of contaminated ready-to-eat meats produced by the same manufacturer [139]. The first two reports of febrile gastroenteritis on South Island were associated with corned beef. Two additional reports of a similar syndrome indicated consumption of products from the same manufacturer. Leftover corned beef samples yielded L. monocytogenes at concentrations of >2.5 × 105 CFU/g, prompting the manufacturer to issue a product recall. On that date and shortly afterward, two foodborne outbreaks were reported among 7 residents of South Island and among 21 of a cohort of 24 residents of North Island; both meals had included ready-to-eat products produced by the same manufacturer. In the latter case, the retailer had failed to remove the product from sale despite the product recall. The two reported outbreaks were characterized by different clinical syndromes; flu-like symptoms including fever, myalgia, and headache predominated in South Island cases, whereas the majority of persons associated with the North Island outbreak also experienced gastrointestinal symptoms including diarrhea, vomiting, and abdominal cramps. Most experienced symptom onset within 24 hours of meal consumption. L. monocytogenes serotype 1/2 was isolated from stool specimens collected from 2 of the 4 ill persons from the initial reports, 2 ill persons associated with the South Island outbreak, and from 20 of 21 specimens collected from 17 ill and 3 well persons associated with the North Island outbreak. Several of the manufacturer’s food items were available for screening. L. monocytogenes serotype 1/2 was isolated from leftover corned beef (as noted above) and ham (North Island outbreak, 1.8 × 107 CFU/g), luncheon ham (85% similarity by PFGE, and were genetically similar according to other characterization methods.

CONTROL

OF

L.

MONOCYTOGENES–ASSOCIATED

FEBRILE GASTROENTERITIS

Strategies outlined for the continued reduction of invasive listeriosis would serve this goal well. As described in the examples above, the sources and route of contamination often parallel scenarios observed for invasive listeriosis outbreaks, with growth to high levels facilitated by subsequent temperature abuse or long periods of storage. In addition to efforts to reduce the frequency and level of L. monocytogenes contamination at the farm, producer, distribution, and retail levels, prevention strategies should include educational programs focused on proper handling and storage of products during preparation and prior to consumption. Such messages should target the general population in addition to persons at higher risk for invasive listeriosis. L. monocytogenes-associated gastroenteritis has been well characterized only recently. Outbreaks are likely underrecognized because of a lack of awareness of this syndrome and because stool samples are infrequently screened for L. monocytogenes. Healthcare workers and public health

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officials should consider L. monocytogenes as a cause of febrile gastroenteritis, particularly in cases where routine screening fails to identify an enteric pathogen. The factors leading to febrile gastroenteritis as opposed to invasive disease are currently poorly understood. As with invasive listeriosis, a better understanding of factors resulting in development of this illness along with the disease process may contribute to improved control and prevention strategies.

SURVEILLANCE OF LISTERIOSIS IN THE UNITED STATES FOODBORNE OUTBREAK SURVEILLANCE In the United States, the CDC has collected reports of foodborne outbreaks since 1973. These reports comprise summary information concerning outbreaks including the number of illnesses, hospitalizations, and deaths, the etiologic agent for illnesses, implicated food vehicles, and other outbreak-associated factors. Officials in all states currently report foodborne outbreaks in this surveillance system, now called the electronic Foodborne Outbreak Reporting System (eFORS). This system was recently enhanced by efforts to ensure complete and accurate reports, including verification of the number and content of reports with state officials and the implementation of Internet-based data entry into a national database. The eFORS system receives between 1,200 and 1,500 outbreak reports annually. Among 6,647 foodborne outbreaks reported to eFORS from 1998 to 2002, 2,168 had an identified etiology; 11 were due to L. monocytogenes, including a total of 256 cases and 38 (14.8%) deaths (CDC unpublished data). These deaths accounted for 48% of all deaths among outbreakassociated cases reported. The number of cases in listeriosis outbreaks ranged from 2 to 101. Four outbreaks occurred in multiple states. Four outbreak reports indicated delicatessen meats as the implicated vehicle (two sliced turkey, one multiple delicatessen meats, and one unspecified delicatessen meat), three implicated hot dogs, one implicated deli sandwiches, one implicated pâté, one implicated queso fresco (cheese), and one implicated potato salad. Foodborne outbreak surveillance provides an opportunity to construct statistical models to assess the burden of illnesses due specific food categories (i.e., poultry). These models incorporate data from case surveillance to estimate the total number of illnesses associated with various foods, as outbreak-related cases comprise only a fraction of the total burden. Investigators in the United Kingdom have published one such model [1]. From 1996 to 2000, an estimated 221 cases and 78 deaths were attributed to listeriosis. Though the authors provided estimates of the total estimated number of illness attributed to food categories due to all etiologies, they did not provide estimates due to listeriosis specifically. Several groups in the United States and other countries are conducting similar analyses; these efforts will undoubtedly enhance our knowledge of the burden of listeriosis due to specific food categories.

SURVEILLANCE

OF

SPORADIC LISTERIOSIS

Although we have learned much about the epidemiology of listeriosis through outbreak investigation, timely, effective surveillance of listeriosis is critical to the overall goals of control and prevention of this disease. Reliable surveillance data are critical in estimating the burden of listeriosis, determining disease trends, guiding development of targeted intervention strategies, providing a framework for their assessment, and developing risk assessment models used to guide regulatory actions. Furthermore, determining the expected incidence of sporadic listeriosis in a population through surveillance is critical for efficient outbreak detection and response, as both local and diffuse outbreaks are often detected by an increase of cases over the baseline rate. Prior to the mid-1980s, when the Los Angeles County outbreak solidified the public health significance of foodborne listeriosis, the incidence of sporadic human disease in the United States was poorly understood. Listeriosis was monitored exclusively by passive surveillance, which relies

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upon voluntary reporting by physicians and clinical laboratories to state health officials, who then voluntarily report to CDC [114], and by analysis of hospital discharge data [47]. The low sensitivity of these methods likely greatly underestimated its true incidence in the population. The 1985 outbreak underscored the importance of improved surveillance for listeriosis in the United States. An active surveillance program, for which public health officials routinely contacted personnel at all clinical laboratories and acute-care hospitals in five states and Los Angeles County to ascertain listeriosis cases, was initiated in 1986 in an effort to more precisely estimate the incidence of laboratory-confirmed disease [68,133]. The resulting estimates are detailed in Chapter 4. In an effort to improve nationwide reporting in the United States, listeriosis was made a nationally notifiable disease (one for which routine and timely reporting is considered necessary for prevention and control measures) in 2000. Although disease reporting is mandated only at the state level, and laws and regulations regarding which diseases and conditions must be reported to public health officials vary slightly from state to state, most currently require reporting of listeriosis. CDC provides uniform case criteria to increase specificity and data comparability [7], and reports the data weekly in its Morbidity and Mortality Weekly Report. Comprehensive annual summaries of nationally notifiable diseases, including listeriosis, present data stratified by important risk markers including geographic distribution, gender, age group, and race/ethnicity. The incidence and trends in listeriosis, as estimated by this surveillance program, along with international listeriosis estimates, are detailed in Chapter 4. Last, as discussed earlier in this chapter and in Chapter 9, participating PulseNet laboratories routinely subtype L. monocytogenes isolates by PFGE to facilitate rapid detection of clusters of listeriosis that may have a common source. In an effort to address emerging infectious diseases in the United States, CDC launched the Emerging Infections Program (EIP) in collaboration with selected state health departments, local health departments, academic institutions, and additional partner agencies in 1994. The Foodborne Diseases Active Surveillance Network (FoodNet), a collaboration among CDC, the USDA, FDA, and the EIP sites, is the principle foodborne disease component of the EIP [111]. FoodNet’s primary goals include (1) determining more precisely the burden of foodborne diseases in the United States, (2) monitoring trends in foodborne diseases, (3) determining the proportion of foodborne disease attributable to specific foods, and (4) developing and assessing interventions to reduce the burden of foodborne illness. In 1996, FoodNet began active, laboratory-based surveillance of selected foodborne diseases including listeriosis. As described for the active surveillance program initiated in 1986, which was continued and expanded as a component of the EIP, public health officials in FoodNet sites contact laboratory directors frequently to ascertain new cases of laboratory-confirmed foodborne disease. As a result, the burden of specific foodborne diseases in the United States can be more precisely estimated over time. Surveillance began in 5 geographically diverse sites with a catchment population of 14.3 million people. By 2003, FoodNet had expanded considerably to include 10 sites, more than 650 clinical laboratories, and a catchment population of 41.9 million people (14.4% of the U.S. population) [15]. By 2004, the catchment population had expanded to 44.5 million (15.1% of the U.S. population) (Figure 10.12). The FoodNet population under surveillance is comparable to the U.S. population, with few limitations [80]. A demographic comparison of the FoodNet population under surveillance and the U.S. population, conducted in 2000, found little variation in age and gender distributions. However, among race/ ethnic groups, the Hispanic population in FoodNet was underrepresented at 6% compared to 12% of the U.S. population as indicated by census data. Figure 10.13, in which relative rates of listeriosis are shown in comparison with a 1996–1998 baseline estimate, summarizes trends in the incidence of listeriosis since the initiation of FoodNet surveillance. The use of an average annual incidence for the 3 years (1996 to 1998) as the baseline period provides the most stable, precise relative rate estimates [15]. To account for the increase in the number of FoodNet sites, the corresponding increase in the population under surveillance and variations in the incidence of listeriosis among sites, a main effects, log-linear Poisson regression (negative binomial) model was used to estimate statistically significant changes in incidence. As shown in

346

FIGURE 10.12 Map showing Emerging Infections Program sites (
) participating in the Foodborne Diseases Active Surveillance Network, United States, 2004. The population under surveillance is 44.5 million persons, representing 15.1% of the total population.

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Figure 10.13, the relative rate (RR) of listeriosis among the FoodNet population declined significantly by 45% from the 1996–1998 baseline period to 2002 (95% CI = 31%–57%, RR = 0.55). The relative rate increased in 2003 compared to 2002 (RR = 0.71, 29% decrease from 1996 to 1998, 95% CI = 12%–42%). These data may indeed reflect a true increase in the incidence of listeriosis in 2003. However, the widely publicized, multistate outbreak that occurred during late 2002 [70] may have resulted in an increased awareness of listeriosis among health providers, leading to enhanced diagnosis and reporting. This would, in turn, contribute to an apparent increase in incidence. Health officials hypothesized that a similar phenomenon may have contributed to statistically significant geographic differences in 1986 rates of perinatal listeriosis [68], estimated using active surveillance data collected following the large, highly publicized 1985 outbreak in Los Angeles County. The rate in Los Angeles County (24.3/100,000 live births) was over three times the combined rate in other surveillance areas. As shown in Figure 10.13, the relative rate of listeriosis declined again in 2004 to rates similar to 2002 (RR = 0.60, 40% decrease from 1996 to 1998, 95% CI = 25% to 52%) [15]. The incidence of 0.27 cases/100,000 persons approaches the 2005 National Health Objective of 0.25 cases/100,000 persons. Analyses of FoodNet data have revealed ethnic disparities in the incidence of listeriosis, which correlate well with nationwide trends discussed in Chapter 4. Among 523 listeriosis cases reported from 1996 to 2003 among individuals of known race/ethnicity, the incidence among Hispanics was significantly higher than in non-Hispanics (RR = 1.7, 95% CI = 1.2–2.4) (56). Among Hispanic casepatients, 58% were female and 63% were of childbearing age (15 to 39 years). These findings correspond well with the findings from outbreak investigations in which Hispanics were overrepresented among outbreak cases and infections were associated with the consumption of Hispanic-style cheeses. The substantial overall decline in the incidence of listeriosis can be attributed to several factors. The FDA and USDA have focused significant effort on regulatory measures, industry guidance, and initiatives on L. monocytogenes, including FDA and USDA–FSIS’s 1989 zero-tolerance rulings for ready-to-eat foods, the joint USDA–FSIS/FDA risk assessment focused on ready-to-eat foods, and the USDA’s more recent completion of a risk assessment for deli meats [67]. In October 2003, the USDA published an interim final rule (detailed in the discussion of meat and poultry productassociated outbreaks) requiring processors to enhance and verify measures to reduce the prevalence of L. monocytogenes in ready-to-eat products [8]. The food industry has responded with significant

Relative Rate

2

1 –29% (–42% –12%)

0.8 0.7 0.6

–40% (–52% –25%)

0.5 –45% (–57% –31%) 1996–1998

1999

2000

2001

2002

2003

2004

2005

Year

FIGURE 10.13 Relative rates of laboratory-diagnosed Listeria infections in humans compared with 1996–1998 baseline period, by year—Foodborne Diseases Active Surveillance Network, 1996–2004.

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effort to develop strategies to reduce the levels of L. monocytogenes in the food processing environment and prevent post-processing contamination of food items. For example, subsequent to the 2003 USDA interim rule, a survey of more than 2,900 establishments producing ready-toeat meat and poultry products found that more than 87% had made improvements to L. monocytogenes controls [6]. Improvements in the timeliness of listeriosis outbreak detection, greatly facilitated by the routine molecular characterization of L. monocytogenes human isolates, allows public health and regulatory officials to investigate and control outbreaks more efficiently. Enhanced consumer education efforts, such as the “Fight BAC” educational campaign (www.fightbac.org) developed by the Partnership for Food Safety Education in conjunction with the President’s National Food Safety Initiative and programs of the Food Safety Training and Education Alliance have likely contributed to a reduction in listeriosis incidence as well. FoodNet data have been used to provide the most accurate estimates of the incidence of listeriosis. However, these and estimates derived from passive surveillance data must be interpreted with limitations in mind. First, FoodNet captures exclusively laboratory-confirmed infections. A primary outcome of listeriosis during pregnancy is fetal loss; however, bacterial cultures are not routinely requested for spontaneously aborted fetuses or stillborn neonates. This results in reduced laboratory diagnosis [19]. Thus, whereas FoodNet surveillance provides the most robust estimates available, the true incidence of listeriosis in the United States is most certainly higher. Second, it is notable that the Hispanic population is underrepresented in the population under surveillance by FoodNet. Although it is clear that the incidence of listeriosis is disproportionately high among Hispanics compared to other racial-ethnic groups, it is likely that FoodNet data underestimate the true incidence for this population. This, in turn, would contribute to underestimation of listeriosis overall.

IMPROVED ESTIMATES

OF THE

BURDEN

OF

LISTERIOSIS

IN THE

UNITED STATES

In an ongoing effort to provide more accurate estimates of foodborne diseases including listeriosis, which are critical to guide prevention efforts and assess the effectiveness of food safety regulations, Mead et al. reported foodborne disease estimates in 1999 that were derived and validated using data from multiple sources [110]. Although L. monocytogenes causes only about 2,500 of the estimated 76,000,000 annual cases of food-related disease in the United States, it is responsible for an estimated 500 deaths annually, or one-third of the deaths caused by known foodborne pathogens. The hospitalization rate was estimated to be 92%, with a case fatality rate of 20%. Several data sources contributed to the listeriosis estimates. The annual number of food-related cases was calculated using an average of the 1996–1997 FoodNet incidence applied to the total U.S. population, and previous estimates resulting from a comparable sentinel surveillance system [143]. A multiplier of two was applied to account for underreporting based upon the assumption that the severity of listeriosis results in medical attention for most cases and, in turn, a higher rate of diagnosis and reporting as compared to a disease with less severity. Similarly, hospitalization rates were drawn from FoodNet data indicating that nearly 90% of listeriosis cases result in hospitalization. The case fatality rate was estimated using FoodNet data, previous estimates of the incidence of listeriosis in the United States [143], and outbreak data. Accounting for the demonstrated potential for nosocomial transmission [134], calculations were performed based upon the estimate that 99% of listeriosis cases are transmitted by food. Although some assumptions were required for generation of these estimates, such as those used to arrive at the multiplier accounting for underreporting, they are generally accepted as the most precise estimates of the burden of listeriosis currently available. L. monocytogenes is a foodborne pathogen of significant public health concern. Though relatively rare compared to other foodborne pathogens, it is responsible for almost one-third of foodborne disease-related deaths caused annually by known pathogens. Despite the challenges encountered during the investigation of listeriosis outbreaks, much has been learned from investigations

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resulting in the successful implication of food vehicles. These implicated foods tend to be those commercially prepared and consumed without cooking by the consumer. Environmental contamination of the processing facilities, which can be sustained for prolonged periods, often leads to contamination of the implicated processed foods. The incidence of listeriosis has been decreasing in the United States and other countries. Results of surveillance and successful outbreak investigations, as described in this chapter, have focused regulatory and food industry actions to control food contamination. However, despite documented progress, foodborne listeriosis outbreaks and sporadic cases leading to severe disease and death continue to occur. Further efforts are required to enhance surveillance, outbreak identification, investigation, regulatory interventions, industry controls, and consumer education to achieve food safety objectives nationally and internationally.

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143. Tappero, J., A. Schuchat, K. Deaver, L. Mascola, and J.D. Wenger 1995. Reduction in the incidence of human listeriosis in the United States: effectiveness of prevention efforts? J Am Med Assoc 273: 1118–1122. 144. Tham, W., H. Ericsson, S. Loncarevic, H. Unnerstad, and M.-L. Danielsson-Tham 2000. Lessons from an outbreak of listeriosis related to vacuum-packed gravad and cold-smoked fish. Int J Food Microbiol 62: 173–175. 145. Tulzer, G., R. Bauer, W.D. Daubek-Puza, F. Eitelberger, C. Grabner, E. Heinrich, L. Hohenauer, M. Stojakovic, and F. Wilk 1987. A local epidemic of neonatal listeriosis in Austria—report of 20 cases. Klin Pädiatr 199: 325–328. 146. Urbach, H. and G.I. Schabinski 1955. Zur Listeriose des Menschen. Z Hyg 141: 239–248. 147. Villar, R.C., M.D. Macek, S. Simons, P.S. Hayes, M.J. Goldoft, J.H. Lewis, L.L. Rowan, D. Hursh, M. Patnode, and P.S. Mead 1999. Investigation of multidrug-resistant Salmonella serotype Typhimurium DT104 infections linked to raw-milk cheese in Washington State. J Am Med Assoc 281: 1811–1816. 148. Watson, C. and K. Ott 1990. Listeria outbreak in Western Australia. Commun Dis Intelligence 24: 9–12. 149. Wenger, J.D., B. Swaminathan, P.S. Hayes, S.S. Green, M. Pratt, R.W. Pinner, A. Schuchat, and C.V. Broome 1990. Listeria monocytogenes contamination of turkey franks: evaluation of a production facility. J Food Prot 53: 1015–1019. 150. Wesley, I.V. and F. Ashton 1991. Restriction enzyme analysis of Listeria monocytogenes strains associated with food-borne epidemics. Appl Environ Microbiol 57: 969–975. 151. Wiedmann, M., J.L. Bruce, C. Keating, A.E. Johnson, P L. McDonough, and C.A. Batt 1997. Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential. Infect Immun 65: 2707–2716.

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and Behavior 11 Incidence of Listeria monocytogenes in Unfermented Dairy Products Elliot T. Ryser CONTENTS Introduction ....................................................................................................................................357 Incidence of Listeria spp. in Unfermented Dairy Products ..........................................................358 Raw Cow’s Milk...................................................................................................................358 Raw Ewe’s and Goat’s Milk ................................................................................................366 Pasteurized Milk and Other Unfermented Dairy Products..................................................367 Behavior of L. monocytogenes in Unfermented Dairy Products ..................................................376 Raw Milk ..............................................................................................................................377 Pasteurized and Intensively Pasteurized Milk .....................................................................379 Autoclaved Milk, Cream, and Chocolate Milk....................................................................380 Sweetened Condensed and Evaporated Milk.......................................................................387 Ultrafiltered Milk..................................................................................................................388 Growth of L. monocytogenes in Mixed Cultures ..........................................................................388 Nonfluid Dairy Products ................................................................................................................391 Ice Cream..............................................................................................................................391 Butter ....................................................................................................................................392 Nonfat Dry Milk...................................................................................................................393 References ......................................................................................................................................394

INTRODUCTION Recognition of raw milk as a potential source of Listeria monocytogenes led to speculation that consumption of such milk was at least partly responsible for the previously described listeriosis outbreak in post–World War II Germany. After this listeriosis epidemic, sporadic reports of individuals drinking raw milk, along with assurances that raw milk was being properly pasteurized, virtually eliminated the threat of any further outbreaks of milkborne listeriosis. Consequently, research in this area also decreased. However, in 1983, concerns about the possibility of milkborne listeriosis were rekindled when consumption of pasteurized milk was epidemiologically linked to an outbreak of listeriosis in Massachusetts. Two events, namely, publication of an article in the New England Journal of Medicine detailing this outbreak in Massachusetts and a report in June of 1985 that as many as 300 people in California had acquired listeriosis after eating Mexican-style cheese contaminated with L. monocytogenes, caused considerable concern in the United States about the presence of Listeria in dairy products. This problem subsequently took on international proportions with the 1987 report of another cheese-related outbreak in which consumption of tainted

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Vacherin Mont d’Or soft-ripened cheese was directly linked to numerous cases of listeriosis in Switzerland. Years later, one outbreak of Listeria gastroenteritis was traced to consumption of contaminated chocolate milk in the United States [201]. Despite considerable progress, L. monocytogenes outbreaks continue to plague the dairy industry, with soft cheeses, particularly the Mexican and surface-ripened varieties, being responsible for continued sporadic listeriosis outbreaks in North America and Europe. In response to questions raised by milk producers, dairy processors, health officials, and the general public, a plethora of work has been conducted worldwide since 1983 to determine the incidence and behavior of L. monocytogenes in unfermented (raw milk, pasteurized milk, chocolate milk, cream, butter, ice cream, other frozen dairy desserts), as well as fermented (cheese, yogurt, cultured milk) dairy products. The incidence and behavior of L. monocytogenes in unfermented dairy products will be dealt with in this chapter; similar information about fermented dairy products appears in Chapter 12.

INCIDENCE OF LISTERIA SPP. IN UNFERMENTED DAIRY PRODUCTS The dairy-related listeriosis outbreaks reported during the mid-1980s (see Chapter 10) prompted scientists worldwide to determine the extent of Listeria contamination in raw milk and in pasteurized dairy products including milk, ice cream, ice cream novelties, frozen desserts, nonfat dry milk, and casein. Listeria monocytogenes can readily enter dairy processing facilities in the raw milk supply, which can in turn lead to contamination of the factory environment. The occasional appearance of listeriae in pasteurized dairy products nearly always has been associated with contamination of the product after pasteurization. Thus, it is fitting to begin this discussion by examining the incidence of Listeria spp. in raw milk, which is a major source of this bacterium in dairy factory environments.

RAW COW’S MILK As you will recall from the discussion of animal listeriosis in Chapter 2, dairy cattle can intermittently shed L. monocytogenes in their milk as a consequence of listerial mastitis, encephalitis, or a Listeria-related abortion. Although milk from animals showing obvious signs of listeriosis is unlikely to reach consumers, the scientific literature contains numerous accounts in which mildly infected and apparently healthy dairy cattle, sheep, and goats have shed L. monocytogenes intermittently in their milk for many months. Thus, it appears that such asymptomatic carriers of listeriae pose the greatest threat to public health. The 1983 listeriosis outbreak in Massachusetts that was supposedly associated with drinking a particular brand of pasteurized milk raised numerous milk safety questions. The well-publicized outbreak of 1985 in which consumption of contaminated Mexican-style cheese was directly linked to at least 40 deaths in California prompted additional concerns about the safety of dairy products manufactured in the United States. Because raw milk is a potential source of L. monocytogenes, recalls of Listeria-contaminated pasteurized dairy products (i.e., milk, chocolate milk, ice cream) and imported soft-ripened cheeses prompted more than 80 surveys worldwide to determine the extent of Listeria contamination in raw milk. Results of these surveys, which will now be described in some detail, have been summarized in Table 11.1 and Table 11.2. The first large-scale survey of raw milk for Listeria spp. was prompted by the 1983 listeriosis outbreak in Massachusetts [127]. During the 3-week period immediately following the outbreak, Hayes et al. [151] examined 121 raw milk samples collected from milk trucks (40 samples), milk cooperatives (72 samples), and bulk tanks from four farms on which bovine listeriosis was diagnosed (9 samples), as well as 14 milk socks used to remove debris but not leukocytes from milk. All samples were analyzed for L. monocytogenes using a multiple two-stage enrichment procedure. Although investigators at the US Centers for Disease Control and Prevention (CDC) isolated the epidemic serotype along with other serotypes of L. monocytogenes from 15 of 121 (12.4%)

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TABLE 11.1 Incidence of Listeria spp. in Raw Milk Produced in the United States and Canada Location United States Northwest West Midwest Northeast Southeast California Massachusetts Massachusetts, Vermont Minnesota Nebraska South Dakota /Minnesota Ohio, Kentucky, and Indiana Pennsylvania Tennessee Wisconsin Total Canada Alberta Manitoba Ontario Ontario Total

Number of Samples

948 176 361 363 61 200 100 121 939 300 84 200 131 350 2511 292 50 55 7227 426 252 256 1720 445 315 3414

Number of Positive Samples (%) L. monocytogenes

56 7 18 32 6 14 0 15 15 9 0 8 12 13

(5.9) (4.0) (5.0) (8.8) (9.8) (7.0) (12.4) (1.6) (3.0) (4.0) (9.2) (3.7)

79 (3.1) 12 (4.1) 0 0 288 (4.0) 8 4 4 47 6 17 86

(1.9) (1.6) (1.6) (2.7) (1.3) (5.4) (2.5)

L. innocua

L. welshimeri

Others

Reference

ND ND ND ND ND 19 (9.5) 4 (4.0) ND ND 77 (25.7) 6 (7.1) 10 (5.0) ND 27 (7.7)

ND ND ND ND ND

ND ND ND ND ND

186 236 236 236 236 168 168 151 101 171 193 165 158 168

ND ND ND

0 0 ND ND 5 (1.7) 1 (1.2) 0 ND 6 (1.5) ND ND ND

0 1 (1.0)a ND ND 0 0 0 ND 3 (0.9)a ND ND ND

0 143 (11.1)

0 12 (0.9)

0 4 (0.3)

ND ND ND ND 43 (9.7) 26 (8.2) 69 (9.1)

ND ND ND ND 6 (1.3) 1 (0.3) 7 (0.9)

ND ND ND ND 0 0 0 (0)

102 210 103 237

123 235 93 227 112 223

Note: ND = Not determined (omitted from total). a

Two L. ivanovii and one L. seeligeri.

(including 1 of 9 bulk tank samples) [150] and 2 of 14 (14%) raw milk and milk sock samples, respectively, the epidemic phage type was never detected. Between October 1984 and August 1985, US Food and Drug Administration (FDA) officials surveyed 650 raw milk samples that were collected from bulk tanks in Massachusetts, Vermont, California, and the tristate area of Kentucky, Ohio, and Indiana. The samples were examined for Listeria spp. using the original FDA method [168]. Low levels of various Listeria spp., including L. monocytogenes, were detected in raw milk samples obtained from all states except California. Overall, 82 of 650 (12.6%) samples contained Listeria spp., with L. monocytogenes being found in 27 of 650 (4.2%) samples. Of the 27 L. monocytogenes strains isolated from raw milk, 16 were serotype 1, 10 were serotype 4, and 1 was nontypeable. In addition, only 2 of the 27 L. monocytogenes strains proved to be nonpathogenic to mice, and both were of serotype 4. In 1988, Donnelly et al. [101] used flow cytometry to analyze 939 raw milk samples obtained from 54 farms in California. Unlike the FDA study just described [168], string samples (milk pooled

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TABLE 11.2 Incidence of Listeria spp. in Raw Cow’s Milk Collected outside North America Number of Positive Samples (%) Location Europe Austria Belgium Czechoslovakia Denmark Finland France

Germany Hungary Ireland Italy

The Netherlands Poland Portugal Spain Sweden Switzerland Turkey United Kingdom Great Britain England/Wales Scotland

Northern Ireland Total

Number of Samples

L. monocytogenes

201 143 177 123 1,227,053 256 59 1787 1409 561 337 51 635 80 50 589 50 290 142 98 85 50 40 32 137 134 81 54 340 774 294 4046 340 77

0 9 6 4 278 13 1 60 85 21 14 10 2 3 2 29 4 8 0 2 0 0 0 0 6 2 6 3 23 28 3 14 2 14

350 2009 361 640 560 540 176 113 18,271

13 102 13 90 14 14 27 6 653

L. innocua

L. welshimeri

Reference

(4.4) (1.5) (7.4) (5.6) (6.8) (3.6) (1.0) (0.4) (0.6) (18.2)

2 (1.0) ND ND ND ND ND ND ND ND ND 5 (1.5) 10 (19.6) ND ND ND 20 (3.4) ND 16 (5.6) 1 (0.6) ND 0 1 (2.0) 0 2 (6.3) ND ND ND ND 24 (7.0) 21 (2.7) 7 (2.3) ND ND ND

ND ND ND ND ND ND ND ND ND 0 18 (35.3) ND ND ND ND ND 0 0 ND 0 0 0 ND ND ND ND ND 1 (0.3) 0 0 ND ND ND

2 (1.0) ND ND ND ND ND ND ND ND ND 0 9 (17.6) ND ND ND ND ND 2 (0.7) 0 ND 0 0 0 ND ND ND ND ND 6 (1.8) 0 0 ND ND ND

107 97 181 182 200 200 200 180 45 51 139, 140 139, 140 133, 232 207 207 206 157 96 226 164 136 234 175 130 78 161 211 146 240 135 241 73 73 220

(3.6) (5.1) (3.6) (14.1) (2.5) (2.6) (15.3) (5.3) (3.6)

ND ND ND ND 7 (1.3) ND 18 (10.2) ND 134 (3.4)

ND ND ND ND 0 ND 0 ND 19 (0.6)

ND ND ND ND 1 (0.2) ND 5 (2.8)a ND 35 (1.0)

137 188 145 125 124 124 149 138

(6.3) (3.3) (3.3) (0.02) (5.1) (1.7) (3.4) (6.0) (3.8) (4.2) (19.6) (0.3) (3.8) (4.0) (4.9) (8.0) (2.8) (2.0)

0

Others

(continued)

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TABLE 11.2 (CONTINUED) Incidence of Listeria spp. in Raw Cow’s Milk Collected outside North America Number of Positive Samples (%) Location Elsewhere Argentina Australia Brazil

Costa Rica Egypt Iran Japan

Jordan Korea Malaysia Mexico Morocco New Zealand South Africa Taiwan Total

Number of Samples

208 169 150 220 20 12 220 236 190 362 120 150 170 50 45 930 1300 100 30 71 982 80 5815

L. monocytogenes

1 1 0 11 0 1 0 7 4 3 0 6 2 1 2 18 162 0 3 0 67 5 304

(0.5) (0.6) (4.8) (8.3) (3.0) (2.1) (0.8) (0.4) (7.1) (2.0) (4.4) (1.9) (12.5) (10.0) (6.8) (6.3) (5.2)

L. innocua

L. welshimeri

Others

Reference

5 (2.4) ND 4 (2.7) 21 (9.5) ND 2 (16.7) 10 (4.5) 20 (8.5) ND ND ND 2 (1.3) ND ND ND 20 (2.1) 9 (0.7) 7 (7.0) ND 10 (14.1) ND ND 110 (3.1)

1 (0.5) ND 0 2 (0.9) ND 0 3 (1.4) 0 ND ND ND 0 ND ND ND 6 (0.6) 0 2 (2.0) ND 1 (1.4) ND ND 15 (0.4)

0 ND 0 1 (0.4) ND 0 1 (0.5)b 0 ND ND ND 0 ND ND ND 0 129 (9.9) 0 ND 7 (9.9) ND ND 138 (3.8)

162 110 154 185 83 129 70 108 205 244 231 219 121 147 74 85 238 169 109 229 243 84

Note: ND = Not determined (omitted from total). a

L. seeligeri. L. grayi.

b

from 25 to 40 cows), combination samples (milk pooled from 200 cows), and samples of raw milk from bulk tanks were tested for L. monocytogenes. Using this method, L. monocytogenes was detected in 15 of 939 (1.5%) samples. Researchers in Minnesota [193] and Wisconsin [103,237] failed to detect L. monocytogenes in raw milk during three small surveys. However, the pathogen was found in 4.0 and 2.8% of raw milk samples obtained from bulk storage tanks and tank trucks in Nebraska [166] and Pennsylvania [102], respectively, with approximately equal numbers of L. monocytogenes isolates being classified as serotype 1, 4, or nontypeable (non–serotype 1 or 4) in the latter study. More recent findings indicate a relatively stable incidence for L. monocytogenes in bulk tank raw milk, with 3.0 and 4.1% of such samples from Minnesota [171] and Tennessee [210], respectively, being positive. In the Minnesota survey, Listeria-positive samples also tended to have higher bacterial and somatic cell counts, which are indicative of less stringent sanitation and mastitis control practices. As part of the National Animal Health Monitoring System, a large-scale national survey was conducted in 2002 during which 861 raw milk bulk tank samples were analyzed for various microbial contaminants including L. monocytogenes [236]. The incidence of L. monocytogenes contamination was 4.0, 5.0, 8.8, and 9.8% for milk samples collected in the West, Midwest,

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Northeast, and Southeast (average of 6.5%), respectively, with 93% of the isolates belonging to serotypes 1/2a, 1/2b, and 4b. One year earlier, Muraoka et al. [186] reported an overall incidence rate of 5.9% for L. monocytogenes in bulk tank samples from the Pacific Northwest with strains of serotype 1/2a predominating. In one additional survey [149], milk filters rather than samples of bulk tank milk were collected from 404 farms throughout New York State from April 1998 to March 1999 and assessed for L. monocytogenes. Fifty-one (12.6%) of these milk filters through which an average of 1351 kg of milk had passed yielded L. monocytogenes with this substantially higher contamination rate compared to bulk tank milk related to presence of debris in the filters at the time of sampling. Additional observations from this study included regional differences in percentage of positive filters (6.5 to 19.0%), with the highest contamination rate found in central New York State and a higher isolation rate during spring (18%) compared to summer, fall, and winter (6–8%). The overall findings from Table 11.1 indicate that L. monocytogenes was present in 0–12.4% of the US raw milk samples examined. When the results are averaged, 4.0% of all raw milk processed in the United States can be expected to contain low levels (i.e., 100 or 1000 CFU/g or mL), particularly for products in which Listeria is unable to grow. Until recently, the Canadian government confined its recalls to only those foods that have been linked to major outbreaks of listeriosis, with the role of pasteurized milk in foodborne listeriosis still being highly debated [157]. Hence, no recalls were issued when investigators at the Health Protection Branch of Health and Welfare Canada (analogous to the U.S. FDA) identified L. monocytogenes in 1 of 394 (0.25%) and 1 of 51 (2.0%) samples of ice cream and ice cream novelties, respectively [111], during their own federal inspection program. Although subsequent investigations were presumably conducted to identify (1) the source of contamination, (2) proper corrective measures, and (3) possible links to human illness, Canadian officials maintained that recalling the two contaminated lots would be inappropriate without proof that consumption of Listeria-contaminated ice cream could lead to listeriosis. Many individuals and most manufacturers have argued in favor of the more relaxed Canadian position. When one considers the numerous recalls of Listeria-contaminated ice cream in the United States, the fact that worldwide only one case of listeriosis has been positively linked to ice cream containing unusually high numbers of listeriae, the inability of L. monocytogenes to grow in this product during frozen storage, and the normal exposure rate of the human population to listeriae, it is clear that the risk of contracting listeriosis from contaminated ice cream is extremely low, as will be discussed later in regard to several risk assessments. Although current regulations mandate immediate removal of fluid dairy products and cheeses that support growth of L. monocytogenes, a scientifically valid argument can now be made against recalling certain dairy products in which listeriae will not proliferate, such as ice cream and dried goods which, if contaminated, typically contain very low numbers of listeriae as postpasteurization contaminants. As a result of several large recalls of French Brie cheese and a listeriosis outbreak in Switzerland that was traced to consumption of Vacherin Mont d’Or soft-ripened cheese, European scientists have logically focused their attention on the incidence of listeriae in soft cheese. However, numerous recalls of unfermented dairy products in the United States also have heightened public health concerns about the presence of listeriae in pasteurized dairy products manufactured outside North America [41]. In one of the first European surveys of finished products reported in 1988, researchers in Germany [232] failed to isolate Listeria spp. from pasteurized milk (39 samples), nonfat dry milk (11 samples), casein/caseinate (30 samples), and various dried products, including baby food (Table 10.6). During the same year, investigators in Hungary [115] and The Netherlands [79] also failed to recover L. monocytogenes from samples of pasteurized milk, with similar negative findings being obtained from most other subsequent surveys of pasteurized milk and cream produced elsewhere (Table 11.6). However, L. monocytogenes was eventually demonstrated in 11 of 1039 (1.1%), 4 of 115 (3.5%), and 1 of 95 (1.1%) pasteurized milk samples examined in the United Kingdom [138,145,215] for a combined contamination rate of 1.3%, these findings generally being similar to those observed in the United States. According to Garayzabal et al. [131], 21.4, 89.2, 10.7, and 3.6% of pasteurized milk samples from one particular milk processing facility in Madrid contained L. monocytogenes, L. grayi, L. innocua, and L. welshimeri, respectively. These authors [132,209] previously reported similar Listeria contamination rates for raw milk entering the same processing facility. Further, after pasteurization these same samples had a total mesophilic aerobic plate count of 2.5 × 107 CFU/mL, which is well above the maximum allowable limit of 1 × 104 CFU/mL for properly pasteurized milk in the United States. Hence, improper pasteurization caused

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TABLE 11.6 Incidence of Listeria spp. in Pasteurized Dairy Products Produced outside the United States and Canada Number of Positive Samples (%) Product

Country of Origin

Milk

Australia Brazil Czechoslovakia Germany Hungary Italy Korea

Chocolate milk Flavored milk Ice cream

Cream

Butter Nonfat dry milk Casein/caseinate Dry infant formula

Morocco The Netherlands Poland Portugal Turkey United Arab Emirates United Kingdom England/Wales Scotland Northern Ireland Hungary Australia Australia Chile Costa Rica England/Wales Korea Turkey Australia England/Wales Hungary Morocco Hungary Italy Germany Germany Germany

Note: ND = Not determined. a

Seven non-L. monocytogenes isolates. One non-L. monocytogenes.

b

Number of Samples

L. monocytogenes

L. innocua

L. welshimeri

Other

Reference

77 33 220 20 30 15 39 100 50 348 26 50 20 41 73 28 22 182

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 ND 2(0.9) 0 ND ND 0 ND 0 0 ND 0 ND 0 0 ND ND 0

0 ND 0 0 ND ND 0 ND 0 0 ND 0 ND 0 0 ND ND 0

0 ND 0 0 ND ND 0 ND 0 0 ND 0 ND 0 7a ND ND 0

144 72 185 83 181 182 232 155 115 136 74 234 109 79 211 179 220 141

ND ND ND 0 ND ND ND ND ND ND 6 (12.0) 0 ND ND ND 0 ND 0 0 0

ND ND ND 0 ND ND ND ND ND ND 0 0 ND ND ND 0 ND 0 0 0

ND ND ND 0 ND ND ND ND ND ND 0 0 ND ND ND 1b ND 0 0 0

145 215 138 155 239 72 90 184 137 74 86 144 145 155 109 155 202 232 232 232

1039 115 95 60 206 166 603 50 40 132 50 12 40 15 20 15 130 11 30 120

11 4 1 7 1 23 21 1 8 5

1 5

(1.1) (3.5) (1.1) (11.6) (0.5) (13.9) (3.5) (2.0) 0 (6.1) (10.0) 0 0 0 0 (6.7) (3.8) 0 0 0

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by leaking pasteurizer plates, as suggested by Northolt et al. [187], or postpasteurization contamination from the factory environment appear to be most likely responsible for the unusually high incidence of listeriae in “pasteurized” milk samples from this particular dairy factory. Although results from these aforementioned surveys of pasteurized milk, cream, and dried products are very encouraging, the isolation methods used in these studies were generally unable to detect sublethally injured listeriae. Hence, the true incidence of listeriae in pasteurized milk, cream, and dried products may well be somewhat higher. To enhance recovery of injured cells, the International Dairy Federation has recommended that such dairy products undergo preenrichment in a nonselective medium (i.e., buffered peptone water) before primary enrichment in various selective broths and plating on Listeria-selective media [39,233]. Further details concerning recovery of sublethally injured listeriae can be found in Chapter 7. Results from a 1989 International Dairy Federation survey [157] indicated that public health issues regarding the presence of listeriae in pasteurized milk were clearly spreading beyond the continental boundaries of Europe and North America, with the many aforementioned surveys from Table 10.6 attesting to these concerns. More recently, the safety of several additional dairy products, including flavored milks, chocolate milk, ice cream, and particularly butter, has attracted international attention with the FDA Initiatives Program, the many Class I recalls of Listeria-contaminated dairy products, and fears of international trade embargoes fueling these concerns. Following the 1987 discovery of L. monocytogenes in Australian ricotta cheese, New Zealand and Australian officials instituted Listeria-monitoring programs for casein/caseinate products as well as high-moisture cheese, pasteurized milk, ice cream, and milk powders. Results from one 10-month survey begun in April 1988 [239] revealed the presence of L. monocytogenes in 1 of 206 (0.48%) samples of pasteurized flavored/unflavored milk processed in and around Melbourne. Subsequent identification of heat-labile alkaline phosphatase in the contaminated product (pasteurized milk to which a pasteurized flavored syrup was added) suggested that improper pasteurization was most likely responsible for the presence of L. monocytogenes in the final product. However, unsatisfactory storage of the flavored syrup also may have contributed to contamination. In keeping with Listeria policies developed in the United States and Canada, Australian officials withdrew the affected product from the marketplace and prohibited the sale of all subsequently produced product until 12 consecutive lots of Listeria-free pasteurized flavored milk could be produced from the same product line. As in the United States, recent foreign surveys also have shown a higher incidence of L. monocytogenes in chocolate milk (11.6%), ice cream (2.0–13.9%), and butter (3.8–6.7%) as compared to pasteurized milk and dried products that are seldom contaminated (Table 11.6). The increased incidence of listeriae in ice cream and butter is clearly the result of postpasteurization contamination during handling and packaging, as evidenced by the highest contamination rates in ice cream bars and novelties. The fact that Listeria spp. are more commonly found in chocolate milk, as opposed to unflavored milk, is also not surprising given that the added ingredients can serve as another source of listeriae.

BEHAVIOR OF L. MONOCYTOGENES IN UNFERMENTED DAIRY PRODUCTS Although the psychrotrophic nature of L. monocytogenes and the ability of both normal and diseased animals to shed this pathogen in their milk have been recognized for many years, behavior of L. monocytogenes in raw milk and unfermented dairy products did not receive serious attention until 1983 when an outbreak of “milkborne” listeriosis was reported in Massachusetts. Research efforts prompted by this and two other dairy-related outbreaks in the United States and Switzerland have given us an understanding of the behavior of L. monocytogenes in raw and pasteurized milk as well as in chocolate milk, cream, nonfat dry milk, and butter. The remainder of this chapter will describe results from these studies along with information concerning behavior of this organism in ultrafiltered milk and ice cream mix.

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RAW MILK Despite longstanding recognition of L. monocytogenes as a raw milk contaminant, relatively few studies assessing the behavior of this organism in raw milk can be found in the literature. In 1958, Dedie [95] found that L. monocytogenes survived 210 days in naturally contaminated raw milk stored in an ice chest. Thirteen years later, Dijkstra [98] reported results from a much longer storage study in which 36 samples of naturally contaminated raw milk (obtained from cows that experienced Listeria-related abortions) were held at 5°C and examined for viable L. monocytogenes over a period of 9 years. Although 4 of 36 (11%) samples were free of L. monocytogenes within 6 months, the pathogen was still detected in 16 of 36 (44%) samples following 2 years of refrigerated storage. The number of samples from which listeriae could be isolated continued to decrease, with 9 of 36 (25%) samples being positive after 4 years of storage. However, the pathogen was still present in 4 of 36 (11%) raw milk samples after 8–9 years of storage. These early findings emphasize the importance of establishing proper cleaning and sanitizing programs for all phases of milk production. If routinely used, such programs will likely prevent this organism from finding an appropriate niche within the farm or dairy factory environment and greatly reduce the threat of this pathogen surviving long term. The studies just described adequately demonstrate that L. monocytogenes can persist in raw milk for long periods; however, until several outbreaks of milkborne and cheeseborne listeriosis were reported in the 1980s, little attention had been given to the potential for growth of L. monocytogenes in raw milk. In 1988, Northolt et al. [187] examined the behavior of listeriae in samples of freshly drawn raw milk that were inoculated to contain approximately 500 L. monocytogenes CFU/mL and incubated at 4 and 7°C. As shown in Figure 11.4, Listeria populations decreased approximately 4- and 8.5-fold in raw milk during the first 2 days of incubation at 4 and 7°C, respectively. These authors suggested that naturally occurring antibacterial substances in raw milk (i.e., lactoperoxidase and lysozyme) may have partially inhibited growth of listeriae during the first 2 days of incubation as has been more recently confirmed by two other investigators [199,245]. However, in a Canadian study that will be discussed shortly [113], no such decrease was observed when incubated samples of naturally contaminated raw milk were surface-plated on FDA Modified McBride Listeria Agar. Hence, a more likely explanation is that the plating medium—Trypaflavine Nalidixic Acid Serum Agar—used by Northolt et al. [187] was less than ideal for recovering listeriae, as also was observed during concurrent work with pasteurized milk. Although L. monocytogenes failed to grow in raw milk samples incubated at 4°C for up to 7 days, Listeria populations increased approximately 10-fold during this period when the incubation temperature was raised to 7°C. Following 3 days of incubation at 4 and 7°C, Listeria populations began doubling every 3.5 and 1.0 days, respectively. Two years later, Wenzel and Marth [242] reported that populations of L. monocytogenes strain V7 remained constant in inoculated raw milk during 5 days of storage at 4 and 7°C, with numbers of listeriae also being unaffected by the presence of a commercial raw milk lactic acid bacteria inoculant designed to suppress growth of primarily Gram-negative psychrotrophic bacteria. L. monocytogenes failed to grow during 3–5 days of incubation at 7°C. It appears that the 3-day period during which raw milk is sometimes held in farm bulk tanks is insufficient to allow growth of the organism. However, the temperature of raw milk in farm bulk tanks fluctuates every time freshly drawn raw milk at 37°C is commingled with bulk tank milk at 4°C from previous milkings. In 1985, Oz and Farnsworth [190] found that raw milk in farm bulk tanks attained temperatures of 30–31°C, 10–14°C, 12°C, and 9°C when freshly drawn raw milk was added after the first, second, third, and fourth milking periods, respectively. Moreover, 6 h were generally needed for the milk to cool to 4°C after each milking period. In view of these findings, it appears that temperatures obtained after adding warm milk to farm bulk tanks may be sufficient to allow at least limited growth of L. monocytogenes, particularly when raw milk from early milkings enters the bulk tank. Although the temperature of bulk tank milk will eventually decrease to 4°C, exposure

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104

L. monocytogenes CFU/mL

7°C

103

4°C

102

Raw Milk 0

0

2

4

6

Days

FIGURE 11.4 Growth of L. monocytogenes strains in raw milk incubated at 4 and 7°C (enumerated on Trypaflavine Nalidixic Acid Serum Agar). (Adapted from Northolt, M.D., H.J. Beckers, U. Vecht, L. Toepoel, P.S.S. Soentoro, and H.J. Wisselink. 1988. Listeria monocytogenes: heat resistance and behavior during storage of milk and whey and making of Dutch types of cheese. Neth. Milk Dairy J. 42: 207–219.)

to temperatures as high as 9°C when raw milk is trucked to processing facilities during summer [114] also may lead to some multiplication of the pathogen. Discovery of a naturally infected cow in Canada that shed freely suspended and phagocytized cells of L. monocytogenes in milk (maximum of 104 CFU/mL in milk from one of four quarters of the mammary gland) continuously for nearly 3 years provided Farber et al. [113] with a unique opportunity to study growth of L. monocytogenes in naturally rather than artificially contaminated raw milk during extended storage. When raw milk from this cow was analyzed for numbers of L. monocytogenes, no appreciable growth of the pathogen was observed during the first 3 days and 1 day of incubation at 4 and 10°C, respectively (Figure 11.5). The delay in onset of growth was less than 1 day at 15°C. Immunological staining of milk smears indicated that some multiplication of L. monocytogenes had occurred within macrophages after 1 and 2 days of incubation at 15 and 10°C, respectively, with 10–50% of the macrophages containing 1–20 intracellular listeriae. Nonetheless, as previously noted by Doyle et al. [105], rapid deterioration of macrophages shortly thereafter was followed by appearance of freely suspended listeriae in milk with few intact macrophages remaining after 5 days, regardless of incubation temperature. Following the lag phase, L. monocytogenes entered a period of logarithmic growth, with generation or doubling times of 25.3, 10.8, and 7.4 h being calculated for raw milk samples held at 4, 10, and 15°C, respectively.

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7.0

10

L. monocytogenes log CFU/mL

Incidence and Behavior of Listeria monocytogenes in Unfermented Dairy Products

6.0

4°C 10°C 15°C

5.0

4.0 0

2

4

6

8

10

12

14

FIGURE 11.5 Growth of L. monocytogenes in naturally contaminated raw milk during incubation at 4, 10, and 15°C. (Adapted from Farber, J.M., G.W. Sanders, and J.I. Speirs. 1990. Growth of Listeria monocytogenes in naturally-contaminated raw milk. Lebensm. Wiss. Technol. 23: 252–254.)

Although maximum L. monocytogenes populations were approximately 2 × 107 CFU/mL after 10, 7, and 3 days of incubation at 4, 10, and 15°C, respectively, the highest achievable population in raw milk was independent of incubation temperature (Figure 11.6). As in the previous study by Northolt et al. [187], these findings again stress the importance of maintaining raw milk at 4°C during storage and transport to milk processing facilities. Despite an increasing market for both ewe’s and goat’s milk and the use of these milk types in a wide range of ethnic and specialty cheeses and, to a lesser extent, yogurt, most work on growth and survival of Listeria in raw milk has been conducted using cow’s milk. Reports of ovine listeriosis in Europe dating back to the 1940s prompted an early study by Ikonomov and Todorov [156] to examine the behavior of L. monocytogenes in raw ewe’s milk inoculated with the pathogen. In their work, L. monocytogenes remained viable for long periods and persisted in the milk even after coagulation at 10 and 20°C. Information on the behavior of L. monocytogenes in goat’s milk is limited to one 2002 report by Leuchner et al. [164a] in which samples of raw and pasteurized goat’s milk were inoculated with a 3-strain “cocktail” of L. monocytogenes at 103 CFU/mL and then stored at 4, 10, and 15°C for 58 days. The pathogen survived at least 58 days when raw goat’s milk was held at 4 and 10°C and up to 44 days when the same milk was stored at 15°C. Populations of Listeria in pasteurized milk increased about 3 logs after 58 days of storage at 4°C and about 4 logs after 58 days at 10 and 15°C. Given these findings and the fact that many varieties of ethnic- and specialty-type cheeses are now being manufactured by many small cheese makers from ewe’s or goat’s milk, the safety of these cheeses is likely to receive increased attention in the future.

PASTEURIZED

AND INTENSIVELY

PASTEURIZED MILK

In addition to defining the growth pattern of L. monocytogenes in artificially contaminated raw milk (Figure 10.4), Northolt et al. [187] also examined behavior of this organism in pasteurized (72°C/15 sec) and intensively pasteurized whole milk (Figure 11.6). Although L. monocytogenes failed to grow in raw milk incubated at 4°C (Figure 11.4), Listeria populations in pasteurized milk increased nearly 10-fold during 7 days of incubation at the same temperature. The organism also grew markedly faster in pasteurized than in raw milk when both products were incubated

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3

7°C

L. monocytogenes CFU/mL

7°C 2

4°C

4°C

1

Intensively Pasteurized Milk

HTST-Pasteurized Milk

0

2

4

6

0

2

4

6

FIGURE 11.6 Growth of L. monocytogenes in high-temperature, short-time (HTST)-pasteurized and intensively pasteurized milk incubated at 4 and 7°C. —: Enumerated from samples at 4°C on Trypaflavine Nalidixic Acid Serum Agar; ---: enumerated on Nutrient Agar. (Adapted from Northolt, M.D., H.J. Beckers, U. Vecht, L. Toepoel, P.S.S. Soentoro, and H.J. Wisselink. 1988. Listeria monocytogenes: heat resistance and behavior during storage of milk and whey and making of Dutch types of cheese. Neth. Milk Dairy J. 42: 207–219.)

at 7°C. In contrast to their data for raw and pasteurized milk, lag times for L. monocytogenes were reduced considerably when the organism was grown in intensively pasteurized milk incubated at 4 and 7°C. Further, numbers of listeriae in intensively pasteurized milk increased approximately 100-fold following 3 and 6 days of incubation at 7 and 4°C, respectively. When L. monocytogenes was later grown in ultrahigh temperature (UHT) sterilized milk, Rajikowski et al. [204] reported generation times of 4.7, 1.7, 1.0, and 0.9 h for samples incubated at 12, 19, 28, and 37°C, respectively. Hence, these findings suggest that the growth rate for L. monocytogenes in milk is directly related to the degree of heat applied to milk, as was also reported by Mathew et al. [176]. Further work is needed to define more clearly the effect of competing microorganisms on growth of listeriae in raw and pasteurized milk as compared to intensively pasteurized and UHT-sterilized milk, with biochemical changes that occur in milk during thermal processing (i.e., protein denaturation, enzyme inactivation, carmelization) also likely influencing listeriae growth in these products. However, the aforementioned studies all indicate the potential for L. monocytogenes to reach potentially hazardous levels in pasteurized milk during the now normal 2-week refrigerated shelf life, with at least one risk assessment to be discussed in Chapter 18 also suggesting that consumption of pasteurized milk is likely responsible for the largest percentage of listeriosis cases.

AUTOCLAVED MILK, CREAM,

AND

CHOCOLATE MILK

Except for the three studies just mentioned [113,187,176] and an initial attempt by Pine et al. [198] to quantify growth of L. monocytogenes in inoculated samples of pasteurized milk, all remaining

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9

Strain CA log10 CFU/mL

8

7

6 Skim Milk 5

Whole Milk Chocolate Milk

4

Cream 3

2 0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

Days

FIGURE 11.7 Growth of L. monocytogenes strain California in fluid dairy products at 4°C. (Adapted from Rosenow, E.M. and E.H. Marth. 1987. Growth of Listeria monocytogenes in skim, whole and chocolate milk, and in whipping cream during incubation at 4, 8, 13, 21, and 35°C. J. Food Prot. 50: 452–459.)

work dealing with behavior of Listeria in fluid dairy products has been done using autoclaved samples. Although using such sterile products as growth media for listeriae offers several major advantages, including the ability to accurately quantify both stressed and unstressed listeriae on nonselective plating media in the absence of other microbial competitors, readers should keep in mind that growth rates of L. monocytogenes are somewhat faster in autoclaved than in pasteurized, and especially in raw milk, products. Nevertheless, L. monocytogenes clearly can grow to dangerously high levels in all three types of milk during extended refrigeration. In 1987, Rosenow and Marth [213] published results from a definitive study in which autoclaved (121°C/15 min) samples of whole, skim, and chocolate milk, as well as whipping cream, were each inoculated separately with four strains of L. monocytogenes (Scott A, V7, V37CE, or California), incubated at 4, 8, 13, 21, or 35°C, and examined for numbers of listeriae at suitable intervals by surface-plating appropriate dilutions on Tryptose Agar. Growth rates for L. monocytogenes were generally similar in all four products at a given temperature and increased with an increase in incubation temperature. At 4°C, listeriae grew after an initial delay of approximately 5–10 days depending on the bacterial strain and type of product (see Figure 11.7). All four strains generally attained maximum populations of 107 CFU/mL after 30–40 days of incubation, with little change in numbers occurring after 30–40 days of additional storage. Overall, chocolate milk supported development of the highest Listeria populations followed by skim milk, whole milk, and whipping cream. Generation times at 4°C ranged between 28.16 and 45.55 h with average generation times for L. monocytogenes in all four products shown in Table 11.7. Although these results clearly demonstrate the ability of L. monocytogenes to reach potentially hazardous levels in fluid dairy products held at 4°C, more recent data suggest that slow growth of this organism can even occur in milk held at 0°C. Thus, the only way to avoid a public health problem with fluid dairy products is to prevent L. monocytogenes from entering such products before, during, and after manufacture. Increasing the incubation temperature from 4 to 8°C decreased the lag period to 1.5–2 days (Figure 11.8) and nearly tripled the growth rate for L. monocytogenes in all four products (Table 11.7) [213,214]. After 10–14 days of incubation, the growth curves at 4 and 8°C were similar,

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TABLE 11.7 Generation Times for L. monocytogenes in Autoclaved Samples of Various Dairy Products Generation Time (h) at 4°Ca 33.27 34.52 33.46 36.30

Product Whole milk Skim milk Chocolate milk Whipping cream

8°Ca 13.06 12.49 10.56 11.93

13°Ca 5.82 6.03 5.16 5.56

21°Cb 1.86 1.92 1.72 1.80

35°Cb 0.692 0.693 0.678 0.683

a

Average generation times for four strains of L. monocytogenes. Strain V7 only.

b

Source: Adapted from Meyer-Broseta, S., A. Diot, S. Bastian, J. Riviere, and O. Cerf. 2003. Estimation of low bacterial concentration: Listeria monocytogenes in raw milk. Int. J. Food Microbiol. 80: 1–15.

with highest Listeria populations again being found in chocolate milk. Theoretical calculations based on these data indicate that Listeria populations could increase from 10 to 4.2 × 106 organisms/qt (947 mL) of milk during 10 days of storage at 8°C (46°F), a temperature that commonly occurs in some home and commercial refrigerators. These findings, which have since been confirmed by Siswanto and Richard [221] using skim milk, raise additional safety concerns about reclaiming and reprocessing returned products that have likely undergone some degree of temperature abuse. As is true for 8°C, 13°C (55°F) also represents a temperature that dairy products occasionally encounter during transportation and storage. Following a 12-h lag period, all four Listeria strains grew nearly twice as fast at 13°C as at 8°C (see Table 11.7) and generally attained levels of

9

.

Strain CA log10 CFU/mL

8.

7.

6. Skim Milk 5.

Whole Milk Chocolate Milk

4.

Cream 3.

2. 0

2

4

6

8

10

12

14

16

18

20

22

Days

FIGURE 11.8 Growth of L. monocytogenes strain California in fluid dairy products at 8°C. (Adapted from Rosso, L., S. Bajard, J.P. Flandrois, C. Lahellec, J. Fournaud, and P. Veit. 1996. Differential growth of Listeria monocytogenes at 4 and 8°C: Consequences for the shelf life of chilled products. J. Food Prot. 59: 944–949.)

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106 CFU/mL in all four products by the third day [213]. These generation times are somewhat longer than those observed by Farber et al. [113] when naturally contaminated raw milk was incubated at 4 (25.3 h), 10 (10.8 h), and 15°C (7.4 h). L. monocytogenes also attained maximum populations that were approximately 10-fold lower in raw than in sterile milk, which in turn suggests possible depletion of essential nutrients by raw milk contaminants or production of substances inhibitory to growth of the pathogen. Maximum Listeria populations of 109 CFU/mL were again observed in chocolate milk, with numbers generally being 10-fold lower in skim milk, whole milk, and whipping cream [213]. Increasing the incubation temperature to 21°C doubled the growth rate (see Table 10.7) and led to maximum Listeria populations of 108–109 CFU/mL within 48 h. As expected, L. monocytogenes grew most rapidly at 35°C, with populations of 108–109 CFU/mL being observed after only 24 h of incubation. In another study examining the influence of temperature and milk composition on growth of listeriae, Donnelly and Briggs [100] found that five L. monocytogenes strains began growing in inoculated samples of autoclaved (121°C/10 min) whole, skim, and reconstituted nonfat dry milk (11% total solids) after approximately 24–48, 2–24, 4–12, and 0.5–4.0 h of incubation at 4, 10, 22, and 37°C, respectively. Although growth rates for all Listeria strains were primarily determined by the incubation temperature, two strains of L. monocytogenes serotype 4b grew considerably faster in whole rather than skim or reconstituted nonfat dry milk during incubation at 4 and 10°C. These observations led Donnelly and Briggs [100] to suggest a possible relationship between levels of milk fat and growth rate of L. monocytogenes in milk during refrigerated storage. Furthermore, these authors suggested that enhanced psychrotrophic growth in whole milk may be related to a listerial lipase produced by both hemolytic strains of L. monocytogenes serotype 4b. Unlike both of these strains, the three remaining L. monocytogenes strains of serotypes 1 and 3 failed to exhibit enhanced growth in whole milk at 10°C and had little if any hemolytic activity on McBride Listeria Agar containing sheep blood. In contrast to what might be expected from the study just described, Rosenow and Marth [213] failed to observe any significant difference in growth rates among four strains of L. monocytogenes (two serotype 4b, two serotype 1) when they were incubated in autoclaved samples of whole and skim milk at 4, 8, 13, 21, and 35°C. The pathogen also attained lower maximum populations in whipping cream than in whole, skim, or chocolate milk at all incubation temperatures. In support of these findings, Marshall and Schmidt [174] failed to observe enhanced growth of L. monocytogenes strain Scott A (serotype 4b) in whole rather than skim milk during 8 days of incubation at 10°C. Finally, in a study to be discussed in greater detail in Chapter 12 [218], four strains of L. monocytogenes (three serotype 4b and one serotype 1) frequently attained higher maximum populations in whey samples that were defatted by centrifugation, filter sterilized, and incubated at 6°C than would be expected to occur in autoclaved skim milk, whole milk, or whipping cream after prolonged incubation at 8°C. Thus, although some L. monocytogenes strains are lipolytic as reported by Marshall and Schmidt [174], one must presently conclude that psychrotrophic growth of L. monocytogenes is not generally enhanced by the normal level of milk fat found in fluid milk. Recognizing the vital importance of carbohydrates in microbial metabolism, researchers at the CDC [198] attempted to define growth of Listeria spp. in terms of sugar utilization. An initial experiment using aerobically incubated broth media indicated that five strains of L. monocytogenes and one strain each of L. innocua, L. seeligeri, and L. ivanovii utilized only the glucose moiety of lactose, whereas single strains of L. grayi and L. murrayi utilized both the glucose and galactose of lactose. Overall, maximum cell populations, as determined by optical density, were directly proportional to the concentration of glucose (0.125%) in the growth medium. However, marked differences were observed in the ability of L. monocytogenes and L. innocua to utilize lactose, with three strains of L. monocytogenes (isolated from Mexican-style cheese in connection with the 1985 listeriosis outbreak in California) unable to grow in a medium containing lactose as the only carbohydrate. Although these observations agree with several reports [117,173,174] indicating that the pH of fluid milk is unaffected by L. monocytogenes growth, Quinto et al. [203] did report a

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sharp pH decrease in such milk after 16 and 24 days of incubation at 14 and 7°C, respectively, these differences being most likely related to strain variation. Growth of L. monocytogenes in autoclaved samples of whole and skim milk was generally similar to that previously observed by Rosenow and Marth [213], with maximum populations of 5 × 108 CFU/mL developing after extended incubation at 5 and 25°C. Except for L. seeligeri, the behavior of L. innocua and L. ivanovii did not differ markedly from that of L. monocytogenes in these samples (Figure 11.9). However, as noted by Northolt et al. [187], higher maximum populations and increased survival rates were again observed when these organisms were grown in autoclaved rather than pasteurized whole milk. Examination of milk by gas-liquid chromatography indicated that lactic, acetic, isobutyric, isovaleric, and 2-hydroxy isocaproic acids were formed during incubation. Because this milk initially contained 81–85 mg of glucose/L, the aforementioned acids likely resulted, at least in part, from fermentation of glucose. Considerably lower populations of L. monocytogenes as well as L. innocua, L. grayi, and L. murrayi also developed in glucoseoxidase-treated (an enzyme that degrades glucose) rather than untreated milk during both aerobic and anaerobic incubation, and so it is evident that glucose is one of the major substrates for growth of listeriae in milk. However, when incubated anaerobically in glucose-oxidase-treated milk, two lactose-negative L. monocytogenes isolates from Mexican-style cheese still attained final populations of 108 CFU/mL, thus suggesting the involvement of other as yet unidentified growth factors.

L. monocytogenes L. seeligeri L. ivanovil L. innocua

Listeria log10 CFU/mL

9.0

8.0

7.0

6.0 0

4

8 12 16 20 24 Days

FIGURE 11.9 Growth of Listeria spp. in pasteurized (open symbols) and autoclaved whole milk (solid symbols) incubated at 5°C. (Adapted from Pine, L., G. B. Malcolm, J.B. Brooks, and M.I. Daneshvar. 1989. Physiological studies on the growth and utilization of sugars by Listeria species. Can. J. Microbiol. 35: 245–254.)

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10 9

Strain V7 log10 CFU/mL

8 7 6 2% milk (m) 5

2%m+sugar (s)

4

2%m+cocoa(c)+carr. 2%m+c+s+carr.

3 2 0

20

40

60

80

100

120

140

160

180

200

Hours

FIGURE 11.10 Growth of L. monocytogenes strain V7 in 2% fat milk with added sugar, cocoa, and carrageenan (carr.) at 13°C. (Adapted from Rosenow, E.M. and E.H. Marth. 1987. Addition of cocoa powder, cane sugar, and carrageenan to milk enhances growth of Listeria monocytogenes. J. Food Prot. 50: 726–729, 732.)

In the aforementioned study by Rosenow and Marth [213], maximum populations of L. monocytogenes were typically about 10-fold higher in chocolate milk than in other fluid dairy products. To explain the enhanced growth of L. monocytogenes in chocolate milk, several investigators at the University of Wisconsin examined the effect of major chocolate milk constituents (i.e., cocoa, sugar, and carrageenan) on growth of this organism in autoclaved skim milk and laboratory media. Rosenow and Marth [212] found that growth of L. monocytogenes at 13°C was only slightly enhanced in skim milk containing 5% cane sugar, and that the organism attained higher final populations when commercial cocoa power (1.3%) and carrageenan stabilizer (0.5%) were used in place of cane sugar (Figure 11.10). Carrageenan also enhanced the growth rate of L. monocytogenes in the presence of cocoa; however, the organism attained similar maximum populations regardless of the presence or absence of carrageenan. These findings suggest that carrageenan may be more important in increasing contact between cocoa particles and Listeria than as a source of nutrients. Highest final populations and shortest generation times were observed when L. monocytogenes was grown in skim milk containing cocoa, sugar, and carrageenan. In addition, maximum Listeria populations obtained in skim milk containing all three ingredients (see Figure 11.10) were similar to populations observed in initial work with commercially produced chocolate milk (see Figure 11.7 and Figure 11.8). Subsequently, Pearson and Marth [194] examined growth of L. monocytogenes strain V7 at 13°C in skim milk containing various concentrations of cocoa, sugar, and carrageenan. Because some Listeria strains can utilize sucrose, it is not surprising that L. monocytogenes developed significantly higher final populations (see Figure 11.11) and had shorter generation times (5.05 vs. 5.17 h) as the concentration of cane sugar (sucrose) in skim milk was increased from 0 to 12%. (Peters and Liewen [197] also reported that addition of 7% sucrose to ultrafiltered (concentrated) skim milk caused maximum L. monocytogenes populations to increase rather than decrease.) A near-linear relationship between increasing sugar concentration and maximum attainable populations of L. monocytogenes also was observed for all but one combination of sugar, cocoa, and

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8.90

Maximum population (log10 CFU/mL)

8.80 8.70 8.60

8.50 8.40

8.30 0

3

9 6 Cane Sugar (%, W/V)

12

FIGURE 11.11 Maximum L. monocytogenes populations in skim milk alone (
), skim milk + carrageenan (), skim milk + cocoa (), and skim milk + cocoa + carrageenan () with 0, 6.5, and 12.0% cane sugar after 36 h of incubation at 13°C. Any two points differing by 0.07 log10 CFU/mL are significantly different (P < .05). (Adapted from Pearson, L.J. and E.H. Marth. 1990. Behavior of Listeria monocytogenes in the presence of cocoa, carrageenan, and sugar in a milk medium incubated with and without agitation. J. Food Prot. 53: 30–37.)

carrageenan tested; that was 12% sugar and 0.03% carrageenan (see Figure 11.11). Although addition of 0.03% carrageenan significantly lengthened generation times and decreased maximum populations compared to those observed in skim milk without carrageenan, L. monocytogenes achieved highest populations in skim milk containing 0.75% cocoa with or without carrageenan, which in turn indicates that the apparent ability of cocoa to stimulate growth of this organism in skim milk containing 0–12% sugar is independent of carrageenan. Because cocoa contains only trace amounts of fermentable carbohydrates, these authors theorized that cocoa enhanced growth of L. monocytogenes in skim milk by providing increased levels of peptides and amino acids, particularly valine, leucine, and cysteine, which are reportedly essential for growth. Additional work showed that agitation, combined with the presence of cocoa, sugar, and/or carrageenan in skim milk, enhanced growth of the pathogen at 30°C when compared to growth in the same medium that was incubated quiescently. However, growth of Listeria in skim milk alone was better without rather than with agitation. Thus, agitation most likely increased the availability of extractable nutrients from cocoa, which in turn led to enhanced growth of the pathogen. In 1968, anthocyanins in cocoa were reported to inhibit growth of salmonellae in laboratory media; however, the inhibitory effect of cocoa could be neutralized with casein [82]. These early findings prompted Pearson and Marth [196] to investigate the effect of cocoa with and without casein on growth of L. monocytogenes strain V7. Using Modified Tryptose Phosphate Broth containing 0.2% tryptose, addition of 0.75–10% cocoa increased the generation time for L. monocytogenes at 30°C (1.02–1.12 h) as compared to samples without cocoa (0.94 h). However, the pathogen generally attained higher populations when grown in media with (1.1–1.5 × 109 CFU/mL) rather than without (6.4 × 108 CFU/mL) cocoa. Interestingly, when the same medium was inoculated to contain 105 L. monocytogenes CFU/mL and agitated, the pathogen decreased to nondetectable levels in samples containing 5–10% cocoa after 15–24 h of incubation at 30°C. Nevertheless, the

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organism readily grew in the presence of 0.75% cocoa and attained higher maximum populations in media with (1.9 × 109 CFU/mL) rather than without (7.6 × 108 CFU/mL) cocoa during agitated incubation at 30°C. As previously reported for salmonellae, the presence of 1.5 or 3.0% casein neutralized the inhibitory effect of cocoa toward L. monocytogenes, the pathogen exhibiting shorter lag phases and higher maximum populations in media containing both casein and 5.0% cocoa rather than cocoa alone, and incubated quiescently at 30°C. However, results obtained during agitated incubation of cultures containing 5% cocoa were far more dramatic, with L. monocytogenes populations of 2.9 × 109 rather than 42 days in sweetened condensed milk and grow rapidly in evaporated milk, special precautions should be taken to prevent listeriae from entering these products during packaging, storage, and subsequent use.

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ULTRAFILTERED MILK Ultrafiltration, a mechanical process by which milk is filtered under pressure and concentrated, results in major compositional changes in the finished product when compared to the starting material. During ultrafiltration, 94–100% of the milk proteins and protein-bound vitamins (i.e., vitamin B12 and folic acid) remain in the retentate along with milk fat, whereas lactose is equally divided between the retentate and permeate. Increased use of ultrafiltered milk in cheesemaking prompted El-Gazzar et al. [106] to investigate the growth characteristics of L. monocytogenes in 2X and 4X retentate as well as the corresponding permeate obtained from ultrafiltered pasteurized milk. When samples were inoculated to contain about 104 CFU/mL of L. monocytogenes strain V7 or CA and incubated at 4°C, growth of both organisms was enhanced 10- to 100-fold in retentate as compared to unfiltered skim milk. Increasing the concentration of ultrafiltered skim milk retentate from 2X to 5X also resulted in faster growth, the pathogen attaining a population of 106 CFU/mL in 5X and 2X retentate after approximately 7 and 10–12 days of refrigerated storage, respectively. L. monocytogenes also grew to dangerous levels in permeate with maximum levels of 106 CFU/mL as compared to 108 CFU/mL in retentate following 30 days of incubation. When identical tyndalized samples were incubated at 32 and 40°C, both Listeria strains grew similarly in skim milk and retentate, with populations of 108 CFU/mL generally being reported after 24 h of incubation. However, as was true for samples incubated at 4°C, maximum numbers of listeriae were again 10to 100-fold lower in permeate than in retentate and unfiltered skim milk. Hence, the same care should be given to production of unfiltered milk to prevent contamination and subsequent growth of Listeria in the product during cold storage.

GROWTH OF L. MONOCYTOGENES IN MIXED CULTURES Except for several early works assessing the behavior of L. monocytogenes in raw and pasteurized milk, all studies described thus far have dealt with Listeria growth in the absence of competitive microorganisms. Although results from these studies have been of great value to the dairy industry, one should remember that pasteurized dairy products are not sterile. Psychrotrophic bacteria belonging to the genera Pseudomonas and Flavobacterium are typically present in raw milk and, similar to L. monocytogenes, can grow in milk at refrigeration temperatures both before and after milk is pasteurized. Although readily destroyed during pasteurization, these organisms universally appear in pasteurized dairy products as postpasteurization contaminants, often at levels >100 CFU/mL. Because L. monocytogenes is thought to enter dairy products primarily after pasteurization, products that contain low levels of listeriae (probably 104 CFU/g following 70 days of refrigerated storage. Although freezing the contaminated butter prevented growth of L. monocytogenes, the organism was still present at levels of 103 CFU/g after 70 days of storage at −18°C, as was also reported by Slavchev et al. [224]. Following a 2000 report from Finland linking consumption of butter to 25 listeriosis cases including 6 fatalities [172], researchers at the University of Georgia [152,153] assessed the fate of L. monocytogenes in butter and other related fat spreads as a postmanufacturing contaminant. In the first of these studies [153], sweet cream whipped unsalted butter (pH 4.51), salted light butter (pH 4.58), two yellow fat spreads (pH 4.05 and 5.37), and a light margarine (pH 5.34) were either surface-inoculated with a 6-strain cocktail of L. monocytogenes and then stored at 4.4 or 21°C under high relative humidity for 21 days, or uniformly contaminated by mixing the inoculum into the product followed by storage at 4.4 or 21°C for 14 days. For surface-inoculated samples, growth of L. monocytogenes was only seen on sweet cream whipped salted butter (pH 6.40) with populations increasing < 1 log during 21 days of storage. On all other surface-inoculated samples, numbers of Listeria decreased 1.7 to > 5 logs and > 5 logs during storage at 4.4 and 21°C, respectively. In uniformly contaminated samples of sweet cream whipped salted butter (pH 6.40), Listeria populations remained relatively constant during 14 and 7 days of storage at 4.4 and 21°C, respectively, with decreases of 1.5 to > 2.0 logs reported for the remaining products. Holliday and Beuchat [152] later assessed the impact of storage temperature on L. monocytogenes growth in seven different yellow fat spreads. In this study, one margarine, one butter–margarine blend, and five dairy/nondairy spreads and toppings were inoculated with a 6-strain cocktail of L. monocytogenes at ~106 CFU per 3.5 mL or 4.0 g and then stored at 4.4, 10 and 21°C for up to 94 days. Overall, L. monocytogenes persisted 10–14 to >94 days in the seven fat spreads examined, with longer survival seen in samples stored at 4.4 as opposed to 10 and 21°C. The only growth of Listeria was seen when the butter–margarine blend (pH 6.66) was stored at 21°C with numbers of Listeria increasing ~1.5 logs after 2 weeks and then slowly decreasing to populations near the original inoculation level after 94 days of storage. These findings are also consistent with those of Cirigliano and Keller [87] who reported that L. monocytogenes failed to grow in margarine, yellow fat spreads, or topping held at 5, 10, or 23°C. Lack of Listeria growth in these non-butter-containing yellow fat spreads has been attributed to acidity (pH < 5.60) along with the presence of preservatives (potassium sorbate, sodium benzoate, salt) and the unfavorable growth environment provided by some emulsions. Addition of garlic to butter is also reportedly somewhat effective in reducing Listeria survival when the product is stored at 10 and 37 but not at refrigeration temperatures [3]. Thus far L. monocytogenes has not been isolated from pasteurized cream manufactured in the United States; however, given the massive Listeria recall of Texas-produced fluid dairy products, including half-and-half and whipping cream, in May 1986 (see Table 11.5), one cannot assume that all pasteurized cream and butter manufactured in the United States and elsewhere will be universally free of listeriae. Hence, because at least six Class I recalls have been issued for L. monocytogenes-contaminated butter, and also because growth of L. monocytogenes has been demonstrated experimentally in both cream and butter during refrigerated storage, it is necessary to ensure that cream is pasteurized and that recontamination of pasteurized cream is prevented before and during its churning into butter. However, margarine and other non-dairy-based fat spreads are generally unable to support growth of Listeria and therefore pose a lesser risk for consumers.

NONFAT DRY MILK Dried dairy products, including nonfat dry milk, whey, and casein, also may become contaminated with pathogenic microorganisms both before and after drying. Such concerns have been raised

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recently in Australia and New Zealand [157]. Although all dry dairy products examined thus far have been Listeria-free, methods used to detect listeriae in these surveys were generally unable to recover cells that may have been injured during the drying process. Two factors, namely, the unusual thermal resistance of L. monocytogenes and the report of a milkborne listeriosis outbreak in Massachusetts during 1983, prompted Doyle et al. [104] to examine behavior of L. monocytogenes during manufacture and storage of nonfat dry milk. Samples of concentrated (30% solids) and unconcentrated (10% solids) skim milk were inoculated to contain 105–106 L. monocytogenes (strain Scott A or V7) CFU/mL and dried to moisture contents of 3.6–6.4% in a gas-fired pilot-plant-sized spray dryer with inlet and outlet air temperatures of 165 ± 2 and 67 ± 2°C, respectively. All samples of nonfat dry milk were stored at 25°C for up to 16 weeks and periodically analyzed for listeriae, using both direct plating on McBride Listeria Agar (detects uninjured cells) and cold enrichment in Tryptose Broth (detects injured and uninjured cells). Listeria populations decreased approximately 1.0–1.5 orders of magnitude during spray drying regardless of whether or not nonfat dry milk was prepared from concentrated or unconcentrated skim milk. Strain V7 was generally hardier than strain Scott A during both spray drying and storage of nonfat dry milk. Twelve to 16 weeks of storage at room temperature were required to decrease populations of strain V7 >1000-fold in nonfat dry milk, whereas only 6 weeks of storage were necessary to obtain similar decreases in numbers of strain Scott A. Overall, strains Scott A and V7 survived a maximum of 8 and 12 weeks in nonfat dry milk, respectively. Although strain Scott A generally survived equally well in nonfat dry milk prepared from concentrated and unconcentrated skim milk, strain V7 survived 2 weeks longer in nonfat dry milk manufactured from concentrated rather than unconcentrated skim milk. The higher moisture content of nonfat dry milk (i.e., 5.7 and 6.4%) prepared from concentrated skim milk may have enhanced survival of listeriae in this product during extended storage. Overall, populations of L. monocytogenes decreased >10,000-fold in nonfat dry milk during 16 weeks of storage at room temperature. Hence, if commercially produced nonfat dry milk is ever found to contain L. monocytogenes, presumably at very low levels, it may be possible to eliminate this pathogen by holding the product at room temperature for several months.

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236. Van Kessel, J.S., J.S. Karns, L. Gorski, B.J. McCluskey, and M.L. Purdue. 2004. Prevalence of salmonellae, Listeria monocytogenes, and fecal coliforms in bulk tank milk on US dairies. J. Dairy Sci. 87: 2822–2830. 237. Vassau, N. 1988. Personal communication. 238. Vazquez-Salinas, C., O. Rodas-Suarez, and E.I. Quinones-Ramirez. 2001. Occurrence of Listeria species in raw milk in farms on the outskirts of Mexico City. Food Microbiol. 18: 177–181. 239. Venables, L.J. 1989. Listeria monocytogenes in dairy products—the Victorian experience. Food Aust. 41: 942–943. 240. Vitas, A.I., V. Aguado, and I. Garcia-Jalon. 2004. Occurrence of Listeria monocytogenes in fresh and processed foods in Navarra (Spain). Int. J. Food Microbiol. 90: 349–356. 241. Waak, E., W. Tham, and M.L. Danielsson-Tham. 2002. Prevalence and fingerprinting of Listeria monocytogenes strains isolated from raw whole milk in farm bulk tanks and in dairy plant receiving tanks. Appl. Environ. Microbiol. 68: 3366–3370. 242. Wenzel, J.M. and E.H. Marth. 1990. Behavior of Listeria monocytogenes at 4 and 7°C in raw milk inoculated with a commercial culture of lactic acid bacteria. Milchwissenschaft 45: 772–774. 243. Wnorowski, T. 1990. The prevalence of Listeria species in raw milk from the Transvaal region. Sud.-Afrik. Tydsk. Suiwelk. 22: 15–21. 244. Yoshida, T., M. Sato, and K. Hirai. 1998. Prevalence of Listeria species in raw milk from farm bulk tanks in Nagano prefecture. J. Vet. Med. Sci. 60: 311–314. 245. Zapico, P., M. Medina, P. Gaya, and M. Nunez. 1998. Synergistic effect of nisin and the lactoperoxidase system on Listeria monocytogenes in skim milk. Int. J. Food Microbiol. 40: 35–42.

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and Behavior 12 Incidence of Listeria monocytogenes in Cheese and Other Fermented Dairy Products Elliot T. Ryser CONTENTS Introduction ....................................................................................................................................406 U.S. Surveillance Programs and Recalls for L. monocytogenes in Domestic and Imported Cheese ................................................................................................407 Domestic Cheese ..................................................................................................................407 Imported Cheese...................................................................................................................414 France .......................................................................................................................414 Other Western European Countries..........................................................................418 Surveys and Monitoring Programs for Listeria spp. in Cheese Produced outside the United States ............................................................................................................................419 Canada ..................................................................................................................................419 France ...................................................................................................................................421 Germany ...............................................................................................................................428 Italy .......................................................................................................................................430 Switzerland ...........................................................................................................................430 Other European Countries....................................................................................................432 Other Countries ....................................................................................................................435 Behavior of L. monocytogenes in Fermented Milks .....................................................................436 Starter Cultures, Cultured Milks, and Cream ......................................................................436 Mesophilic Starter Cultures......................................................................................436 Cultured Buttermilk..................................................................................................439 Cultured Cream ........................................................................................................441 Thermophilic Starter Cultures..................................................................................441 Yogurt .......................................................................................................................443 Kefir ..........................................................................................................................445 Traditional Fermented Milk Products ..................................................................................445 Behavior of L. monocytogenes in Cheese .....................................................................................446 Coagulants ............................................................................................................................447 Coloring Agents and Starter Distillates ...............................................................................448 Mold-Ripened Cheeses.........................................................................................................449 Camembert Cheese...................................................................................................449 Blue Cheese ..............................................................................................................453

405

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Bacterial Surface–Ripened Cheeses.....................................................................................454 Brick Cheese.............................................................................................................455 Taleggio Cheese........................................................................................................456 Tilsiter Cheese ..........................................................................................................456 Trappist Cheese ........................................................................................................456 Soft Italian Cheeses..............................................................................................................457 Mozzarella ................................................................................................................457 Semisoft and Hard Cheeses .................................................................................................457 Gouda and Maasdam Cheeses .................................................................................458 Colby Cheese............................................................................................................458 Cheddar Cheese ........................................................................................................459 Swiss Cheese ............................................................................................................462 Parmesan Cheese ......................................................................................................462 Hard Italian-Type Cheese.........................................................................................463 Hispanic Cheeses..................................................................................................................464 Queso Blanco Cheese...............................................................................................464 Queso de los Ibores Cheese .....................................................................................464 Mexican Manchego ..................................................................................................464 Chihuahua .................................................................................................................465 Pickled Cheeses ....................................................................................................................465 Feta ...........................................................................................................................465 Turkish White-Brined Cheese ..................................................................................467 Domiati Cheese ........................................................................................................467 Bulgarian White-Pickled Cheese..............................................................................467 Sudanese White-Pickled Cheese ..............................................................................467 Yugoslavian White-Brined Cheese...........................................................................468 Ewe’s and Goat’s Milk Cheese ............................................................................................468 Kachkaval Cheese.....................................................................................................468 Manchego Cheese.....................................................................................................469 Goat’s Milk Cheese ..................................................................................................469 Soft Unripened Cheese.........................................................................................................471 Cottage Cheese .........................................................................................................471 Cream Cheese...........................................................................................................474 Whey Cheeses ......................................................................................................................474 Cold-Pack Cheese Food ...........................................................................................476 Pasteurized Processed Cheese..............................................................................................477 Behavior of L. monocytogenes in Cheese as Affected by Cheese Composition ................478 Feasibility of Preparing Cheese from Raw Milk.................................................................481 Whey.....................................................................................................................................482 Brine Solutions .....................................................................................................................483 References ......................................................................................................................................486

INTRODUCTION On June 14, 1985, Listeria monocytogenes emerged from relative obscurity to the front page of many American newspapers because of a large listeriosis outbreak in California that was directly linked to consumption of Mexican-style cheese manufactured in metropolitan Los Angeles. By the time this outbreak subsided in August 1985, as many as 300 cases of listeriosis had been reported, including 85 deaths—at least 40 of which were traced to the tainted cheese. In response

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to this foodborne outbreak of listeriosis, U.S. Food and Drug Administration (FDA) officials added L. monocytogenes to their list of pathogenic organisms that should be of concern to cheesemakers and began surveying various soft domestic cheeses for listeriae. Approximately 6 months later, isolation of L. monocytogenes from several imported Brie cheeses purchased at a supermarket led to the eventual recall of approximately 300,000 tons of Brie cheese imported from France and to a real concern about the incidence of this pathogen in other European cheeses. Recall of this cheese prompted two corrective measures: (1) adoption of a cheese certification program by the United States and France to prevent importation of Listeriacontaminated cheese and (2) initiation of numerous large-scale surveys to determine the extent of Listeria contamination in virtually all types of cheese manufactured in the United States, Canada, and Western Europe. Throughout 1986 and most of 1987, the impact of Listeria on European cheesemakers was primarily in the form of economic losses from destruction of contaminated product. However, L. monocytogenes struck again late in 1987 with the report of a large listeriosis outbreak in Switzerland (see Chapter 11) in which Vacherin Mont d’Or soft-ripened cheese was incriminated as the vehicle of infection. This pathogen has continued to plague consumers of cheese with a series of smaller outbreaks traced to commercially produced European soft cheeses and, most recently, several small outbreaks in the United States that involved soft homemade Mexican-style cheese prepared from raw milk. These cheeseborne listeriosis outbreaks have prompted ongoing worldwide efforts to better understand the incidence of and behavior of L. monocytogenes during manufacture and storage of numerous cheese varieties as well as other fermented dairy products. The first half of this chapter summarizes Listeria-related recalls of cheese in the United States and results from surveys dealing with the incidence of listeriae in domestic and imported fermented dairy products. The second half of this chapter addresses the fate of L. monocytogenes during manufacture and storage of buttermilk, yogurt, and various cheeses (including whey) and the potential for cheese ingredients, such as rennet, salt brine, and coloring agents, to serve as vehicles of contamination during cheesemaking.

U.S. SURVEILLANCE PROGRAMS AND RECALLS FOR L. MONOCYTOGENES IN DOMESTIC AND IMPORTED CHEESE DOMESTIC CHEESE The concept of listeriosis as a foodborne illness is not new. Consumption of contaminated raw milk was believed to have caused several cases of listeriosis in post–World War II Germany. In 1961, Seeliger [358] also suggested sour milk, cream, and cottage cheese as possible vehicles of infection in this outbreak. Although results from two Yugoslavian studies concerned with behavior of L. monocytogenes in various fermented dairy products (i.e., cultured cream, unsalted skim milk cheese, Kachkaval cheese, and yogurt) were published in 1964 [246] and 1981 [367], no surveys dealing with the incidence of listeriae in fermented dairy products were made before contaminated Mexicanstyle cheese was linked to the California listeriosis outbreak in June 1985. Public health concerns about presence of L. monocytogenes in domestic and imported fermented dairy products as well as other foods, such as meat, poultry, seafood, fruits, and vegetables, can be traced either directly or indirectly to the 1985 listeriosis outbreak in California. Less than 1 month after the first nationwide Class I Listeria-associated recall was issued for 22 varieties (∼500,000 lb) of Mexican-style cheese contaminated with L. monocytogenes (Table 12.1), the FDA developed a series of programs designed to prevent the recurrence of such an outbreak [365] (Figure 12.1). The Domestic Soft Cheese Surveillance Program—the first of the dairy factory initiative programs—was instituted by the FDA in July 1985 and involved on-site inspection of firms manufacturing soft cheese [15]. Priority was given to manufacturers of Mexican-style soft cheese, followed by firms producing other ethnic-type soft cheeses, such as Edam, Gouda, Liederkranz,

California

11/6/1990 2/1/1991 2/14/1991

7/11/1991 10/28/1991 3/10/1992

10/14/1992

Cheese spread Mozzarella

Ricotta Jack Cold-pack cheese food

Queso fresco

Washington

New York Wisconsin Wisconsin

Arizona, Arkansas, California, Colorado, Georgia, Guam, Hawaii, Idaho, Illinois, Kansas, Louisiana, Marshall Islands, Massachusetts, Nevada, New Jersey, New Mexico, New York, Oklahoma, Oregon, Rhode Island, Samoa, Texas, Utah, Washington State Nationwide, Puerto Rico Arizona, California, Oregon, Texas Virginia, Washington, DC Illinois, North Carolina, Ohio, Pennsylvania Nationwide California, Washington State Arizona, California, Florida, Texas, Washington State Arizona, California, Idaho, Nevada, Oregon, Washington State Southeastern United States Connecticut, Georgia, Illinois, Michigan, New York, Ohio, Pennsylvania, Texas, West Virginia, Wisconsin Florida, New York Iowa, Minnesota, Wisconsin Arizona, California, Colorado, Florida, Georgia, Illinois, Indiana, Maryland, Michigan, Minnesota, New York, Ohio, Pennsylvania, Tennessee, Texas, Vermont, Virginia, Wisconsin Oregon, Washington State

Distribution

Unknown

1109 12,500 Unknown

~1362 >89,000

500,000

~10,000 127,607 10,850 1150 ~13,800 ~1400 Unknown

~500,000

Quantity (lb)

87

84 81 83 85

83 82

80

9,10,13 21,39 43,55 53 52 54,365 65

11,12

Reference FDA Enforcement Report

408

Florida Wisconsin

Ohio California Virginia Illinois Kentucky Wisconsin California

8/14/1985 3/5/1986 9/11/1986 4/17/1987 5/6/1987 8/21/1987 1/29/1988

Liederkranz (Brie,a Camembert) Soft Mexican-style: Queso fresco and 5 other varieties Semisoft Salvador-style white Soft-ripened: Old Heidelberg Soft-ripened: Bonbel and Gouda Raw milk sharp Cheddar Soft Mexican-style: Cotija, Queso fresco, and 8 other varieties; Baby Jack and Monterey Jack Mexican-style soft cheese

California

Origin

6/13/1985

Date Recall Initiated

Jalisco-brand soft Mexican-style: Cotija, Queso fresco, and 20 other varieties

Type of Cheese

TABLE 12.1 Chronological List of Class I Recalls in the United States for Domestic Cheese Contaminated with L. monocytogenes

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12/18/1992 3/4/1993 10/19/1993

4/15/1994 5/11/1994 5/20/1994 5/21/1994 5/23/1994 5/24/1994 6/15/1994

8/11/1994 8/11/1994 10/28/1994 2/2/1996

10/30/1997

11/14/1997

2/4/1998

Limburger Cheese spread

Cream cheese

Queso Prensado Cream cheese and lox Mexican-style soft white Mexican-style soft white Mexican-style soft white Queso blanco Goat milk cheese

Torte loaf cheese

Swiss cold-pack cheese food Swiss Gorgonzola

Cream cheese with vegetables

Cream cheese

Queso fresco

Wisconsin

Massachusetts

Massachusetts

Wisconsin Ohio Wisconsin

Missouri

Wisconsin Massachusetts Texas Texas Texas Wisconsin California

Wisconsin

Wisconsin Tennessee Wisconsin Alabama, Illinois, Indiana, Kentucky, Mississippi, Tennessee California, Florida, Georgia, Illinois, Indiana, Iowa, Minnesota, Nebraska, North Carolina, Ohio, South Carolina, South Dakota, Tennessee, Wisconsin Florida, New Jersey, Wisconsin Connecticut, Georgia, Massachusetts Texas Texas Texas New Jersey California, Colorado, Georgia, Illinois, Massachusetts, Michigan, New York, Oregon, Texas Illinois, Indiana, Kansas, Louisiana, Missouri, Texas Missouri, Ohio Pennsylvania California, Colorado, Florida, Georgia, Illinois, Minnesota, New Jersey, New York, North Carolina, Pennsylvania, Tennessee, Washington State, Wisconsin Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Pennsylvania, Vermont Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont Nationwide 248,938

Unknown

7340

510 2270 4500

301

1429 20 Unknown Unknown Unknown 1220 ~5682

3075

1500 11,789

(continued)

107,108

104

103

97 99 100

98

96 91 94 93 93 95 92

90

86,88 86

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Incidence and Behavior of Listeria monocytogenes 409

Date Recall Initiated 3/20/1998 3/23/1998 3/26/1998 3/27/1998 4/10/1998 4/11/1998 5/1/1998 5/26/1998 5/26/1998 7/27/1998 3/29/1999 5/22/1999

6/1/1999 6/11/1999

7/24/2000 10/17/2000 10/27/2000 11/1/2000 11/1/2000 11/2/2000 11/4/2000 11/6/2000

Type of Cheese

Queso blanco Queso fresco

Queso blanco Blue cheese Mozzarella Blue cheese Blue cheese salad dressing Queso fresco Spanish white Mexican-style fresh white Shredded Monterey Jack Raw milk Colby

Queso fresco Raw milk mild Cheddar

Cheddar Cheddar, Monterey Jack, and Muenster (19 varieties) Shredded Colby and Monterey Jack Cheddar Cheddar Jack, cheese sticks Cheddar Colby and Monterey Jack Oregon Wisconsin Wisconsin Wisconsin Wisconsin Wisconsin Mississippi Wisconsin

Nationwide Alabama, Florida, Georgia, North Carolina, South Carolina, Tennessee, Virginia Florida Nationwide California, Colorado, Nevada Nationwide Nationwide New York New York New York Texas Arizona, California, Colorado, Georgia, Illinois, Maryland, Missouri, New York, Pennsylvania, South Carolina, Tennessee, Utah New Jersey, New York Alabama, Arkansas, Florida Georgia, Michigan, Missouri, North Carolina, Pennsylvania, Tennessee, Washington State Alaska, California, Oregon, Washington State Nationwide Nationwide Nationwide Georgia, Oklahoma, Wisconsin States east of the Rocky Mountains Alabama, Florida, Louisiana, Mississippi Nationwide (except far western United States)

Distribution

1260 1,325,000 62,180 33,963 159,850 74,302 17,362 70,216

500 228

Unknown 123,000 Unknown Unknown Unknown Unknown Unknown Unknown 6000 135

248,938 Unknown

Quantity (lb)

389 389 389 389 389 389 389 389

389 389

389 389 389 103 106 389 389 389 389 389

389 109

Reference FDA Enforcement Report

410

New York Missouri

Wisconsin Wisconsin California Wisconsin Louisiana New York New York New York Iowa Missouri

Wisconsin Domestic

Origin

TABLE 12.1 (CONTINUED) Chronological List of Class I Recalls in the United States for Domestic Cheese Contaminated with L. monocytogenes

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12/8/2000 5/1/2001 12/3/2001 2/27/2002 4/25/2003 9/12/2003 10/3/2003 10/16/2003 10/22/2003 11/9/2003 12/12/2003 12/17/2004 8/22/05 12/10/05 12/14/05

Mozzarella string cheese Cheddar, Jack, and spread cheese Cheddar, Colby, and American cheese cubes Ackawi Queso fresco Blue cheese Queijo Minerio Queso fresco Raw milk Cheddar Sharp Cheddar, cold-pack cheese food Queso fresco Manouri Greek cheese

Queso seco American pasteurized processed cheese

Bleu cheese Later found to contain only L. innocua.

a

11/6/2000 11/6/2000 11/8/2000

Cheddar Colby and Monterey Jack Sharp Cheddar

Massachusetts

Florida Wisconsin

Wisconsin California Ohio Michigan New York Colorado Pennsylvania North Carolina Oregon Wisconsin New York New Jersey

Wisconsin Wisconsin Wisconsin Alabama Nationwide California, Florida, Illinois, Maryland, North Carolina, Tennessee, Virginia Minnesota, Ohio, Oklahoma, Wisconsin Nationwide Maryland, Pennsylvania Michigan New York, New Jersey Nationwide New Jersey, New York Illinois Oregon Illinois, Minnesota, Wisconsin New Jersey, New York California, Illinois, Michigan, New Jersey, New York, Pennsylvania, Virginia Florida Alabama, Illinois, Minnesota, Missouri, Texas, Washington State Massachusetts 36

2000 550,254

8688 Unknown 6200 Unknown 350 360 4758 4266 46 2736 Unknown 32

18,000 1,431,539 17,712

389

389 389

389 389 389 389 389 389 389 389 389 389 389 389

389 389 389

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Incidence and Behavior of Listeria monocytogenes 411

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Listeria, Listeriosis, and Food Safety

DOMESTIC

California listeriosis outbreak — January–August 1985

Domestic soft cheese surveillance program — begun July 1985 L. monocytogenes accidentally isolated from French Brie cheese — January 1986

IMPORTED Imported soft cheese surveillance program — begun March 1986

Aged and ripened cheese survey — begun January 1986

Temporary FDA/French soft-ripened cheese testing program — begun April 1986

Continuation of soft cheese survey — March 1987

French certification program — begun April 1987 Survey of cheese manufactured from raw milk — begun April 1987

Cheese under general pathogen surveillance program — 1988

L. monocytogenes isolated from Italian Romano cheese — June 1987

Italian cheese surveillance program — begun July 1987 Survey of import cheese in domestic status — begun December 1987

FIGURE 12.1 Surveillance programs for Listeria spp. in domestic and imported cheese. (Adapted from Archer, D. L. 1988. Review of the latest FDA information on the presence of Listeria in foods. WHO Working Group on Foodborne Listeriosis. Geneva, Switzerland, February 15–19.)

Limburger, Monterey Jack, Muenster, and Port du Salut, made from raw, heat-treated (