Pathogens and Toxins in Foods Challenges and Interventions | Foodborne Illness | Bacillus

Pathogens and Toxins in Foods
CHALLENGES AND INTERVENTIONS

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Pathogens and Toxins in Foods
CHALLENGES AND INTERVENTIONS
EDITORS

VIJAY K. JUNEJA
Microbial Food Safety Research Unit, Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, Pennsylvania

JOHN N. SOFOS
Center for Meat Safety & Quality, Food Safety Cluster of Infectious Diseases Supercluster, Department of Animal Sciences, Colorado State University, Fort Collins, Colorado

Washington, DC

Address editorial correspondence to ASM Press, 1752 N St. NW, Washington, DC 20036-2904, USA Send orders to ASM Press, P.O. Box 605, Herndon, VA 20172, USA Phone: 800-546-2416; 703-661-1593 Fax: 703-661-1501 E-mail: books@asmusa.org Online: estore.asm.org Copyright © 2010 ASM Press American Society for Microbiology 1752 N St. NW Washington, DC 20036-2904 Library of Congress Cataloging-in-Publication Data Pathogens and toxins in foods : challenges and interventions / editors, Vijay K. Juneja, John N. Sofos. p. ; cm. Includes bibliographical references and index. ISBN 978-1-55581-459-5 (hardcover) 1. Food—Microbiology. 2. Food—Toxicology. I. Juneja, Vijay K., 1956- II. Sofos, John Nikolaos. [DNLM: 1. Food Microbiology. 2. Food Contamination—prevention & control. 3. Food Handling. QW 85 P297 2009] QR115.P383 2009 664.001Ј579—dc22 2009017076 Current printing (last digit) 10 9 8 7 6 5 4 3 2 1 All Rights Reserved Printed in the United States of America Cover illustration: Fluorescent microscopic image of green fluorescent protein (GFP)-expressing Escherichia coli O157:H7 cells. Image has been converted to red. (Provided by John Sofos and his collaborators.)

CONTENTS

Contributors Preface •

• xi

vii

10. Pathogenic Vibrios in Seafood Anita C. Wright and Keith R. Schneider

146

1. Bacillus cereus and Other Bacillus spp. • 1 Mansel W. Griffiths 2. Campylobacter jejuni and Other Campylobacters • 20 Nelson A. Cox, L. Jason Richardson, and Michael T. Musgrove 3. Clostridium botulinum Michael W. Peck • 31

11. Yersinia enterocolitica and Yersinia pseudotuberculosis • 164 Maria Fredriksson-Ahomaa, Miia Lindström, and Hannu Korkeala 12. Other Bacterial Pathogens: Aeromonas, Arcobacter, Helicobacter, Mycobacterium, Plesiomonas, and Streptococcus • 181 Elaine M. D’Sa and Mark A. Harrison 13. Food-Borne Parasites • Dolores E. Hill and J. P. Dubey 195

4. Clostridium perfringens • Vijay K. Juneja, John S. Novak, and Ronald J. Labbe

53

14. Human Pathogenic Viruses in Food • 218 Lee-Ann Jaykus and Blanca Escudero-Abarca 71 15. Seafood Toxins Sherwood Hall • 233

5. Diarrheagenic Escherichia coli • Catherine S. Beauchamp and John N. Sofos 6. Listeria monocytogenes • Anna C. S. Porto-Fett, Jeffrey E. Call, Peter M. Muriana, Timothy A. Freier, and John B. Luchansky 95

16. Biogenic Amines in Foods • 248 K. Koutsoumanis, C. Tassou, and G.-J. E. Nychas 17. Fungal and Mushroom Toxins Charlene Wolf-Hall • 275

7. Salmonella • 108 Stan Bailey, L. Jason Richardson, Nelson A. Cox, and Douglas E. Cosby 8. Staphylococcal Food Poisoning Keun Seok Seo and Gregory A. Bohach 9. Shigella Keith A. Lampel • 131 • 119

18. Critical Evaluation of Uncertainties of Gluten Testing: Issues and Solutions for Food Allergen Detection • 286 Carmen Diaz-Amigo and Jupiter M. Yeung 19. Naturally Occurring Toxins in Plants • 301 Andrea R. Ottesen and Bernadene A. Magnuson
v

vi

CONTENTS

20. Chemical Residues: Incidence in the United States • 314 Stanley E. Katz and Paula Marie L. Ward 21. A European Food Safety Perspective on Residues of Veterinary Drugs and Growth-Promoting Agents • 326 Martin Danaher and Deirdre M. Prendergast 22. Prions and Prion Diseases Dragan Momcilovic • 343

26. Interventions for Hazard Control in Retail-Handled Ready-To-Eat Foods • 411 Alexandra Lianou and John N. Sofos 27. Interventions for Hazard Control at Food Service • 436 O. Peter Snyder, Jr. 28. Recent Developments in Rapid Detection Methods • 450 Lawrence D. Goodridge and Mansel W. Griffiths 29. Molecular Subtyping and Tracking of Food-Borne Bacterial Pathogens • 460 Brandon A. Carlson and Kendra K. Nightingale 30. Food Safety Management Systems • 478 Virginia N. Scott and Yuhuan Chen

23. Interventions for Hazard Control in Foods Preharvest • 357 Jarret D. Stopforth, Balasubrahmanyam Kottapalli, and John N. Sofos 24. Interventions for Hazard Control in Foods during Harvesting • 379 Mayra Márquez-González, Kerri B. Harris, and Alejandro Castillo 25. Interventions for Hazard Control during Food Processing • 396 Ifigenia Geornaras and John N. Sofos

Index

493

CONTRIBUTORS

Stan Bailey Biomerieux, Inc., Hazelwood, MI 63042 Catherine S. Beauchamp Colorado Premium Foods, 2035 2nd Ave., Greeley, CO 80631 Gregory A. Bohach Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID 83844 Jeffrey E. Call Microbial Food Safety Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038 Brandon A. Carlson Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523 Alejandro Castillo Department of Animal Science, Texas A&M University, College Station, TX 77843-2471 Yuhuan Chen Grocery Manufacturers Association, 1350 I St. N.W., Suite 300, Washington, DC 20005 Douglas E. Cosby USDA-ARS-Bacterial Epidemiology and Antimicrobial Resistant Research Unit, Russell Research Center, Athens, GA 30605 Nelson A. Cox USDA-ARS-Poultry Microbiological Safety Research Unit, Russell Research Center, Athens, GA 30605
vii

Martin Danaher Food Safety Department, Teagasc, Ashtown Food Research Centre, Ashtown, Dublin 15, Ireland Carmen Diaz-Amigo Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Laurel, MD 20708 Elaine M. D’Sa Department of Foods and Nutrition, The University of Georgia, Athens, GA 30602 J. P. Dubey U.S. Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Building 1001, BARC-East, Beltsville, MD 20705-2350 Blanca Escudero-Abarca Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695-7624 Maria Fredriksson-Ahomaa Institute of Hygiene and Technologie of Food of Animal Origin, Ludwig-Maximilians University Munich, Schoenleutnerstrasse 8, D-85764 Oberschleissheim, Germany Timothy A. Freier Corporate Food Safety and Regulatory Affairs, Cargill, Inc., Minneapolis, MN 55440-9300 Ifigenia Geornaras Center for Meat Safety & Quality, Food Safety Cluster of Infectious Diseases Supercluster, Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523-1171

viii

CONTRIBUTORS

Lawrence D. Goodridge Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523-1171 Mansel W. Griffiths Canadian Research Institute for Food Safety and Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Sherwood Hall Chemical Contaminants Branch HFS-716, Division of Bioanalytical Chemistry, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. FDA, College Park, MD 20740 Kerri B. Harris Department of Animal Science, Texas A&M University, College Station, TX 77843-2471 Mark A. Harrison Department of Food Science and Technology, The University of Georgia, Athens, GA 30602 Dolores E. Hill U.S. Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Building 1044, BARC-East, Beltsville, MD 20705-2350 Lee-Ann Jaykus Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695-7624 Vijay K. Juneja Microbial Food Safety Research Unit, Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 E. Mermaid Lane, Wyndmoor, PA 19038 Stanley E. Katz Department of Biochemistry and Microbiology, Rutgers, The State University of New Jersey, School of Environmental & Biological Sciences, 76 Lipman Drive, New Brunswick, NJ 08901-8525 Hannu Korkeala Department of Food and Environmental Hygiene, University of Helsinki, P.O. Box 66, 00014 Helsinki University, Finland Balasubrahmanyam Kottapalli Kraft Foods, 200 Deforest Ave., East Hanover, NJ 07936

K. Koutsoumanis Faculty of Agriculture, Department of Food Science and Technology, Laboratory of Food Microbiology and Hygiene, Aristotle University of Thessaloniki, P.O. Box 265, 54124, Thessaloniki, Greece Ronald J. Labbe Department of Food Science, University of Massachusetts, Amherst, MA 01003-1021 Keith A. Lampel Food and Drug Administration, HFS-710, 5100 Paint Branch Parkway, College Park, MD 20740 Alexandra Lianou Laboratory of Food Microbiology and Hygiene, Department of Food Science and Technology, Faculty of Agriculture, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece Miia Lindström Department of Food and Environmental Hygiene, University of Helsinki, P.O. Box 66, 00014 Helsinki University, Finland John B. Luchansky Microbial Food Safety Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038 Bernadene A. Magnuson Cantox Health Sciences International, 2233 Argentia Road, Suite 308, Mississauga, Ontario, Canada L5N 2X7 Mayra Márquez-González Department of Animal Science, Texas A&M University, College Station, TX 77843-2471 Dragan Momcilovic Center for Veterinary Medicine, Food and Drug Administration, Rockville, MD 20855 Peter M. Muriana Department of Animal Science, Food and Agricultural Products Research and Technology Center, Oklahoma State University, Stillwater, OK 74078 Michael T. Musgrove Egg Safety and Quality Research Unit, Russell Research Center, Athens, GA 30605

CONTRIBUTORS

ix

Kendra K. Nightingale Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523 John S. Novak American Air Liquide, Delaware Research & Technology Center, 200 GBC Drive, Newark, DE 19702-2462 G.-J. E. Nychas Agricultural University of Athens, Department of Food Science and Technology, Laboratory of Microbiology & Biotechnology of Foods, Iera Odos 75, Athens 11855, Hellas Andrea R. Ottesen Plant Sciences and Landscape Architecture, University of Maryland, College Park, MD 20742 Michael W. Peck Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom Anna C. S. Porto-Fett Microbial Food Safety Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038 Deirdre M. Prendergast Food Safety Department, Teagasc, Ashtown Food Research Centre, Ashtown, Dublin 15, Ireland L. Jason Richardson USDA-ARS-Poultry Microbiological Safety Research Unit, Russell Research Center, Athens, GA 30605 Keith R. Schneider University of Florida, Food Science and Human Nutrition Department, 359 FSHN Bldg., Newell Drive, Gainesville, FL 32611 Virginia N. Scott Grocery Manufacturers Association, 1350 I St. N.W., Suite 300, Washington, DC 20005

Keun Seok Seo Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID 83844 O. Peter Snyder, Jr. Hospitality Institute of Technology and Management, 670 Transfer Road, Suite 21A, St. Paul, MN 55114 John N. Sofos Center for Meat Safety & Quality, Food Safety Cluster of Infectious Diseases Supercluster, Department of Animal Sciences, Colorado State University, 1171 Campus Delivery, Fort Collins, CO 80523-1171 Jarret D. Stopforth PURAC, Arkelsedijk 46, 4206 AC, Gorinchem, The Netherlands C. Tassou National Agricultural Research Foundation, Institute of Technology of Agricultural Products, S. Venizelou 1, Lycovrisi 14123, Athens, Hellas Paula Marie L. Ward Department of Biochemistry and Microbiology, Rutgers, The State University of New Jersey, School of Environmental & Biological Sciences, 76 Lipman Drive, New Brunswick, NJ 08901-8525 Charlene Wolf-Hall Great Plains Institute of Food Safety, 1523 Centennial Blvd., 114A Van Es Hall, North Dakota State University, Fargo, ND 58105 Anita C. Wright University of Florida, Food Science and Human Nutrition Department, 359 FSHN Bldg., Newell Drive, Gainesville, FL 32611 Jupiter M. Yeung Grocery Manufacturers Association, Washington, DC 20005

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intrinsic and extrinsic factors that affect survival and growth in food products and contribute to illness. and postharvest intervention technologies. and postharvest environments makes it challenging to trace or control all potential sources of microbial contamination in foods. and that offer convenience and require minimum preparation before consumption. globalization of the food system. and challenges. These food safety concerns are magnified because of increased international travel and trade. safe. covers current. recent advances in biological. and factual material on topics of practical worldwide importance. Applicable and effective preharvest controls to minimize microbial contamination sources of food products of animal and plant origin. including microbial pathogens and various toxic agents. written by select experts who represent the best in the field of food safety. sources. spread. has increased significantly in the last 2 decades. Accordingly. such information has been presented in a variety of sources which are not always readily available. Until now. during harvesting. low-cost food products to consumers. and at food service have been addressed. Microorganisms previously unknown or not considered to be causes of food-borne illnesses. The emphasis of each chapter is on the type of illness and characteristics of the hazard. in conjunction with the tracking of food-borne pathogens with appropriate and effective diagnostic methods.PREFACE Our understanding of the contamination of food matrices with human health hazards. and discriminative detection methods for confirmation and trace-back of contaminated products. Foods previously thought not to be involved in foodborne illnesses or believed to be infrequent sources of specific food-borne hazards have been associated with major outbreaks or sporadic episodes of sometimes fatal illnesses. This knowledge contributes to the development of regulations and optimization of HACCP. chemical. food processing. as well as the reasons for their occurrence in foods. are continually being recognized and linked to documented outbreaks of illnesses. this compilation. can assist in developing and implementing effective hazard analysis critical control point (HACCP) plans. It is necessary for the food industry and regulatory agencies to have personnel who are knowledgeable about methodologies applied in the control or inactivation of microorganisms that may be present in foods. and incidence of the hazard in the environment and foods. food processing operations that influence the level. controlling toxins. Hence. definitive. or characteristics of the hazard. such as food-borne hazard issues. or reducing pathogens. The complexity of the preharvest. removing. with the primary objective of delivering safe foods to consumers. and consumer preferences for natural. and physical interventions to guard against food-borne hazards. nutritious. harvest. and retail handling of ready-to-eat foods. minimally processed foods that are prepared with minimum preservatives. as well as food processing operations and interventions to control hazards. These developments and interest by regulatory and xi public health agencies. Authors explore different intervention approaches to killing. concerns. Accordingly. that are safe and easily available. recent developments in intervention strategies for control of food-borne hazards preharvest. . led us to the conclusion that a comprehensive book that provides the reader with the latest research advances and insights into the safety of foods is timely. as well as our involvement through the years in food safety research. and offering quality. there has been a concerted effort by the industry and researchers to continually make advances in food processing and preservation and apply antimicrobial interventions at all stages of the food chain in order to control pathogens and toxic agents in foods and ensure their safety.

should find the book helpful in their work. handling.xii PREFACE Accordingly. private. control of hazards and spoilage of food products. Vijay K. Those in academic. and serving of food. inspection of food processing facilities. we look forward to the breakthrough discoveries that will emerge in the future as a result of the information presented in this book. as well as food consultants and lobbyists. The credit for making this book a reality goes to the authors and coauthors of various chapters. and local agencies. distribution. this book brings together these latest advances and should be of special benefit to those looking for a resource along with or in place of additional classroom training. processing. and government institutions. Sofos . industrial. Furthermore. We hope that the excellent work of the contributors will serve as a basis for new and innovative approaches to controlling food-borne hazards and significantly contributing to technologies that decrease the incidence of foodborne illnesses. Juneja John N. state. This book should be a valuable tool for those who are directly or indirectly involved in the production. or research studies on microbial control or inactivation. We commend the authors for their endeavor in compiling the necessary information in their chapters. including federal.

thuringiensis. Ontario. and ellipsoidal spores. Sofos © 2010 ASM Press. endospore-forming facultatively anaerobic bacteria. suggesting the presence of even more species (or subspecies) within this group. anthracis is exclusively lysed by the gamma phage. (1999) found an extensive genotypic diversity among the species. and this has contributed significantly to the evolution of toxin-encoding genes. There is still significant controversy surrounding the taxonomy of the B. The latter has led to the separation of groups of species from the core genus Bacillus to form new genera (Fritze. Cells of these organisms are wider than 1 ␮m. the taxonomy of these organisms has been restructured based on the application of better enrichment and isolation procedures that take into account their physiology and nutritional and cultural requirements and on the development of molecular methods. These species are generally mesophilic. pumilus.. While the members of this group are easily distinguished from other spore-formers by their inability to produce acid from mannitol and their production of lecithinase. The ubiquitous nature of these organisms makes contamination of food materials a common occurrence. University of Guelph. cereus group. with cells less than 1 ␮m wide.. Mansel W. weihenstephanensis. 1980). DC Chapter 1 Bacillus cereus and Other Bacillus spp. A psychrotrophic species. B. The genus Bacillus can be split into two groups: the B. 2008).. Since 1980. and the pathogenicity of B. 2004). B. In 1980. Griffiths BACILLUS SPP. anthracis. mycoides. are gram-positive. cereus. soil. 1998). Multiple locus sequence typing of strains of B. licheniformis.Pathogens and Toxins in Foods: Challenges and Interventions Edited by V. anthracis. cereus group. 38 species of aerobic. and further study may result in the identification of additional genospecies. B. subtilis. and B. and B. B. cereus group. and B. Guelph. taxonomic experts cooperated in establishing a comprehensive and agreed-upon list of accepted names of bacterial species. cereus has revealed three lineages. Canada N1G 2W1. and water. mycoides are hemolytic and penicillin resistant. endospore-forming bacteria were listed. mycoides. cereus and B. All members of the group are placed in 16S rRNA/DNA group 1. while Jackson et al. has also been isolated (Lechner et al. At this time. they are difficult to distinguish from each other. Mansel W. This work also demonstrated that there is widespread horizontal gene transfer among the strains. and their spores are ellipsoidal. B. Only some of the species in this group can be differentiated based on classical phenotypic tests. especially 16S rRNA/DNA sequence analysis. thuringiensis. B. Griffiths • Canadian Research Institute for Food Safety and Department of Food Science. of which 31 were allocated to the genus Bacillus (Skerman et al. cereus. cereus. K. (2000) have proposed that B. with the majority of food-related isolates belonging to lineage I (Cardazzo et al. and B. whereas B. as well as animal and plant material. thuringiensis should be regarded as one species with subspecies. However. Many of the socalled typical reactions. Helgason et al. B. B. sporangia are not swollen. the formation of crystalline parasporal inclusion bodies by B. subtilis group are closely related and include B. The resistance of their spores to adverse conditions has resulted in widespread distribution of the organism. 1 . thuringiensis. A second group of species of major interest is the B. subtilis group and the B. sporangia not swollen. They have been isolated from air. thuringiensis are usually motile. such as the rhizoid growth of B. Species of the B. with the publication of the Approved Lists of Bacterial Names. which is comprised of B. anthracis are culture dependent and/or plasmid encoded. Bacillus spp. Juneja and J. N. Washington. all species can be differentiated by molecular techniques.

The ability of spores to survive transit through the intestinal tract also depends on the food matrix and stomach acidity (Clavel et al. including epithelial cells (Faille et al. not host adapted. Generation times at 7ЊC lie between 9. or those that prevent the action of. 2002) and germination and outgrowth (Gaillard et al. However. 1991).. Germination can be inhibited by a variety of compounds. with other strains showing a wider range of heat resistance (D95 of 1. 2007). 2004). cereus (Wijnands et al... cereus spores have been generated. but cells may become gram variable when in late log or stationary phase.93. that is.3. but vitamins are not required. or in response to physical treatments.. enables spores to adhere to several types of surfaces. A surface structure of B. food preservatives. cereus are ellipsoidal and central to subterminal and do not distend the sporangia (Griffiths and Phillips. and other environmental challenges. The spores of B. However. Colonies on agar media have a dull or frosted appearance. which they sense by receptors encoded by the gerA family of operons (Hornstra et al. coupled with the presence of appendages on their surfaces.25 and 4 kGy. which have been classified as Bacillus weihenstephanensis. and their spores are widely distributed throughout nature. 2008). Meer et al. 2001). 2004)... but in boiled rice at 30ЊC the generation times can range between 26 and 57 minutes (Dufrenne et al. There was little difference in the heat resistances of spores in the presence and absence of nisin. there appears to be substantial heterogeneity among spore populations in their response to heat treatment (Cronin and Wilkinson. cereus has an absolute requirement for amino acids as growth factors. Klavenes et al.. 2005). and the dose for a 90% reduction in count lies between 1. Stalheim and Granum. CHARACTERISTICS OF BACILLUS CEREUS FOOD POISONING Two distinct types of illness have been attributed to the consumption of foods contaminated with . 1990b). 2006b). 2007).. B.4 to 9.. but this phenomenon appears to be strain dependent (Fermanian et al. Bacillus cereus spores germinate in response to particular nutrients. cereus cells to tolerate bile depends on strain and the food in which the cells are present (Clavel et al.. 1999. 2002. The corresponding dose for vegetative cells is 0. which is known to prevent spore outgrowth. one nutrient receptor on the spore. These limits for growth are not absolute and are dependent on several factors. Spore germination can occur over the temperature range of 5ЊC to 50ЊC in cooked rice and between Ϫ1ЊC and 59ЊC in laboratory media (Griffiths and Schraft. Organic acids can be used to control germination and outgrowth of B. 2000). The spore is also more resistant to irradiation.. damage to the exosporium. several or all of the spore’s nutrient receptors (Cortezzo et al. 2008)..65 kGy (De Lara et al.. 1995. or response to. with a minimum water activity (aw) for growth between 0. is involved in the adhesion of the organism to host cells and increased radiation resistance (Kotiranta et al. 1991). 1998). 1993). Several strains can grow slowly at sodium chloride concentrations of 10%.. 2002. drying. 2001. 1997b). such as glycine or a neutral l-amino acid and purine ribosides..2 GRIFFITHS Characteristics of Bacillus cereus Arguably the most important characteristic of Bacillus spp. The z value varies between about 6ЊC and 9ЊC (Byrne et al. Models describing the effects of temperature and pH on thermal inactivation (Fernandez et al. The organism grows over a pH range of approximately 4. and these can be assigned to either of two broad groups: those that inhibit the action of..17 to 0. Vegetative growth Early in the growth cycle vegetative cells are gram positive. These spores are more resistant than vegetative cells to heat. such as temperature (Griffiths and Phillips. cereus cells. now means that the temperature range for growth is between approximately 5ЊC and 50ЊC (Lechner et al. cereus spores in foods. 2001. It has been demonstrated that simulation of passage through the gastrointestinal tract at 37ЊC resulted in better germination of spores of mesophilic rather than psychrotrophic strains of B. but only when the storage temperature is Յ8ЊC (Del Torre et al. 2006). The organism can produce toxin at low temperatures. Their hydrophobic nature.. but spores were 10 times more sensitive to high-pressure treatments (517 MPa) when nisin was present (Cruz and Montville..5 to 36 minutes) (Meer et al.. Tauveron et al. The bacteria of the genus Bacillus are usually free living. or response to.4 and 75 hours.. results in a decrease in the ability of the spore to adhere to surfaces (Faille et al. 2006). the S-layer. The spore Spores from strains commonly associated with food poisoning have a decimal reduction time at 95ЊC (D95) of approximately 24 minutes. such as that which would occur during sporulation under unfavorable conditions or by shear stress that may occur during processes such as microfluidization (Feijoo et al. 2002). The emergence of psychrotrophic strains of the organism. 2002). 2005) of B.. 1990b) and high pressures of 500 MPa (Black et al.. is their ability to form refractile endospores.91 and 0. 1997). including strain (Baker and Griffiths. 2007).. Peng et al. It has also been shown that the ability of B.

The HBL enterotoxin complex produces a unique discontinuous pattern of hemolysis on blood agar (Beecher and Macmillan. Initial attempts to purify the enterotoxin of B. 1993.. (1972) were the first to relate the mechanism of pathogenicity of B. fluid replacement therapy may be required. The genes encoding the Nhe complex are situated in an operon containing three open reading frames (ORFs). 1999). Although a rare event. 1994) and cytotoxic (Beecher et al. In some B. resulting in 26 to 84 hospitalizations (Vaillant et al. the food involved will typically contain 5 8 10 to 10 cells/g. In contrast. The nucleotide and deduced amino acid sequences have been reported for all three components of HBL (Heinrichs et al. 1997). meningitis. termed B.000 cases of B. and no treatment is necessary. which has an incubation time of 1 to 5 h. cereus is selflimiting. and some B. NheB is the only . To cause emetic B. 1972). 3 B. cereus to a possible enterotoxin responsible for fluid accumulation in the ligated rabbit ileal loop (LRIL) test measured after injection of B. Bacillus cereus Diarrheal Syndrome The disease The diarrheal illness caused by B. where it oligomerizes to form pores (Beecher and Wong. The organism is also commonly associated with eye infections. cereus gastrointestinal infections may occur annually (Mead et al.. In France it has been estimated that there are 219 to 701 cases of B. which involves the B and L1 components. the Centers for Disease Control and Prevention reported 90 outbreaks and 1. 1989). septicemia. and nheC (Granum et al. and it has been proposed that the systemic complications observed with these cases are associated with enterotoxins (Girisch et al. consisting of three proteins.. Ryan et al. 1999). it has been reported that outbreaks due to B. 1997) and Taiwan (Chang and Chen. reports on the incidence of B. Since that first publication.. cereus revealed a protein complex. cereus strains may produce more than one set of the three HBL components (Beecher and Wong. As well as causing enteric illness. Schoeni and Wong. 1997. 2005). This type of B. All three components were apparently required to produce maximal fluid accumulation in the LRIL assay. Lund and Granum.. In severe cases. 1997). The proteins exhibit 20 to 24% homology and have a structure that is almost entirely ϰ-helix. This accounted for 3. cereus. Pathogenesis of the diarrheal syndrome Goepfert et al.. 2003). cereus cultures or culture supernatants (Spira and Goepfert. and can very quickly result in irreversible tissue damage (Schoeni and Wong. 2000. The toxin acts by binding to the membrane of eukaryotes. and pneumonia. 1994. such as posttraumatic endophthalmitis and metastatic endophthalmitis.. among other symptoms (Drobniewski. cereus varies from country to country. cereus. hemolysin BL (HBL). 1996.. The 105-kDa protein is a protease that is not part of the Nhe complex (Lund and Granum. cereus-associated food-borne illness annually.6% of cases of bacterial food-borne illness reported during this period (Anonymous. such as The Netherlands (Simone et al. 2005). especially in immunocompromised patients. cereus outbreaks there appears to be a clear overlap of the diarrheal and emetic syndromes (Kramer and Gilbert. cereus has been responsible for postoperative infections. 1997). 45. and 105 kDa).. 1994. 2005). cereus diarrheal disease have increased worldwide. which demonstrate homology with each other and the components of HBL (Schoeni and Wong. 1990. cereus food poisoning was first described in detail by Hauge (1955). For the period between 1998 and 2006.623 cases of infection caused by Bacillus spp. Granum’s research group in Norway has purified and characterized a nonhemolytic enterotoxin (Nhe) (Granum et al. This toxin is also composed of three protein components (39. A possible pathway of pore formation has been suggested based on the X-ray crystal structure of the B component (Madegowda et al. Okstad et al. cereus can be the most common cause of food-borne illness in some countries. 2005). Ryan et al. The HBL enterotoxin is very heterogeneous. causing nausea and vomiting that last for 6 to 24 h. As well as causing fluid accumulation in the LRIL assay. 2003). cereus food poisoning. L1. Beecher and Wong. B.CHAPTER 1 • BACILLUS CEREUS AND OTHER BACILLUS SPP. HBL is dermonecrotic (Beecher and Wong. There have been some reports of neonatal infections due to B. cereus: (i) the diarrheal syndrome.4% of outbreaks and 1. 1999). 1996). Data for the United States suggest that more than 27.. 1999). 1997). such an infection can result in bacteremia. which has an incubation time of 4 to 16 h and is manifested as abdominal pain and diarrhea that usually subsides 5 within 12 to 24 h (the infective dose is reported as 10 7 to 10 cells ingested) and (ii) the emetic syndrome... indicating that they have evolved from the duplication of a single gene (Schoeni and Wong. 2005). Schoeni and Wong. 2008). 1999. 1995). The incidence of food-borne illness caused by B. who found that an outbreak of gastroenteritis in a hospital was associated with vanilla pudding contaminated with high 8 numbers (up to 10 CFU/ml) of B. and L2. Although information on the extent of the problem is scant. nheB. 1993. nheA. that exhibited positive results in the LRIL and vascular permeability assays (Beecher and Wong. endocarditis. 2008).

cereus enterotoxin T (BcET). A cytotoxic protein (CytK) implicated in necrotic enteritis has been isolated from a B. cereus in the food. in which PCR primer pairs targeting hblC... nheB. Whereas the exact mode of action is unclear. The authors concluded that the enterotoxic activity of the original cloned bceT construct could be due to either this fusion gene or the fragment with homology to ORF 101. 1998. 2006).4 GRIFFITHS protein of the complex that binds to Vero cells. The function of NheC is not yet understood. cereus isolates involved in diarrheal disease. (1997).. hblA. respectively. 2004). 2002. these studies were conducted using enterotoxin produced in defined or semidefined media. (i) Most strains produce enterotoxin in significant amounts only after reaching cell 7 concentrations of 10 /ml. 2006). Both nhe and entFM genes were present in all isolates tested (Ngamwongsatit et al. with maximum enterotoxin activity produced during late exponential or early stationary growth (Granum et al.. and this is too long for simple enterotoxin action and may reflect the time required for spore germination. Early work suggested that enterotoxigenic B. Griffiths and Schraft. because of their common structural and functional properties. 1993). A fourth possible B.” either by bringing NheA and NheB together following binding of NheB to the target cells or by enhancing conformational changes (Lindback et al. (ii) The toxin is rapidly degraded at pH 3 (a pH similar to that found in the stomach) and hydrolyzed by the proteolytic enzymes of the digestive tract (Granum et al. all eight genes were present. and entFM were used to analyze 411 B. groups II and III contained isolates devoid of all hbl genes and cytK genes. However. Subsequently. which resulted in three deaths. 1999). hblD. However. which was thought to be similar to the gene encoding the 45-kDa cytotoxic protein described by Shinagawa and colleagues (Shinagawa. 2006). The protein was very similar to ß-barrel channel-forming toxins found in Staphylococcus aureus and Clostridium perfringens (Lund et al.. due to the ingestion of toxin produced during growth of B.. Sequencing and gene expression assays show that the genes responsible for both HBL and Nhe are transcribed from one operon. 1991). ligated DNA fragments (Hansen et al. but it probably functions as a “catalyst. cereus linked to human illness than in a reference strain and that cytK was controlled by the global regulator PlcR. confirmed this finding and identified four groups. 1990. Schoeni and Wong. 1995). thuringiensis strains. cereus. 1997). cereus strain involved in a severe outbreak of B. It was originally thought that the B. Brillard and Lereclus (2004) reported that cytK was transcribed more strongly in an isolate of B. 2002. In group I. and it should be noted that enterotoxin produced and ingested in a food matrix may be protected from damage through low pH and enzymes (Baker and Griffiths.. cereus enterotoxin (EntFM). However. cereus diarrheal syndrome was the result of intoxication. subtilis (Margot et al. nheA. Shinagawa et al. 2008).. The purified Nhe components also combined to form large pores in lipid bilayers.. 2000). 2003). but their toxicity was about 20% of that of CytK1 (Fagerlund et al. but all three proteins are needed for activity. several B. Agata et al. 2008). 2004. The isolated CytK2 proteins were hemolytic.. This claim is based on three observations. which led to the hypothesis that. not a single gene but four independent. and capable of forming membrane pores. have been identified. Schraft and Griffiths. (1995) described yet another protein. However. has been cloned and sequenced by Asano et al. 2002) and that the bceT gene was. 2005). cereus cells that grow and produce enterotoxin within the intestinal tract of the patient (toxico-infection) (Granum and Lund. cytK. and group IV strains lacked both the hbl and cytK genes. Guinebretiere et al. cereus isolates are able to grow well under anaerobic . cereus strains and 205 B. A more extensive study. cereus isolates produce either both Nhe and HBL toxins or only one (Lund and Granum. cereus diarrheal disease. cytotoxic... the HBL/Nhe and ClyA families of toxins belong to a superfamily of pore-forming cytotoxins. (iii) Incubation times of more than 24 h have been observed for many outbreaks. Again this protein was capable of forming pores in lipid bilayers (Hardy et al. nheC. It has been suggested that EntFM has similar properties to a phosphatase-associated protein with cell wall hydrolase activity from B. B. two variants of CytK. it has been postulated that the disease results from ingestion of B. in fact. plasma membrane disruption was observed with epithelia exposed to Nhe from culture supernatants of B. The cytK gene was detected in 73% of B. but only in 37% of isolates from food not implicated in disease (Guinebretiere et al. The protein and the encoding DNA sequence are not related to HBL or Nhe. One of these fragments had 93% homology to an ORF (ORF 101) located within the pathogenic island of the Bacillus anthracis pXO1 virulence plasmid. while the infectious dose in many food-borne outbreaks has been determined to 3 4 be 10 to 10 /ml. 1997. based on the presence of virulence genes. which showed positive reactions in vascular permeability reaction VPR and LRIL tests.. additional research indicated that this protein is not likely to cause diarrheal disease (Choma and Granum. CytK1 and CytK2. but not with those exposed to supernatants from a mutant strain lacking NheB and NheC (Fagerlund et al. 2001).

comprising osmotic lysis following pore formation in the plasma membrane. ResDE. 2007). This response is controlled by a twocomponent regulatory system. The enterotoxins identified for B. cereus gastroenteritis results from adhesion of spores to the epithelial cells. there is a strong stimulation of HBL production and. 5 conditions at 37ЊC and produce significant levels of enterotoxin within 6 h (Glatz and Goepfert. In one outbreak.CHAPTER 1 • BACILLUS CEREUS AND OTHER BACILLUS SPP. the spores were able to germinate and produce enterotoxins.. In another outbreak. Nhe production. the incubation period was greater than 24 hours in some cases and symptoms persisted for between 2 and several days. and HBL was synthesized later. Andersson et al. cereus strains with high hydrophobicity are more adherent to CaCo-2 cells than spores with low hydrophobicity and that two of these highly hydrophobic strains were isolated during the outbreak involving meat stew (Anderson et al. also longer than commonly observed for B. Nhe was produced first. The symptoms are usually selflimiting. has been proposed by Fagerlund et al. cereus are capable of forming pores in lipid bilayers. After adhesion. cereus diarrhegenic syndrome described by Granum (1997). Na . 1989). For many years the structure of the emetic toxin was elusive. 1976. It has been suggested that the effects on fluid absorption are due to stimulation of adenylate cyclase (Kramer and Gilbert. cereus was also cultured from both intestinal contents and pan residues. A subsequent outbreak linked to emetic B. An outbreak was described in which a 17-year-old boy exhibited B.2 kDa and the following composition: [d-O-leu-d-ala-l-O-val-l-val]3. followed by germination and enterotoxin production within the intestinal tract... alkaline and acid pH. competitors at a skiing event in Norway became ill after drinking milk. cereus emetic toxin were found in the pan used to reheat the food and in the boy’s liver and bile. 1989). cereus emetic syndrome and died of liver failure within 2 days (Mahler et al. Pathogenesis of the emetic syndrome Early experiments relying on monkey feeding trials identified the cause of the emetic syndrome as a toxin because cell-free supernatants produced the same symptoms as cell cultures (Kramer and Gilbert. to a lesser extent. and by a protein similar to the Fur redox regulator of B. It may well be possible that both modes of pathogenesis may be possible. especially since several diarrhegenic enterotoxins have been described. cereus and involving five children from a family in Belgium who had eaten a contaminated pasta salad led to the death of the youngest child due to liver failure (Dierick et al. Diarrhea is observed infrequently.. (2008). High levels of B. and Cl by epithelial cells and cause malabsorption of glucose and amino acids. Again. The infective dose observed in this 4 5 outbreak was 10 to 10 cells. 2006). as well as necrosis and mucosal damage. 1997). but he recovered. 2005). the food involved will typically contain 10 to 8 10 cells/g. and 3 of the patients were hospitalized. Bacillus cereus Emetic Syndrome The disease A second type of B. B.. which is lower than that usually associated with B. Granum et al. emetic B. At low oxidoreduction potentials. 2002). cereus enteroϩ Ϫ toxins reverse absorption of fluid. during the early-stationary-growth phase (Zigha et al. The emetic toxin has now been identified as a ring-structured peptide composed of three repeat sequences of four amino and/or oxy-acids.. For example.. 17 out of 24 people were affected after eating stews. cereus may also cause more severe disease. termed cereulide. a toxico-infection might be the mode of action for a more severe form of B. To cause 5 emesis. 1998a). B. subtilis (Duport et al. and treatment is seldom necessary. partly because there was no convenient assay. Other studies have shown that enterotoxin was not produced in defined medium during anaerobic growth (Beattie and Williams. Only limited studies have been carried out on the mode of action of Bacillus enterotoxin. was first identified in the 1970s and was associated with the consumption of fried rice (Kramer and Gilbert. It was thought to be a lipid and was known to be nonantigenic and extremely resistant to heat. 2006). Zigha et al. 2006.. 1993). cereus gastroenteritis. It was concluded that liver failure was due to the emetic toxin causing inhibition of hepatic mitochondrial fatty-acid oxidation. and a mechanism of cytotoxicity for Nhe. The dodecadepsipeptide. . the emetic syndrome. The illness was restricted to young skiers between the ages of 16 and 19. The emetic illness has an incubation time of 1 to 5 h and is manifested by nausea and vomiting that last for 6 to 24 h. The time to onset of symptoms for these patients was more than 24 hours. 1 for 3 weeks. The longer incubation time seen in these cases may be due to the time required for germination of the spores. On rare occasions. It has been demonstrated that spores of B. cereus. and proteolysis. while the older coaches and officials did not exhibit symptoms. (1998a) postulate that this more severe form of B. has a molecular mass of 1. 1989). early in the exponential growth phase. His father suffered hyperbilirubinemia and rhabdomyolysis. cereus. Both HBL and Nhe production are influenced by carbon source and growth rate of the organism during anaerobic growth (Ouhib et al.

2006).. cream. the production of emetic toxin was as high as that in the foods implicated in illness. 1997. 1994).. This has led to the conclusion that. 2002). 2002).. The emetic syndrome is most frequently associated with starchy foods. and a small quantity of toxin was detectable in milk and soy milk. 1991)..953 and pH values of Ͼ5. and eggs. higher cereulide concentrations were achieved (4 ␮g/g in puree and 3. These genes are found only in emetic strains of B.2 ␮g/g in penne) than in boiled rice (2 ␮g/g) (Rajkovic et al. 1988). 2002). cereus and stored without shaking. anthracis.. 1994. (2005) have described a quantitative assay for cereulide based on uncoupling of the respiratory activity of rat liver mitochondria. The emetic toxin can be assayed by determining vacuolation induced in HEp-2 cells (Hughes et al..28 ␮g/g (Agata et al. Regions flanking the ces gene locus have high homology to virulence plasmids of B. and an ABC transporter (downstream).. withstood heating at 121ЊC for 90 min. When potato puree and penne pasta were inoculated with cereulide-producing strains of B. especially rice dishes (Kramer and Gilbert.6 (Jaaskelainen et al. Cytotoxicity is accompanied by inhibition of RNA synthesis and cell proliferation at cereulide concentrations of 2 nM (Andersson et al..4%) have a clearly lower incidence. Low levels of emetic toxin were detectable in eggs and meat and their products.. 2004). and B. 1994). 1994. Cereulide was not observed following . which suggests that the toxin may possess immunomodulating properties (Paananen et al. 8 2006).. At high doses. cereus distinguishes between emetic and diarrheal syndromes.6 GRIFFITHS It closely resembles the potassium ionophore valinomycin (Agata et al. cereus and are located on a 208-kb megaplasmid. The toxin also inhibits natural killer cells in vitro. 2005b). England and Wales.. which is based on high-performance liquid chromatography and mass spectroscopy (Haggblom et al. When challenge studies were performed using an emetic toxin-producing strain of B. besides the insecticidal and anthrax toxins of B. with toxic effects due to potassium-ionophoretic properties (Agata et al. cereus. Unlike the diarrheal strains of B.01 to 1. poultry. 2002). Szabo et al. The reasons for these differences are not known. 2005a).. cereulide was not formed in products stored at 4 to 8ЊC (Jaaskelainen et al. despite counts in excess of 10 CFU/g being attained in all three products. thuringiensis and B.. 2003). and the United States (1. cereulide production in these products was more than 10-fold lower. Oxygen is required for the production of the toxin (Jaaskelainen et al. a third type of B.. The ces gene cluster (24 kb) comprises seven coding sequences (CDSs). anthracis. 1999). cereus. cereus group toxins exists.. cereulide can cause a massive degeneration of hepatocytes (Yokoyama et al. 2007). mayonnaise. cereus emetic strains were present at cell 6 numbers of Ն10 CFU/g in products with aw values of Ͼ0. and these toxins are encoded on megaplasmids. presumably due to the low pH generated by acetic acid (Agata et al. when not aerated.5 ␮g/g) were produced during growth of B. Incidence and Transmission through Food The mild and transient character of the B. 1995. encoding a phosphopantetheinyl transferase and two enzymes for the activation and incorporation of monomers in the growing peptide chain. vegetables. which tests toxicity of cereulide to boar spermatozoa (Andersson et al.. thuringiensis. The amount of emetic toxin in 13 of 14 food samples implicated in emetic-type illness ranged from 0. B. Cereulide is produced by a nonribosomal peptide synthetase (Ehling-Schulz et al. In farinaceous foods.3 to 5.. 1999. emetic strains have very low diversity and consist of a single. A rapid bioassay. Cereulide was formed when B. However. distinct cluster of isolates that are unable to degrade starch and do not ferment salicin (Ehling-Schulz et al. Sakurai et al. Foods most often implicated as vehicles for transmission of the diarrheal syndrome include meat and meat dishes. Japan. which is encoded by a cereulide synthetase (ces) gene cluster (Ehling-Schulz et al. The toxin was stable at pH 2 to 11. None of the reports on foodborne disease outbreaks caused by B. spices. and a chemical assay. cereus in rice-containing pastries at ambient temperatures (21 to 23ЊC). cereus food-borne diseases seems to be higher in countries of northern Europe (20 to 33%) and in Canada (14%). 2004). 1998b.. and was not inactivated by treatment with trypsin and pepsin (Shinagawa et al.. High levels of toxin (0. 1996). but it is likely that the outbreaks reflect the diarrheal syndrome.. the organism was capable of rapid growth in boiled rice and produced emetic toxin at both 30 and 35ЊC... and catsup. a CDS encoding a putative hydrolase (upstream region). The incidence of B. 2003). cereus food-borne diseases likely results in the disease being vastly underreported. respectively. Mikami et al. Kawamura-Sato et al. These include the typical nonribosomal peptide synthetase genes.2 to 4. With aeration. cereus that are extremely heterogeneous. 1989). and this has been aided by the development of colorimetric modifications (Finlay et al. are now available.. Cereulide stimulates the vagus afferent by binding to 5-HT3 receptors and induces swelling of mitochondria. Mikkola et al. Bacterial growth and toxin production were inhibited in foods cooked with vinegar.

2002). a number of factors. cereus. but they should not pose a risk in refrigerated foods. This led the investigators to suggest that the cause of the illness was due either to a new cereulidetype toxin or to the high levels of B. however. cereus illness (Lund et al. cereus strains isolated from dairy products are not highly cytotoxic (Arnesen et al. but cereulide was not detected. In general.. meat and meat products. The dairy industry faces problems due to B. and one strain was psychrotrophic. short holding time processing.. The last can be a source of contamination of prepared meals (Banerjee and Sarkar. subtilis present. It has been reported that infant food containing both cereal and milk powder are able to support greater levels of cereulide production than other infant formulas and that mishandling and temperature abuse of reconstituted formula were high-risk activities (Shaheen et al. but levels reached 1. Of seven strains isolated from these products. animal feces. cereus spores. Hanson et al.. An infant food containing cereal and dried infant milk formula was associated with illness in two infants. 2005). 2008). . te Giffel et al. Fortunately. The organism is also frequently isolated from milk and dairy products. cereus enterotoxin. temperature. suggested that these properties make emetic strains a special risk in heat-processed foods or preheated foods that are kept warm. subtilis and B.. which has resulted in shelf-life issues with milks pasteurized at temperatures in excess of the recommended minimum for high-temperature. Thus. which presented as rapid projectile vomiting (Duc et al... Similarly. cereus can easily contaminate various types of foods. including food type. 2000). Altayar and Sutherland (2006) found that 45. CEREUS IN FOODS Presence of B. 2006). and pasteurization does not eliminate them (Andersson et al. Indeed. and spices (te Giffel et al.. aeration. their spores readily attach to processing equipment. Cereulide is also remarkably resistant to heat and alkaline pH (Rajkovic et al. All the positive isolates came from washed or unwashed potato skins. cereus and that about 75% of these were enterotoxigenic.8% of 271 samples of soils. Griffiths and Phillips. Because of the ubiquitous distribution of the organism. and strain. with a prevalence of 9 to 37% in raw milk and 2 to 35% in pasteurized milk (te Giffel et al. it is virtually impossible to obtain raw products that are free from B. Studies indicate that both raw and pasteurized milk will harbor psychrotrophic B. No spores were detected on the processing equipment. cereus in Foods Members of the B. pasteurization may result in activation and germination of spores (Griffiths and Phillips. 1989... cereus group are ubiquitously distributed in the environment. (1997) reported that 40% of pasteurized milks sampled from household refrigerators in The Netherlands were contaminated with B. only 3 were positive for emetic toxin production. Carlin et al. 1990. used in the formulation (Guinebretiere et al. 2004). The appearance of psychrotrophic strains in the dairy industry has added a new dimension to B. cereus surveillance in food. it has been determined that the level of cereulide retained in paper is too low to present a risk to human health (Hoornstra et al. such as milk protein and starch. cereus were isolated from the product. Griffiths.. in many cases. PRESENCE. GROWTH. It has also been shown that emetic strains of B.. 1995). Subsequent work has suggested that the presence of B. cereus in aerated milk.CHAPTER 1 • BACILLUS CEREUS AND OTHER BACILLUS SPP. 1997a). and the source of the contamination is the raw vegetable and the texturing agents.1 ␮g/ml in statically incubated milk. ready-to-eat vegetables. 2003). cereus because it is virtually impossible to exclude the organism from milk. 1995). 2007). mainly because of their spore-forming capabilities. B. and that the source of the contamination of raw milk was primarily silage (te Giffel et al. Odumeru et al. 7 growth of emetic strains of B.. cereus could be present in paper pulp and the products made from it. cereus was also isolated from vacuum-packaged and film-packaged soyaand cereal-based vegetarian foods (Fang et al. Vegetable purees have been implicated in outbreaks of B. cereus. For example. spores of emetic strains also were more resistant to heat (90ЊC) (Carlin et al. 2001). determine the amount of cereulide that will be produced.. cereus were not able to grow at temperatures below 10ЊC but were able to grow at 48ЊC. 1996). pH. 2005). These psychrotrophic strains have an average generation time of 17 h at 6ЊC and produce enterotoxin during extended storage at slightly abusive temperatures (Christiansson et al.. one produced diarrheal toxin and four caused lethality in mice.. In coffee creamers stored at ambient temperatures. pasteurized liquid egg.. Both B. 1990a). B. it seems that the majority of B. Thus. 1990b. and raw and processed vegetables were positive for B. 2006). especially products of plant origin. cereus in pasteurized milk may be the result of postpasteurization contamination at the filler (Eneroth et al. 1999). Of the 325 isolates obtained. (1997) found that 38% of pasteurized milks stored at 10ЊC until their expiry date contained B.. cereus can grow to levels of public health concern within 11 h (Feijoo et al. B. 2006). AND SURVIVAL OF B. rice. It was also postulated that B.

1994.93. In the product groups’ flavorings. meat and meat products. milk and milk products. Heat Resistance and Germination of Spores Effective destruction of B. Growth and Survival An excellent overview of the factors affecting growth and survival of B. A similar wide range for D95 (2. cereus.. fish and fish products. 2000. cereus cells in each of the food groups by using mannitol-egg yolk-polymyxin agar and characterized the isolates for growth temperature range and the presence of toxin genes by PCR. of the 39 Bacillus isolates obtained from whipping cream. The incidence of the latter was greatest in the milk-and-milk products’ group. 1998a. Cereulide was produced by 8. Most of the strains isolated during the study were mesophilic (89. cereus counts 5 of Ͼ10 CFU/g.0 to 7. cereus in pasta and rice products. In the presence of NaCl as a humectant.3 minutes) was observed for spores in milk-based infant formula. The optimum pH for B. when glycerol was used as a humectant. such as infant formula (Becker et al. with growth possible between 4 and 55ЊC. but no toxin production was observed with cocoa powder (Cooper and McKillip. such as heat.1 value of 0. and oil and fat products.03 to 2. Psychrotrophic and mesophilic strains of Bacillus can be distinguished based on PCR targeting of genes responsible for encoding the major cold shock protein (Francis et al. and CytK were present in Ͼ97%. cereus to produce enterotoxin in infant milk formula as well as coffee creamer was confirmed. 2004).8 GRIFFITHS The psychrotrophic B. the D95 value ranged from 1. and 50% of the isolates. but all those isolated also had genes for one or more of the other virulence factors.0. The most important aspects are summarized below. cereus associated with food-borne illness are more resistant to adverse pH environments than those that did not produce gastrointestinal infections (Garcia-Arribas and Kramer. milk and milk products.0. ready-to-eat foods. The minimum pH for growth is 5. the organism was isolated from evaporators and drip trays of chilling units in catering plants and in plants processing cooked meats (Evans et al. Rowan and Anderson. However. and ready-to-eat foods. The response of B.9%). only 4 were identified as B. They determined the presence and number of B.93. 1990). 2001).4% of the isolates were psychrotrophic. vegetable(s) and vegetable products.2% of the isolates.8 to 19. 0. Perhaps surprisingly.8. 0.. This species has also been isolated from liquid egg (Baron et al.39% potassium sorbate. acid. For example. and the maximum pH is 8. 1998b). There is evidence that B. cereus has an optimum growth temperature of 30 to 40ЊC. Butylated hydroxyanisole at concentrations of 0.. and pastry. 1998)..0.29%. Mesophilic B.19%.92. As well as the foods themselves.5% inhibited B. growth was possible at an aw of 0. 1998a).16%. processing plant equipment can harbor B. Bacillus cereus has also been isolated from highly controlled foods. pastry. The ability of B. 0. A relatively low percentage of isolates from the flavorings group contained genes encoding Nhe.14%. while only 4. weihenstephanensis and none of these were cytotoxic. 2006). 1998b). cereus growth at 32ЊC over 24 h. (2006a) categorized randomly selected foods into eight product groups: oils. The genes for Nhe. 1998). cereus can be found in the monograph Microorganisms in Foods 5 published by the International Committee on Microbiological Specifications of Foods (ICMSF) in 1996. However. Strains that grow at 7ЊC or below are identified as psychrotrophic and will not grow above 43ЊC. B. cereus isolates described in these earlier reports may be strains of B. 0. and 0.. cereus spores is the ultimate goal for safe foods with an extended shelflife.1 to 0.9ЊC) has been reported for spores suspended in phosphate buffer at pH 7. Wijnands et al. Under ideal conditions B.. cereus usually grows at temperatures of 15ЊC to 50Њor 55ЊC. the isolates obtained from these products did not exhibit an elevated prevalence of the cereulide gene. respectively. 1997). It has been shown that strains of B. In rice filling held at 23ЊC. flavorings. 2007).1 min.04%. dietary supplements for hospitalized human immunodeficiency virus patients (Rowan and Anderson. Dufrenne et al. contained B. and osmotic stress. although all possessed at least one enterotoxin (Stenfors et al. A D121. cereus (Faille et al.7 to 15.26% sorbic acid and by 0. 2002). HBL. cereus will not grow at an aw of 0. with generation times of approximately 23 minutes at 30ЊC. has been described by Browne and Dowds (2001. but not 0. growth was inhibited by 0. 2002). fats.. For spores suspended in milk. cereus growth has been reported as 6.35 minutes (z value ϭ 7.9 to 9. Only a few reports are available on the effects of organic acids and chemical preservatives on B. approximately 66%. cereus can demonstrate a cold adaptation response following repeated exposure to low temperatures (Foegeding and Berry. vegetables and vegetable products. cereus to a number of stresses. weihenstephanensis (Lechner et al. and airline meals (Hatakka. (1995) reported that D90 values for . although there was a relatively high incidence of B. respectively. and a higher-than-average number of isolates in the pastry group possessed the genes encoding both HBL and Nhe.

CHAPTER 1 • BACILLUS CEREUS AND OTHER BACILLUS SPP. which is based on the detection of the phosphatidylinositolspecific phospholipase C enzyme present in B. cereus strains.2 to 9. A method for the detection of B. 2003) and the effect of heat shock and germinant concentration on germination (Collado et al. cereus at different pH values and temperatures (Collado et al. but the value for one strain was Ͼ100 min. a nutrient-rich broth. and growth in 0. has been described. VRM. spores of psychrotrophic B. because the use of conventional media could result in substantial misidentification and underestimation of B. Both rely on the presence of lecithinase (phospholipase C) and the absence of mannitol fermentation in B. the API 50 CHB test. However.. and methods for the detection of B. (1997). If enrichment is required for detection of low numbers in foods. They showed that the chromogenic media performed as well as the conventional standard media and were useful if staff were not highly trained in identification of the organism. l-tyrosine degradation. cereus. Molecular-based assays PCR is now routinely used for the detection of food-borne pathogens. D values of B. megaterium and many other competitive bacteria present in foods (DeVasconellos and Rabinovitch. Germination of spores requires the presence of purine ribosides and glycine or a neutral l-amino acid. and detection of the organism is achieved primarily by direct plating onto selective agar media. such as brain heart infusion broth or trypticase soy broth. A biochemical Bacillus identification system. the chromogenic media could not detect some B. The procedure includes a heat treatment at 72ЊC and incubation with trypsin and Triton X-100 at 55ЊC. is available from bioMérieux. cereus spores in milk has been described by Christiansson et al. a pleiotropic regulator of various virulence factors and B. Two selective media are widely used for the isolation and presumptive identification of B. In general. Presumptive colonies isolated from selective media are characterized by various confirmatory tests. in cooked rice. cereus are less heat resistant than spores of mesophilic strains (Dufrenne et al. One group of these assays aims at detecting B. 1998). Noncultural Detection Methods With the advent of rapid microbiological methods for the detection of bacteria in foods. The samples are filtered through a membrane filter to concentrate the spores. revealed a significant correlation between atypical colony appearance and specific variances within the plcR gene of those strains.. Tris and resazurin are included in VRM to inhibit growth of B. Fricker et al. l-alanine is the most effective amino acid stimulating germination. ISOLATION AND IDENTIFICATION Cultural Methods Both emetic and diarrhegenic B. cereus spores ranged from about 32 min at 85ЊC to about 2 min at 95ЊC (z value ϭ 6. 9 spores of 12 psychrotrophic strains of B.2 min. In addition. (2008) evaluated two chromogenic media (chromogenic B. cereus-specific enzymes. 2006). cereus . cereus and closely related species will be gram-positive.. cereus. 2006) have been published. germination temperatures ranged from 5ЊC to 50ЊC (Kramer and Gilbert. Mathematical models to describe the heat resistance of B. In laboratory media.6) (Byrne et al. thuringiensis chromogenic agar plating medium. VogesProskauer assays. In pork luncheon meat. several culture-independent approaches have been proposed. cereus. Notermans and Batt. The filter is then placed onto solid agar media to allow for growth of the spores.000 IU/liter) is recommended (Van Netten and Kramer. cereus ranged from 2. cereus.001% lysozyme. Spores are able to sense the presence of these germinants in the environment by receptors encoded by the gerA family of operons (Hornstra et al. germination has been observed at Ϫ1ЊC to 59ЊC. 1995). catalase-positive rods with spores that do not swell the sporangium.. A medium that requires no polymyxin. The test determines the ability of an isolate to assimilate 49 carbohydrates and has become a reference method for the biochemical identification of Bacillus species. cereus have a relatively high infective dose. Germination temperatures vary greatly between strains and are strongly influenced by the substratum. 1992). cereus agar and BCM) and two standard media (PEMBA and MYP) for the isolation and identification of B. 1994. Both media contain polymyxin to inhibit growth of competitive organisms (van Netten and Kramer. (2001) developed the BCM B. cereus: mannitol-egg yolk-polymyxin (MYP) agar and polymyxin-pyruvate-egg yolk-mannitolbromothymol blue agar (PEMBA). cereus and related organisms have been described. they will produce acid from glucose anaerobically and show a positive reaction for nitrate reduction. 1989). Sequence analysis of the plcR gene. cereus/B. 2005). supplemented with polymyxin (1. Peng et al. Presumptive colonies are surrounded by an egg yolk precipitate and have a violet-red background on MYP and a turquoise or peacock blue color on PEMBA.. 1992). B.

cereus group. However. The authors used a DNA purification procedure that allowed the detection of less than 1 CFU per ml of milk without a preenrichment step.. 2008) and a piezoelectric excited cantilever (Campbell and Mutharasan. the assay was capable of removing more than 90% of the spores from phosphate buffer and 37% from whole milk. Yamada et al. 1999). 2007). PCR protocols have been described that allow the selective detection of psychrotrophic B. cereus and other species. 1998) and for Nhe and CytE (Granum et al. As described above. 2007).. Applying principal component analysis to these spectra reveals clear distinctions between different bacterial strains. Schraft and Griffiths (1995) reported a sensitive test for the detection of B.. Real time PCR-based methods for the identification of emetic strains of B. some B. 1999. such as nucleic acid sequence-based amplification. (Gore et al. More recently. Antibodies against spores and vegetative cells Koo et al. DNA microarrays have been used to detect the presence of genes encoding the B. this magnetic capture hybridization PCR assay might be useful for the detection of B. cereus. McGovern et al.. even in food matrices (AlHoly et al. 2006). 1998). assays that simultaneously target all potential enterotoxin genes would seem to have the greatest potential. there are several different proteins or protein complexes that have been implicated in the B. 1998. when they are present both as vegetative cells (Lin et al. 2002) have been described... Although no lower detection limits were reported. cereus have been described based on amplification of sequences of the cereulide synthetase genes (Fricker et al. Other amplification technologies.. They evaluated the assay using experimentally inoculated rice homogenates and found that a detection limit of less than 1 CFU/g could be achieved when a 15-h preenrichment and two-step filtration were applied before the PCR amplification. phage-based biosensor capable of detecting 10 cells/ml within 8 h has been developed and measures ␣-glucosidase activity released from cells upon phage lysis (Yemini et al. (1998b) have engineered a monoclonal single-chain antibody.. anthracis spores using evanescent waves (Love et al. cereus in foods. cereus. Matrix-assisted laser desorption ionization– time of flight (mass spectrometry) can also be used to . cereus (Kretzer et al. 2001). cereus. while other assays target specific subgroups of B. cereus T spores. but not with spores. These tests are based on the gene sequence of the toxin to be detected. Guinebretiere et al. cereus toxins (Liu et al. The technique is able to differentiate between B. Charni et al. 2007. Damgaard et al. 2004) and as spores (Perkins et al. which targets 16S rRNA genes. cereus vegetative cells. an amperometric. cell wall-binding domains of bacteriophage endolysins have been used to capture cells of B. 2008) for detection have been described. cereus.. When samples were inoculated 4 with 5 ϫ 10 B. such as psychrotrophic or enterotoxigenic strains (Hsieh et al.. (1996a) combined magnetic capture hybridization with a nested PCR assay targeting the phospholipase gene of B. have been used to detect hblC expression in Bacillus spp. cereus. Fourier transform infrared spectroscopy can provide a way to differentiate bacterial genera and species through unique Fourier transform infrared vibrational combination bands produced from active components of bacterial cells. High-affinity. real-time. (2000) reported the production of monoclonal antibodies which reacted only with B. Several PCR assays have been described for the detection of enterotoxigenic B.. has been developed by Hansen et al. The antibody was fused with a streptavidin molecule and the fusion protein expressed in Escherichia coli (Koo et al.. This antibodystreptavidin protein was then used in a magnetic beadbased immunoassay to concentrate B. which is reported to be specific for B. thuringiensis in naturally contaminated rhizosphere samples. cereus strains that do not possess these rRNA gene signature sequences can grow at low temperatures (Stenfors and Granum. 2007).. cereus diarrheal syndrome. 2003). 1998a). 2005). A PCR-based assay for the detection of all members of the B. antibody-based biosensors for B. cereus in milk.. Protocols for the detection of the HBL and BcET toxin complex (Mantynen and Lindstrom.. This gene encodes the enzyme responsible for the positive egg yolk reaction used for the presumptive identification of the organism on selective plating media. cereus and B.. 2007). 1998) and gene sequences for the major B. which is based on primers targeting the phospholipase gene of B. Winder and Goodacre.10 GRIFFITHS per se. Other detection methods Methods for the detection of Bacillus using bacteriophage in a variety of ways have been reported. cereus spores. cereus spores from liquid samples. More recently. In addition. The primers used in the assay target the psychrotrophic and mesophilic rRNA gene signature sequences (von Stetten et al. (1999) designed a primer set for a PCR assay based on the gyrase B gene (gyrB) of B. cereus cold shock proteins CspF and CspA (Francis et al. thuringiensis to detect B. (2001).

Helgason et al.. biochemical profiles. cereus strains (Helyer et al. which is based on the use of primers designed for conserved regions of housekeeping genes.CHAPTER 1 • BACILLUS CEREUS AND OTHER BACILLUS SPP.e. The specificity. genotypic methods are frequently used for epidemiological typing of B. than the biochemical Vitek system (42. serology. anthracis but was of limited value for differentiation of B. RAPD typing also allowed the separation of mesophilic from psychrotrophic B. proportion of reference negative strains not identified as B. 2007). i. the subunit L2 of the tripartite enterotoxin complex (HBL) described by Beecher and Wong (1994). Mostly phage typing and fatty acid profiling have been applied in more recent studies. cereus group.. e. (1991) found phage typing could be used to exclude packaging materials as a potential source of B. was very good (97.5%. 1998. 1997. the RiboPrinter. 1994). adk. All isolates could be typed with high reproducibility. Traditionally. It detects a 41-kDa protein (NheA). and it has been used to differentiate strains of the B. ftsA. cereus strain may produce either HBL or Nhe or both. This kit is marketed under the name Bacillus diarrhoeal enterotoxin visual immunoassay kit (Tecra). ccpA. 1996). Andersson et al.5%.. 1998). For example... and sequencing of 330. cereus isolates (Lechner et al. cereus with either RAPD or AFLP (Nilsson et al. cereus food poisoning.e. The second assay uses the enzymelinked immunosorbent assay format. B. Typing Methods Fast and accurate identification methods for B. the microbial identification system described above. randomly amplified polymorphic DNA (RAPD) has been applied to type over 2. Newark. anthracis (Lasch et al. 2008). cereus diarrhegenic toxins. (1995) described a 12-phage typing scheme which could be used in epidemiological investigations for the identification of sources of B.. The fatty acid-based microbial identification system (MIDI Inc. phage typing.. it has not found widespread application in the food industry. cereus are also important in the food industry.to 504-bp internal fragments of seven of these genes. pyrE. respectively. cereus. cereus with 21 cultures from seven different outbreaks. When the definition of a correct identification was expanded to include closely related members of the B. Detection of Toxins Two commercial kits are available for the detection of B.. Pyrolysis mass spectrometry is capable of differentiating strains of B.. it is . Amplified fragment length polymorphism (AFLP) is a genotyping method with significantly higher reproducibility than that of RAPD. Ripabelli et al. The reverse passive latex agglutination enterotoxin assay produced by Oxoid reacts with a 43-kDa protein. Odumeru et al.. cereus strains colonizing the processing environment of dairy plants (Pirttijarvi et al. glpT. mycoides and B. Fatty acid analysis can now be performed with a fully automated system. 2003). 2006. but they detect different antigens (Day et al. cereus group (Hill et al.000 mesophilic and thermophilic Bacillus species isolated from an industrial setting (Ronimus et al. thuringiensis. Thus. for identification. (2000) evaluated AFLP for epidemiological typing of B. (1999) evaluated two automated microbial identification systems for their ability to accurately and reproducibly identify 40 B. and Ahmed et al. Väisänen et al. Since phage typing requires continuous phage propagation. An automated ribotyping instrument.5%) for both systems. DE) had a slightly better (55%) sensitivity. the sensitivity of the systems increased to 82. which is part of the nonhemolytic enterotoxin complex (Nhe) described by Lund and Granum (1996).g. methods for tracing possible contamination sources throughout the production chain are of crucial importance.. Whole-cell fatty acid analysis has been used to trace and identify B.. in milk (Durak et al. The introduction of a rapid DNA preparation method for Bacillus cereus will facilitate large-scale typing of B. When B. In addition to phenotypic typing. The API 50 CHB biochemical test was used as a reference test. 1997). The authors were able to trace individual species from the feedstock to the finished product in a food processing plant.. Any enterotoxigenic B. the proportion of reference positive strains correctly identified. i. cereus group. and fatty acid analysis have been used for typing. A molecular typing method based on sequencing of the rpoB gene has been applied for the differentiation of Bacillus spp. cereus is repeatedly isolated in finished products or suspected of causing a food-borne disease outbreak. 2004).. Schraft et al. 11 detect spores and vegetative cells of B. (1999) compared RAPD typing with automated ribotyping and found the latter to be a fast and reliable method. cereus in dairy products. Huck et al. recF.5% and 67. and a unique AFLP pattern was generated for isolates of each outbreak. 1998). Hazelwood. cereus isolates.. cereus. MO). and sucC. Both kits are immunoassays. has been developed by Qualicon Inc. which was only slightly less discriminatory than the more labor-intensive RAPD technique. (2004) have described a multilocus sequence typing scheme for the B. bioMérieux Vitek.

mojavensis. cereus enterotoxins. The agent was nonproteinaceous. cereus group have been reported to cause foodborne disease (Kramer and Gilbert. pumilus were detected in cooked. depleted cellular ATP. Symptoms included dizziness. cereus and still widely used as an insecticide. F. 1989). protease. The isolates associated with illness were ␤-hemolytic and grew anaerobically and at temperatures of 55ЊC. and back pain. produced by B.. 2005. subtilis. licheniformis obtained from foods involved in outbreaks. An outbreak involving three people who became ill after eating dinner in a Chinese restaurant was described by From et al. The bioassay using boar spermatozoa and the high-pressure liquid chromatography–mass spectrometry procedure described in Bacillus cereus Emetic Syndrome: pathogenesis of the emetic syndrome are the only tests reported for the detection of the emetic toxin cereulide. and acid or alkali. 2008). Cell-free cultures of toxin-producing isolates of B. 1999). but no mitochondrial damage was observed (SalkinojaSalonen et al. cereus enterotoxins (Gaviria Rivera et al. B. OUTBREAKS CAUSED BY BACILLUS SPP. 1999. with an incuba7 tion time of about 8 h. Ped-2E9. chills. lichenysin.. thuringiensis. 2005). 2007b). The B. seafood with rice. and they called for a reevaluation of the role of these compounds in food-borne illness (From et al.. was demonstrated for approximately 50% of B. licheniformis cells isolated from food (Salkinoja-Salonen et al. indicating that these Bacillus species may potentially cause food-borne diarrhegenic disease (Rowan et al. OTHER THAN B. and caused swelling of the acrosomes of spermatozoa. B. reheated rice. 2005). licheniformis isolated from an infant milk formula was thought to be responsible for the death of an infant (Salkinoja-Salonen et al.12 GRIFFITHS not surprising that early comparisons of the two commercial test kits have triggered contradicting reports on their accuracy in detecting the enterotoxin. 2001). B. and B. licheniformis.. Based on today’s knowledge about B. and 7 pastry)... The pumilacidin complex isolated from the B. High levels of B. a few hours later. 2000. from raw milk. licheniformis has caused diarrheal illness. they were indistinguishable from the type strain of B. High numbers of B. These effects were due to a molecule smaller than 10 kDa. circulans. and from industrially produced baby food. 2007b). 1999).. subtilis cells (10 CFU/g) were isolated from the patients’ vomitus. and resistant to heat. CEREUS Bacillus species that do not belong to the B. Final confirmation of enterotoxigenicity of an isolate can be obtained by cytotoxicity tests using Vero cells. with physicochemical properties resembling those of cereulide. C. firmus (Taylor et al. fusiformis produce lipopeptides with cytotoxic activity. 1999).. 2007a. licheniformis. megaterium. B.. pumilus associated with the restaurant outbreak was active in the boar sperm assay and was cytotoxic (From et al. poultry or vegetable pies. B. by stomach cramps and diarrhea which lasted for several days. any isolate that reacts positively with one or both of the commercial kits should be considered enterotoxigenic (Granum and Lund. and G. B. weihenstephanensis have been shown to produce cereulide at temperatures as low as 8ЊC (Thorsen et al. B. and this organism was found to produce a complex of lipopeptides. The B. or human embryonic lung cells (Fletcher and Logan. Jackson et al. which developed during the meal. simplex. subtilis has caused vomiting within a few hours of ingesting the incriminated food (meat. Similar results have been observed with B. these were followed. cereus isolates has been confirmed. These lipopeptides have previously been regarded as harmless to humans (Pedersen et al. 1991. and stews). damaged cell membrane integrity. inhibited sperm motility.. Dietrich et al. Inhibition of boar sperm motility. . and B. Honda et al. From and colleagues also demonstrated that B. 1997). cereus emetic toxin. High numbers (10 CFU/g) of B.. (1999) have described monoclonal antibodies that target each of the proteins in the HBL complex.. D. Phenotypically. 2002). pumilacidins. 1999). A cell-based biosensor containing a B-cell hybridoma. Production of toxins by non-B. (2007a). cereus enterotoxin reverse passive latex agglutination assay (specific for the L2 subunit of the HBL enterotoxin) was positive for isolates of B. megaterium. pumilus isolate produced a sevencomponent complex identical in molecular mass to pumilacidins A. licheniformis cells were isolated from the implicated foods (meat. closely related to B. an indicator for B.. but not at 10ЊC. has been reported to contain B. soluble in 50 and 100% methanol. encapsulated in a collagen matrix has been described for the rapid detection of enterotoxin from Bacillus species. 2006). E. headache. Chinese hamster ovary (CHO) cells. Strains of the psychrotolerant B. The sensor measures the alkaline phosphatase released from infected Ped-2E9 cells using a colorimetric assay (Banerjee et al. but a lipopeptide.. and B. B. subtilis. The same group has developed polyclonal and monoclonal antibodies against the proteins of the Nhe complex (Dietrich et al.

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. has an extremely important role in virulence because it is required for the bacterium to reach the attachment sites and penetrate into the intestinal cells. but some important virulence factors include motility. state (Rollins and Colwell. Cox and L. and energy is derived from amino acids or tricarboxylic acid cycle intermediates due to their inability to oxidize or ferment carbohydrates. Campylobacter spp.. and in length from 0. Washington. K. which grow optimally at 42°C. Athens. Arcobacter. Athens. causing enteric and extraintestinal illnesses.9 µm. The cells can form spherical or coccoid bodies as cultures age. 1986. The flagellum of Campylobacter spp. C. but not in all cases.Pathogens and Toxins in Foods: Challenges and Interventions Edited by V. GA 30605. Michael T. microaerophilic. have typical biochemical characteristics. Cox. GA 30605. and Michael T. Sofos © 2010 ASM Press. several are considered pathogenic to humans. negative hippurate hydrolysis (except for most C. The pathogenic mechanisms by which campylobacteriosis occurs are not totally understood. 1987). and of these species. A few strains can grow aerobically or anaerobically. Motility. and information on species other than C. If the bacterium loses its motility. darting. allowing access to Nelson A. a negative reaction to methyl red. flagellin has been proposed as an adhesin in the binding to culture cells. Ketley. Russell Research Center. plays a much more important function than just motility. reduce nitrate and nitrite. its ability to colonize the gastrointestinal tract and cause infection is lost. except for the thermophilic Campylobacter spp. 20 . 1986. reciprocating motility (corkscrew-like motion by means of a single polar flagellum at one or both ends of the cell) and can occur in short or long chains. Moran and Upton. Juneja and J. jejuni strains).2 to 0. which include the reduction of fumarate to succinate. ability to translocate. the wild-type bacterium expresses flaA only. can be either catalase positive or negative. In regard to the genus Campylobacter. jejuni is scarce. 2000). acetone. C.. which is achieved by means of a single flagellum at one or both ends of the bacterium. and production of toxins (Walker et al. Campylobacter species have a chemoorganotrophic metabolism. are most often associated with human disease. jejuni contains two flagellin genes. DC Chapter 2 Campylobacter jejuni and Other Campylobacters Nelson A. but to date the family Campylobacteraceae includes the genera Campylobacter. It appears that the virulence factors involved in the infection greatly influence the symptoms of the disease. chemotaxis. and pathogenesis results from several different bacterial properties and host defenses. Sellars et al. jejuni is able to become internalized within human intestinal epithelial cells and traverse monolayers of polarized human colonic carcinoma cells. 2000. nonspore-forming organisms with curved or small spiralshaped cells that have characteristic rapid.5 to 5 µm. and positivity for oxidase activity (Vandamme. Musgrove • Egg Safety and Quality Research Unit. and it has been postulated that certain species can have the characteristics of a viable. It has been shown that C. flaA and flaB. and most species have an optimum temperature range for growth of 30 to 37°C. 1997. 2002). catalase-positive Campylobacter spp. Musgrove CHARACTERISTICS OF THE ORGANISM AND TYPES OF ILLNESS The taxonomy has changed considerably over the years and could change in the future. Jason Richardson. and indole production. there are 14 species. but flaB can be expressed under certain conditions. but not culturable. N. Campylobacter spp. In addition. An atmosphere containing increased nitrogen may be needed for optimum growth of certain strains. The majority of Campylobacter spp. Campylobacter species are gram-negative. jejuni flagella may also play a role in the dissemination and internalization of the organism. They range in width from 0. and Sulfurospirillum and the generically misclassified Bacteroides ureolyticus (Vandamme. L. Broadly speaking. Wassenaar. Jason Richardson • USDA-ARS-Poultry Microbiological Safety Research Unit. 1997). Russell Research Center.

bloody stools containing leukocytes and fever and (ii) noninflammatory diarrhea. which are similar to certain human gangliosides (Ang et al. can be found in review articles by Walker et al. their mechanism of action and importance in regard to disease still remain unclear. Even though the synthesis of several toxins has been reported. jejuni. young adults aged 15 to 25 years. Both of these syndromes are characterized by being acute or subacute. 1997). 2001. the core oligosaccharides of its LPS contain ganglioside-like structures. C. Ketley. 1997. causing mononuclear infiltration of peripheral nerves. 2001). motor paralysis. 1998). Molecular mimicry is believed to be the cause of GBS because a few peripheral nerves of the human neurological system share molecules similar to those of antigens on the surface of C. Campylobacter spp. Campylobacter spp. classified mainly as enterotoxins or cytotoxins (Wassenaar. headaches. contributing to the appearance of neurological symptoms (Lindsay. However. In humans. jejuni infections are GBS and Miller Fisher syndrome. intense abdominal pain. have limited spread from host to host. Since C. myocarditis. and immunosuppressed individuals (Keener et al. GBS occurs in approximately 1 out of 1. C. the genus Campylobacter has had increased focus as a threat to food safety. and this eventually leads to primary axonal degeneration or demyelination. in some instances symptoms can persist longer than 2 weeks. jejuni. and upon exposure to C. upsaliensis) are known as “thermophilic campylobacters” and are clinically significant due to their roles as dominant causative agents of human campylobacteriosis (Blaser et al. jejuni is the predominant species that causes bacterial gastroenteritis in the United States and in many other developed countries. jejuni cells (Winer. 2004). Campylobacter . 2004). osteomyelitis. 2000. Willison and O’Hanlon (2000). and they are intestinal and extraintestinal infections. 1997). which leads to tissue inflammation and damage (Grant et al.000 cases (Winer. Guillain-Barré syndrome (GBS). there are two types of illnesses associated with Campylobacter infections.. Keener et al. and C. Although campylobacteriosis does not usually lead to death. 1982. antibodies attack the gangliosides on the neuromuscular junction. 2001). and vomiting can occur. and Wassenaar (1997). to humans generally occurs by either ingestion of contaminated food or water or by direct contact with fecal material from infected animals or persons. after the infection. Ang et al.. the immune system produces antibodies against the LPS structure as an attempt to fight the infection. 2001). C. In the United States. can arise (Altekruse et al.CHAPTER 2 • CAMPYLOBACTER JEJUNI AND OTHER CAMPYLOBACTERS 21 submucosal tissue. Winer. For approximately 3 decades. may not be recovered by conventional cultural methods outside of the host if exposed to dry conditions or atmospheric oxygen levels for extended periods of time.. In the vast majority of cases. Two types of diarrhea are usually observed with campylobacteriosis: (i) an inflammatory diarrhea. septic abortion. Four species (C. JacobsReitsma. Transmission of Campylobacter spp. Keener et al. the organism synthesizes several toxins.. The problem in determining the aforementioned is that researchers have not been able to detect toxin produced by Campylobacter spp. acellular increase in the total protein content in the cerebrospinal fluid. Two neuropathies associated with C. Ketley (1997). and bacteremia in certain cases. this is characterized by alexia. In regard to C. Serious complications. due to the rise in enteritis in humans caused by consumption or handling of foods contaminated with the organisms. and inflammatory demyelinating polyradiculoneuropathy. it has been estimated that as many as 730 people in the United States with Campylobacter infections die annually. A concern for those suffering from campylobacteriosis is that they will suffer from neurological sequelae months or even years afterward. Due to the similarity of the core oligosaccharides of the LPS and the gangliosides.. jejuni contains a lipopolysaccharide structure (LPS) attached to the outer membrane. such as Reiter’s syndrome. In some cases. GBS cases are associated with nerve roots. SOURCES AND INCIDENCE IN THE ENVIRONMENT AND FOODS Unlike many other enteric pathogens. C. 1999. and recovery is completed in approximately 8 days. immunemediated neuropathies. with watery stools and the absence of blood and leukocytes (Wassenaar. with slimy. coli. Spikes in levels of all immunoglobulin classes have been shown with humans after infection. campylobacteriosis is mainly a self-limiting bacterial gastroenteritis. Toxin production also plays a role in pathogenicity. cramping.. and Winer (2001).. often due to secondary complications (Saleha et al. A more comprehensive review of the pathogenesis of Campylobacter spp. jejuni. cystitis. In regard to GBS. 2004).. lari. coli is responsible for the majority of other cases of illness. 1997). (1986). 1993. nephritis. either spontaneously or after appropriate antimicrobial therapy. A more detailed review of GBS can be found in review articles by Lindsay (1997). pancreatitis. campylobacteriosis and salmonellosis go back and forth as the leading cause of bacterial food-borne illness. 2001). The population that is most susceptible to illness includes children less than 1 year of age. The LPS structure is very antigenic..

liver.. ovarian follicles. distribution. Richardson et al. Friedman et al. Keener et al. and within a week Campylobacter prevalence in the flock can reach 100% (Beery et al. suggesting it is not limited to the digestive tract. have been isolated from the circulating blood. Campylobacter spp. In poultry and cattle. eggs. coastal waters. colonize the mucus layer of the intestinal tract but have been recovered from numerous tissues and organs within the bird. 2004). 1998). and 71% of whole chickens. were isolated from 82%. In regard to the digestive tract. C.. then as much as 70% to 100% of the samples can be positive for Campylobacter. swine. unabsorbed yolk sacs.. breast with skin attached. Campylobacter spp. to the extent that they can be overgrown by organisms that were meant to be suppressed (Musgrove et al. It has been shown that if a single bird in a flock is colonized.. particularly poultry. in poultry is not fully elucidated. Generally speaking.. and contact with farm animals and pets (Altekruse et al. thymus. therefore.22 COX ET AL.. 2004). 2007a). However. This presents a risk to food safety due to the contamination of carcasses at slaughter and other foodstuffs by cross-contamination when raw or undercooked meat is handled. Campylobacter spp. 2001). This includes.. Increased attention has been given to reducing the level of Campylobacter spp. cattle. are the most common host for Campylobacter spp. fruits. rodents. Certain species of Campylobacter are routinely associated with certain species of animals. Campylobacter spp.. enteritis can be classified as a zoonosis. Kemp et al. such as direct plating onto selective agar. The majority of Campylobacter infections are sporadic.. 2006. jejuni is the predominant species.. 9 .. Studies have shown that as much as 70% of human illnesses due to Campylobacter spp. but on average greater than 70% of the birds are Campylobacter positive (Maekin et al... gain entry into poultry flocks so that more effective intervention strategies can be employed. spleen. and C. For example. 2005). if a survey is being conducted on the prevalence of Campylobacter on poultry carcasses postchill and enrichment of the sample is utilized. if a less sensitive method is utilized. Gregory et al. Byrd et al. 1999). 2004... Even though the enrichment used is designed to be selective for Campylobacter spp. 1999. 2000. 2007. 2004. but is not limited to. raw milk. Friedman et al. production. beef. and it is critical to consider the cultural procedures utilized and the impact those choices have on sensitivity to recover or detect the organism. are caused by the consumption or handling of raw or undercooked poultry or poultry products. A question often asked is whether the injured or stressed cells could have the ability to infect humans and cause illness. The prevalence of Campylobacter contamination of carcasses and poultry products can vary greatly. The reported prevalence rate for the farm continuum varies between studies. poultry. 1997. processing. The ecology of Campylobacter spp. In a study of supermarkets. 2008. 2001. then the spread to adjacent rearing mates is rapid. Numerous studies are being conducted to determine when and how Campylobacter spp. are fastidious organisms that are capable of existing in a broad range of environments and have been sporadically recovered from rivers. 2001). Campylobacter incidence within the farm continuum does not generally relate to an increase in retail positives in other types of animal products. However. 2004). 2007). gallbladder. 1988. handling. and pieces. because animals are the main reservoir of these organisms. and avian species (Jacobs-Reitsma. and vegetables but routinely recovered from sheep.. depending on the sensitivity of the cultural procedures utilized and by the point along the process chain at which sampling is being conducted. coli is the common species recovered from swine.. 2007). Wallace et al. Including both direct plating and enrichment often allows the best probability for recovery. the primary source of contamination of the environment and foods is believed to be from animal feces (Brown et al. respectively (Harrison et al. then the number of samples detected as positive could be greatly reduced. and outbreaks are rare but have been traced back to contaminated water. which may exclude sublethally injured cells. 2005.. contamination can be found in retail poultry and poultry products and is often directly related to the prevalence rate at the farm. and reproductive tracts of commercial poultry (Cox et al. 2004). from carcasses at the final stages of processing. the organisms can be culturally fragile. levels up to 10 CFU/g of fecal content have been shown (Altekruse et al. shellfish.. and preparation.and postharvest to reduce the level and incidence of raw product contamination (Allos. In addition to the digestive tract. 82%. Two modes of transmission of Campylobacter into poultry flocks occur and they are horizontal and vertical transmission (Keener et al. Contamination with this pathogen can occur at numerous stages along the food chain. Allen et al. The type of methodology employed significantly affects prevalence rates of Campylobacter spp. exist as commensals in many wild and domestic animals (Keener et al. A significantly high prevalence rate of Campylobacter spp. poultry is considered the main source of human illness. in poultry pre.. Avian species. Campylobacter spp. This is one reason why studies on the incidence of Campylobacter in poultry processing plants vary..

1991. jejuni. 1991. The VBNC state arises from exposure to sublethal adverse environmental conditions. but the rate of death at this temperature was much less than that at 25°C. In addition. At 4ºC. SPREAD. 2003. but as the understanding of this phenomenon unfolds. particularly after a feed withdrawal prior to transport to the processing facility. 1998). a number of factors such as commensal level in poultry.. and low pH (less than or equal to 4. C.0% or more NaCl. this could shed light on how Campylobacter spp. survive and persist in certain food commodities and go undetected in dry environments.. 2001).5% (Doyle and Roman. 2001). coli after subjection to acid stress were shown to be viable by injecting the cultures into the amniotic fluid and yolk sac of fertilized eggs (Chaveerach et al. have been found in these meat products at the retail level (Zhao et al. with an optimum growth concentration of 0. they are still one of the biggest causes of gastroenteritis. have been shown to survive for several weeks in groundwater (Buswell et al. Campylobacter spp.. OR CHARACTERISTICS While outbreaks of human campylobacteriosis have been associated with raw milk and untreated water.CHAPTER 2 • CAMPYLOBACTER JEJUNI AND OTHER CAMPYLOBACTERS 23 Campylobacter prevalence rates in one study reported 47% of cattle and 46% of swine harbored the organism within the farm continuum (Nielsen et al.1 days at 25°C. may harbor large numbers of Campylobacter bacteria (Musgrove et al. The cells are metabolically active and show signs of respiratory activity but are unable to be cultured through conventional methodology procedures. The VBNC stage was first described by Rollins and Colwell (1986). which is frequently contaminated with the organism. 1991). and recovery occurs by passage of the organism to a susceptible host. 2007b). Even though Campylobacter spp. high oxygen concentration. 1982a). though it has also been demonstrated that broiler crops. can be sensitive to environmental conditions outside of an animal’s intestinal tract.. poultry meat.. Richardson et al.7). are sensitive to sodium chloride.. INTRINSIC AND EXTRINSIC FACTORS THAT AFFECT SURVIVAL AND GROWTH IN FOOD PRODUCTS AND CONTRIBUTE TO OUTBREAKS Cross-contamination of food products is a major factor that contributes to human illness. acetic. processing procedures utilized for poultry carcasses. can enter into a viable but nonculturable (VBNC) state. ascorbic. freezethaw-injured C. FOOD PROCESSING OPERATIONS THAT INFLUENCE THE NUMBERS. Campylobacter cells were subjected to dry stress on filter and chick paper pads and after becoming nonculturable were determined to be viable utilizing a chick bioassay (Richardson et al. Several studies have shown that strong acids. Nonculturable C.. Campylobacter spp. comparatively low prevalence rates (less than 2%) of Campylobacter spp. 1997). Studies have shown that the potential for survival decreases to a few hours at temperatures of 37°C and increases to a few days at temperatures of 4°C. which is followed by a slower rate of inactivation. In a separate study. skin removal from the carcasses of other animals during processing. varies. Sensitivity to salt also shows a temperature-dependent effect. may be responsible for as much as 70% of sporadic campylobacteriosis (Skirrow. This could be due to. and lactic acids. A 3 log10 decrease of cells occurred in 4. and the sheer number of poultry carcasses being processed in the plant each day. depending on the type of food product. 2007b). jejuni was sensitive to 1. Campylobacter spp. 1997. Several studies have explored the recovery of VBNC forms of Campylobacter cells (Jones et al. Saha and Sanyel. When environments become unfavorable for growth. C.5% NaCl after 1. This may explain the high survival rate of Campylobacter spp. The significance of the VBNC state remains unclear and controversial. are sensitive to drying. jejuni cells that were nonculturable were converted back to culturable after passage through the rat gut (Saha et al. unlike poultry.25 to 2. who postulated that it could play a role in human infection and illness.. rapidly inhibit the growth and survival of Campylobacter spp. However. Contamination is thought to originate from the intestinal tract of primarily avian species (mainly poultry) and then spread to the meat during transport and processing.. The decimal reduction time for Campylobacter spp. such as formic. 2003). on poultry carcasses due to the high levels of the organisms in the bird’s digestive tract at the time of processing.. but a similar reduction took about 2 weeks at the same salt concentration when a temperature of 4°C was maintained. jejuni and C. 1991). but is not limited to. it is postulated. Chaveerach et al. Berrang et al.. However. Crops burst more often than cecal pouches or other parts of the gut and can contaminate previously . but survival kinetics generally follow a rapid decline in numbers.

External contamination often increases during transport from grow-out houses to the processing plant.5%) and pooled crushed egg shells/membranes (4. Gibbens et al. adding new equipment. Muir et al. the presence of the bacteria bears consideration. Generally. and many researchers report a decreased incidence of Campylobacter contamination on many poultry products.. Commercial products. were not recovered from either egg contents or shells.. Oosterom et al. 1998. Campylobacter counts decrease in the scalding tank.. 2005. Musgrove and Jones (2006) sampled packer head brushes at two commercial shell egg processing facilities. 2000). This part of the process has a tendency to dilute Campylobacter numbers. the amount of water used during processing has increased dramatically. jejuni whole-cell vaccine was shown to reduce colonization in immunized chickens by 16% to 93%.24 COX ET AL. undefined cultures made from mucosal scrapings have been the most effective. and antimicrobial packaging. and even treatment with other microorganisms or their products (Humphrey et al. ensuring clean water for processing. and are at their highest immediately following evisceration (Berrang and Cason. Competitive exclusion products can be supplied to hatching chicks to prevent enteric colonization by human pathogens. 2001. carcasses are chilled in a series of tanks using recirculated cool water. Chantarapagant et al. levels of Campylobacter in the intestinal tract 7 can be as high as 10 CFU/g cecal contents. 1985). Generally. maintaining increased water during processing. jejuni only during the first few hours. They detected an initial drop but determined . In most commercial processing facilities. and knowledge of Campylobacter have seen many changes in the last 15 to 20 years. Campylobacter is a culturally fastidious organism. are now available. While Campylobacter spp. Samples analyzed included eggs. 2006). Campylobacter-free carcasses. Cox sampled spent hens and a small number of eggs from a commercial shell egg washing facility.. Though low rates of recovery were observed with packer head brushes (1. AND PHYSICAL INTERVENTIONS TO GUARD AGAINST THE PATHOGEN In developed countries worldwide a great deal of effort is being expended on developing interventions in Campylobacter contamination of poultry and poultry products. In order to reduce the chances of cross-contamination. and purification of flagellin antigens by preservation of conformational epitopes may enhance their use as vaccines (Widders et al. 10 CFU/ml of rinse can be recovered (Berrang et al. 1993. Postharvest efforts include making plants sanitary.. Other interventions have included use of other microorganisms. Northcutt. providing carcasses frozen instead of fresh. most of the hens were positive for the organism. increase during removal of feathers (picking). irradiating. Wagenaar et al. cultural media for. 2002). many of which are of a defined nature. A formalin-inactivated C. Prior to further processing or transport to a retail market. expertise in.. Since passage of hazard analysis and critical control points. Recovering this organism from samples which are low in biologically available moisture can be especially challenging. carcasses and other poultry products may be stored under refrigerated or frozen conditions. particularly since an egg-borne outbreak of campylobacteriosis has been reported (Finch and Blake. This has contributed to the perception that Campylobacter contamination of egg may not be important. Campylobacter colonization of broilers has been affected by application of bacteriophages (Carillo et al.. 2001). These brushes assist in gently transferring eggs from the weighing/grading equipment to a series of belts.. 2005). amendments such as sodium hypochlorite. and surfaces within the facilities. evaluating chill tank water sanitizers. RECENT ADVANTAGES IN BIOLOGICAL. As birds enter the plant. some of the last surfaces to touch the eggs before they reach retail packaging.1% of layer hens shed Campylobacter chronically and found ~1% of shell-contaminated table eggs. including the reproductive tract (Cox et al. and trisodium phosphate may be added to the inside-outside bird washer or to the chill tank (Kemp et al. However. egg products. training of farm personnel. However. CHEMICAL... but some degree of protection has been observed for various formulations. Campylobacter was never recovered from any of the sample types.. and 6 when whole carcasses with feathers are rinsed. Survivability of the organism is favored at refrigerated temperatures but may also occur under frozen conditions. 2000). egg wash water. more success has been achieved in suppressing Salmonella than Campylobacter. Truly effective vaccines and application strategies have yet to be developed for prevention of avian intestinal colonization. drinking water amendments. acidified sodium chlorite. (1983) reported that freezing affected C. especially on those carcasses that are highly contaminated. 2004. 2005). Doyle (1984) reported that only 8. Izat and Gardner (1988) sampled two commercial egg processing facilities.2%). Preharvest efforts include improved biosecurity. such as Pseudomonas aeruginosa or excretory-secretory products of Trichuris suis to affect attachment to skin or intestinal epithelial cells (Abner et al. 2002).

A variety of other automated kits and systems based on either immunological or molecular criteria are now available.. Presumptive Campylobacter isolates should be observed using dark-field or phase-contrast microscopy for characteristic morphology and motility. while at 55°C they ranged from 0. with a distinct edge. however. including flagellin typing.. jejuni in a skim-milk heat menstruum. bacterial restriction endonuclease DNA analysis (BRENDA). Campylobacter spp. particularly marination. pepper. 1996). Typing methods can be divided into categories.2 to 12. pulsed-field gel electrophoresis of chromosomal DNA.. 2000). 2001. and confirmation of Campylobacter isolates by traditional methods require plating enrichment broth samples after incubation onto selective agar plates.8 min. and 42°C over a 48-h period. A 3. die faster at 25°C than at either 4 or 30°C. cells from cultures older than 24 h may appear coccoidal and nonmotile (Musgrove et al. and spreading. it appears that phage typing should be used together with another typing method to confidently discriminate among isolates. Growth may be confluent without distinct colonies.74 to 1. and glistening. relatedness to other isolates. These data indicate that ordinary cooking temperatures should be sufficient to destroy campylobacters contaminating poultry or other meat samples. though the variety of approaches published in studies worldwide make interlaboratory comparisons difficult (Wassenaar and Newell. A second category consists of the various genotyping analyses. shiny. (1985) originally described a set of 14 phage types which discriminated about 90% of 255 human isolates tested. with an irregular edge. and ground cloves on the growth of C. DISCRIMINATIVE DETECTION METHODS FOR CONFIRMATION AND TRACE-BACK OF CONTAMINATED PRODUCTS Isolation. In the last 15 years. such as heat-stable or heat-labile antigens or antimicrobial susceptibility patterns. Heat injury of C. such as sodium chloride.. Genotyping techniques can be used to determine sources of infection and routes of transmission in humans and animals. Typing of Campylobacter strains is needed for epidemiological purposes.CHAPTER 2 • CAMPYLOBACTER JEJUNI AND OTHER CAMPYLOBACTERS 25 that the organism could survive on chicken carcasses and chicken livers at Ϫ20°C for more than 64 and 84 days. to be of value. can be confirmed using latex agglutination assays. 1982). Campylobacter spp.0 log10 reductions below the respective controls were noted for the maximum concentrations of garlic. In addition. 2001). as well as the openness of management to embrace new and sometimes daunting technologies. one of which is typing based on phenotypic characteristics. By 1982.9 log10 decrease was noted for the broth containing onion. 25°C. and death occurs at 48°C. Lior et al. Deibel and Banwart (1994) conducted a study on the effects of various concentrations of oregano.. more than a 3 log10 decrease in cell numbers was observed with the suspensions containing sage or oregano. 2002). At 25°C. The usefulness of any of these approaches depends upon availability of resources and personnel time and reproducibility and discriminatory capability of the method. and ribotyping.. D values ranged from 7. in terms of antimicrobial resistance. black pepper.. and oregano. Padungtod et al. and 3. are sensitive to certain spices and food ingredients. sage. Koidis and Doyle (1983) analyzed the inhibitory effects of garlic. 2001). jejuni in a liquid growth medium incubated at 4°C. speciation. or flat.00 min. and at 48°C. though some information can be obtained without obtaining a specific isolate (Lu et al. Another phenotypic assay used to discriminate between isolates involves the analysis . onion. Immunomagnetic separation of Campylobacter spp. less than a 1 log10 reduction was observed for any of the three spices. multiplex PCR-RFLP. Campylobacter spp. randomly amplified polymorphic DNA. such as done with ribotyping or other genomes. Grajewski et al. strain differentiation has shifted to highly specific genotyping analyses. and some methods combine various identification approaches (Cloak et al. may create a more hostile environment for Campylobacter spp. 1980. coupled with PCR-based detection systems are being developed for detection with food samples (Docherty et al. They are colorless or grayish or light cream and may range from pinpoint to 4 to 5 mm in diameter. respectively. convex. and pulsed-field gel electrophoresis. This may suggest that further processing. At 4°C. the method used for isolation can affect genotyping results (Newell et al. Therefore. Included in this category are restriction fragment length polymorphism (RFLP) of selected genomes. and oregano on C. Typical colonies are smooth. identification. translucent. 2003). which are commercially available. particularly on wet agar. the most widely used serological schemes were published and in use (Penner and Hennessy. jejuni occurs at 46°C. Doyle (1982b) determined rates of thermal inactivation for five strains of C. or pinpointing sources for epidemiological purposes. jejuni in an enrichment nutrient broth stored at 4ºC. An isolated colony can be useful in further characterization.

Many continue to say that there is no evidence to suggest egg transmission and hatchery contamination when the evidence is not only present.26 COX ET AL. If the research community continues to ignore published facts. and additional evidence continues to mount. The method uses a PCR and RFLP. There is conflicting evidence in the published literature as to whether Campylobacter can pass from breeder hen to progeny through the fertile egg. and observing patterns were revealed by UV illumination following ethidium bromide staining. Upon electrophoresis of the digest product. such as “Campylobacter cannot be transmitted through the egg.. (1997) have described a system that allows differentiation of strains based on differences in flaA gene sequencing.. Although discrimination is possible. (2006) stated that they could not find any evidence for vertical transmission of Campylobacter when only 13 . Techniques such as these allow for a greater level of discrimination than was previously possible. of the outer membrane protein contents of Campylobacter by sodium dodecyl sulfate polyacrylamide gel electrophoresis. 1996. and in order to reduce the contamination level of poultry products. whether it is vertical or horizontal contamination of the egg and its developing embryo. Modification to the method by Patton et al. and then strong conclusions.. Hiett et al. 1985. are widespread in poultry.. BRENDA. they were able to distinguish 32 different band profiles that allowed discrimination between the strains analyzed. Pearson et al.. considerable technician time is required for such analyses. 2007). restriction digests of selected bacterial chromosomal DNA (ribotyping). 2002. but it is definitely a source. 1997. BRENDAs entailed electrophoresing DNA fragments. 1990. CONCLUDING REMARKS Commercial poultry have been shown to be a major source of Campylobacter gastroenteritis in humans. limitations of sodium dodecyl sulfate-polyacrylamide gel electrophoresis to discriminate between isolates have also been reported (Derclay et al... Chuma et al. (1991) employed use of the HaeIII enzyme to digest the isolated chromosomal DNA.. however. For example. It has never been said that vertical transmission is the only source of contamination of a flock. It is easy to take issue with some of the studies which support the hypothesis that Campylobacter is not transmitted by the egg. 1991). In addition. allowing for recombinations within loci used in typing. (1996) analyzed 39 strains of Campylobacter by ribotyping and confirmed the above favorable conclusions. Meinersmann et al. Ribotyping analysis required characterizing the strains for restriction patterns resulting from the digest of the ribosomal DNA. for differences or similarities between strains. This genome plasticity. then this source will always be present and the level of contamination of commercial poultry will never be eliminated in reference to Campylobacter. Hernandez et al. with sampling methods that were not very sensitive. and interlaboratory reproducibility has been poor. for many methods. The number of samples tested in some studies is very small. having a natural ability to take up DNA. 1989). 2003. 2005. jejuni and C. Campylobacter spp. coli are the species most often associated with human illness and isolated from poultry. and plasmid profile analysis. An early comparison of 10 methods for subgrouping Campylobacter strains was published in 1991 (Patton et al. 1994. (1982) stated that no Campylobacter could be found in turkey eggs or young turkey poults. Acevedo.. In order for this to be accomplished. 2002. isolates were assayed for enzymatic activity and the patterns were then statistically analyzed to determine the genetic distances between strains.. a large portion of microbiologists will have to stop arguing whether Campylobacter is transmitted via the egg from parent birds to progeny. 1986. Maruyama and Katsube. jejuni is partly nonclonal. The authors observed that MEE and ribotyping had several advantages over the other methods because they measured relatively stable and significant chromosomal differences and were applicable to other species and genera. flaA. C. has been described (Nachamkin et al. For the MEE approach. A typing scheme for Campylobacter spp. 2004). and ribotyping were the most sensitive methods that were capable of identifying nine types among 22 strains. reductions within the preharvest continuum will have to be accomplished in order to reduce the level of contamination entering processing facilities. Methods such as these are now the standard means for determining outbreak strains and for more in-depth analysis of epidemiological data. but in recent years overwhelming (Clarke and Bueschkens. Each ribotyping pattern comprised between 3 and 11 fragments of 1 to 10 kb. makes for complex population genetics and further complicates interpretations of typing data (Wassenaar. Acuff et al. Cox et al. However. Maruyama et al. Multilocus sequence typing has shown that C. genotype instability has also been demonstrated. only 20 samples of each were tested. Byrd et al.” are made. Callicott et al. The authors concluded that MEE. 1996) and entails probing the flagellin gene. BRENDA. 1995. The genotyping methods studied included multilocus enzyme electrophoresis (MEE). Lindblom et al.

Microbiol. B. intestinal tracts of developing embryos. N. and T. J. etc. P. Gonzalez. Allos. Domingue. vertical transmission will not be debated. obvious deficiencies in the standard cultural methodologies prevent this hypothesis from being universally accepted. 2005. J.. Universidad de Puerto Rico.. Puerto Rico. 1994). Parasitol.. 2003). E. contamination of chicken carcasses during processing in relation to flock colonization. Stern. Y. R. As methodology procedures improve and our understanding of the Campylobacter ecology improves. Also. Environ. A. R. R. These studies conclusively prove that Campylobacter is present in the hatchery. J.. S. S. M. F. Studies using a sensitive detection method (colony DNA hybridization) indicated the carrier rate of Campylobacter in the cecal contents of newly hatched chicks to be as much as 35%. Swerdlow. 2006). Campylobacter isolates from two independent commercial broiler breeder flocks. M. 1997. 1988.. Perhaps. B. D. V.. and M. E. 2001. Acuff. REFERENCES Abner. (2007). suggesting that the chicks were already contaminated with Campylobacter before they were delivered to the farm (Chuma et al. For example. Allen. 1994. . and the breeders providing eggs to these hatcheries were also positive for Campylobacter. breeder flocks. Campylobacter spp. Ang. Petersen et al. Examination of turkey eggs. J.. Endtz. Beery. B. as well as from their respective progeny. Jacobs. Golan. and F. 2002. F. Whyte. de Klerk. P.CHAPTER 2 • CAMPYLOBACTER JEJUNI AND OTHER CAMPYLOBACTERS 27 pooled fluff samples were tested out of 60. the main reason is the inability of routine cultural methods to consistently recover these organisms from many dry types of samples (e. Modifications in standard laboratory procedures have led to recent discoveries. Response of intestinal epithelial cells to Trichuris suis excretory-secretory products on the influence on Campylobacter jejuni invasion under in vitro conditions. it will be accepted as fact that the fertile egg is a significant source of Campylobacter spp. Through molecular testing. when fertile chicken eggs were inoculated with Campylobacter by pressure differential. Altekruse. J. M. 88:738–745. numerous advances and discoveries can be achieved. 69:2462– 2469. R.. P. C. Urban. 32:1201–1206. when Campylobacter actually is present. More recently. 2005). such as E. the majority of the published data support just the opposite conclusion. C.S. van der Meché. Laman. Studies such as these leave the impression that transmission via the egg is highly improbable. San Juan. but presently nonculturable. van Doorn.).. A. coli and Salmonella spp. Byrd et al. Bull. In addition. GuillainBarré syndrome and Miller Fisher syndrome-associated Campylobacter jejuni lipopolysaccharides induce anti-GM1 and anti-GQ1b antibodies in rabbits.. A. J. using a modified methodology procedure. A. L. Mansfield. Black. Frost. A. 5:28–35. but will become part of a more focused and effective set of intervention strategies. G. vision. However. 45:1279– 1281. and L. E. 1986). poults. Humphrey. hatching debris. N. Campylobacter jejuni infections: update on emerging issues and trends. both viable and dry-stressed viable.. Dis. J. Gardner. T. W. D. F. thesis. A. L. J. were able to culture Campylobacter from three different commercial hatchery chick pads. 54:2365–2370. epidemiological surveys have traced the source of broiler flock infections to hatcheries (Pearson et al. F. Hugdahl. J. and D. Dis. 2001. Chickens raised in a laboratory environment without exposure to any farm environment still became colonized by Campylobacter (Lindblom et al.. Some recent studies support this hypothesis.. Campylobacter jejuni—an emerging foodborne pathogen. Clin. 1991. 2002. and chicks prior to exposure to any possible environmental source. egg shells. Int. were shown to be clonal in origin using both ribotyping and DNA sequencing analysis (Cox et al. Campylobacter has been found in hatchery fluff. 11% of these chicks at hatch had the inoculated microorganism in their intestinal tract (Clarke and Bueschkens. Deteccion de Campylobacter jejuni en las Incumadoras de una planta procesadora de pollos parrilleros de Puerto Rico. 1965. Doyle.000 parent breeders. 2002). and brooder house facilities for Campylobacter jejuni. 2007.. Infect.. and P.. C. S. Hiett et al. Acevedo. M. Also. P. even in light of these numerous peer-reviewed published studies demonstrating the transmission of Campylobacter from breeders and hatcheries to broiler flocks. G. Appl. A. S. H. G. Immun. Food Microbiol. J. Colonization of gastrointestinal tracts of chicks by Campylobacter jejuni. Bartlett. M. I.. and persistence. 1982. Corry. 1990). Vanderzandt. J. it seems strange that some scientists refuse to consider that Campylobacter is also doing the same. and newly hatched chicks (Chuma et al. and with knowledge. 1985). 1999.g. however. D. this provides an excellent means of studying the ecology of Campylobacter spp. Infect. Emerg. Campylobacter could be transmitted to the offspring via the egg following oral inoculation of Japanese quails (Maruyama and Katsube. 1996). have been demonstrated to transmit from one generation of chickens to another via fertile eggs (Gordon and Tucker. Humphrey et al. F. Infect. 113:45–61. Jørgensen. A. The actual samples may be called negative utilizing traditional cultural detection methods. Given that other microorganisms. Turner. Elviss. A. Hill. but in low numbers or in a VBNC state. Food Prot. Campylobacter was cultured from a commercial incubator and from the interior egg content and egg surfaces of fertile commercial breeder eggs (Acevedo. Fields. As we continue to improve our laboratory methods to detect Campylobacter. Due to the array of environmental conditions in the poultry continuum.

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dried fish. While there is a low genetic relatedness between the genomes of proteolytic C. botulinum group II (nonproteolytic C. tetani). perfringens. botulinum group II. botulinum) is actually more closely related to C. In general terms. but a collection of four physiologically and genetically distinct bacteria (Hatheway. respectively. baratii and C. difficile. It forms spores with high heat resistance and is of particular concern in the production of canned foods.6 to 4. 1984. Energy is derived from the degradation of sugars. Nonproteolytic C. There is a significant lack of recently acquired DNA.3 kb). Strains form either type B. which carry 3. The name C. C. This contrasts with the genome of C. botulinum group III.650 and 19 predicted genes. botulinum form either type A. or F neurotoxin. Norwich NR4 7UA. This pathogen is a mesophile with growth not reported at 10°C or below. 2006). which is highly mobile (Sebaihia et al. K. botulinum group IV. botulinum). NCTC 13319) has revealed Michael W.. Peck • new information on the biology of this organism (Sebaihia et al. botulinum and nonproteolytic C. 26% to 28%). and C.Pathogens and Toxins in Foods: Challenges and Interventions Edited by V. botulinum group I (proteolytic C. while many singleneurotoxin-forming strains also carry an incomplete neurotoxin gene. have a similar genome size (3. there are also genes encoding enzymes involved in chitin degradation (Sebaihia et al. botulinum type A strain Hall A (ATCC 3502. C. 2006. it is well adapted to a saprophytic lifestyle. a nonneurotoxigenic phylogenetically equivalent organism is described (Table 1). botulinum is a highly dangerous pathogen. N. Peck CHARACTERISTICS OF CLOSTRIDIUM BOTULINUM AND ITS NEUROTOXINS Characteristics of Clostridium botulinum The ability to form botulinum neurotoxin is restricted to strains of Clostridium botulinum and some strains of C..5°C. C. 1993). Juneja and J. The plasmid encodes a bacteriocin (called a boticin). endospore-forming anaerobes. DC Chapter 3 Clostridium botulinum Michael W. they share some common features. botulinum is retained for this heterogeneous species in order to emphasize the importance of neurotoxin formation.1 Mb). There appears to be an absence of dual-neurotoxin-forming strains or strains carrying a second incomplete neurotoxin gene. botulinum group I (proteolytic C. The genome consists of a chromosome (3. For example. Lindström and Korkeala.9 Mb) and a plasmid (16. 31 . Washington.. and neurotoxigenic strains of C. C. botulinum group III is associated with botulism in birds and animals. sporogenes than to C. All are gram-positive. Sequencing of the genome of proteolytic C. C. and possess plasmids (Strom et al. butyricum. The foods most commonly involved in outbreaks are fermented marine products. argentinense) have been isolated from the environment but not associated with botulism. botulinum is a psychrotroph. while strains of C. for example. type E. botulinum group IV (C. while only 16% of the predicted genes are present in all five sequenced clostridia. Dual-neurotoxin-forming strains have been reported. In addition to the expected large number of genes encoding secreted proteases and enzymes involved in uptake and metabolism of amino acids. botulinum). Colney. botulinum. Although proteolytic C. Thus.0°C but not at 2. butyricum. 2007). Spores formed by nonproteolytic C. acetobutylicum. difficile. or C. B. C. Approximately 43% of the predicted genes present in the Hall A genome are absent from other sequenced clostridia (C.. and vacuum-packed fish. botulinum are of moderate heat resistance. Lund and Peck. 2000. indicating a stable genomic content. four neurotoxigenic clostridia are associated with botulism in humans: C. with growth and neurotoxin formation reported at 3. both genomes are AT-rich (GC content. C. 2007). or type F neurotoxin. United Kingdom. Sebaihia Institute of Food Research. Strains of proteolytic C. For each of the six neurotoxigenic clostridia. Norwich Research Park. Sofos © 2010 ASM Press. botulinum is not a homogeneous species. baratii and C.

Each light chain cleaves a specific protein in the synaptic vesicle docking/fusion complex at a unique site. and can take many months or even longer. may require mechanical ventilation. D G F ⌭ Equivalent nonneurotoxigenic clostridia C. 2006. descending bilateral flaccid paralysis. sporogenes No name given C. botulinum strain Eklund 17B was applied to the microarray. botulinum strains (Lindström and Korkeala. Jahn. 2004. The genetic diversity of nonproteolytic C. The botulinum neurotoxins are 150-kDa proteins and comprise a heavy chain (100 kDa) and a light chain (50 kDa) linked by a disulfide bond. The robustness of this scheme has been confirmed by sequencing of neurotoxin genes and studies of the site of action of the neurotoxins. Symptoms of botulism are primarily neurological and frequently commence with blurred vision. botulinum strain Hall A. D. and some foods afford protection (Siegel. Recently. The six physiologically and phylogenetically distinct clostridia that form the botulinum neurotoxin Neurotoxigenic clostridia C. unpublished data).. The subdivision into seven distinguishable serotypes was originally made on the basis that polyclonal antibodies raised against purified neurotoxins neutralized the toxicity of neurotoxins of the same serotype. botulinum) C. Mahrhold et al. Dong et al.000. 2005. The light chains of type A and E neurotoxins target the synaptosome-associated 25-kDa protein (SNAP-25). in the mouse test. butyricum Neurotoxins formed A. nausea/vomiting.32 PECK Table 1. W.. subterminale All typical strains All typical strains et al. 2007). When DNA from the nonproteolytic C. A2. botulinum) C. the presence of a number of botulinum neurotoxin subtypes has been reported. 2005). Freezing does not inactivate botulinum neurotoxins. 2006). for example. Stringer and M. Arndt et al. Rossetto et al. 2006. The light chains possess zinc endopeptidase activity and cleave protein components of the acetylcholine-containing synaptic vesicle docking/fusion complex within the cytosol of the nerve. 2004. VAMP. argentinense) C. 2006). four subtypes of type A neurotoxin (termed A1. botulinum group III C. Both are mesophiles and have a minimum growth temperature within the range of 10°C to 15°C (S. C. butyricum form type F or type E neurotoxin. 1993). . novyi C. F. The neurotoxins are commonly associated with other proteins. The heavy chain has two functional domains. Characteristics of Botulinum Neurotoxins The botulinum neurotoxins are heat-labile proteins. type B.. F C. but not those of different serotypes. Neurotoxigenic strains of C. There are seven major botulinum neurotoxins (types A to G). although complete recovery can be slow. Flaccid paralysis of the respiratory or cardiac muscles can result in death if not treated. B. a C-terminal binding domain that facilitates binding to synaptic membrane vesicle proteins (synaptotagmins or SV2) at the nerve terminal and an N-terminal translocation domain that delivers the light chain into the cytosol of the nerve cell following receptor-mediated endocytosis (Simpson.. Evidence for the low genetic relatedness comes from studies of DNA homology (Hatheway. In many countries. and G light chains cleave the vesicle-associated membrane protein (VAMP). botulinum group IV (C. such as hemagglutinin and nontoxin-nonhemagglutinin. 2007). botulinum group II (nonproteolytic C. equine antitoxin is administered to patients suffering from food-borne or wound botulism (but not infant botulism)... that serve to protect the neurotoxin and assist in its absorption into the body. baratii C. Variability within the various subtypes is between 3% and 32% at the amino acid level. it was too genetically diverse to give a meaningful hybridization (Sebaihia et al. botulinum strains appears greater than that of proteolytic C. 2006). 1993) and tests with a DNA microarray based on the genome sequence of the proteolytic C. F B. Peck. paralysis of face muscles. and the type C light chain cleaves both SNAP-25 and syntaxin (Simpson. generalized weakness. The physiological properties of these two organisms have not been studied extensively.. Cleavage of any one of SNAP-25. 2005. Montecucco and Molgo. and muscle weakness.. leading to flaccid paralysis of the muscle. baratii and C. botulinum group I (proteolytic C. Heat inactivation is rapid at temperatures greater than 80°C but nonlinear. dysphonia (difficulty speaking). Rapid treatment with equine antitoxin and supportive therapy has led to a reduction in the fatality rate to approximately 5 to 10% of cases. A3. and syntaxin prevents binding of acetylcholine-containing synaptic vesicles and consequently neurotransmitter release. 2006). compared to up to 70% between different botulinum neurotoxins (Smith et al. and A4) have been distinguished (Smith et al. E. Heating at 85°C for 5 min reduced the concentration of active neurotoxin by a factor of 10. dizziness/ vertigo. often leading to dysphagia (difficulty swallowing). respectively.

000 human lethal doses of neurotoxin per gram. breathing difficulties.. Infants aged between 2 weeks and 6 months are most susceptible (Arnon.g. In 1897. Neurotoxins of types A. but more than 100 suspected cases were reported between 2000 and 2005. The isolate is now lost.. 2000. Botulism outbreaks involving commercial processing are less common but can be associated with significant medical and economic consequences (e. Infectious botulism is a similar but rarer disease that affects adults. botulinum neurotoxin type A or B occurs in a wound in the body.. more rarely. soil and dust) have been identified as sources of spores (Lund and Peck. Since the early 1990s.. Since some strains of C. consuming only a few micrograms of food in which C. Work to develop the use of botulinum neurotoxin as a potential biological weapon began more than half a century ago. B. an immature intestinal flora fails to prevent colonization. Once spores enter the body. 2005. neurotoxigenic strains of C. E.” meaning sausage. 2002. Equine antitoxin is not given in cases of infant botulism. 1998. however.. and was given to a disease associated with consumption of blood sausage. 2000. there has been a significant increase in reports of wound botulism in many countries associated with intravenous drug users (Passaro et al. Through the identification and implementation of appropriate effective control measures. Typical symptoms include extended constipation and flaccid paralysis. prior to 2000. crush injury) to an extremity (Passaro et al. and initially most cases involved gross trauma (e. It is estimated that between 10 and 100 spores are sufficient to bring about infection (Arnon. 2004). baratii or C. 2002). the use of human botulism immune globulin has been developed (Arnon et al. 2005. This is more than a million-fold lower than the fatal oral dose for alkali cyanides.. the cost per botulism case in the United States has been estimated at $30 million [Setlow and Johnson.. outbreaks are often associated with home-prepared foods. This treatment has been shown to be safe and effective in bringing about a significant reduction in the length and cost of the hospital stay and the severity of illness. Aureli et al. Honey and general environmental contamination (e. 2006). but reports of its properties suggest that it would be classified as nonproteolytic C. 1977). Wound botulism is also an infection. Sandrock and Murin. and occasionally F are associated with botulism in humans.g. The disease is rarely fatal. Infant botulism has been reported in many countries and has been suggested as one possible cause of sudden infant death syndrome (Fox et al. botulinum or other neurotoxin-producing clostridia have grown could result in food-borne botulism. For example. for which known control measures have not been implemented. botulinum type B.. 1997]). This association has led to recommendations in several countries that honey jars should carry a warning label indicating that the product is not suitable for infants less than 12 months of age.. The botulinum neurotoxin is the most potent substance known...g. by antibiotic treatment). 2006). Although the 1972 Biological and Toxin Weapons Convention prohibited offensive research and production of biological .. 1998. 2004).CHAPTER 3 • CLOSTRIDIUM BOTULINUM 33 Botulism — the Disease Food-borne botulism is a severe but rare neuroparalytic intoxication resulting from consumption of preformed botulinum neurotoxin. the incidence of foodborne botulism is lower than a century ago. 1979). There is anecdotal evidence that food-borne botulism may have occurred in ancient cultures and that some dietary laws and taboos may have evolved to prevent the disease (Smith.. 2001. Infection and then colonization of the gastrointestinal tracts of susceptible infants by proteolytic C.. 1979) and 5 to 70 spores per gram (Arnon et al. Lund and Peck. Today. Brett et al. although subsequent investigations revealed earlier cases (Arnon. botulinum form as much as 1. which is estimated to be in the region of 50 to 100 mg (CDC. van Ermengem first isolated a causative organism from raw homemade salted ham and the spleen of a man who later died of botulism. This disease was first described in 1943. Arnon. The word “botulism” is derived from the Latin word “botulus. 2000). As an alternative. Approximately half of the European cases of infant botulism have been associated with honey consumption (Aureli et al.. in central Europe in the 18th and 19th century. 2000). Werner et al. 2004). all involving heroin injection (Akbulut et al. This is based on reports that honey samples that have been associated with infant botulism contain 5 to 25 spores per gram (Midura et al. CDSC. in which growth and neurotoxin formation by proteolytic C.. wound botulism had not been reported in the United Kingdom. butyricum) can lead to infant botulism. botulinum (or. 2005a). 2007). The first clinical cases were described in 1976. with as little as 30 ng of neurotoxin sufficient to cause illness and even death (Lund and Peck. Spore germination is followed by cell multiplication and neurotoxin formation. Nevas et al. characterized by muscle paralysis. and a high fatality rate. 2004).g. A great number of botulism outbreaks occurred in the early part of the 20th century and were often associated with commercial and home canning processes. 2004). It occurs when competing bacteria in the normal intestinal flora of adults have been suppressed (e.

and the implications of a bioterrorism attack in which botulinum neurotoxin is deliberately added to milk have been assessed (Wein and Liu. The injections were given for cosmetic purposes. Harvey et al.. Therre (1999). 2001). neurotoxigenic strains of C. Dysport. thus.857 times the human lethal dose may have been delivered (Chertow et al. of cases Total 32 439 4. with a higher incidence in China and Poland (Table 2). and the United States. butyricum are implicated (Anniballi et al. Botox. Italy. 1999). the Soviet Union and Iraq) further developments continued (Arnon et al.377 18 1. however. One large outbreak at a wedding in 1994 affected 173 people and was associated with consumption of contaminated fish. although these attacks apparently failed (Arnon et al. the extent of reporting and investigation of cases varies from country to country (Therre. Georgia. Recorded food-borne botulism in different countries Country Belgium Canada China Denmark France Georgia Germany Italy Japan Poland Spain Sweden United Kingdom United States a a Period 1982–2000 1971–2005 1958–1983 1984–2000 1971–2003 1980–2002 1983–2000 1979–2000 1951–1987 1971–2000 1971–1998 1969–2000 1971–2005 1971–2003 No. highly concentrated botulinum neurotoxin preparation. 2002. Occasionally. CDC (2005). Poland. Azerbaijan.. 2006). Austin (personal communication). and Myobloc/NeuroBloc) have been approved in various countries for therapeutic and cosmetic use.. More than 2. 2006). and dried fish and home-canned vegetables (Peck. aerosols of botulinum neurotoxin were dispersed by the Aum Shinrikyo cult in Japan. for which known control measures have not been implemented. Turkey. J. . and Uzbekistan. Lund and Peck (2000).. Hauschild (1993).500 cases of food-borne botulism were reported in Europe in 1999 and 2000 (Peck. it is highly desirable to Table 2. EPIDEMIOLOGY OF FOOD-BORNE BOTULISM General Overview Food-borne botulism is not a reportable disease in all countries. In recent years. W. in some countries (e. 2004). A total of 887 cases were reported in Russia in 1999 and 2000 and were often associated with consumption of smoked. 2001). The use of an unlicensed. McLauchlin et al. The use of botulinum neurotoxin as a weapon by bioterrorists remains a concern. baratii and C. Data may include some cases of human botulism that are not food borne. botulinum or nonproteolytic C.34 PECK weapons. Galazka and Przbylska (1999). A number of botulinum neurotoxin preparations (e. led to four cases of botulism in the United States in 2004. 2001). reflecting difficult economic conditions at the time. 2003). Belarus. On at least three occasions between 1990 and 1995.. lead to three cases of botulism in Germany in 1962 (Arnon et al. botulinum. WHO (2000. (2004). (2006). Inadvertent inhalation of aerosolized botulinum neurotoxin type A by veterinary personnel disposing of contaminated animal fur did..g. Many recent food-borne botulism outbreaks have been associated with homeprepared foods. between 20 and 40 cases of food-borne botulism have been recorded annually in France. In some countries with a particularly high incidence. Varma et al. Georgia. 2005). with most cases attributed to home-preserved vegetables and fish (Varma et al. however. 2002). 2006).. Germany.g. Most cases of food-borne botulism are associated with proteolytic C.. Reported data are therefore likely to be far from complete. Georgia has one of the highest nationally reported rates of food-borne botulism in the world.286 879 376 750 479 9. Popoff and Carlier (2004).. this has often been associated with an increased reliance on home preservation/bottling/canning of foods. and it is estimated that 2. salted. In order to understand the causes of botulism outbreaks and to prevent any recurrence. Countries with a high reported incidence include Armenia.219 277 13 38 934 Avg per yr 2 13 168 1 39 40 22 34 13 307 10 1 1 28 Table compiled from information in works by Hauschild and Gauvreau (1985). Russia.

permitting growth and neurotoxin formation by proteolytic C. Louis et al. 2007).. where 209 villagers contracted botulism and 134 were hospitalized. botulinum are physiologically distinct and present different hazard scenarios. Both products were purchased from the refrigeration section of a supermarket. Since proteolytic C. In some botulism outbreaks. It is likely that there would have been significant mortality if the Thai authorities had not rapidly mobilized 42 ventilators. botulinum type A to grow and form neurotoxin (Carlier et al. (2002) Espie and Popoff (2003) Cawthorne et al. botulinum in these foods appears to be refrigerated storage. botulinum type A. 1990). 2006. 1988). 1995). were labeled “keep refrigerated” but stored at ambient temperature by consumers. Food-borne botulism has also occurred when ingredients containing preformed botulinum neurotoxin have been added to a correctly refrigerated product. both on a commercial scale and at home (Table 4). An outbreak in September 2006 in Canada (Toronto) and the United States (Florida and Georgia) was associated with growth and neurotoxin formation by proteolytic C. Examples of recent incidents of food-borne botulism in which the neurotoxigenic clostridia and/or neurotoxin have not been reported Yr 1998 1999 2003 2004 1994 1998 1999 Country Croatia Morocco France Italy Georgia Algeria Azerbaijan Product Ham Commercial mortadella sausage Commercial halal sausage Restaurant-preserved green olives in saline Fish consumed at wedding Kashir (meat dish) Fish consumed in restaurant Toxin type B B B B ? ? ? No. the type of neurotoxin formed is determined but not the organism. The largest recorded botulism outbreak in the United Kingdom occurred in 1989 (27 cases. This soup was temperature-abused in the home.. The spores subsequently germinated. The only barrier to controlling neurotoxin formation by proteolytic C.. 2006. One case in France in 1999 involved commercial chilled fish soup in a carton. it is appropriate to consider the epidemiology of food-borne botulism for each separately. botulinum has also been associated with botulism outbreaks in which foods intended for refrigerated storage have been held at ambient temperature (Table 4). The bamboo shoots were consumed at a local religious rite. botulinum is responsible (Table 3). when it is not established whether proteolytic C. This outbreak was severe. (2005) Varma et al.400 (17) 90 (4) Reference Pavic et al. leading to growth and neurotoxin formation in the hazelnut conserve. and this allowed proteolytic C. (2001) Ouagari et al. CDC 2006b). botulinum type A led to botulism outbreaks associated with vacuumpacked clam chowder and with a black bean dip in the United States in 1994 (Anonymous. Growth and neurotoxin formation by strains of proteolytic C. 2006a). 1998). The neurotoxin-containing potatoes were then added to yogurt to make skordalia. botulinum type B grew and formed neurotoxin in a bottle of chopped garlic in soybean oil stored at ambient temperature (and not refrigerated. botulinum or nonproteolytic C.CHAPTER 3 • CLOSTRIDIUM BOTULINUM 35 identify which of the neurotoxigenic clostridia is responsible. Proteolytic C. CDC.. botulinum type B. botulinum Outbreaks of food-borne botulism have often been associated with the inadequate processing of canned or bottled foods. 2006b). with all six cases requiring mechanical ventilation (Anonymous. (2004) Anonymous (1998a) Anonymous (1999) . This has happened most frequently when type B neurotoxin has been involved. Potatoes wrapped in foil were baked and then stored at ambient temperature. botulinum type A (CDC. of cases (deaths) 20 78 (20) 4 16 173 1. Examples include the large outbreaks associated with hazelnut yogurt in the United Kingdom and skordalia in the United States (Table 4). Proteolytic C. one fatal) and involved yogurt prepared with hazelnut conserve contaminated with type B neurotoxin (O’Mahony et al. 1995). Heat processing of the hazelnut conserve was insufficient to destroy spores of proteolytic C.. the type of neurotoxin is also not reported (Table 3). botulinum type A in commercial chilled carrot juice (Anonymous. Food-Borne Botulism Outbreaks Associated with Proteolytic C. and were consumed despite seeming spoiled (Anonymous. with 42 requiring mechanical ventilation (Ungchusak et al. Table 3. A large restaurant-associated outbreak in the United States in 1994 (30 cases) involved skordalia prepared with potato contaminated with type A neurotoxin (Angulo et al. The garlic was subsequently used in sandwiches (St. 2001). as indicated on the label) for 8 months in a restaurant in Canada. botulinum and nonproteolytic C. Subsequent ambient storage led to growth and neurotoxin formation by proteolytic C. In some food-borne botulism outbreaks. One very large outbreak in rural Thailand in March 2006 was associated with inadequately home-canned bamboo shoots.

Roberts et al.8. (1997) Gotz et al. (2005) McLauchlin et al. McLean et al. (1987) O’Mahony et al. Food-Borne Botulism Outbreaks Associated with Nonproteolytic C. CDC (2006b) 2006 2006 United States Canada/ United States A A 2 6 Dangerous process Temperature abuse a aw. potato dip (“skordalia”) and aubergine dip (“meligianoslata”) Commercial clam chowder Commercial black bean dip Commercial mascarpone cheese Homemade pesto/oil Home-prepared beans Traditional cheese preserved in oil Home-canned bamboo shoots Meat roll (“matambre”) Toxin type B A B A B A No. or salted fish. (1996) CDC (1995) Angulo et al. (1998) CDC (1999) Villar et al. improper acidification Baked potatoes held at room temperature Reference(s) St. (1999). (2006) Akdeniz et al. 1993). (2003) Kalluri et al. permitted secondary contamination Baked potato held at room temperature (?) Not known Dangerous process Inadequate processing Anonymous (1995) Anonymous (1995) Franciosa et al. (2003) Frean et al. in . temperature abuse Underprocessing and/or inadequate acidification Hazelnut conserve underprocessed Contaminated after opening. temperature abuse No secondary barrier. no preservatives. temperature abuse Unsafe process Temperature abuse at home Inadequate processing (?) Temperature abuse at salvage store Corrosion of tin. Aureli et al. botulinum Recorded outbreaks of botulism involving nonproteolytic C. (2000) Chiorboli et al. water activity. (2007) CDC (2006a). (1998) Carlier et al. (2002) Pourshafie et al. Examples of recent incidents of food-borne botulism involving proteolytic C. temperature abuse pH 5. aw 0. botulinum are generally associated with strains forming type E neurotoxin (Table 5). botulinum Yr 1985 1987 1989 1993 1993 1994 Country Canada Canada United Kingdom United States Italy United States Product Commercial garlic in oil Bottled mushrooms Commercial hazelnut yogurt Restaurant. Hauschild. (2004) Bhutani et al. of cases (deaths) 36 11 27 (1) 8 (1) 7 30 Factors contributing to a botulism outbreak Bottled.97 Poor preparation Unsafe process Inadequate processing (?) Cooked and heat-shrunk plastic wrap. (2007) Meyers et al. dried. temperature abuse No competitive microflora. (2007) Anonymous (2006). commercial process cheese sauce Commercial canned roasted aubergine in oil Restaurant. it appears likely that many European outbreaks associated with type B neurotoxin are probably due to nonproteolytic C. (1999) 1998 1999 2000 2001 2002 2002 2005 2005 2006 United Kingdom France France Home-bottled mushrooms in oil (imported from Italy) Commercial chilled fish soup Homemade asparagus soup B A B A A A A A A 2 (1) 1 9 16 2 (2) 1 1 10 (2) 209 United Commercial frozen chilli States sauce South Africa Commercial tinned pilchards Canada United Kingdom Turkey Thailand Restaurant-baked potato in aluminium foil Not known (travel from Georgia) Homemade suzme (condensed) yogurt Home-canned bamboo shoots Home-fermented tofu Commercial refrigerated carrot juice CDSC (1998). Louis et al. However. Other recent botulism outbreaks involving smoked. pH Ͼ6. (1998) 1994 1994 1996 1997 1997 1997 1998 1998 United States United States Italy Italy Germany Iran Thailand Argentina A A A B A A A A 2 1 8 (1) 3 1 27 (1) 13 (2) 9 No secondary barrier. (1988) CDC (1987). (2001) Abgueguen et al. (1990) Townes et al. then temperature abused Insufficient heat treatment.36 PECK Table 4. Ungchusak et al. 1984. botulinum type B (Lucke.

salted. air-dried fish (“kapchunka”) Vacuum-packed.CHAPTER 3 • CLOSTRIDIUM BOTULINUM 37 Table 5. (1998) Weber et al. (2001) Jahkola and Korkeala (1997). of cases (deaths) 1 Factors contributing to botulism outbreak Reference(s) CSHD (1981) Viscens et al. Korkeala et al. (2004) Dressler (2005) Lindström et al.g. (2006) Poorly controlled salting. fermented seal or walrus. vacuum. Time and/ . (2004) Eriksen et al. smoked. are also likely to be associated with nonproteolytic C. salted fish (“faseikh”) Commercial uneviscerated. lack of refrigeration 60 (30) Inadequate preservation 2 (2) 8 (1) Poorly controlled salting. fermented beaver tail and paw. (1989) Korkeala et al. (2001) Boyer et al. vacuum-packed fish (“Lachsforellen”) Commercial smoked. (2004) Boyer et al. lack of refrigeration Poorly controlled salting. salted fish (“moloha”) Vacuum-packed. vacuumpacked scallops Commercial frozen. (1985) CDC (1985) Slater et al. botulinum Yr 1981 1982 1985 1987 1991 1991 1992 1994 1995 1997 1997 Country United States Madagascar United States United States/ Israel Sweden Egypt United States Sweden Canada France Germany Product Uneviscerated. or dried) • meat (e. hot-smoked rainbow trout Commercial uneviscerated. (2001) Lindström et al. salted. (2006a) Vahdani et al.. (2001) Mackle et al. vacuumpacked fish Commercial frozen. vacuum-packed fish (“Raucherfisch”) Home-cured ham Home-smoked. botulinum have been associated most frequently with • fish (e. salted. Examples of recent incidents of food-borne botulism involving nonproteolytic C. salted. (1998) Proulx et al. fermented salmon roe. air-dried fish (“kapchunka”) Commercial uneviscerated. vacuumpacked prawns Whitefish eggs Salmon or fish soup Grey mullet Reheated chicken Homemade fermented beaver tail and paw Homemade fermented salmon roe (2 outbreaks) Homemade “muktuk” (from Beluga whale) Home-salted.. Food-borne botulism outbreaks associated with nonproteolytic C. and “muktuk”) A large outbreak involving consumption of commercially produced uneviscerated salted fish (“faseikh”) was recorded in Egypt in 1991. (2001) Boyer et al. air-dried fish (“kapchunka”) Commercial pork sausage Uneviscerated. hot-smoked rainbow trout “Fermented” seal or walrus (4 outbreaks) Fish Commercial hot-smoked.g. (1998) Rosetti et al. (2001) CDC (2001) Anonymous (2002) McLaughlin et al. (1997) Boyer et al. (1999) Anonymous (1998b) Therre (1999) Boyer et al. (1993) CDC (1992) Korkeala et al. lack of refrigeration Not known Not known Ͼ 91 (18) Putrefaction of fish before salting 8 Insufficient salt Not Not known known 9 Unsafe process 1 2 Not known Suspected temperature abuse Not known Temperature abuse Not known Temperature abuse (?) Temperature abuse (?) Temperature abuse Not known Temperature abuse (?) Poor temperature control Temperature abuse Unsafe process Unsafe process Temperature abuse (?) Consumed after “use-by date” Temperature abuse (?) Not known 1997 1997 1998 1998 1998 1999 1999 1999 2001 2001 2001 2002 2003 2004 2006 2006 Argentina Germany Germany France France Finland France France Australia United States Canada United States Germany Germany Finland Iran E E E E E E E E E E E E E E E E 6 4 4 1 1 1 1 1 1 3 4 12 3 1 1 11 which the responsible organism/toxin has not been identified.g. botulinum.. sausage and home-cured ham) • homemade foods prepared by the peoples of Alaska and north Canada (e. air-dried fish Commercial vacuum-packed smoked salmon Commercial vacuum-packed smoked whitefish Traditional soup (Ashmast) Toxin type B E E E E E E E E E E No.

g.e. This subject has been reviewed extensively previously (Dodds. For example. botulinum).. In surveys of soil and sediment in Europe. ingredients taken from other sources may contain lower numbers of spores (e. 2000). and Argentina. In similar surveys in North America. In response. botulinum. the prevalence of spores and their concentration in the environment and hence in foods are related to the incidence of botulism. an underestimate of the extent of contamination may arise because (i) the detection limit is not as low as assumed (for example. and 3 reports of more than 1. nonproteolytic C. spores may be inadvertently added to foods (e. the position might be more complicated. Conversely. botulinum. albeit generally at a low concentration. however. botulinum type B appears to predominate in much of Europe. consequently. There are no reports of food-borne botulism that involved neurotoxin formation by nonproteolytic C.. it should be recognized that some of these surveys may be subject to various limitations (Lund and Peck. INCIDENCE OF CLOSTRIDIUM BOTULINUM SPORES IN THE ENVIRONMENT AND FOODS In order to assess the risk presented by C. For example. Lund and Peck. A change in the etiology of food-borne botulism in France has been reported over the last few years. or other inhibitors in the test samples [i.000 spores/kg. and Japan (Dodds.. 2004). or processing conditions and plant hygiene may influence the contamination of foods.38 PECK or temperature abuse of commercial refrigerated foods has led to food-borne botulism (Table 5). Home-prepared foods have also been associated with botulism (Table 5). Nonproteolytic C. difficult to establish whether proteolytic C. The overall picture is that of a widespread but generally low contamination. In view of this problem and other issues. detection of one spore per bottle is not achieved]) and/or (ii) the use of heat treatments may inactivate spores of nonproteolytic C. botulinum and nonproteolytic C. 2008). botulinum in commercial chilled foods for which the shelf-life and storage temperature were maintained as specified by manufacturers (Peck et al. Boyer and Salah.g. from spices). While proteolytic C. honey).000 spores/kg. One difficulty with such work is the need to detect the four physiologically different clostridia that comprise the C.. proteolytic C. botulinum type E is associated primarily with marine and freshwater sediments in temperate regions of the world and is the predominant cause of food-borne botulism in Scandinavia. 1993a). it can be . vegetables and fish). In some circumstances. meat and dairy products). there have been many surveys of the incidence of C. Outbreaks of botulism have been associated with temperature abuse of commercial products intended for frozen storage in France in 1998 (Table 5). Canada. Food ingredients originating from soils or sediments in geographic regions of high spore incidence may themselves have high loadings (e.. Minimally heat-processed refrigerated foods have an excellent safety record with respect to foodborne botulism. there were 23 reports of less than 100 spores/kg. Popoff and Carlier. botulinum or nonproteolytic C. 2000). botulinum is present (e.. if type B or type F neurotoxin is formed). however. 2001. these surveys present a consistent picture of widespread contamination of the environment and foods. Alaska. Brazil.g. botulinum. botulinum in the environment.. however. 1993a. botulinum species. bacteriophages or bacteriocins. botulinum type B appears more prevalent in soils in the eastern continental United States. Incidence of Clostridium botulinum in the Environment The natural habitat of proteolytic C. foods may consist of a blend from a variety of geographic regions (e. it is necessary to have quantitative data on its incidence in the environment and foods. 1993b. In many circumstances. botulinum type A is reported to be prevalent in soils in the western continental United States. It has been suggested that these changes might be associated with an increased consumption of vacuum-packed foods (Boyer et al. that safety is maintained as new types of food are developed. 7 reports of 100 to 1. A most-probable-number approach was used by Dodds (1993a) to summarize data on the incidence of C. This can be a severe limitation if attempting to evaluate the hazard presented by just one of the two (proteolytic C. For example. China.. botulinum and nonproteolytic C. there were 16 reports of less than 100 spores/kg. outbreaks in Germany (1997) and Finland (2006) were associated with suspected temperature abuse of commercial hot-smoked vacuum-packed fish. It is essential. 2002. botulinum is soil and sediments. There has been an increased association with type E neurotoxin compared to type B neurotoxin and with commercial foods compared to home-prepared foods. For example. A further complicating factor is that many studies have ascertained only the type of neurotoxin formed. because of the presence of competing bacteria.g. Nonetheless. and most cases of food-borne botulism in these locations are associated with this organism.g. An outbreak in Germany in 2004 was associated with consumption of commercial vacuum-packed salmon after the use-by date.

combase.21 min D ϭ 2. CONTROL OF PROTEOLYTIC CLOSTRIDIUM BOTULINUM IN FOOD PROCESSING OPERATIONS The minimum temperature at which growth and neurotoxin production occurs is within the range of 10°C to 12°C (Peck and Stringer. 1993a). Extremely high loadings of honey with spores of C. giving the user confidence that the models can be used to target.6 hours (Peck. F 10–12°C 37°C 4.1°C for 3 min (or Table 6.94 0. B. E. F 2. as humectants (Table 6). respectively.. botulinum and must therefore be subjected to treatments that destroy spores or stored under conditions that prevent growth and neurotoxin formation. botulinum. 2006). . The largest most-probable-number estimate was 410 spores/kg. Lund and Peck.000 spores/kg. or can become.usda.4/231 min D ϭ 1. botulinum produces spores with high heat resistance and is the principal concern for the safe production of low-acid canned foods (Table 6). and 1 report of more than 1. Foods which are. 2000). botulinum have been reported in surveys carried out in Japan and North America. Growth and neurotoxin formation at 3 to 4 weeks at 12°C with a large inoculum have been reported. Carlin et al.000 spores/kg. 2004]). htm?docidϭ6796 [both accessed 22 May 2007]). botulinum are freely available through software packages. faulty commercial processing Nonproteolytic C.. Growth of proteolytic C.cc [accessed 22 May 2007]). Lund and Peck. the highest being 5.000 spores/kg. albeit often at low numbers. Published and unpublished original growth and death curves are available free of charge in ComBase (www. Effect of environmental factors on the growth and survival of proteolytic C. While many studies have reported a low contamination (e.93 with NaCl and glycerol. botulinum Factor Neurotoxins formed Minimum growth temperature Optimum growth temperature Minimum pH for growth NaCl concentration preventing growth (%) Minimum water activity for growth NaCl as humectant Glycerol as humectant Spore heat resistance Spore radiation resistance Foods involved in botulism outbreaks a b Proteolytic C. Hauschild et al. Incidence in Foods Thirteen surveys of meat found a consistent but very low contamination. In view of the ubiquitous nature of spores of C.5–3. Proteolytic C. botulinum type A (Dodds. dried fish.combase. Some predictive models for proteolytic C. 2002. with 4 out of 14 reports indicating more than 1. vacuum-packed fish b Heat resistance data without/with lysozyme during recovery.html.. botulinum has been described. Modified from the work of Lund and Peck (2000). botulinum and nonproteolytic C. One survey in China reported 25. anaerobic may allow growth of C.000 spores/kg. and the minimum water activity permitting growth is 0.CHAPTER 3 • CLOSTRIDIUM BOTULINUM 39 7 reports of 100 to 1. The optimum growth temperature is 37°C. the time to a three-decimal (3-D) increase from a spore inoculum is 12. botulinum.0°C 25°C 5. Garcia and Genigeorgis. 2000). Predictions generally compare well with observed growth and neurotoxin formation in independent tests.0–4. and the highest mostprobable-number estimate was 7 spores/kg. botulinum A. 2000). 1990.97 0. 1974.0 kGy Fermented marine products. Sixteen surveys of fruits and vegetables found a consistent but low contamination (Lund and Peck.94 a D82.000 spores/kg of proteolytic C. it is apparent that raw products cannot be guaranteed free of spores. A heat treatment at 121. such as ComBase Predictor and the Pathogen Modeling Program (www.6 10 0.6.g. Baker et al.94 and 0.ars.. 2005).300 spores/kg (Huss et al. 1993c. and predictive models have been developed (Dodds. 1975. challenge tests. cc/predictor. Fish and seafood appear to be more heavily contaminated.5 kGy Home-canned foods.2°C ϭ 2. A similar picture has been presented in other parts of the world. Lindroth and Genigeorgis. At this temperature it is estimated that under good growth conditions. www.. with the most probable spore-loading estimated at Ͻ10 spores/kg [Fach et al. Rouhbakhsh-Khaleghdoust. or by 10% NaCl. botulinum is prevented at a pH value of Ͻ4. 1986. effectively. several studies reported more than 100 spores/kg and two reported more than 1. 1987.0 5 0. 1975.93 D121°C ϭ 0.0–2. The use of other factors and combinations of factors to control or prevent growth of proteolytic C. botulinum B..gov/Services/docs. 38 out of 283 French fish/ shellfish samples were positive for C.

and 95°C for 6 15 min each prevented an inoculum of 10 spores of nonproteolytic C. time to neurotoxin formation will be closely related to the distribution of individual lag times within the population. 2006). 1997). however. 1994). 1975). Fernandez and Peck. 92. 1994) may facilitate recovery of sublethally damaged spores.2°C for 35 min. 1995. as the humectants (Hauschild. Lag time. 14.. Naturally occurring lysozyme in crabmeat (estimated at 200 µg/g prior to heating) (Lund and Peck. botulinum). Published and unpublished original data and some of these predictive models are freely available through the Internet (as noted above for proteolytic C. In the case of nonproteolytic C. Eklund et al. 1998). Peck and Stringer. At this temperature. and time to two cells). botulinum and other food-borne pathogens (Baranyi. and although the safety of this heat treatment has been demonstrated over many decades. the time to a 3-D increase from a spore inoculum is estimated to be 10. 2007). and predictive models have been developed (Graham et al. they had a longer lag time and lower probability of reaching one cell than spores formed in medium without added NaCl (Webb et al. Recent studies have begun to extend understanding and prediction of lag time in nonproteolytic C.1 to 1%) of these sublethally heat-damaged spores inducing germination by hydrolyzing peptidoglycan in the cortex... The effect of other preservative factors (alone and in combination) on growth and neurotoxin formation has been reported previously. Webb et al. leading to growth and neurotoxin formation at 25°C in 60 days in a model food with no added lysozyme. 1996a.. Peck and Stringer. when hen egg white lysozyme was added prior to heating at 85°C for 84 min.g. 2000). botulinum are of moderate heat resistance (Table 6). and 32 days. 2005. or 94. Lund and Peck. 2001. CONTROL OF NONPROTEOLYTIC CLOSTRIDIUM BOTULINUM IN FOOD PROCESSING OPERATIONS Growth and neurotoxin have been reported at 3. this variability is particularly important. as lysozyme is present in many foods and relatively heat stable (Stringer et al. when other environmental factors are also optimal. Spores of nonproteolytic C. The application of this heat treatment has ensured the safe production of low-acid canned foods. The large variability in lag time was found to result from variability in each stage of lag (germination. 2005). respectively. emergence. Schmidt et al.. botulinum.4 hours (Peck. Developments in predictive microbiology over the last few decades have enabled reliable and accurate predictions to be made of the effect of environmental conditions on the growth rate of nonproteolytic C. 1996). time for maturation of one cell. Lysozyme. Spores formed in medium to which 3% NaCl was added were sublethally damaged.. The optimum growth temperature is 25°C. 1994..3°C at 5 to 7 weeks but not at 2.97 and 0. botulinum. It is influenced by spore history (e. 1997. sublethal damage) as well as current growth conditions. as growth is likely to result from one or a small number of spores. 1967b. In growth media with and without added NaCl. growth was observed at 25°C after 13. Lund and Peck. and botulism outbreaks have occurred only when the full heat treatment has not been appropriately delivered for home-prepared or commercial ambient-stored foods (Table 4). Graham et al. Anderson et al. resulting in sublethal injury (Peck et al. Variability in the population lag time is dependent on variability in the lag times of individual spores. 1961. 90°C for 34 min. 90°C for 10 min. 2000. 2007). botulinum.. In these circumstances.. or 95°C for 15 min. Webb et al.5°C at 12 weeks (Ohye and Scott. 1995. In these circumstances the duration of all stages of lag was extended. The minimum water activity permitting growth is 0. 1992. Peck and Fernandez.40 PECK equivalent heat treatment at another temperature. 1999. and the duration of these various stages was not correlated. 2005). Lysozyme-permeable spores are approximately 100 times more heat resistant than those that are not permeable (Table 6).4°C for 15 min were required to prevent growth and neurotoxin . giving growth and biphasic survival curves (Peck and Stringer. however.. 2007). Fernandez et al. Lund and Peck. This effect of lysozyme may be important for food safety. 1957. Growth and neurotoxin formation are not reported below a pH of 5. and the first spore to germinate was not the first to two cells (Stringer et al. 1967a. is able to diffuse through the coat of a fraction (typically 0. especially for sporeforming bacteria.0 or at a NaCl concentration above 5%. Whiting and Oriente.. 2005. 1989. For example.. Cawley et al. The lag time of individual spores could not be predicted from their germination time.. 1996b. 1997. it has been questioned whether a 12-D reduction is actually delivered (Casolari.1°C to 2. based on a z value of 10°C) has been adopted as the minimum standard for a “botulinum cook” for canned foods (Stumbo et al.94 with NaCl and glycerol. botulinum (Stringer et al. 2005. since heat treatments at 90. 1999).. is variable and more difficult to predict reliably. This heat treatment is intended to deliver a 12-D reduction in the number of spores of proteolytic C..0°C to 3. 2006)... Graham et al.. while heat treatments of 85°C for 36 min. Heating for a few minutes at 80°C to 85°C inactivates the germination system. respectively (Peck et al.6°C for 65 min.

97 throughout the food Storage at chill temperature combined with a combination of heat treatment and other preservative factors which can be shown consistently to prevent growth and neurotoxin production by C.5% throughout the food Storage at chill temperature combined with an aw Յ 0. It 10 is estimated that more than 1. botulinum a b PYGS. yeast extract. fruit juices) first became commercialized in Japan in the early 1980s.g. Low irradiation doses (1 to 3 kGy). Effect of heat treatment at 85°C/60 min on the inactivation of spores of nonproteolytic C.5 ϫ 10 of these meals have been sold in Europe over the last 20 years. (1999) reported that a combination of a pressure of 827 MPa and 40°C/10 min or 50°C/5 min delivered a 5-D reduction in the number of spores of nonproteolytic C. Indeed in some tests. These foods are not sterile and are often packed under a modified low-oxygen atmosphere or vacuum.8 4.0 throughout the food Storage at chill temperature combined with a salt concentration of Ն3. ‡. botulinum is not related to the type of neurotoxin formed (Table 7). presumably Table 7. are summarized in Table 8. †. Nonproteolytic C. and it is essential that the risk of neurotoxin formation by nonproteolytic C. botulinum.7 3. Peck.0°C Storage at Յ8°C and a shelf-life of Յ10 days † Storage at chill temperature combined with heat treatment of 90°C for 10 min or equivalent lethality (e. 1996. 1997).0 Storage at Ͻ3. alternative heat treatments of 80°C for 270 min and 85°C for 52 min are recommended by the European Chilled Food Federation (ECFF. botulinum are more sensitive to irradiation than those of proteolytic C. with sales increasing by about 10% per annum. the first treatment to germinate the spores and the second to kill germinated spores (Heinz and Knorr. an acidic environment prevented subsequent growth. and although viable spores remained in these high-acid foods after pressure treatment. however. creating intrinsically safe products. botulinum has been identified as the principal microbiological safety hazard in minimally heated refrigerated foods (also known as refrigerated processed foods of extended durability.1 3.7 3. 2006).8 3. chill temperature is specified as 8°C in England and Wales.g. 2006). botulinum and the associated food-borne botulism hazard. Highhydrostatic-pressure-treated high-acid foods (e.. with respect to C.” had little effect on the control of neurotoxin formation by nonproteolytic C. and ready meals) (Peck. 80°C for 129 min. A proposed alternative approach is to apply a two-stage high-hydrostatic pressure treatment...0 4. as a small proportion of spores are likely to resist pressure-induced germination. 2005.6 3.2 5. irradiation-pasteurization led to more rapid neurotoxin formation. glucose. botulinum at 27°C in 150 days (Peterson et al. botulinum Recommendation b Neurotoxin type E B B B E E B E E F F B Log kill after 60 min/85°C 1. Safety and quality are dependent on a combination of • minimal heat treatment (typical maximum of 70°C to 95°C) • refrigerated storage temperature (typically Յ8°C) • restricted shelf-life (which can be up to 42 days) • other intrinsic/extrinsic factors that may also be important (e. .g.9 2. Peck and Stringer. Recommended procedures to ensure the safety of minimally heated refrigerated foods with respect to a nonproteolytic C.CHAPTER 3 • CLOSTRIDIUM BOTULINUM 41 formation from 10 spores of nonproteolytic C. peptone. aw water activity. It is recommended that the heat treatments or combina6 tion processes deliver a safety factor of 10 (a 6-D Table 8. The product range has now been extended to include products with a high pH and water activity. starch medium (Stringer et al. Spores of nonproteolytic C.. ‡ 85°C for 36 min) Storage at chill temperature combined with pH Յ 5. 2001). 2006). 1982). Information derived from the work of the ACMSF (1992. The heat resistance of spores of nonproteolytic C. botulinum in fish (Eklund. cook-chill foods. Bacterial spores are particularly pressure resistant. a process sometimes called “irradiation-pasteurization.7 3. botulinum recovered a on PYGS medium with lysozyme Strain Foster B96 Eklund 17B Eklund 2B 2129B Hazen 36208 Sebald P34 Hobbs FT50 Dolman VH Beluga Eklund 202F Craig 610 Colworth 151 a 6 because the competing microflora were inactivated or damaged (Eklund. botulinum is controlled. 1995. CFA. pH and water activity) Recommendations from the United Kingdom Advisory Committee on the Microbiological Safety of Food (ACMSF) on the safe production of vacuum and modified atmosphere packed chilled foods. botulinum (Table 6). 1997. Such an approach requires caution. 1999). Reddy et al. sous-vide foods. botulinum. 1982).. Alternative processing technologies may also be effective in controlling growth and neurotoxin formation by nonproteolytic C.7 3.

botulinum (ACMSF. Trypsin is added to inactivate bacteriocins that may be produced by closely related clostridia. with respect to temperature of storage and time prior to consumption. botulinum type E that it was necessary 6 to add 10 spores of this bacterium to one gram of sediment before type E neurotoxin could be detected in enrichment culture. botulinum hazards (Barker et al. Cultural Methods for Proteolytic C. it was demonstrated that gnocchi is particularly safe with respect to nonproteolytic C. from production to consumption.. Competing vegetative bacteria can be eliminated. Barker and colleagues have used a probabilistic modeling approach to assess the risk of food-borne botulism presented by nonproteolytic C. botulinum. with regard to spores of nonproteolytic C. 2002. 1996. 2004). 1994). An extended period of incubation (7 days or longer) should be allowed for growth. botulinum. 1970. delivery of a minimal heat treatment. This approach considers the entire food process. botulinum in gnocchi. A probabilistic risk model has been developed for the exposure of consumers to neurotoxin formed by nonproteolytic C. in attempts to culture C. Sebald and Petit. 2005). it is important to determine the efficiency of the method(s) used by demonstrating that strains of all relevant groups of C.. 2005). Solomon and Lilly.. botulinum from the environment or foods. It must be ensured that all appropriate safety guidelines are in place before work is started. Conditions that are optimum for enrichment and isolation of one are often not the best for the other. Output from the process risk model was consistent with an assessment of the prevalence and behavior of nonproteolytic C. ECFF.. It is essential that the enrichment media is fully anaerobic (Hatheway. Unheated samples may also be tested in order to isolate vegetative bacteria or spores that are not fully heat resistant..g. 2004). Fach et al. 1992. botulinum may be required for studies of the extent of contamination of the environment or foods and for detection of the organisms in foods or clinical samples suspected of being implicated in botulism outbreaks... botulinum.. while for nonproteolytic C. CFA. The type of problems that can be encountered is illustrated by the work of Sugiyama et al. botulinum and nonproteolytic C. 1989) or trypticase-peptoneglucose-yeast extract broth containing trypsin is suggested. and packaging. a minimally heated refrigerated potato product with an extended shelf-life (Barker et al. While heating at 75°C to 80°C for 10 to 15 min is useful in culturing from spores of proteolytic C. samples should be inoculated into cooked meat medium and incubated at 35°C. To enrich for proteolytic C. Account is taken of consumer behavior.. All the distributions are then incorporated in a belief network to give a clear picture of information dependencies and system complexity.. 2005). The production process includes mixing of ingredients (e. Furthermore. A portion of the enriched culture can be plated onto a suitable solid medium. botulinum can be isolated following addition of low numbers of bacterial spores to test samples (Sugiyama et al. Del Torre et al. 1992. Solomon and Lilly. botulinum in gnocchi (Del Torre et al. botulinum. 2006). practical work is restricted to containment laboratories offering an appropriate degree of protection. and neurotoxin formation. with incubation at 26°C (Solomon and Lilly. cooling. A probability distribution is established for each variable (e. as a series of linked operations and utilizes recent developments in mathematics and computing to quantify the magnitude of the risks and to identify hazard scenarios.g. botulinum are physiologically distinct. a single enrichment procedure cannot be relied on for both organisms. botulinum Culture of C. 2001). 1984). Egg-yolk agar . 1988. 1993. Sebald and Petit. As proteolytic C. prior to enrichment. By combining data and information from several sources. 2001). by the use of a heat treatment. heating at 60°C is more appropriate for use during isolation of nonproteolytic C. and the redox dye resazurin can be a useful indicator (Lund and Wyatt. These authors found that sediment samples from the Fox River in the United States were so inhibitory to nonproteolytic C. raw potato flakes and starch). chopped meat glucose starch medium (Hauschild. The use of ethanol is an alternative to heat treatment (Smith and Sugiyama. Outputs from these probabilistic risk models can be used to identify and prioritize steps that minimize the effects of detrimental events and that maximize awareness of process control options. Kautter et al. 2001). sporulation. initial contamination load) and incorporates an intrinsic representation of uncertainties. botulinum and its potent neurotoxins. 1994. 1988. and the addition of lysozyme to the enrichment medium can increase recovery of heated spores (Peck et al. and another portion used to test for botulinum neurotoxin or for neurotoxin gene by PCR. (1970). 2002. botulinum and Nonproteolytic C. botulinum (Barker et al.42 PECK process).. METHODS FOR THE DETECTION OF CLOSTRIDIUM BOTULINUM AND ITS NEUROTOXINS In view of the danger presented by C.

. Doellgast et al. Trypsin is required to convert the single-chain neurotoxin to the more toxic dichain form.. some of which are nonlethal (Sesardic et al. 1988. Disadvantages of the mouse test are the ethical issues concerning the use of animals. B. however... 1998. 2005. requiring 2 hours to complete. The first assays to be developed used an immunochemical . (2006) reported on an amplified ELISA that detected Ͻ5 50% minimum lethal doses (MLD50)/ml of type A. Specificity is achieved by the use of specific antisera and by observations of typical symptoms of botulism in the mice prior to death. Solomon and Lilly. the need to wait several days before a sample can be judged negative. 2004) offers independent detection of types A. 1985). 2005. The test permits rapid throughput of samples. Additionally. there is the potential to detect previously undescribed neurotoxins.. 2006). 1987. and F neurotoxins [Sharma et al. Hatheway. 2005. as colonies of proteolytic C. Shone et al. and some have the same sensitivity and specificity. Huhtanen et al. Sharma and Whiting. or enrichment media. Immunochemical methods for the detection of botulinum neurotoxins include enzyme-linked immunosorbent assays (ELISA). Selective plating media include C. 1981) and botulinum selective medium (Mills et al..CHAPTER 3 • CLOSTRIDIUM BOTULINUM 43 is a favored nonselective solid medium. 1993. so that the tests may not be specific for neurotoxins (Sakaguchi. and the need for skilled personnel.. 1977. 1993). 2005. Colony immunoblot techniques have been used to identify colonies of proteolytic C. 1996. botulinum. and F neurotoxins in casein buffer and Ͻ60 MLD50/ml in various foods.. 2001. botulinum neurotoxins (Table 9). 2005). 2001. 1985.. (ii) some may react with biologically inactive neurotoxin. 1979. A number of alternative animal methods have been described. 2005). Goodnough et al. chemiluminescent slot blot immunoassay has been used for the quantification of type E neurotoxin (Cadieux et al. such as fiber-optic evanescent-wave immunosensors (Scarlatos et al. Smith et al... botulinum (Hatheway. Gessler et al. Sharma et al.. clinical samples. Lindström and Korkeala. Capps et al. atypical neurotoxins. Ekong et al. botulinum type E (Dezfulian. ELISAs and other immunochemical methods have been used widely for detection of C. 1995.000 to 2.. botulinum and nonproteolytic C. 2005). 2006).. The various immunochemical methods may be subject to various limitations. lateral flow immunoassays. 1992.. 1988). Sharma and Whiting. E. since these neurotoxins may differ in antigenicity (Gibson et al. 2006). 2005.. rapid. 2007]). 1993. The standard method for detection and identification of botulinum neurotoxin has been intraperitoneal injection into mice (Hatheway. botulinum have a typical appearance associated with their lipase activity. 2001.. Modi et al. Ferreira. E. botulinum Neurotoxins It may be necessary to test for botulinum neurotoxin in samples of food. A number of other immunochemical methods have been developed for quantification of botulinum neurotoxins. and F neurotoxins ranged from 2 to 70 MLD50/ml in clinically relevant matrices and Ͻ1 to 70 MLD50/ml in selected food matrices (Rivera et al. These assays are qualitative and appear to show promise only as an initial screening tool for botulinum neurotoxin. The mouse test is extremely sensitive (5 to 10 pg of neurotoxin). A further method uses paramagnetic bead-based electrochemiluminescence to detect botulinum neurotoxins. These methods are generally cheaper and much easier to use than the mouse test. Potter et al. 2004).. A number of in vitro assays have been developed that quantify the highly specific endopeptidase activity of the botulinum neurotoxin light chains.. 2004. E. cheap. and is both repeatable and reproducible (Kautter and Solomon. Ferreira et al. 1992. Detection limits for type A. A simple. 1993). and antigenic variants. measures the biological activity of the neurotoxin. B. It is necessary to treat samples with trypsin to detect neurotoxin formed by nonproteolytic C.. 1997). In this test the signal is generated after capture.000 MLD50/ml for type A. the expense. 2003. (i) some may react differently with neurotoxins of a specific type produced by different strains. Pearce et al. Doellgast et al. Despite these limitations. Detection and Quantification of C. typically in 15 to 30 min (Aldus et al. 1993). and various other tests (Scarlatos et al. The test is not as sensitive as the mouse test. and (iv) many tests require complex and expensive amplification systems to achieve the sensitivity of the mouse test (e. (iii) some use antibodies that were raised to preparations containing a mixture of antigens. B. Huhtanen et al.. Lateral flow immunoassays are extremely easy to use and provide a very rapid detection of the target. botulinum isolation agar (Dezfulian et al. by a magnet.... 1993) and nonproteolytic C.g. Sharma et al. of complexes containing paramagnetic beads complexed with capture and detection antibodies and botulinum neurotoxin. and F neurotoxins and has been used to detect neurotoxin in food associated with botulism outbreaks in the United States (Ferreira et al.. Lindström and Korkeala. B. sensitive. botulinum types A and B (Goodnough et al. The trimethoprim in these media may inhibit nonproteolytic C. for example. 2001). 1988). An ELISA developed by Ferreira and colleagues (Ferreira and Crawford. due to their relatively high detection limit (1.... 1988. 1986.

Carlin and Peck (1995). The sample containing neurotoxin is then added. High specificity. B (1). F (Ͻ1) Ferreira and Crawford (1998). ELISA. botulinum neurotoxins Neurotoxins detected (MLD50/ml) A (5–10) Comments ELISA. (1994) Ekong et al. Roman et al. Wictome et al. Used with foods. 1997). Cross-reaction with other clostridia. (2006) specific detection in food and clinically relevant matrices. 1999a. denatured neurotoxin. The concentrations of all seven neurotoxin types were measured simultaneously in a single test sample. Ferreira (2001). Schmidt and Stafford.. E (1) Potter et al. 2001. E (5). Advantages over ELISA procedures follow: (i) the tests measure the biological activity of the neurotoxin light chain (but not the heavy chain). botulinum and nonproteolytic C. Used to quantify neurotoxin present in food samples associated with botulism outbreaks. a highly sensitive assay for type B neurotoxin has been developed that captures the neurotoxin on an immunoaffinity column or microtiter plate. prior to an endopeptidase assay (Wictome and Shone. Stringer et al. The endopeptidase activity of botulinum neurotoxins has also been quantified using mass spectrometry. In these assays. E (10). (1986).. 2003). Reference(s) Shone et al. (1993. Correlation between the response from ELISA and mouse test not always consistent. Weak reaction with neurotoxin from some strains.. Examples of sensitive quantitative immunochemical methods for detection of C. and after a period of incubation. a fragment of the target protein (SNAP-25 or VAMP) is attached to a microtiter plate and serves as the substrate. 2004) Chemiluminescent slot blot immunoassay. fish.2 to 1. F (Ͻ1) A (50). E (5). inoculated fish. B (2). Complex amplification system. (ii) variations in the antigenicity of neurotoxins of a specific type do not influence the response. B. Gibson et al. ELISA. and with samples in buffer. (2006) ELISA. 2005. ELISA. This method worked well with foods including meat. B (Ͻ1). did not cross-react with each other or other neurotoxins. Used extensively to measure formation of type A. (1987) Modi et al. F (10) E (4) A (2). Sensitive and Rivera et al. botulinum in fish fillets. Used with foods. 1999b).. and cheese and was able to distinguish between type B neurotoxin formed by proteolytic C.44 PECK Table 9. (2001.. In a slightly different format. Used with foods. Used to measure neurotoxin production by nonproteolytic C. higher in food samples (60 MLD50/ml). May respond to antigens with no neurotoxicity. B (10). Synthetic substrates are incubated with the test sample. had detection limits of 30 to 40 MLD50/ml (Hallis et al. (1995) Wong (1996) A (1–20) E (1–10) A (10). The endopeptidase activity of the botulinum neurotoxins leads to cleavage of these peptides and a release of fluorescence (Schmidt et al. Complex amplification system. Tested in a ring trial. No cross-reaction with other neurotoxins or other clostridia. 1996). (1985). specific antibodies are added that bind to the cleaved target protein and then secondary antibodies and a detection system are added. No cross-reaction with other clostridia. and the product peptides identified on the basis of their mass. this method has been expanded to include an antibody capture method . and after signal amplification. Low detection limit in casein buffer. this assay was more sensitive than the mouse test (Barr et al. No cross-reaction with other clostridia or other neurotoxin. Complex amplification system. ELISA. Failed to detect neurotoxin produced by one type A strain. Gibson et al. (1992) B (20) A (1–32). Paramagnetic bead-based electrochemiluminescence. A highly sensitive endopeptidase assay developed for therapeutic preparations of type A neurotoxin correlated well with the mouse test and had a detection limit of 0. ELISA. and E neurotoxin in meat and in vegetable preparations. (1988) Huhtanen et al. and (iii) the problem resulting from antibodies having been raised to a mixture of antigens is eliminated. Fernandez and Peck (1999). Failed to detect neurotoxin produced by one type B strain. (1999) Doellgast et al. Ferreira et al. botulinum. or other neurotoxin types. Used with bacterial cultures. To avoid problems encountered with nonspecific proteases. Some cross-reaction.0 MLD50/ml (Ekong et al. ELISA. (1993). E (Ͻ1) A (1). Used with foods. 1994). 2005). 1998. approach to detect cleavage of SNAP-25 or VAMP by botulinum neurotoxins and were often as sensitive as the mouse test. B (70). B (Ͻ1–16) A (9). Alternative assays employ a synthetic peptide (to mimic SNAP-25 or VAMP) labeled with quenched fluorophores as the substrate. Also reacted with type F neurotoxin. Boyer et al. Cadieux et al. Endopeptidase assays for type A neurotoxin and type B neurotoxin were specific. Developed for therapeutic preparations.. (2005) naturally contaminated soil. Sharma et al. ELISA.

g. Comparative genomic indexing using a DNA microarray based on the Hall A genome sequence was an effective tool to discriminate strains of proteolytic C. 2006). The entire method could be performed in 4 hours.. botulinum strain Hall A (ATCC 3502. botulinum has been reviewed recently (Lindström and Korkeala.. 2006b.. 1995. from serum or stool). PCR is targeted at conservative repetitive extragenic elements.. 1993. 1998. botulinum. and F neurotoxin genes in a single reaction (Lindström et al.. Probes have been constructed that enable PCR tests for the nonspecific detection of all botulinum neurotoxin genes (Campbell et al.. Carlin et al. while strain-specific differentiation is limited. Fach et al. 2007). botulinum in food. It was found that 87% to 96% of the Hall A presumptive genes were possessed by nine other strains of proteolytic C. Gauthier et al. 2006). these methods correlated well with tests for neurotoxin using mice and. Fach et al. with the inclusion of a most-probable-number series of dilutions in the cultural enrichment. Wu et al.. Myllykoski et al. for example. PCR-based methods (e. A number of typing tools have been used for the molecular characterization of strains of C. Alsallami and Kotlowski. and F neurotoxins were Ͻ1 to 20 MLD50/ml for human serum samples and Ͻ1 to 200 MLD50/ml for stool samples. this technique was used to investigate the diversity of 55 strains of proteolytic C. 2001. Lindström and Korkeala. botulinum (Lindström and Korkeala. B.. Following a cultural enrichment. botulinum (Nevas et al. 2006.. botulinum but not nonproteolytic C. (because the endopeptidase assays relate to the biological activity of the light chain and would not be affected by inactivation of the heavy chain). Detection in these tests was achieved by gel electrophoresis confirmation of product size or by further hybridization onto a polyester cloth membrane coated with cDNA probes to the PCR products. 2005b. 2001. E.. have been used for quantitative detection of bacteria containing these genes in relevant samples (e. genomic DNA that has been digested with rare-cutting restriction enzymes is separated by electrophoresis. Unlike other typing methods. botulinum is in progress. 2005c). and randomly amplified polymorphic DNA analysis). E.. botulinum (Sebaihia et al.... Nevas et al. 2001. PFGE has a higher discriminatory power than ribotyping (Lindström and Korkeala. An important step forward has been the development of multiplex PCR methods for simultaneous detection of type A. 2004. 2007). Franciosa et al.. information is also provided on the genome content of the tested strains. A species-specific fingerprint can be derived from the size and number of amplification products. 2006)... 2004.g. Molecular Methods for the Detection and Characterization of C. botulinum. For PFGE. 1997. The genome sequence of proteolytic C. (1999) used this method to characterize strains of proteolytic C.CHAPTER 3 • CLOSTRIDIUM BOTULINUM 45 to partially purify and concentrate neurotoxin (e. For repetitive element sequencebased PCR. botulinum and nonproteolytic C. In some of these tests. 1993) and for the specific detection of genes encoding each neurotoxin (e. For AFLP. 1996. 1995. and the sequencing of genomes of other strains of proteolytic C. Two prophages present in the Hall A strain were absent in the 11 test strains. NCTC 13319) has now been published (Sebaihia et al. Ferreira and Hamdy. The use of molecular methods for the detection of C. Szabo et al. pulsed-field gel electrophoresis (PFGE).. Detection limits for samples spiked with type A.. 2006).. by sequencing of the PCR product.. These include ribotyping. Merivirta et al.. botulinum A number of tests have been developed that use PCR to detect neurotoxin genes. While these tests do not detect the neurotoxin. amplified fragment length polymorphism [AFLP]. prior to quantification of endopeptidase activity (Kalb et al. In a recent study.g. and focal plane arrayFourier transform infrared spectroscopy. repetitive element sequence-based PCR. botulinum and that 84% to 87% of the presumptive genes were shared with two strains of C. Potential limitations of all endopeptidase activity tests include (i) interference by other proteases (although in some tests this has been addressed by capture of the neurotoxin prior to measurement of endopeptidase activity) and (ii) positive reaction with neurotoxin that is inactive in vivo. Lindström et al. Hyytiä et al. Carlin et al. 2002. retargeting of the PCR primers may be required in order to ensure the detection of all recently described neurotoxin subtypes (Smith et al. 2005). Ribotyping is based on analysis of conservative ribosomal genes and is widely used for molecular typing of unknown bacteria and distinguished strains of proteolytic C. 2006).g. 1994. they have been shown to be of considerable utility in the investigation of botulism outbreaks and in the testing of enrichment media during surveys for the presence of C. DNA microarrays.. botulinum and nonproteolytic C. 2006. B.. There is also merit in confirming the specificity of a positive PCR result. but limitations are the high equipment costs and the need for highly trained personnel. Hielm et al. 2005). The use of a cultural enrichment ensures good sensitivity. Aranda et al. 2006). botulinum and nonproteolytic C. DNA sequencing. sporogenes.. clinical. 2001. Lindström and Korkeala. genomic . and environmental samples. while minimizing possible problems due to the presence of extracellular DNA or dead bacteria.

it is necessary to apply treatments that destroy spores or store the food under conditions that prevent growth and neurotoxin formation. botulinum has been identified as the principal safety hazard in minimally heated refrigerated foods. J. L. In comparison. In view of the severity of food-borne botulism. Barth. 1998. J. Annual Report 1995. H. C. Nonproteolytic C. A. J. 2002. Food Prot. Her Majesty’s Stationery Office. A. E. 1996. P. Infect. O. Ariens.2193. C. Her Majesty’s Stationery Office.. Infect. Vithani. A. 2001. A. 95: 380–389. I am grateful for funding from the Competitive Strategic Grant of the BBSRC and other funders of the Institute of Food Research. Buzgan. Chennebault. J. Dairy Food Environ. J.. botulinum does not become an emerging pathogen. Commun. This method provided rapid discrimination of the two C. F. C. J. M. ACMSF. C. 80:283–290. botulinum and was suitable for typing at the strain level. E. 2002.46 PECK DNA that has been digested with a pair of restriction enzymes is ligated with restriction site-specific adaptors. previously there were only four United Kingdom outbreaks between 1956 and 2001 (McLauchlin et al. Johnson. M. Report on Vacuum Packaging and Associated Processes. Infect.g.1393.. and a subset of the fragments amplified by PCR. Dis. A large outbreak of botulism: the hazardous baked potato. McLauchlin. 2006). S. K.. Influence of pH and temperature on the growth of and toxin production by neurotoxingenic strains of Clostridium butyricum type E. Cole. 19980723. 1995. Microbiol. This method clearly differentiated between proteolytic C. Kotlowski. J. J. REFERENCES Abgueguen. through globalization of trade. botulinum (Kirkwood et al. 2003. J. Aldus. and A. www. Fenicia.. J. www. J. S. 2006). Fanello. ProMED archive no. Wound botulism in injectors of drugs: upsurge in cases in England during 2004. W. Delbos. 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Aureli.uk/science/ ouradvisors/microbiogsafety/acmsfmeets/acmsf2006/ acmsfmeet080606/acmsfmin080606. Randomly amplified polymorphic DNA analysis involves PCR amplification with randomly annealing universal primers under conditions of low stringency. botulinum type E from the Canadian arctic (Leclair et al. Bull. 2006). N. J. C. V. United Kingdom. H. M. www. and L. Karsen. botulinum and 37 strains of nonproteolytic C. V. Nine cases of foodborne botulism type B in France and literature review. Akbulut. J. and R. K. 2005. Botulism. Anonymous. and M..Azerbaijan (Baju). F. Wyatt. L. Foodborne outbreaks in California. 28/06:1–4. botulinum are ubiquitous in food. and P. Hatheway. A. United Kindgom. however. Nickey. Selection of primers for specific detection of Clostridium botulinum types B and E neurotoxin genes using a PCR method. Dis. O. van Amerogen. Tekin. 15:611–615. Int. botulinum and nonproteolytic C. the principle of equivalence with existing safe processes is adopted. P. H. Angulo. 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the rods are long.. perfringens clearly stresses the importance of cooling foods quickly after cooking. Most cases of C. epsilon. Delaware Research & Technology Center. DC Chapter 4 Clostridium perfringens Vijay K. Major Vijay K. the rods are short. 1995). John S. Spores are oval and subterminal. raw ingredients. and ferment lactose (stormy fermentation of lactose in milk). Lecithinase (alpha-toxin) activity also characterizes the isolates and is demonstrated by a zone of hemolysis (often a double zone) on blood agar or a pearly opalescence around colonies on egg yolk agar. and Ronald J. The estimated large number of illnesses due to C. Mermaid Lane. and E) are based upon the production of four extracellular toxins: alpha. C. In 1994. Juneja and J. Taormina et al. C. Eastern Regional Research Center. 200 GBC Drive. This activity can be inhibited by type A antitoxin (Nagler reaction). Washington.2% of the cases of food-borne illnesses (CDC. C. such as spices used in food processing. In glucose-rich medium. 2000. Agricultural Research Service.. the total cost of illnesses due to C. perfringens. Ronald J.7% in U. In another report from 1993 to 1997.Pathogens and Toxins in Foods: Challenges and Interventions Edited by V. 1996. gram-positive. Juneja et al. The types (A. perfringens isolates from 131 retail food samples in the United States were enterotoxin-positive C..S. Juneja • Microbial Food Safety Research Unit. D. whereas Wen and McClane (2004) found a prevalence rate of 1. or food service level. 1990). D. N. such as inadequate cooling at the home. 1990). with 100% of these cases being due to food-borne transmission of the pathogen (Mead et al. University of Massachusetts. Novak. Bean et al. Sofos © 2010 ASM Press. beta.. Microbiological testing for confirmation is based upon the ability of the pathogen to hydrolyze gelatin. The incidence of C. CDC. K. The length of the rods depends upon the growth environment. Types B. MA 01003-1021. perfringens also produces a wide variety of extracellular toxins (soluble antigens) (Table 1). Juneja. with proper refrigeration during shelf-life storage. Labbe CHARACTERISTICS OF THE ORGANISM AND TYPE OF ILLNESS Clostridium perfringens is an anaerobic (microaerophilic). C. reduce nitrate to nitrite. nonmotile.S.1% of the outbreaks and 3. and D are pathogenic to humans. perfringens accounted for 2. rod-shaped bacterium.000 cases per year. 1999) and with an estimated cost of $200 per case (Todd. perfringens food poisoning are mild and are not reported. perfringens is ubiquitous in the environment and is found in soil. Bean and Griffin. 2006a). Wyndmoor. However. whereas in starch-based sporulation medium. leading to 41 hospitalizations and 7 deaths each year. Types A. In a survey conducted by Lin and Labbe (2003). Novak • American Air Liquide. U. The enterotoxin produced by type A and C is distinct from the exotoxins and is responsible for the typical symptoms of food poisoning. and maybe A affect animals. 2000). 1999). dust. and iota (Petit et al. none of 40 C. perfringens was estimated at $123 million in the United States (Anonymous. rarely involving commercial meat processors (Bryan. perfringens outbreaks usually result from improper handling and preparation of foods. perfringens gastrointestinal illnesses in the United States has been estimated by the Centers for Disease Control and Prevention to be 248. retail food. PA 19038. These toxins are not associated with food poisoning. perfringens found in raw foods is enterotoxin negative.. retail. perfringens. 1989). perfringens organisms. raw protein foods of animal origin are frequently contaminated with C. C. a large proportion of C. 1988. the colonies appear black due to sulfite reduction. Labbe • Department of Food Science. 2003. and in the intestines of humans and animals (ICMSF. B. 1997. John S. Amherst. DE 19702-2462. spore-forming. E. Foods that are associated with food poisoning outbreaks contain large numbers of enterotoxin-positive C. 53 . On sulfite-containing agars. Newark. C. lacking cpe (Saito. 600 E. Department of Agriculture. containing cpe. C. Thus.

.... The enterotoxin is responsible for the pathological effects in humans... 1972)....... D)...... such as spices......... Hemolytic.................................. 1988).. lethal Epsilon-toxin (B..... B............. Hyaluronidase Nu-toxin (A........ C.. 66.. improper cooling (40. 1988).......... 1989) 6 and occurs typically from the ingestion of Ͼ10 viable vegetative cells of the organism in temperatureabused foods (Labbe and Juneja...54 JUNEJA ET AL. E) .... D) . E) ........... and acute symptoms usually last less than 24 hours.... D..... respectively (Tschirdewahn et al. perfringens enteritis outbreaks in the United States from 1973 to 1987 and 33................... 1969..... necrotizing..... outgrow........ gelatinase) Lambda-toxin (B.. and multiply at a very rapid rate during postcook handling........ as well as the typical symptoms of acute diarrhea with severe abdominal cramps and pain (Duncan and Strong... perfringens....... 2002).. Germination and outgrowth of C. C............ Toxins of Clostridium perfringens Lethal toxin (types) Activity of toxins Alpha-toxin (A. Collagenase (lethal................. perfringens food poisoning is one of the most common types of food-borne illness (Labbe... lethal (activated by trypsin) Kappa-toxin (A. poultry...... Lethal (validity questionable) Theta-toxin (A...... Accordingly.. Hemolytic (oxygen labile.. and pigs........ generally through fecal contamination of carcasses... C.... although poultry products were also commonly implicated (Bean and Griffin..... Complex with plasma membrane contributing factors leading to food poisoning associated with C................... turkey.................... Duncan et al.. The organism was found on 29. 2002)... E) ..... C) .......... and other meat dishes have been associated with C...... the vegetative cells sporulate. most outbreaks of C. Brynestad and Granum.... C.......... Full recovery within 24 to 48 hours is normal... The typical incubation period before onset of symptoms is 8 to 24 hours.............. D) .... Lethal.. Lethal Delta-toxin (B........ meat-containing Mexican foods..... Proteinase (disintegrates Azocoll and hide powder but not collagen........... D.... 2002... pork........ Deoxyribonuclease Enterotoxin (A.. Roast beef and other types of cooked beef were implicated primarily as vehicles of transmission for 26... as well as the ability to germinate... respectively ... necrotizing (activated by trypsin) Eta-toxin (A) ................... C................8% of 190 C........... Lethal... Meat and poultry products were associated with the vast majority of C........ 1990....... hemolytic lecithinase C Beta-toxin (B............... and 35% of beef.......... lethal) Iota-toxin (E) ....................... 1991)...... E) ..... perfringens can be confirmed if Ͼ10 CFU/g of the organism or Ͼ10 spores/g are detected in the implicated food or feces.... E) .. necrotizing................ 1998).. C) ...... Acidic conditions encountered in the stomach may actually trigger the initial stages of sporulation of the vegetative cells of C........... perfringens outbreaks in the United States.... releasing an enterotoxin upon sporangial autolysis.................... necrotizing Gamma-toxin (B............... perfringens food poisoning outbreaks (Bryan.. The organism is found frequently in meats........ and 2% of fecal samples from cattle... perfringens include its ability to form heat-resistant spores that can survive commercial cooking operations........................................ perfringens spores during cooling of thermally processed meat products have been reported extensively (Juneja et al. primarily under conditions conducive to germination....9%) of food products has been cited as the most common cause of C......... 1994c........ contamination from other ingredients.... Lethal. gelatinase) Mu-toxin (A... and lamb carcasses...... B....... perfringens food poisoning can be avoided by adequate cooking of meat products followed by holding at hot temperatures or rapid cooling...... 1999). Such conditions occur when the cooling of large batches of cooked foods is not fast enough to inhibit bacterial growth or when the foods are held at room temperature for an extended period or are temperature abused.... Pyrexia and vomiting are usually not encountered in affected individuals.... Once in the small intestine... Bryan.. C....... D............9% of 115 outbreaks from 1977 to 1984. C... C) . Necrotizing. Food-borne outbreaks of 5 C....... or postprocessing contamination... Roast beef........... Fatalities are rare in healthy individuals.................. perfringens outbreaks (Angulo et al... 80%... Table 1....... perfringens was detected in 36%......... B.... 6 SOURCES AND INCIDENCE IN THE ENVIRONMENT AND FOODS Clostridium perfringens is commonly found on vegetable products and in other raw and processed foods..... D......... In retrospect.. B. probably due to the fastidious requirement of more than a dozen amino acids and several vitamins for the organism to grow in these products (Labbe and Juneja.. D......... respectively (Labbe and Juneja. 1969..... 2002)..

1996). It is currently estimated that the incidence of foodborne illness from C. 2003). perfringens isolates in these two environments and reported that neither soil nor home kitchen surfaces represent major reservoirs for type A isolates with chromosomal cpe that cause food poisoning. fish.1% of processed and unprocessed meat samples tested in one study. These surveys indicate a low incidence of C.9% of commercial pork sausage samples (Bauer et al. Populations of vegetative cells and spores did not exceed 2. ground. perfringens in raw and processed foods. Li et al. 1979). perfringens. predominantly C.3 and 66 spores per g. as was previously explained in this chapter. Rahmati and Labbe (2008) reported 17 samples positive for C. and the incidence of C. 48. In the United States from 1983 to 1997. 1981) and on raw beef. In another survey. Growth outside this range of temperatures may be characterized by extended lag and longer generation times and might require strict anaerobic conditions. and cooked beef in food service establishments (Bryan and McKinley. 2008). Taormina et al. 1989). 1979). The optimal growth temperature range for the organism is 43 to 45°C. C. combined with an absence of active surveillance for this microorganism. emulsified meats and in raw comminuted meat samples.. and a mean level of 2 45. perfringens spores were isolated from 80% of 54 different spices and herbs (Deboer et al. C. veal. 2001). and chicken products (Hall and Angelotti. perfringens vegetative cells and spores.72 and 2. (2007) surveyed soils and home kitchen surfaces in the Pittsburgh. perfringens was the third most common cause of confirmed outbreaks and cases of food-borne illness (CDC. respectively. About 50% of raw or frozen meat and poultry contains C.. equipment. 1999). perfringens on raw poultry ranges from 10 to 80% (Waldroup. early surveys of foods did not determine the enterotoxin-producing ability of isolates. 2002). In recent surveys on the incidence of C. In a survey conducted in the United Kingdom by Hobbs et al. perfringens is quite aerotolerant. Underreporting. all but 2 samples had undetectable levels (Ͻ3 spores per g) and 2 ground pork samples contained 3. one possessing the enterotoxin gene.3°C was observed only under strict anaerobic conditions (Shoemaker and Pierson. cured. However. C. 1976). In a national survey of the retail meats conducted in Australia.. out of 347 fresh and processed seafood samples examined. This presents a public health hazard since spices and herbs are commonly used in meat and meat products. 2004). Many areas within broiler chicken processing plants are contaminated with the organism (Craven. although soil does appear to be a reservoir for cpe-positive isolates causing non-food-borne gastrointestinal diseases. Out of 194 cured whole-muscle samples examined.CHAPTER 4 • CLOSTRIDIUM PERFRINGENS 55 (Smart et al. (2003) reported that out of 197 raw comminuted meat samples analyzed. These studies suggest a low incidence of spores in raw. 2000). 1985). INTRINSIC AND EXTRINSIC FACTORS THAT AFFECT SURVIVAL AND GROWTH IN FOOD PRODUCTS AND CONTRIBUTE TO OUTBREAKS Temperature Although technically an anaerobe. 1965). The mild symptoms of the illness result in underreporting. perfringens was detected on 38.00 log10 CFU/g.1 C. leads to only a select number of cases that are voluntarily reported to the Centers for Disease Control and Prevention in any given year. There is no evidence to suggest that the contrary exists today. perfringens was detected in 47. C. whole-muscle ham.. 67% of the vacuum-packaged fish product samples were positive for clostridia. as well as low levels of spores in cured. Kalinowski et al. C. 1974). perfringens in retail meats.. C.. beef. C. pork. 1996). a survey of American retail foods did report that approximately 1. perfringens is a factor of 10 to 350 times the number of cases actually reported (Mead et al. perfringens. PA (United States) area to determine the prevalence of cpe-positive C. Populations of C.7% of raw meat. Determining the level of contamination. perfringens (Labbe.6% were positive for vegetative cells. perfringens was not recovered from any of the 94 ground beef samples and was isolated from 1 of 92 samples of diced lamb (Phillips et al. and poultry items sold in retail food stores contain type A isolates carrying a chromosomal cpe gene. an incidence level of 30 to 80% has been found (Lin and Labbe. including beef. Growth at 52. and spores were not detected. and chicken product mixtures acquired from industry sources for C. although growth can occur between 15 and 50°C (Labbe and Juneja. (1965). and no plasmid cpe gene was found in any of those surveyed retail foods.3% were positive for vegetative cells and spores. lamb. suggesting that the pathogen does not grow at proper refrigeration . Out of 152 cured ground or emulsified samples. perfringens at temperatures below 10°C have been reported to decline or remain stable. perfringens was isolated from 43.4% of raw ground beef samples (Ladiges et al. 1.7% and 5.. In another study (Wen and McClane. (2003) examined a total of 445 whole-muscle and ground or emulsified raw pork. perfringens CFU per cm was detected on raw beef carcass surface samples (Sheridan et al.

One strain of C. Curing Salts The sodium chloride level in a food affects the ability of C. soups. In general.5 logs by day 8 for both strains (Juneja et al. 1988). Labbe (1989) suggested that an acidic pH in foods can act synergistically with other hurdles. 1994b). mean log10 CFU/g increased anaerobically by 4 to 4. perfringens does not grow very well in the presence of oxygen. However. perfringens growth sufficiently to cause illness. perfringens vegetative cells in frankfurters increased by 3 log10 cycles in 3 days at 15°C and in 5 days at 12°C. respectively (Strong et al. While growth was not inhibited by 4% (wt/vol) NaCl.. although growth was faster in both products held under anaerobic conditions. These data suggest that low-aw foods are unlikely to support C. 1970. Regardless of packaging. some strains do not grow in 5 to 6% NaCl. Similarly. perfringens population at an aw of 0. 1970).8 log CFU/ml by 5 h and 4. While germination of spores was inhibited on laboratory medium at an aw value of 0. perfringens is capable of extremely rapid growth in meat systems.0 and Ն8. perfringens grew faster at 45°C in autoclaved ground beef than in broth media at the same temperature (Willardsen et al. and most . (1994a.97 (Kang et al.3 (Hobbs. and the presence of various nutrients (Craven. foods contaminated with low levels of C. perfringens.. Juneja et al.0.1 minutes in autoclaved ground beef held at 41°C. 1969. growth at 15°C in air was both slower and less than that under vacuum. refrigeration of foods contaminated with spores and vegetative cells of C. perfringens growth. 1966). perfringens would not result in substantial growth within 5 h of temperature abuse at 37°C. 2006). even after extended storage. C. perfringens growth (Juneja et al.. 1994a). 1979).. Some growth may be expected at pH values of Յ5.96.. 8. Geopfert and Kim (1975) reported that C. and the range is between pH 5.. perfringens vegetative cells inoculated into meat began multiplying without any lag phase. such as curing salts. can rapidly be reached under optimal conditions in meats and meat products. Labbe 1989). pH Optimum pH for C. Temperature abuse (28°C storage) of refrigerated products for 6 h will not permit C.93. Some sausages. temperature abuse of a product stored under aerobic conditions is unlikely to inhibit C. the organism either declined or did not grow at 4. growth was relatively slow and total viable count increased to Ͼ6 logs within 36 h. Strong et al. 1980). though growth can occur at an aw of 0. Oxygen C. perfringens to grow. hams. 1969). This suggests that aerobic conditions can delay growth of C. aw Growth of C.. and 42°C under anaerobic atmosphere and at 37 and 42°C under aerobic conditions. C. perfringens growth completely. Thus.93.93 to 0.9 log10 CFU/ ml by 12 h at 37°C (Kang et al.0 and 9. a C. to restrict C.. although the mean generation time for an eightstrain mixture ranged from 19. C. and 12°C. certain C. perfringens. 1979. Solberg and Elkind (1970) reported that C. perfringens cells in cooked ground beef and cooked turkey at various temperatures and determined that C. at 45°C. 37. perfringens vegetative cells at 37°C was not observed until 12 and 20 h at aw values of 0. perfringens grew in cooked turkey to about 7 logs within 9 h anaerobically and by 24 h aerobically at 28°C. Because of their rapid growth. acidic foods (pH Յ5.. While aerobic growth was slow at 15°C. salamis. Labbe and Juneja. 1989). pH. Strong et al. that being 6 Ͼ10 cells.0) would not foster growth of C. perfringens growth does not initiate in raw ground beef stored at 15°C or below. At 28°C under aerobic conditions. numbers of C. perfringens will not provide favorable conditions for growth. perfringens is inhibited by water activity (aw) ranging from 0.965 and 0. C.5 minutes at 33°C to 8. Therefore.95 showed an increase of 1. but the growth was restricted at 10 and 5°C. perfringens growth is between 6. perfringens strains have been found to be relatively more acid tolerant than others (de Jong. perfringens can grow in media supplemented with various levels of curing salts. Growth of C. perfringens had a generation time of 7. 1978). oxidation reduction potential. 1994b) examined growth of vegetative C. Nevertheless. Published research suggests that C.0 and 7.56 JUNEJA ET AL. Within this range. However. Hall and Angelotti (1965) found that C.0. respectively. even during temperature abuse. temperatures (Traci and Duncan.8 minutes at 45°C (Willardsen et al. perfringens cells sufficient to cause illness. and chipped and dried beef products have aw values below this level (Holley et al. 1978. which makes the organism a particular concern to meat processors as well as the food service industry. temperature. inhibition is dependent on the solute used to adjust the aw and on factors such as inoculum size. pepperonis. perfringens grew in ground beef to Ͼ7 logs within 12 h at 28.. 1974. perfringens was able to grow in both products under both anaerobic and aerobic conditions.

the inhibitory effect of nitrite can be enhanced under acidic conditions and by prior heating of the medium containing nitrite (Labbe. Further. in cured ham (initial sodium nitrite concentration of 156 ppm). perfringens growth was not inhibited at 8. Therefore. such as NaCl. pH. Mead et al. perfringens from spore inocula from specific meat products (Juneja and Thippareddi.1°C to 7. may negatively impact the product quality and desirable organoleptic attributes of foods. spores are likely to survive the normal pasteurization/cooking temperatures applied to these foods. perfringens at 21 and 17°C after 10 and 21 days. 1965. The differences in the minimum growth temperatures could be a function of product characteristics. Gibson and Roberts. (2004) reported no growth of C. perfringens germinate at a higher rate after heat shock. (1984) found that chicken frankfurters containing 2. perfringens growth from spores (starting inoculum of 4. SPREAD. perfringens spores (Labbe and Duncan. perfringens growth. perfringens spores germinated in raw beef without prior heat shock. 1983). 1981). Cooking of foods can also heat shock C. Perigo and Roberts. Amezquita et al. 1975. 2004a. Further. Labbe and Duncan. 1970. the levels of sodium nitrite necessary to inhibit the strains tested dropped from 300 ppm to 25 ppm when the concentration of NaCl was increased from 3 to 6% (Roberts and Derrick. C.01% sodium nitrite slows germination of both heat-resistant and heat-sensitive C. perfringens spores grew rapidly in frankfurters at 37°C and 23°C. populations increased 2 log CFU/g after 2 days at 15°C and 3 days at 12°C (Solberg and Elkind. 1999. C.. Roberts and Derrick.5°C/hour (Willardsen et al. and concentrations of functional ingredients. and other antimicrobial ingredients. 2003. sodium nitrite has been used as a preservative in cured meats to inhibit Clostridium botulinum growth and subsequent deadly neurotoxin production. For instance. 1968. In another study. Riha and Solberg. 1970). 1977).5°C/hour) from 25°C to 50°C. perfringens spores. These levels are higher than those typically found in ready-to-eat (RTE) foods. respectively. In their study. 2004b. Thus. if designed to inactivate C. 1986). which is ecologically important because C. spores of C.25% salt (Maurer. 1970. C.CHAPTER 4 • CLOSTRIDIUM PERFRINGENS 57 strains failed to grow in 7 to 8% NaCl (Roberts and Derrick. perfringens to grow in many cured meats is well documented.. Sauter et al. long-time cooking will not eliminate C.7 log) over 9 days at 20°C. For instance. 1990). perfringens vegetative cell growth for 74 h at 20°C. 1978).2 in a laboratory medium. Gibson and Roberts (1986) reported that the inhibitory concentrations of sodium nitrite can be lowered if combined with other curing salts. Zaika 2003). These ingredients have been shown to affect the growth of C. Cooking usually increases the anaerobic environment in food and reduces the numbers of competing spoilage organisms. Hallerbach and Potter (1981) reported that frankfurters containing 2. and sodium nitrite is allowed in cured meats at ingoing (initial) levels of 156 ppm.75 and 3. Similar to spores of other bacterial species.. 1986).02% and 0. Anaerobic conditions can also increase the antimicrobial effectiveness of sodium nitrite. such as nitrates/nitrites or salts of organic acids. perfringens growth at 20°C was inhibited by 200 ␮g of nitrite/ml and 3% NaCl or 50 ␮g of nitrite/ml and 4% NaCl at pH 6. with respect to the growth of C. Gough and Alford (1965) reported that C. Most processed meat products contain about 2. Cooking temperatures. Vareltzis et al. and although growth was slower at lower temperatures.000 ppm of sodium nitrite but was inhibited when the concentration was increased to 12. 1989. 1975). Mean generation times in autoclaved ground beef during slow heating from 35 to 52°C ranged from 13 to 30 minutes. since germination activation of C. with temperature increases of 6 to 12. perfringens competes poorly with the spoilage flora of many foods. Traditionally.2% salt and 140 ppm nitrite did not support C. Another study also demonstrated growth of the organism in autoclaved ground beef during linear temperature increases (4. The ability of C. it has been demonstrated that 0. perfringens spores.. Gibson and Roberts. while only 3% of inoculated C. 1980. 1975). OR CHARACTERISTICS Slow cooking associated with low-temperature. such as meat species. It is worth mentioning that the inhibitory effect of sodium nitrite is enhanced at elevated temperatures (Davidson and Juneja.000 ppm. Studies have been conducted with laboratory media on the antimicrobial effects of nitrites. 1978. almost all spores germinated after the beef received a heat treatment . perfringens vegetative cells and germination of spores (Gough and Alford. since heat-injured spores are more sensitive to the effects of nitrite (Chumney and Adams. phosphates. Labbe and Duncan.. 1970). sodium nitrite acts synergistically with NaCl to inhibit C. (Roy et al. perfringens spores can occur at temperatures between 60 and 80°C (Walker. 1978. Riha and Solberg.6% salt and 150 ppm sodium nitrite did not allow C. 1978). FOOD PROCESSING OPERATIONS THAT INFLUENCE THE NUMBERS. perfringens spores. Thippareddi et al. Further. perfringens appears to be more resistant to nitrites.

2°C at an exponential rate. as well as to the food service industry.. 1963). 0. distribution. RECENT ADVANCES IN BIOLOGICAL. The abuse may occur during transportation. 8. (1974) identified the cooling stage as most critical in ensuring the safety of such products. storage. the experimental design included both the vegetative cells and spores of C. germination and outgrowth of spores and C.2°C within 15. or handling in supermarkets. perfringens spores was cooled from 54. or 21 h. Organic acid salts. or during preparation of foods by consumers which includes low-temperature–long-time cooking of foods as well as scenarios in which foods are kept on warming trays before final heating or reheating. Blankenship et al. A limited amount of published research is available regarding growth of the pathogen in cooked cured meats during cooling. 1% sodium acetate. perfringens with a 95% tolerance interval. or 10 h. This study is not totally applicable to typical retail food operations because cooling is not linear.5% sodium lactate. They observed a declining growth rate in the case of 4-h and 6-h cooling times. multiplication of the organism was observed when the rate was less than 15°C/h. a 3. Juneja et al. Taormina et al. . perfringens during cooling of meat products.5% sodium lactate supplemented with sodium diacetate.3% buffered sodium citrate (with or without sodium diacetate). perfringens is capable of rapid growth in meat systems.75 to 1. perfringens grew by 4 to 5 log10 CFU/g if the cooling time was greater than 18 h. 2003). growth of the organism was not detected in any of the products tested during chilling from 54. CHEMICAL. vacuum-packaged.4 to 7. 18. C.4 to 7. As a result of such heat shock conditions. (1974). Recent studies have shown the efficacy of certain antimicrobial agents against the growth of C.0 log10 CFU/g) occurred if cooling took 15 h or less when cooked ground beef inoculated with heat-activated C. In that study. when cooked ground beef gravy inoculated with a mixture of vegetative cells and spores of C. perfringens spores in turkey roasts cooked to an internal temperature of 72°C and then cooled in a walk-in cooler from 48. perfringens vegetative growth are likely to occur in cooked foods if the rate and extent of cooling are not sufficient or if the processed foods are temperature abused. Studies have described growth of C. a physiological response is triggered in organisms. when cooled exponentially from 54. perfringens spores and then subjected them to cooking and either cooling procedures typically used in industry or extended chilling. and 1. when the gravy temperature was in an ideal growth temperature range. perfringens spores inoculated into ground beef at 60°C and cooled to 15°C at a linear cooling rate of 5 to 25°C/h. 1988). making this organism a particular concern to meat processors. The same researchers also demonstrated growth inhibition of the microorganism by oregano in combination with organic acids during cooling of sous-videcooked ground beef products (Sabah et al. However. uncured meat products.8°C did not support C.9°C to 12.1%. perfringens germination and growth in cured hams with NaCl concentrations of 3. This is approximately equivalent to a cooling rate of 12 h and 18 h for temperatures of 54. Shigehisa et al. perfringens during cooling of cooked. Synthesis of HSPs has been observed in bacterial as well as mammalian cells (Lindquist and Craig.5 to 4. the vegetative cells would not likely be present in a cooked product. (2003) inoculated bologna and ham batter with C. To simulate commercial chili cooling procedures. Steele and Wright (2001) evaluated growth of C.8% sodium citrate inhibited growth of C. Zaika (2003) reported complete inhibition of C. Results of that study indicated that an 8.2°C. In a study by Tuomi et al. leading to the synthesis of a specific set of proteins known as heat shock proteins (HSPs). After heat shock.2°C. the total population did not change for the first 150 min. (1994c) reported that no appreciable growth (Ͻ1. They observed that the organism did not grow during exposure to falling temperature rates of 25 to 15°C/h. AND PHYSICAL INTERVENTIONS TO GUARD AGAINST THE PATHOGEN The presence of inhibitory agents in the products can affect germination of C. however. regardless of the cooling rate. (1985) reported on germination and growth characteristics of C. it is exponential.4 to 7. such as 1 to 1. Sabah et al. perfringens spores and may also affect the minimum growth temperatures for the germinated spores. perfringens growth in 6 h. perfringens in cooked.4°C to 7. 2004)..8°C in 6. While the study by Tuomi et al. (1988) conducted exponential cooling experiments in which the cooling time was 4 and 6 h for a temperature decline from 50 to 25°C.2°C within 18 h. that being more rapid cooling at the beginning followed by a slower cooling rate later.9-hour cooling period was adequate to prevent growth of C. Interestingly. perfringens. This implies that C. (2003) found that 0.4 to 7. perfringens NCTC 8239 was cooled in a refrigerator. rapid growth of the organism was reported to occur during the first 6 h of cooling.9 to 12. restructured beef cooled from 54. For instance. (Barnes et al.58 JUNEJA ET AL.07 log increase was observed following a 24-h cooling time (Kalinowski et al. However. Cooked cured turkey cooled from 48..

and carvacrol (1 to 2%).9-fold. 1. or 3% was assessed. in DS medium ranged from 0.21 min (C 1841 isolate) to 1. as well as the biopolymer chitosan (0.6 min for the E13 and F5603 strains. perfringens spores during the chilling of marinated ground turkey breast (Juneja and Thippareddi. (2002) found that spores of enterotoxinpositive C. Heat shocking vegetative cells suspended in beef gravy at 48°C for 10 min allowed the microorganisms to survive longer and increased the heat resistance by as high as 1. Compared to the control (no heat shock). Studies have been conducted to assess the effects and interactions of multiple food formulation factors on the heat resistance of spores and vegetative cells of C. was determined.1 min and 5. expose the contaminating vegetative and spore-forming food-borne pathogens to conditions similar to heat shock. 2006a. perfringens spores by at least 1. perfringens. Bradshaw et al.5 to 2%).3%. 2007. (1997).60 min (F 4969 isolate).0 and salt levels of 0. Sarker et al.2 min (no salt) . carrying either the chromosomal cpe gene or the plasmid cpe gene. (1982) reported that D values at 99°C for C. Juneja and Friedman.40 min (NB 16 isolate) (Juneja et al. although both heat-resistant and heat-sensitive strains can cause food poisoning (Labbe and Juneja.3% sodium pyrophosphate at pH 6. understanding these variations in heat resistance is certainly necessary in order to design adequate cooking regimes to eliminate C. pork ham. perfringens vegetative cells. 2004a.5 to 3%). the D values at 55 or 57°C for the C. perfringens spores (expressed as D values in min) in turkey slurries that included 0.6.1-fold (NCTC 8238 isolate) to 1. perfringens strains were more heat resistant than enterotoxin-negative strains. respectively. perfringens germination and outgrowth during exponential cooling of ground beef and turkey (Juneja et al. the increase in heat resistance of C. respectively) in roast beef. perfringens at 50°C for 30 min and then determined the D values at 85 or 90°C. The thermal resistance (D values in min) of C. the heat shock response. grown at 43°C in fluid thioglycolate medium to an A600 of 0. such as thymol (1 to 2%). 2001a). thus further minimizing risk to the consumer. natural compounds can be used as ingredients in processed meat products to provide an additional measure of safety to address the C. perfringens spores heated in beef gravy at 100°C ranged from 15. perfringens chromosomal cpe isolates were significantly higher (P Ͻ 0. Heredia et al.. Similarly. perfringens vegetative cells in RTE foods. The D values of C. The authors reported that a sublethal heat shock increased the thermotolerance of C.05) than the D values of the C. perfringens hazard during chilling and subsequent refrigeration of meat products. differences in heat resistance levels were not observed at higher temperatures. 2003). were reported. individually inhibited C. the D values at 99°C decreased from 23.5 and 1.5fold (Juneja et al.2-fold (B 40 isolate) to 1. Numerous studies have examined the heat resistance of C. Heat resistance varies among strains of C. food poisoning isolates of the organism are generally more heat resistant than C.50 min (NCTC 8239 isolate) to 21. the increase in heat resistance after heat shocking at 58°C ranged from 1. respectively.to 1. thereby rendering the heatshocked organisms more resistant to subsequent lethal heat treatments. oregano oil (2%). perfringens spores from other sources (Labbe.7. respectively. 1989). 2003). when the thermal resistance of C.4 min. perfringens spores at 100°C after heat shocking ranged from 1.5 min to 124 min.. 2007).CHAPTER 4 • CLOSTRIDIUM PERFRINGENS 59 inhibited germination and outgrowth of C. (1997) heat shocked sporulating cells of C. (2000) reported D values at 100°C for 12 isolates of C. In a study by Juneja and Marmer (1996). perfringens. 2004b). 2006b. who found that a sublethal heat shock at 55°C for 30 minutes increased tolerance of both spores and vegetative cells to a subsequent heat treatment. such as storage and holding temperatures and low-temperature–long-time cooking.5-fold (F 4969 isolate). perfringens cells heated in beef gravy at 58°C ranged from 1.. In another study. thereby ensuring the microbiological safety of cooked foods (Juneja et al. perfringens spores suspended in commercial beef gravy ranged from 26 to 31. 2001a. however. perfringens. Miwa et al. (2003) reported complete inhibition of C. Therefore. Incorporation of plant-derived natural antimicrobials. 2006). cinnamaldehyde (0. the D values at 85°C increased from 24 to 46 min and at 95°C from 46 to 92 min. perfringens spores. (2000) reported D values at 55°C for C.5-fold (NCTC 8239 isolate).5 to 3%) derived from shellfish and green tea catechins (0. respectively. perfringens spores and/or vegetative cells. perfringens spore germination and outgrowth by sodium salts of lactic and citric acids (2. 2. D values at 55°C of 9 and 5 min for the FD-1 and FD1041 strains. In their study. and the induced thermotolerance to assist food processors in designing thermal processes for the inactivation of C. Nevertheless. These authors also reported a strong association of the food poisoning isolates and increased heat resistance. Thippareddi et al. Researchers have reported on the quantitative assessment of heat resistance. Sarker et al. perfringens isolates carrying a plasmid cpe gene.4 to 0. when the heat resistance of C.. Environmental stresses. The D values of the heat-shocked cells were 85 and 10 min. Compared to the control (no heat shock). perfringens cells grown at 37°C in FTG of 12. and injected turkey products. Similar results were obtained by Heredia et al.

15% SPP resulted in a concomitant decrease in heat resistance. perfringens growth from spores for 480 h at 25°C in sous-vide-processed (71. processed meat products. perfringens spores at 99°C. 2.8% NaL restricted C. 57.6. through lag. and 2.. perfringens in extended shelf-life.5. regardless of NaL concentration (Juneja. C. 60. Addition of 0. Juneja et al.6. perfringens spores germinated and grew at 15°C to Ͼ5 log10 CFU/g in turkey with no salt by day 4.5°C. However. and 28°C. Growth from C.1°C) marinated chicken breast. 57. pH 6. 2001).5°C. While addition of increasing levels (1 to 3%) of salt in turkey (Juneja and Marmer. 1995). and 3%. however. mild heat treatments given to minimally processed foods will not eliminate C. as evidenced by reduced bacterial D values.3% SPP and 0. 1996).5°C. After 16 h. perfringens in cooked beef and turkey. and 8. lag times of 7. In a related study. perfringens in cooked-cured and uncured turkey at refrigeration temperatures. Researchers have assessed the efficacy of added preservatives on inhibiting or delaying the growth of C.6 min at 55.75 log10 CFU/g in cured turkey held at 0. and the reductions in levels were similar in uncured turkey. In their study. 10. respectively (Juneja and Marmer 1996). Growth of C. perfringens from spores.5 inhibited C. ranging from 6. 4. While C. Kalinowski et al. When ground turkey containing 0.54. The heat resistance was further decreased when the SPP levels in beef and turkey were increased to 0.1°C and pH 7.1°C) turkey samples. Predictive bacterial growth models that describe C. perfringens and may provide sousvide-processed foods with a degree of protection against this microorganism. the combination of 3% salt and pH 5. perfringens growth from spores in sous-vide-processed ground beef supplemented with 0. particularly if employed in conjunction with adequate refrigeration.7 min (3% salt).52. perfringens spore germination and outgrowth during cooling of food systems have been generated by researchers using constant temperature data.60 JUNEJA ET AL. the addition of 3% salt in sous-videprocessed ground turkey containing 0. delayed growth for a week at 20°C. perfringens growth at 15°C. it delayed growth for 648 h at 19°C. (2003) investigated the fate of C.3.2 min at 55°C to 1.0 h were observed for salt levels of 0. perfringens food poisoning in the absence of proper refrigeration (Juneja et al.3% SPP at 15 and 28°C. perfringens decreased by 2. regardless of the presence or absence of salt (Juneja and Marmer.5 min at 55°C to 1. Thermal death time values from this study should assist institutional food service settings in designing thermal processes that ensure safety against C.9. and had little inhibitory effect at 25°C (Aran. 3% salt) at 99°C (Juneja and Majka 1995). 2. perfringens in a sous-vide-processed food has been assessed. (1996) showed that C. spores germinated and grew at 28°C from 2.2. perfringens cells in ground beef and turkey containing sodium pyrophosphate (SPP). perfringens growth was not observed at 4°C.0) ground beef samples but was delayed until day 8 in the presence of 3% salt at pH 5. (1999) presented a model for predicting the relative growth of C.3. refrigerated. to 17. perfringens spores in cooked ground turkey with added 0. perfringens growth from a spore inoculum at 4°C was not observed with sous-videcooked turkey or beef samples (Juneja and Marmer.5 at 15°C (Juneja and Majka. 1996).5°C. In another study (Juneja and Majka. C. In a beef slurry. 20% O2.4. 2006). the presence of 3% salt in samples at 15°C completely inhibited germination and subsequent multiplication of vegetative cells. The D values for beef that included 0. and 10°C. perfringens growth in cooked turkey can be effectively inhibited in an atmosphere containing 25 to 75% CO2. C. These studies suggest that NaL can have significant bacteriostatic activity against C.5. 1996. or 3% salt was sous vide processed (71. exponential. Juneja and Majka.4. Thus. 3% salt) to 14. Inclusion of 3% NaL in sous vide beef goulash inhibited C.0. and stationary phases of growth. 1995). the values for turkey ranged from 16. and 1. 10. and 1. respectively. 1. and 62.77°C. The efficacy of sodium lactate (NaL) in inhibiting the growth from spores of C. 3.6.1°C) and held at 28°C. While addition of 4.3 min at 62. and 62. respectively. 2.5 in beef (Juneja and Majka. the atmosphere cannot be relied upon to eliminate the risk of C. Juneja and Marmer (1998) examined the heat resistance of vegetative C. 11. even after 7 days of storage (Juneja and Marmer. the D values significantly decreased (P Ͻ 0. at .0 min (pH 5.15% SPP were 17.3%. 1995). perfringens spores if refrigerated products are temperature abused to 28°C for an extended period.3 min (pH 7.5.05) from 23. perfringens spores. Heating such products to an internal temperature of 65°C for 1 min killed Ͼ8 log10 CFU/g. 9. 60.5. perfringens spores occurred within 6 days in sous-vide-processed (71.3% SPP was inhibited for 12 h at 3% salt.0. 1. 3% salt in RTE products will not inhibit germination and growth of C. The z values for beef and turkey for all treatments were similar. respectively. 1996) or a combination of 3% salt and pH 5. 1996).6. Juneja et al.3% SPP delayed growth for 12 h at 28°C and completely inhibited the outgrowth of spores at 15°C (Juneja and Marmer. 5. the values for turkey ranged from 17.22 to 6.1 at 62. The D values for beef that included no SPP were 21. 1996). and a balance of N2 in conjunction with good refrigeration. 1995) can result in a parallel increase in the sensitivity of C.2 min at 55.25 to Ͼ5 log10 CFU/g in sous-vide-processed (71.

5 and 50. When suspended spores . isothermal growth of C. outgrowth.2 log10. exponential. or exposure to ozone.. Smith-Simpson and Schaffner (2005) collected data under changing temperature conditions and developed a model to predict growth of C. and 11. when cooling was extended from 27°C to 4°C in 15 h.5 h. cured chicken (Juneja and Marks. perfringens cells exposed to 3 ppm O3 for 5 min following mild heat exposure (55°C for 30 min) were more susceptible to ozone treatment. respectively. Shelf-life extension of meat processed with 5 ppm O3 for 5 min and containing C.7 log10 and 3. the lethal effect of heat on spores can be enhanced after exposure to ozone. C. time (5 min). 3. For a cooling scenario of 54. 2003c) used different mathematical methods to estimate the growth kinetics of C. the predicted log10 relative growth was about 3. Juneja et al. the models predicted a log10 relative growth of about 1. high hydrostatic pressure. which estimated that exponential cooling from 51 to 11°C in 6. perfringens spores combined with modified atmosphere packaging as a “hurdle” technology was proven to be effective in inhibiting spore germination and outgrowth over 10 days of storage at CO2 concentrations above 30% and 4°C (Novak and Yuan.1 and 0.. 2004).90 log10.6 log10 and 3. Amezquita et al. The kinetic and growth parameters obtained from these studies can be used in evaluating growth of C. with a standard error of about 0. inactivation of spores ranged between 0. 2002). taking into account the larger relative growth. respectively. respectively. (2001b) developed a predictive cooling model for cooked.4 and 2.2 log cycles (Kalchayanand et al. Novak and Yuan (2004) reported that the spores were more sensitive to heat at 55 or 75°C following 5 ppm of aqueous ozone for 5 min. when assuming that the ratio of the mathematical lag time to the generation time for cells in the exponential phase of growth was equal to 8. the efficacy of high pressure is considerably enhanced when combined with heat. According to the coefficient of the model.8 log10 CFU/g. square-waved. and fluctuating cooling temperature profiles. while observed results for two replicates were 0. Recent research has focused on combining traditional inactivation. the estimated theoretical minimum and maximum growth temperatures of C. for a temperature decline from 54. In a model for growth of C.7 log10. and the observed log10 relative growths were 2. still less than stationary levels of about 7 or 8 log10.CHAPTER 4 • CLOSTRIDIUM PERFRINGENS 61 temperatures spanning the entire growth temperature range of about 10 to 50°C. For example. Huang (2003a. and lag time prediction (~1 h) having a very large effect on the predicted final concentration of C. linear. For example. The differences of the estimates obtained for the models were insignificant. 8. perfringens during cooling of cooked uncured beef (Juneja et al. (2006c) developed a model for predicting growth of C.6 log10 (with a standard error of about 0. or ionizing radiation. perfringens in ground beef during isothermal. such as those encountered in meat processing.4°C to 27°C in 1. The effect of the combined intervention strategies is either additive or synergistic. perfringens spores were suspended in peptone solution and exposed to combination treatments of hydrostatic pressure (138 to 483 MPa). cured beef based on growth rates of the organism at different temperatures. the authors described the effect of temperature on the kinetic parameter K (dissociation constant) by the modified Ratkowski model. with small differences in the germination.5 h.1.07). In their study. or 10 h would result in an increase of 1.43 and 0. perfringens from spore populations during dynamically changing temperature conditions.17. 2008). For the latter cooling scenario the levels were greater than 6 log10. perfringens from spore inocula in cured pork ham.068. perfringens during cooling of cured ham and demonstrated that the effective integration of engineering and microbial modeling is a useful quantitative tool for ensuring microbiological safety.43. Likewise. the average observed and predicted log10 relative growths were 3. and growth-limiting factors at subinhibitory levels with emerging novel nonthermal intervention food preservation techniques using ionizing radiation. and temperature (25 to 50°C). perfringens in cooked cured pork were 13.5 log10. in which the interaction leads to a combined effect of greater magnitude than the sum of the constraints applied individually. The above models can be successfully used to design microbiologically “safe” cooling regimes for cooked meat and poultry products. Juneja et al. perfringens in cooked beef during cooling. the average observed and predicted log10 relative growths were 2. respectively.6°C. outgrowth.9°C was evaluated using a methodology that employed a numerical technique to solve a set of differential equations. When C. for the same temperature decline in 3 h. perfringens. antimicrobials. survival. simulating the dynamics of bacterial growth. the predictions were somewhat better. It was suggested that the accuracy of the germination. perfringens at various temperatures from 10 to 48. 2004). (2004) developed an integrated model for heat transfer and dynamic growth of C.08 log10.5 h and 27°C to 4°C in 12. A similar model was later developed for cooked. and lag time model has a profound influence upon the overall prediction. the estimated geometric mean. 2003b. When the cooling scenarios extended to lower temperatures.4°C to 27°C in 1.

producing a distinctive black ferrous sulfide precipitate on tryptose-sulfite-cycloserine (TSC) agar (Harmon et al. 2001). 12 to 52% spores germinated. If the cooling guidelines cannot be achieved. time to results. 2003). 2002). perfringens in foods (Rhodehamel and Harmon. with respect to sensitivity and discrimination against similar related clostridia.4°C should take no longer than 5 h (USDA/FSIS. perfringens based upon the microorganism’s ability to ferment sucrose. The FDA (2001) Food Code dictates that cooked potentially hazardous foods.7°C should take no longer than 1.7°C in 5 h and from 26.. 2001) for chilling of thermally processed meat and poultry products state that these products should be chilled according to the prescribed chilling rates or require that process authorities validate the safety of customized chilling rates to control spore-forming bacteria. ease of analysis. EP1816209. Additionally. Hampshire.7 to 7. which is used for acid phosphatase detection (Araujo. exposure to ammonium hydroxide causes a dark pink color change in phosphataseproducing colonies (Oxoid Ltd. 1998). A recent report found that TSC was still the medium of choice when compared with iron sulfite agar. and additional challenge studies may be necessary to determine whether performance standards have been met. 1998). These methods are based upon the microorganism’s innate ability to reduce sulfite to sulfide. 2007). and from 60 to 5°C within 6 h..4 to 26. Specifically. A recent European patent. it is recommended that uncured cooked meats be cooled from 50 to 12°C within 6 h and from 12 to 5°C within 1 h (Gaze et al. differential clostridial agar (DCA). perfringens can be a potential hazard in processed meat and poultry products.1 log/ml when bacteriocins were supplemented in the recovery medium. Bacteriological Analytical Manual. 2001).4 to 26. and expense of reagents and/or equipment and materials (Table 2). describes a new medium for selective differentiation of C. as a result of lecithinase activity (Rhodehamel and Harmon. The USDA/ FSIS compliance guidelines (USDA/FSIS. fermentation of lactose-producing acid and gas. lack of motility in motility-nitrate medium. 4-methylumbelliferyl phosphate disodium salt. and an ability to reduce nitrates to nitrites (Rhodehamel and Harmon. sulfite cycloserine azide (SCA). perfringens to cause food-borne illness and occasional associated outbreaks necessitates effective discriminatory detection methods for this pathogen in order to ensure reliable and confirmatory epidemiological screening of suspected foods. Safe cooling times for cured meats may be up to 25% longer (Gaze et al.. C. Different media are continually being compared for their use in the enumeration of C. Further presumptive tests include rapid coagulation of an iron-milk medium. perfringens colonies that hydrolyze indoxyl-␤-d-glucoside turn purple. 1998). antigenbased immunological methods. indicating that germination increased both at 4°C and 25°C during 24 h. Each characterization scheme also contains various advantages and disadvantages. C. Oxoid Ltd.to 4-mm opaque white zone surrounding the colonies. perfringens in the finished product. whereas non-C. the guidelines state that cooling from 54.62 JUNEJA ET AL. liquefaction of gelatin in lactose-gelatin medium. 1971). methods are prescribed for the enumeration and identification of C. Food and Drug Administration Center for Food Safety and Applied Nutrition (FDA/CFSAN).2°C in 10 h (USDA/FSIS. 1998). such as meats. computer modeling and/or product sampling can be used to evaluate the severity and microbiological risk of the process deviation. should be cooled from 60 to 21°C within 2 h. (ii) toxin. In the United Kingdom.. Additional guidelines allow for the cooling of certain cured cooked meats from 54. Regulatory Requirements Due to its ubiquitous nature and rapid growth in meat products.5 h and that cooling from 26. The chromogenic m-CP medium differentiates yellow colonies of C.7 to 4. were pressurized at 50°C for 5 min and stored at 4°C or 25°C for 24 h. they must be able to document that the customized or alternative cooling regimen used will result in a less than 1-log10-CFU increase in C. perfringens colonies in water samples and claims to provide increased sensitivity and specificity compared to TSC agar (Oxoid Ltd. These results show that germinated spores at high levels could be killed by using a bacteriocin-based preservative in foods. With added egg yolk emulsion. perfringens bacteria from foods. (Basingstoke. 1998).. perfringens that utilizes sodium bisulfite and ammonium ferric citrate to distinguish sulfite reduction capability combined with a fluorogenic substrate. Detection of C. United Kingdom) has recently developed a chromogenic medium for identification and enumeration of C.. According to the U. Shahidi-Ferguson perfringens agar. perfringens colonies produce a 2. The many available methods of detection can be categorized as (i) metabolite-based biochemical (phenotyping/biotyping) assays. Log10 reductions of spores were 6. and (iii) nucleic acidbased molecular techniques.S. perfringens in Foods The ability of C. 2002). and oleandomycin polymyxin sulfadiazine perfringens agar (de Jong et al. If meat processors are unable to meet the prescribed time-temperature cooling schedule. Although all .

Unfortunately. (2003) Uzal et al. In another study. B. or E) of C. perfringens in food (de Jong et al. TSC still appears to be best for detection and enumeration of C. C. perfringens spores in water (Araujo et al. Whole-cell preparations of isolated C. many other methods that are not listed here may represent only small variations or improvements over the techniques represented here. perfringens bacteria from foods (Emswiler et al.. perfringens provides an ample library of antigenic determinants for subsequent immunological detection schemes. sulfite polymyxin sulfadiazine agar. m-CP agar. 1999.. of the media tested were effective for enumeration studies.. in terms of differential selective media and after more than 30 years of medium innovations. perfringens strains (Sterne and Batty. (1980). 1980). perfringens cultures were used as antigens against antisera raised using confirmed type strains (Stringer et al.CHAPTER 4 • CLOSTRIDIUM PERFRINGENS 63 Table 2. (2004) This list is representative of the many types of methods available.. Comparison of commonly used discriminatory methods for detection of C. In addition. As a result. It was determined that membrane filtration with fluorogenic TSC agar performed better than any of the other tested enumeration media for C. and DCA and SCA were very laborious to prepare (de Jong et al. (2006) Schalch et al. perfringens (Araujo et al. and Wilson-Blair agar for detection and enumeration of C.. Shahidi Ferguson perfringens and oleandomycin polymyxin sulfadiazine perfringens agars produced low counts at times.. (1988) Brett (1998) Baez and Juneja (1995) Heikinheimo et al. The many toxins produced by C. (2004) Maslanka et al. (1997) Al-Khaldi et al.. whereas cooked meat medium was less favorable for observing C. Lactosesulfite broth was suggested to have greater sensitivity than that of TSC for enumerating C. 1975).25 ng/ml toxin 0. 1980).. toxin-type positive results were at the expense of the animal and may be considered inhumane. and although it may not be totally inclusive. and iota (Petit et al. The mouse neutralization protection test was used initially to type C. (2004) Wise and Siragusa (2005) Baums et al. 1977). (1999) Keto-Timonen et al. 2003). The slide agglutination serotyping technique was developed by Stringer et al. tryptose-sulfite-neomycin agar. 2004).. perfringens growth (Xylouri et al. D. perfringens. 2004). 1997). perfringens bacteria but was not exclusively selective for C.075 MLD50/ml 0. Neutralization of toxin-type specific pathological effects was accomplished using appropriate antisera in laboratory mouse models (Sterne and Batty.. perfringens Method Mouse neutralization ELISA 4-Layer ELISA PC-ELISA MC-ELISA CIEP Vero cell assay Reversed passive latex agglutination Colony hybridization Hydrophobic grid membrane filter Real-time PCR Multiplex PCR PFGE AFLP Ribotyping Oligonucleotide microarrays a a Detection limits 6 MLD50/ml 5 ng CPE/g feces 6. epsilon. Iron sulfite agar lacked selectivity (de Jong et al. perfringens is not based on the serologic specificity of C. beta.. 2003). Agglutination to specific antisera resulted in the designation of specific serotypes (Stringer et al. perfringens enterotoxin (CPE)-related food-borne illness but on the many other exotoxins produced by the microorganism and is designated alpha. (2003) Uzal et al. fluorocult-supplemented TSC agar was compared with TSC agar. the toxinogenic typing (type A. perfringens are listed in Table 1. (1985) El Idrissi (1989) Uzal et al. (2003) Berry et al. Therefore. 2003). (2003) Bartholomew et al. 2004). Typically intestinal . 1975).75 MLD50/ml 200 MLD50/ml 40 ng CPE/g feces 50–100 ng CPE/g feces 10 CFU/g food 10 CFU/g food 50 fg genomic DNA 20 cells (pure culture) Enrichment culture DNA preparations from enriched samples DNA preparations from enriched samples DNA preparations from enriched samples DNA preparations from enriched samples Considerations Inhumane and costly Sporulation variability in foods Proteolysis False positives Lower sensitivity False negatives Sensitivity and reproducibility Nonspecific interference 48-h detection Isolation of cpe-positives Unidentified PCR inhibitors False positives Problematic nucleases Multiple genetic elements Costly equipment and existing databases Fluorescently labeled DNA probes hybridized to single-stranded DNA on a chip Reference Uzal et al. perfringens on m-CP medium was explained by the very subjective color differentiation of pink colonies on the agar following exposure to ammonia fumes (Araujo et al. Brown 2000). a low confirmation rate for C. SCA and DCA were less effective at low concentrations of C. Such a range of products specific to C.

which detected cpe in 4.25 ng/ml for purified toxin preparations (El Idrissi. including a polyclonal capture enzyme-linked immunosorbent assay (PC-ELISA).57%). 2003). 2003).. Due to the inconsistencies among the diagnostic tests for enterotoxemia from C. With respect to ability to detect epsilon-toxin in the intestinal contents and body fluids of sheep and goats. 2003). Baez and Juneja (1995) used a filter colony hybridization assay with digoxigeninlabeled DNA specific for the C. 1985. The mouse neutralization test was also used as a standard (6 MLD50/ml) (Uzal et al. The tissue culture assay using Vero cells has been determined to have relatively low sensitivity.21%) (Uzal et al. perfringens and enterotoxigenicity with a dot-blot technique incorporating a cpe-gene digoxigenin-labeled DNA probe. perfringens cells from meat samples with anti-digoxigenin antibody conjugated to alkaline phosphatase. 2004). the PC-ELISA was most sensitive in enabling the detection of a 0.64 JUNEJA ET AL. fluid samples are typed by combined inoculation of known antitoxins into mice. 1995). 1988).. 2005). expensive. The 2-day assay was sensitive enough to detect Յ10 CFU/g in the presence of a 6 heterogeneous bacterial flora containing 10 CFU/g (Baez and Juneja. Current innovations in epidemiological detection of food-borne CPE include a multitude of DNAbased technologies. The sensitivity results were in marked contrast to specificity results for the PC-ELISA (31. Berry et al.. perfringens bacteria from raw beef. Sheng and Cherniak 1998). Another group of researchers has developed a four-layer sandwich ELISA for the detection of type D epsilon-toxin with a sensitivity limit of 6. Type A food-borne CPE production is considered a relatively rare characteristic. these assays depend on the in vitro sporulation of such isolates. a monoclonal capture ELISA (MC-ELISA). present in approximately only 6% of tested C. 2003). and counterimmunoelectrophoresis (CIEP) (Naik and Duncan. The PCR was used to amplify and incorporate digoxigenin dUTP into a 364-bp internal region of the cpe gene (Baez and Juneja.25% of 188 confirmed isolates of C. 1984. their use is of limited practical routine practice due to being laborious. Many strains of C. Western immunoblotting for CPE typically suffers from the disadvantage of isolates poorly sporulating in vitro in lab media. perfringens on selective agar and then amplified using specific primer pairs for a gene encoding a product such as alpha-toxin (Kalender and Ertas. perfringens epsilon-toxin.075 50% mouse lethal dose (MLD50)/ml compared to the MC-ELISA (25 MLD50/ml) and CIEP (50 MLD50/ ml) (Uzal et al.. Although both gas-liquid chromatography and nuclear magnetic resonance spectroscopy have shown the ability to discriminate among strains of C. 1995). 380 samples of spices and herbs were analyzed for C. having a limit of detection of 40 ng enterotoxin/g feces compared to an ELISA (Tech Lab. A sensitive two-site enzyme-linked immunoassay utilizing electrochemical detection has been developed for C. 1994). There are numerous PCR methods for detection of C. A reversed passive latex agglutination assay for CPE quantitation is also available from Oxoid. 1977. Another similar method has been described for the enumeration and facilitated isolation of cpe-positive C. preventing the detection of the sporulation-associated enterotoxin (Kokai-Kun et al. . 1998). perfringens type D epsilon-toxin. In a similar manner.. which allows the mice to survive if protected through the neutralization of only those toxins in the intestinal fluid samples that match the known antitoxins.. DNA samples are isolated from suspected isolates of C. perfringens (RodriguezRomo et al. perfringens sporulate only reluctantly (Hsieh and Labbe. perfringens from fecal samples using a hydrophobic grid membrane filter colony hybridization method (Heikinheimo et al. perfringens type A enterotoxin gene to enumerate enterotoxigenic C. Naylor et al... perfringens isolates carried a chromosomal cpe gene (Collie and McClane. A number of immunological tests have been evaluated for detection of C. 2007). Serological tests that have been used for epsilon-toxin detection include radial immunodiffusion and reverse passive hemagglutination (Beh and Buttery 1978).0 ng/ ml based on use of microtiter plates and polydispersed polymeric microbeads. There are a number of methods used for the detection of CPE in feces.. 1998). perfringens from foods and stool samples. Uzal et al. 1989). Double antibody sandwich techniques have been used previously (Weddell and Worthington. 2005). Inc. It has been reported that all of the food poisoning C. the authors recommended that absolute confirmation should include combined clinical and pathological data as well.89%).) that can detect 5 ng/g (Bartholomew et al.1 and 13. 1987). Following hybridization of filter membranes containing fixed DNA from C.. 1989). perfringens phospholipase C (alpha-toxin) at detection limits of 67. and CIEP (84.. visualization was accomplished with a blue precipitate using the substrates nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (Baez and Juneja. respectively (Cardosi et al. 1995). MC-ELISA (57. perfringens strains (Van Damme-Jongsten et al. In the case of isolates (as opposed to feces). perfringens due to capsular polysaccharide characteristics.. and time consuming (Paine and Cherniak 1975.

. and cpb2. In AFLP analysis the total DNA from an isolate is digested with restriction enzymes. At the 90% correlation level. 1998). 2006).. and enterotoxin toxin genes in C. Other limitations to PCR use include accurate product size determinations and nonspecific target amplifications. perfringens isolates (Lin and Labbe.. perfringens strains of animal origin (Shinya et al. perfringens isolates analyzed (Schalch et al. through the combination of PCR with fluorescently incorporated nucleotide base labels in the DNA amplification process for specific gene targets. but 10. 2005). 2006). resulting in ribopatterns with greater discernibility than that of the profiling of plasmid DNA (Schalch et al.000 C. epsilon-.... EcoRI-digested DNA fragments were probed with digoxigenin-labeled 16S and 23S rRNA from Escherichia coli. and beta-2-toxin genes of C... The presence of alpha-.. perfringens toxin genes. perfringens CFU/g in cecal samples as a result of unidentified PCR inhibitors (Wise and Siragusa. such as cpa. DE) offers an automated RiboPrint system.. iota-. there are reports that amplified fragment length polymorphism (AFLP) analysis can provide an alternative solution to those problems (Keto-Timonen et al.873) (Siragusa et al. beta-. such as the alpha-toxin gene present in all C. beta-2-. 2006). A drawback of the technique was the presence of multiple C.. perfringens genotyping diagnoses (Baums et al. Unlike standard restriction enzyme patterns for DNA agarose gel electrophoresis. perfringens strains that degrade the genomic DNA preparations (Maslanka et al. Twelve distinct ribotype patterns were discovered among 34 C. 2003). The change in the direction of the electric field and current during a preprogrammed electrophoretic migration of genomic DNA allows the separation of very large (Ͼ10-Mb) DNA fragments. perfringens isolates from poultry across Sweden has been analyzed using PCR (Engstrom et al. as well as different biotypes in the same AFLP profile that was attributed to mobile genetic elements. and bacteriophages (Shinya et al. The AFLP procedure does not need to involve a complex mixture of complementary restriction enzymes. perfringens biotypes in the same animal. economic method with good discrimination and reproducibility for C.938) than that with ribotyping (0. enterotoxin.toxin (cpb1). Certainly with respect to specific components of foods. perfringens isolates in a food or fecal sample (Wise and Siragusa. and beta-2. including the genes for iotatoxin (iA). 2004). perfringens (Shinya et al. iota-. 2005).. molecular subtyping of C. 1999). and then adapters that allow amplification of subsets of fragments by PCR are ligated. plasmids. producing a fingerprint that can be used with fluorescently labeled primers to allow computer automated data collection and database searches for comparisons (Vos et al. Pulsed-field gel electrophoresis (PFGE) has been successfully used to type C. insertion sequences. 2006). 2006).CHAPTER 4 • CLOSTRIDIUM PERFRINGENS 65 Specific PCR assays have been developed for the detection of the alpha-. perfringens strains. A quantitative real-time PCR assay. iap. was used to provide a quick. Hind III. multiplex PCR assays have been developed to simultaneously detect more than one gene target. perfringens isolates (Maslanka et al. such as transposons.to 8-bp cutters) are used in order to enhance discriminatory differences in DNA banding patterns. perfringens/g in ileal samples. 2006). The inability to subtype 8% of the C. Dupont Qualicon (Wilmington. and epsilon-toxin genes that offer a reliable and quicker alternative to the biological mouse assay for toxin typing of C. 1995). epsilon-. as a recent assay using a single restriction enzyme. etx. beta-1. perfringens (Kanakaraj et al.. alpha-toxin (cpa).. In addition to single gene PCR assays. 1997). repetitiveelement PCR with Dt primers demonstrated greater discrimination (0. restriction enzymes that cut DNA less frequently (6. beta-. providing very useful C. With respect to extracellular DNase production limiting C.. 1998). A further advancement of multiplex PCR amplification of DNA sequences involves the use of a microarray-based method for the characterization of six C. such as collagen. 1999). Nauerby et al. PFGE has been shown to be an effective means to analyze the genetic diversity among C. 1997). as well as the cpe gene present only in food poisoning isolates of C.toxin . The analytical sensitivity limit of the real-time PCR assay was reported to be 100 CFU of C. perfringens strains (Miserez et al. Ribotyping was used to determine the genetic relationship of C. enterotoxin E (cpe). 1997). perfringens PFGE results. there may be inhibitory barriers to PCR use (Kim et al. perfringens isolates was dramatically improved over PFGE (Siragusa et al. perfringens isolates from foodborne poisoning cases (Schalch et al. Using repetitive-element PCR and EcoRI ribotyping.. cpb. 2003). perfringens isolates analyzed using PFGE could be explained by the presence of strong nucleases from C. epsilon-toxin (etxD). Another application advantage for multiplex PCR would be the comprehensive detection of several toxin genes. The AFLP method allowed for PCR amplification of the alpha-. beta-. enables an even more rapid detection (within a few hours) and enumeration scheme for C... cpe. Ribotyping is a method that utilizes the highly conserved nature of ribosomal DNA sequences as a means of identifying discriminatory strain differences. 2000). 2003.

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Beauchamp • Colorado Premium Foods. N. even though surveillance programs have been improved and include more extensive geographical areas. coli to make its way into and persist in food products.. 1994.. Rangel et al. grapes. as well as discriminative detection methods for confirmation of these pathogens and trace-back of contaminated food products. during which citrate. coleslaw. carrots. and ready-to-eat beef products (Bell et al. Under optimal temperature conditions (35 to 42ЊC and 95 to 104ЊF) the pathogen can replicate at pH values of 4 to 10 and in the presence of up to 8% sodium chloride. 2002). Traditionally. E. coli are not human pathogens. coli Catherine S. All compounds required for the metabolic function of E.html. 71 . butter and soft cheeses made from raw milk. Greeley. 2006. there has. While potentially contaminated beef products continue to be recalled. Fort Collins. Such flexibility is permitted through facultative respiration. DC Chapter 5 Diarrheagenic Escherichia coli Catherine S. 2005.000 deaths to food-borne diseases each year (Mead et al. Beauchamp and John N. that the manipulation of one or more growth factors can influence the minimum or maximum values of other factors involved in microbial survival and/or growth (Bacon and Sofos. While this information may be outdated. 1996. 2005). The six classes of diarrheagenic E. Juneja and J. 1997.. 1995b. NIIDIDCD/MHWJ. coli infections in the young. its incidence in the environment and in food. coli bacteria are mesophilic organisms which replicate at temperatures of 7 to 45ЊC. cheese curds. 1993. Colorado State University. been a shift in the vehicles of pathogen transmission (CDC.. Tuttle et al. 2003). Tilden et al. unpasteurized apple juice and cider. A minimum water activity of 0. Karmali. 1996). 1993. melons. 1988). 1997. Griffin and Tauxe. factors associated with its association. 1980. 1999). 1983.cfsan. 2035 2nd Ave. and recent advances in control measures used in food processing. (ii) chronic urinary tract infections. Johnson et al.fda. CO 80631. coli. THE ORGANISM Characteristics Escherichia coli is a member of the Enterobacteriaceae family.gov/ola/2006/foodsafety1115. Rangel et al. 1997. survival and growth in food products.. the incidence of documented food-borne diarrheagenic Escherichia coli infections has remained fairly constant over the past 30 years. the elderly. 1995a. 1996. CDC. including unpasteurized (raw) milk. Sofos • Center for Meat Safety & Quality. This chapter presents an overview of diarrheagenic E.html). and is known for its ability to adapt and colonize a diverse array of reservoirs. NO2.. and (iii) gastroenteritis (Buchanan and Doyle.95 is required for growth (ICMSF. 1171 Campus Delivery. Morgan et al. such as Salmonella and Shigella. While most strains of E. certain serotypes are responsible for three types of human illness: (i) neonatal meningitis. however. 1991. Sofos © 2010 ASM Press. http://www. Riley et al. Souza et al. Washington. Sofos Existing estimates attribute 76 million domestic illnesses and 5. Department of Animal Sciences. Food Safety Cluster of Infectious Diseases Supercluster. John N. and bagged lettuce and spinach (Besser et al. however.Pathogens and Toxins in Foods: Challenges and Interventions Edited by V.fda. 1997). necessitates the routine assessment and validation of established and novel pathogen mitigation strategies throughout the food production chain. yogurt and ice cream. other types of food products have also been implicated in outbreaks. along with other enteric gram-negative bacteria... radish and alfalfa sprouts. including open-air environments and the gastrointestinal (GI) tracts of mammals and birds (Falkow. 1999). and NO3 take the place of oxygen in the electron transport chain (Stewart. The ability of diarrheagenic E. coli infections were primarily associated with raw. 2002). outbreaks of food-borne diarrheagenic E.. 2000.gov/~comm/ift3-4a. in conjunction with an increased incidence and severity of diarrheagenic E. CDC.. CO 80523-1171. It is important to note.. 1989. undercooked. and immunocompromised individuals.. coli can be synthesized from glucose (Sussman. http://www. K.

cidrap. symptoms. Berger et al.. severe dehydration Dysentery syndrome Infectious dose High. elderly Symptoms Severe diarrhea. coli (EAEC or EAggEC).html.html). 1986). and susceptibility may also be heightened when using oral contraceptives or antibiotics (Begue et al. more prevalent in developing countries Travelers and infants native to developing countries Children.gov/~ebam/bam-4a. http://www.fda. enteroaggregative E. Illness is uncommon in healthy adults. coli (EHEC). herniated bowels. nausea. vomiting Watery or bloody diarrhea.cfsan. Other symptoms may include mild fever.cfsan. coli (EPEC)..html. Nataro and Kaper. coli infection is elevated among healthy adults traveling to foreign destinations.fda. Besser et al. kidney failure Low a Data derived from Bacon and Sofos (2003). gov/~ebam/bam-4a. microangiopathic hemolytic anemia (lysis of red blood cells). Nguyen et al. Mechanisms of Virulence E. Diarrheagenic Illness The characteristics and symptoms associated with each class of diarrheagenic E. and http://www. also characterized by extensive and irreversible neurological damage (Boyce et al. vomiting Incubation. coli (DAEC) (Begue et al. coli infection are found in Table 1.edu/cidrap/content/fs/food-disease/causes/ecolioview. Wittels and Lichtman. HUS. 1997). html). or vomiting. and characteristics of illness a associated with the different classes of diarrheagenic Escherichia coli Class EPEC Classic host Infants (<6 mo).. Begue et al. malnutrition Chronic watery diarrhea.. D-HUS is commonly misdiagnosed as thrombotic thrombocytopenic purpura. Nataro and Kaper (1998). 1998). . 1995). and symptoms generally approximate a mild bout with the flu.. 1998. coli (EIEC). 2000). 2003. as well as renal (kidney) malfunction and failure. duration (days) Variable Acute or chronic presentation Chronic diarrhea. or hemolytic uremic syndrome (HUS). and diffuse adherent E.gov/~mow/chap16. 4–10 Bloody diarrhea (hemorrhagic colitis).html. headache.72 BEAUCHAMP AND SOFOS associated with human gasteroenteritis are enteropathogenic E. Begue et al. watery stools (diarrhea) and mild to severe abdominal pain and/or cramping. bloody diarrhea. enterotoxigenic E. 1998). nausea Diarrhea.. Some individuals may be genetically predisposed to infection due to their blood type.gov/~mow/ chap14. Prolonged or chronic cases of diarrhea may result in severe dehydration and/or malnutrition due to decreased nutrient uptake by damaged intestinal lining (http://www. thrombocytopenia (inadequate platelet count). in which patients exhibit all of these symptoms (Bacon and Sofos. coli (ETEC). 1998. http://www. Most common mode of transmission.umn. more prevalent in developing countries Children. 1999. fever Watery diarrhea. a strikingly similar type of thrombotic microangiopathy. abdominal pain. self-limiting Low ETEC 1–3. Traditional HUS is most commonly observed in children less than 5 years of age. and/or chronic renal malfunction may also accompany severe infections (Buchanan and Doyle.cfsan. Bacon and Sofos. coli infections (Table 1) generally include loose. Buchanan and Doyle. mild fever. 1999).fda. (2004. Seizure. enterohemorrhagic E. abdominal cramping. stroke. 1997. 3–7 Cholera-like High EHEC 1–8. Common symptoms associated with human diarrheagenic E..cfsan. host. fever Watery diarrhea. more prevalent in developing countries Children. coli bacteria are particularly promiscuous microorganisms and continually exchange genetic material with other related and unrelated species of Table 1. (1998). specifically developing countries (http://www.fda. while adults are more likely to develop an atypical form of HUS characterized by a lack of prodromal diarrhea (D-HUS) (McCrae and Cines. enteroinvasive E. abdominal cramping. Persons with immature or impaired immune systems are more prone to both mild and severe illnesses and are also more likely to sustain secondary infections and/or other health complications (Table 1) (Besser et al. The incidence of certain classes of diarrheagenic E. 1989. 2005). fever. Guion et al. low in infants High EAEC Variable EIEC 1–3.. 2008. Symptoms associated with severe infections of certain serotypes include bloody diarrhea (hemorrhagic colitis). 2003.

and pedestal formation (DeVinney et al. Correspondingly.html. Begue et al.. Knutton et al. thin filaments called adhesive fimbriae (Table 2) (Collinson et al. Singleton. Berin et al.. tumor necrosis factor.com/doi/abs/10. (1988). Nataro and Kaper.fda. The type III translocon locates target host cells and inserts Tir into the plasma membrane. 2001. (2004).gov/~ebam/bam-4a. Colonization of Host Cells The formation of attaching and effacing (A/E) lesions on host intestinal cells is characteristic of EPEC and EHEC (Torres et al. 1998. 2004). 1999. Hicks et al. ETEC.. 1994). although not identical in their genetic homology or subsequent host interaction (Blattner et al. including the type III translocon (comprised of EspA... 2002). 1998. http:// www. enterohemolysin. www. which are encoded on the EPEC adherence factor plasmid (Girón et al. Tzipori et al. http://www. EPEC expresses bundle-forming fimbriae (Bfp). Isberg et al.. O’Brien and Holmes (1996). CNF AAF. The resulting migration of neutrophils across the epithelium damages host mucosal lining and results in fluid secretion (Zhou et al. Nataro and Kaper (1998). Host colonization by Ϫ Tir cells is unlikely. 1997. while ETEC expresses fimbrial colonization factors (Torres et al. 1987. Shiga toxins. which provides a surface for bacterial effacement. (1996). II. flagellin. Hartland et al. Schmidt et al. html). Knutton et al. The coupling of Tir and intimin initiates the degradation of host epithelial cell microvilli. However. and evasion of host defenses... other Type III proteins and toxins enter the cell. Shiga-like toxins. serine-protease (SepA) CDT LEE island. and EAEC to host cells is mediated by long.to 100-fold) expression of Shiga toxin receptors on the surface of vascular endothelial cells (van de Kar et al. Pic Enterotoxin LT. Moon et al. Stx. CNF. EAST1. coli infections (Nataro and Kaper. (1994).. 1983. LEE island. 1998. 1991.umn. and damage to the host are observed with all diarrheagenic E... Blanco et al.. Kenny et al. the virulence genes associated with each class of diarrheagenic E.. Once the lesion is formed and the pedestal protrudes into the lumen. cidrap. intimin (EaeA) (DeVinney et al. CDT. Bacon and Sofos (2003).. The attachment of EPEC. http://www. colonization. Torres et al.cidrap. Zychlinsky et al.. Kaper et al. EPEC and EHEC LEE islands are similar. cytolethal distending toxin.edu/cidrap/content/fs/food-disease/causes/ecolioview. tumor necrosis factor induces increased (10. Kenny et al. replication. CDT Flagellin Cell-to-cell spread (IcsA). 1998. 1998). 2004). pO157. or “beacon. differentiate each of the six classes (Table 2). DeVinney et al.umn. EspB.. ␣-hemolysin.2006. Pet. flagellin. (1994). and interleukin-8 (Moon et al. and EspD) and Tir (or EspE). 2003. EAST1 Other virulence factors EAF plasmid. 2003.. 1983. set of three fimbrial adhesions expressed by EAEC and required for formation of distinctive stacked-brick AA to host cells (Nataro et al. 1999.. as well as the organization of the actual adhesive structure. 1998. Taylor et al.html). Interleukin8 is involved in the translocation of host neutrophils (Zhou et al. CDT. bacteria.1111/ j.. (2005).CHAPTER 5 • DIARRHEAGENIC ESCHERICHIA COLI 73 Table 2. In humans. (1996). Elliott et al. (1992). Savarino et al. The type III protein secretion system is a critical component of the LEE island and encodes a number of factors involved in the formation of A/E lesions. Singleton. (2002). coli (Table 2) were acquired through extensive lateral transfers of genetic material (Souza et al. also known as the pathogenic LEE island. Aggregative adhesion (AA) fimbriae (AAFs) I.edu/ cidrap/content/fs/food-disease/causes/ecolioview.. 1999. Tir then functions as a protein receptor. (1996). and http://www.cfsan. 1999. 1998). (2006).. 2005.” for the adhesin protein.00794. Frankel et al. Mechanisms of pathogenicity which differentiate the classes of diarrheagenic Escherichia coli Class EPEC EAEC EIEC ETEC EHEC a a Adhesion site Small intestine Small and large intestine Large intestine (colon) Small intestine Large intestine (colon) Adhesion mediator Intimin AAF Unclear Fimbrial CFs Intimin Invasion potential Moderate None High None Moderate Toxins Possible enterotoxin (EAST1) EAST1. although some forms of intimin can attach to host cells in the absence of Tir (Hartland et al. 1999. (2006). cytotoxic necrotizing factor. 1992). 1998). Robinson et al. or verotoxins.x). The factors involved in the formation of A/E lesions are genetically encoded on the locus of enterocyte effacement (LEE). Nataro and Kaper. Torres et al.. the sites of pathogen adhesion to host GI tissue and mechanism of attachment to host cells. Beutin et al. 2005). 2005).html.cidrap. Data derived from Aragon et al. initiating signal transduction pathways and activating protein kinase C.. enteroaggregative ST toxin. and III are involved in the binding of most EAEC bacteria to host cells.edu/cidrap/content/fs/food-disease/ causes/ecolioview.umn. the reorganization of the host cell cytoskeletal. 1992). 1997). 1997...1462-5822. (1997). ST Stx. (1998).blackwell-synergy. Colonization of host GI tissue is rather methodical. although afimbrial .. Friedrich et al.

1996. Steiner et al. 2000.. 1996). 1994..edu/cidrap/content/fs/fooddisease/causes/ecolioview..html). 2000. coli (STEC). and specificities between different species of hosts (O’Brien and Holmes.. or only Stx1 (Louise and Obrig. and other factors of unknown importance (Brunder et al. and interfere with neutrophile function (Bauer and Welsh.. coli infections are also unique to each class (Table 2). 2003). Obrig et al. While initial host cell invasion occurs through the lumen of the intestine. 1995). and HUS (Lingwood. These toxins are the primary virulence factor of Shiga toxin-producing E. The mechanism by which EIEC adheres to host cells is not well defined... a catalase-peroxidase. Torres et al. Russo et al. The toxB gene. Sansonetti et al. EAEC forms thick. (ii) a higher affinity of human host cells for Stx2 compared to Stx1. also appear to exhibit toxigenic activity during EAEC infections (Table 2) (Bellini et al. extracellular location. STa (or STI) appears to antagonize the intestinal regulation of sodium chloride and STb (or STII) damages the intestinal mucosa and interferes with fluid absorption (Nataro and Kaper. Other Virulence Factors All E. This trend may be due. allowing effector proteins to enter the cell and trigger signal transduction pathways involved in the symptoms of human infection (Small and Falkow. 2001). 1987). There are two forms of ST toxin: STa and STb.. 1996). and action upon host cells (Dallas and Falkow. which may help explain their predisposal to STEC/ EHEC infections. Flagellin aggravates the release of interleukin-8. Louise and Obrig. O’Brien and Holmes. Currently available data indicate that strains that produce only Stx2 are more commonly associated with HUS infections than strains that produce both Stx1 and Stx2. coli. EIEC also expresses another factor (IcsA). Shiga toxins (also known as Shiga-like toxins or verotoxins) are similar to those produced by Shigella dysenteriae (Sandvig. A subsequent increase in systemic iron. structure. 1998). 2005). the EIEC Ipa complex is inserted into and forms pores in the plasma membrane. may help explain this phenomenon (Schmidt and Karch. Zhou et al. may also be involved in pathogenicity. Pet and Pic. toxins.. König et al. which facilitates the spread of the pathogen from cell to cell (Nataro and Kaper. Once associated with host cells. coli O157:NM and O157:H7. 2002. due to the hemolytic activity of the toxin. 2005). 1988..cidrap. 1994).and ␤-hemolysins by biochemical characteristics. Upon interaction with host cell receptors (Gb3 and Gb4). 1998). defined by the presence of flagellar (H) antigens or the fliC gene. in part. LT toxin and the cholera toxin are similar in genotype. The presence of the pO157 plasmid may be correlated to the development of HUS in STEC patients (Schmidt and Karch. ... 1980). 1996). Friedrich et al. pO157.. While many types of E. and (iv) multiple Stx2 toxin variants which induce mild to severe diarrheic symptoms (Beutin et al. 1997. Following fimbrial/afimbrial attachment. and signal transduction pathways initiated by LT toxin eventually trigger the onset of osmotic diarrhea. Two serine protease inhibitors. For example. hemorrhagic colitis. 2005). 2001). While both induce diarrheic symptoms. 2001). renal cells express a greater number of toxin receptors. In the early stages of childhood development. (iii) a greater cytotoxic activity of Stx2 on human target cells. Hemolysins lyse red blood cells and erythrocytes.. While it is not an obligatory factor in the development of HUS. ␣-hemolysin is involved in the majority of HUS infections. 1996. which encodes genes for adherence factors. 1998). Toxins The toxins involved in diarrheagenic E..umn. researchers have described a possible relationship between flagellin proteins (Table 2) and the degree of host intestinal mucosa inflammation (Berin et al. which may indirectly influence pathogenicity. Enterohemolysins can be distinguished from ␣. to the cumulative effects of (i) the higher level of Stx2 production in species which only produce the one toxin. Torres et al. Motility factors. 2003. http://www.. can also be found on the plasmid (Tatsuno et al. 1989. 1996. 2001.. 2003). including E. which is involved in the translocation of neutrophils across the host epithelium (Zhou et al. Shiga toxins interrupt protein synthesis and trigger cell death (Sandvig. 1996).. function. coli include cytolethal distending toxin and cytotoxic necrotizing factor (Table 2) (Aragon et al.74 BEAUCHAMP AND SOFOS modes of attachment have been suggested (Garcia et al. 2002. Schmidt and Karch. in vivo biofilms exhibit an increased resistance to host defense systems and antibiotics (Okeke and Nataro. Marques et al. coli toxins exist. Nataro and Kaper. Other significant yet less common toxins associated with diarrheagenic E. impede cytokine release.. 1995. and the principal cause of hemorrhagic colitis and HUS in humans (Dean-Nystrom et al. 1999. Enterotoxins include heat-labile (LT) and heat-stable (ST) derivatives and the ST EAEC enterotoxin (EAST1). 1988. coli O157:NM and O157:H7 strains possess a distinguishing virulence plasmid. a metabolic requirement of E. dense biofilms at the intestinal epithelium and begins diffusing cytotoxins into host epithelial cells. 2005).

H7. 1984. Relevant information pertaining to DAEC was insufficient for its inclusion in the aforementioned tables. 1993). Keene et al. H8. Mulvey et al. Spano et al. Boerlin et al. H2. but are not limited to. rhamnose.oznet. H7. coli organisms facilitates their ease of entry into the food Table 3. Vaz et al. municipal water sources.. 1996. little is known about the characteristics. but not in infants (Ͻ1 yr) (Gunzberg et al. 1996.edu/library/fntr2/mf2269.. Nataro and Kaper. LDC. CDC. . compost.S.oznet. H2. Canada. 2008).. ranches and livestock feeding operations. and D-HUS. non-O157 STEC infections are more common in Australia..cidrap. Keene et al. lysine decarboxylase. coli O157:H7 (Mead et al. Rice et al. Hiramatsu et al. route of transmission.. 1994. H21. 1997. livestock harvesting facilities. Vernozy-Rosand et al.... A classic human host is not well defined.html).umn. 2001a. Although human illnesses have been reported (Girón et al. Childs et al. Persons with O157 STEC infections are also more likely to experience bloody diarrhea and develop HUS than non-O157 STEC-infected individuals (CDC. Guth et al. 2002.pdf). According to older estimates. 2005. A review of existing information indicates that diarrheagenic E. while O157:NM and O157:H7 are most commonly recovered from U.govt. 1995. 1998. symptoms of illness. HUS. 2002. 2004.000 of which are caused by E. dulcitol. Ara. and Europe (Blanco et al. 1993..CHAPTER 5 • DIARRHEAGENIC ESCHERICHIA COLI 75 2003.. Data derived from Bielaszewska and Karch (2000). 2002. SOURCES OF DIARRHEAGENIC E.. and agricultural fairs. Belongia et al. H11 HNM. and O111 (Table 3). 1993. 1999a.. 1992.. Virulence factors and phenotypic indicators associated with common Shiga toxin-producing E. While all classes of diarrheagenic E. 2008). 2006. http://www.. although DAEC infections are observed generally in young children.gov/ola/2006/ foodsafety1115. O103. patients. 1999. colonized hosts then shed these organisms into the environment. Raf. H11 HNM. coli (STEC) serotypes O group O22 O103 O111 O157 a a Flagellar antigens HNM. 1989. DAEC is the primary class of diarrheagenic E. including hemorrhagic colitis. STEC causes approximately 110. Goldwater and Bettelheim. Moss and Frost. DAEC appears to initiate host colonization via fimbrial attachment mechanisms (Bilge et al. Predominant non-O157 STEC serotypes associated with food-borne human infections include O26. 1999). Wallace et al. 2007). Hussein and Bollinger (2005). Dul. (2002). dogs. 1991. enterohemolysin. sewage treatment effluent. and virulence mechanisms associated with each class of diarrheagenic E. Travena et al.. 2003.. sorbitol. deer.pdf. (2002).edu/cidrap/content/fs/ food-disease/causes/ecolioview. coli organisms are usually nonpathogenic and account for the majority of facultative flora found in the GI tracts of most vertebrates. and wild and domesticated birds (Buchanan and Doyle. 1995. Scaletsky et al. natural and human-made bodies of water. While not commonly recovered from clinical cases in most countries. Kudva et al. COLI Environment E.. 2003.. Animals shown to shed the organism include beef and dairy cattle.. sheep. Swerdlow et al.nz/science/data-sheets/non-o157-stec. coli associated with acute diarrhea in Brazilian children (Spano et al. Sofos et al. coli O157:H7 cause the most severe types of infection. South America. urban and rural soils and landscapes. Yamamoto et al. Suc. pdf). 1999b. Possé et al. or clinical presentation of DAEC infections... 1993) and also induces finger-like protrusions from host HEp-2 cells. which engulf the pathogen and serve as a shield against antimicrobial treatments (Cookson and Nataro. Levine et al. 73. rodents.edu/ library/fntr2/mf2269. http://www.k-state. (2007). Irino et al. 1996. regional disparities are also observed for different STEC serotypes recovered from clinical cases. (2004). and http://nzfsa. including humans (Falkow. as well as from hospitals and day care centers (Barham et al. STEC and specifically E.. http://www. As with DAEC. 1997. swine. horses. coli can cause serious illness in humans. 1998). 2004. Gunzberg et al..html. 1998). arabinose. Rha.k-state. Sor. (2008)... H25 HNM. raffinose.000 domestic food-borne illnesses each year. coli bacteria have been recovered from a plethora of environmental locations that include.fda. Lopez et al. Classification Specific characteristics.. WHO. http://www.. sucrose. 1996. H7 Hyl ϩ ϩ ϩ/Ϫ ϩ Intimin ϩ ϩ ϩ ϩ Sor ϩ ϩ ϩ Ϫ LDC ϩ ϩ Ϫ ϩ Suc ϩ ϩ ϩ/Ϫ ϩ Rha Ϫ ϩ ϩ Raf ϩ ϩ ϩ Dul Ϫ Ϫ ϩ ϩ Ara ϩ Ϫ ϩ ϩ Hyl. 1997. Foods The ubiquitous nature of diarrheagenic E. Keen et al. Callaway et al. coli are found in Tables 1 and 2.. 1994).

soil. (ii) poor personal hygiene. in conjunction with the apparent resistance of cells in biofilm to various control measures. including raw milk. water. multiple types of outbreaks have been attributed to diarrheagenic . which may occur at nearly all processing or handling steps leading up to consumption. especially acid-tolerant strains in acidic products such as fruit juices. much of the available data indicate a higher prevalence during summer and/or fall months.usda. During harvest processes. according to Allos et al. 2006). 2002. FOOD PROCESSING CONDITIONS ASSOCIATED WITH OUTBREAKS AND RECALLS Although outbreaks do not account for all or even the majority of food-borne diarrheagenic E. Stopforth et al.. 2007.5) and/or thermal and osmotic stresses. Other food products derived from cattle. coli can withstand nutrient starvation. Raw produce may become contaminated through intimate contact with contaminated water and/or soil and animal manure during cultivation or harvest. cfsan. 2006. however. Michel et al. Rangel et al. modernization within the food industry has led to increased availability and decreased cost of countless foodstuffs. equipment. have also been associated with food-borne diarrheagenic E. 1989.pdf). salads.. 2004. As mesophiles.. gov/OPPDE/rdad/FSISNotices/63-06. microorganisms from the gastrointestinal tract and feces of animals may contaminate meat and poultry carcasses. In addition. Nychas et al. and workers. coli infections. coli is determined by numerous factors and can vary at any given time. 2004).gov/~dms/retrsk2. In addition. 2004). which may then become a secondary source of contamination or crosscontamination (Scanga.fda. coli infections (Karmali. as well as the processing environment. (iii) contaminated processing equipment. Shelled and chopped nuts may also become contaminated in a manner similar to produce. with peaks in prevalence being determined by regional temperature and climate (Barkocy-Gallagher et al. have undoubtedly contributed to the persistence of environmental and food-borne contamination and subsequent outbreaks of foodborne illness. leading to contamination of associated products. INTRINSIC AND EXTRINSIC FACTORS INVOLVED IN OUTBREAKS AND RECALLS E. prevalence in contaminated food products. 1969).html). Higher levels of environmental contamination. Since 1982. According to the CDC. In reality. leading to transfer of contamination to raw food ingredients. 2005). and coleslaw (Lee et al. the innate characteristics of produce of neutral or slightly acidic pH (i.. 2005). and (v) improper holding temperatures (http://www. such as juices. 2005). (2004). The considerable numbers of persons being fed during such events may also be a contributing factor to the apparent upsurge in outbreaks during warmer months. 2003.. coli (Lee et al. The likelihood of their presence and the lack of a clear understanding of the fate of biofilms when exposed to common intervention technologies. high water activity and high nutrient content) may support survival and growth of diarrheagenic E. nearly all food products (raw and processed) are subject to cross-contamination.76 BEAUCHAMP AND SOFOS supply. processing water.. however. 2003b). 2004). coli outbreaks are reported to the Centers for Disease Control and Prevention (CDC) generally during the summer and fall months (Hedberg et al. (iv) inadequate heat treatments.. 1997.. While risk of exposure to diarrheagenic E.. concerns regarding the development and maintenance of biofilms within the food industry are justified. and has been shown to persist in both predictable and unusual locations for remarkable lengths of time (LeJeune et al.. 1996). more food-borne diarrheagenic E. 46% of the food-borne outbreaks from 1998 to 2002 involved at least one of the aforementioned risk factors (CDC. not all prevalence studies have observed such trends (Alam and Zurek. 1999). Animal feces are the source of contamination for animal hides. 2003a. 1997). The five most significant “foodborne illness risk factors” include (i) the acquisition of products from unsafe sources. Nevertheless. Thus. coli logically should flourish during warmer months and be of less concern during colder seasons (http://www. and frequency of catered/ picnic/outdoor grilling events may contribute to this increase. diarrheagenic E. and inanimate objects.fsis. Childs et al. as well as exposure to low pH (Յ4.e. such details are generally present in more recent publications.. While early prevalence data were based on suboptimal detection methodologies and tended to overlook season and geographical information. the food processing factors implicated in the development of an outbreak may also lead to isolated cases of illness (Rangel et al. and increasing evidence of and repercussions associated with such niches are accumulating. and such contamination may then be transferred to the nutmeat during shell removal (Meyer and Vaughn. Quinto and Cepeda.. The nationwide distribution of contaminated products may lead to an upsurge in food-borne illnesses that emerge as an outbreak or appear to be sporadic (Killalea et al.

2005). Taylor and Perdue. 1996.. should be investigated and more effective controls should be sought. 1982. and its inactivation on such products may not be immediate (Glass et al.. the incidence of beef samples which were positive for E. 1996. 1993. 1982). 2007). coli O157:H7 infections (MacDonald et al. Meat Products In 1982. during a 4-month span of time in 1992 and 1993. 2000). coli O157:H7 (Hammes et al... or antibodies indicating previous exposure. Buchanan and Edelson. coli O157:H7 in dry fermented sausages (Glass et al. pasteurized and fermented dairy products are not a threat to human health. coli O157:H7 was associated with consumption of fresh cheddar cheese curds made from pasteurized milk (CDC. most of these products do not support extended survival or permit the growth of E. including home-level. 1997. and the Food Safety and Inspection Service (FSIS) reported that the number of ground beef samples positive for E.g. were recovered from the feces and/or milk of lactating animals on implicated farms.beefusa. person-to-person. Following many of these outbreaks. pdf). 1983). Yersinia. as well as those with unknown routes of transmission (Rangel et al. As . Staphylococcus intoxication. organisms genetically homologous to those involved in such illnesses.. 1999). multistate. 2000) and published data demonstrating the inability of existing fermentation.. and three deaths (Bell et al. little can be done to dissuade the small communities that continue to prefer raw milk/ dairy foods. coli O157:H7 is tolerant of acidic environments. 1997. coli O157:H7 was first recognized and classified as a human pathogen after being linked to illnesses stemming from consumption of improperly cooked hamburgers served at fast-food restaurants in Oregon and Michigan. such as lymph nodes and food contact surfaces.. coli O157:H7 infections (CDC. In response. United States (Riley et al. 2002. Even so. 2004). Drastic measures have been taken to control the association of E. 1984. and Cryptosporydium infections. For all intents and purposes. coli O157:H7 decreased by 80% between 2001 and 2006 (Ͻ1% of samples were positive in 2001) (http://www. 1992). 2005). For these reasons. 1992). Unpasteurized Dairy Products Ordinarily. additional sources of contamination. Therefore. Taylor et al.. USDA/FSIS. when improperly cooked hamburgers contaminated with E. Potter et al.. 1994). 1989). E. and brucellosis (Bielaszewska et al. 1998. coli. E. pdf). Millard et al. FSIS. 1994. 1990. Campylobacter... 45 cases of HUS. Listeria monocytogenes. it has been suggested that cells which survive the fermentation process may not be as susceptible to ensuing heat (56°C) treatments during processing (Ryu and Beuchat.. coli O157:H7 with beef products. Unfortunately. Unpasteurized Juices Documented outbreaks of human STEC infection have been attributed to unpasteurized apple juice and cider (Besser et al. 1979. and in developed countries consumption of raw milk and dairy products is limited to an unconventional (or unsuspecting) minority... Despite such achievements. Williams et al. Keene et al. coli O157:NM and O157:H7.. http://www. 1995. drying. Another landmark outbreak was documented 10 years later. org/PorkScience/documents/SEMIDRYSAUSAGE.. Aside from educating/reminding milk producers and consumers of the substantial risks associated with these products.pork. Raw milk and cheese derived from raw milk have been implicated in repeated outbreaks of human E. only those outbreaks associated with contamination introduced at or before the commercial food processing level are discussed in detail. terminal HUS.CHAPTER 5 • DIARRHEAGENIC ESCHERICHIA COLI 77 E. 2002. Doyle and Roman. the pathogen may survive long enough to cause food-borne illness when contaminated sausages are consumed shortly after production (Naim et al. One outbreak of E.. and water-borne outbreaks. ground and mechanically tenderized beef products were associated with multiple outbreaks of E. In response to international outbreaks (1994 to 1998) of E. laboratory-related. CDC. From 1995 to 2004. Steele et al. animal-to-person. 1982. the FSIS mandated that all processors implement a combination of treatments/processes capable of inactivating Ն5 log cycles of E. 2000). coli O157:H7 in dry fermented sausage before retail distribution (FSIS.org/uDocs/ecolinumbersdecline. However. coli O157:H7 were again linked to 501 illnesses. 1996). coli O157:H7 increased in 2007. Tilden et al. and multistate outbreaks and large recalls continue (CDC. Salmonella. fermented sausages continue to be associated with outbreaks of food-borne E. 2004). 1997. coli O157:H7 infections associated with dry fermented sausages (Alexander et al. slicing and repackaging) at the retail level.. Blaser et al. tuberculosis.. Outbreak-related contamination events may also occur during postprocessing handling (e.. and/or storage methods to inactivate 4 log cycles of E. It was eventually determined that the curds were stored in a nonsanitized vat which had previously held cheddar cheese curds made with unpasteurized milk (CDC.

and the market share for these products continues to grow (http://www. 2006). 2001).gov/ola/2006/ foodsafety1115. and clover sprouts contaminated with E. cfsan. orange juice produced and sold by a small roadside vendor also caused a small outbreak of ETEC infections in 1992 (http://www.fda. 2002a. was again implicated in a multistate outbreak of E. php). and the immunocompromised are encouraged not to eat sprouts (CDC.. served at multiple restaurants from a single fast-food chain. gov/~comm/ift3-4a.. coli O157:H7 infections and three deaths (Michino et al.fda.gov/ola/2006/foodsafety1115. While apple juice/ cider has been implicated in the majority of juicerelated outbreaks.. the elderly. coli O157:H7 infections eventually traced back to freshcut bagged spinach (http://www.gov/~comm/ift3-4a. 31 cases of HUS.html).gov/~dms/prodgui4. Processes/control measures designed to address microbial contamination associated with seeds or sprouts were not in place prior to the series of international sproutassociated outbreaks of E. Temperature (25 to 30ЊC) and humidity conditions during sprouting may then facilitate the growth of existing STEC contamination (Hara-Kudo et al. in combination with the addition of preservatives and the acidic pH (3 to 4) of apple juice/cider. coli O157:H7 infections (http:// www. NIIDIDCD/MHWJ.. coli can be incorporated into the root systems (at the root junctions or rhizosphere) of traditionally and hydroponically grown spinach seedlings in the early stages of cultivation and recovered from the leaves. stems. According to Solomon et al. Produce In 1996.. including E.html).gov/~comm/ift3-4a. In late 2006. Over the next 2 years. CDC. coli O157:H7 infections were caused by the consumption of unpasteurized apple cider made from both hanging and fallen (drop) apples (CDC.html).html). 1997). and three deaths were associated with a highly publicized outbreak of E. and root system of plants at harvest (Warriner et al. 1997).fda. 2000b). fresh-cut produce sales have generated about $12 billion in annual revenue. with raw herbs and vegetables being implicated in the majority of such outbreaks (Beatty et al. 1997). fresh-cut leafy green produce has fallen under intense scrutiny due to the size of recent outbreaks and recalls associated with these products. Between 1995 and 2005. STEC. Seeds which are contaminated before germination or exposed to contaminated soil/water may develop into sprouts with pathogens attached to the root system or internalized into the inner tissue of the young plant (Solomon et al. All apples had been brushed and washed in potable water before pressing. Cut fruit and fruit salads have also been implicated in outbreaks of food-borne E. radish sprouts fed to Japanese school children caused over 6. 2004).1% potassium sorbate was added to the cider before bottling (CDC. cfsan.cfsan.fda. smaller outbreaks in Japan and the United States were also traced back to radish.gov/~comm/ift3-4a. coli O157:H7 was recovered from water sources used to irrigate the spinach and from the feces of hogs which had reportedly broken into the spinach fields (http:// www. 1999.cfsan. as a genetically homologous strain of E. cfsan.000 E. These outbreaks further reinforce the acid-tolerant nature of E. Similar findings were reported for pears and apples irrigated with E. coli O157:H7 and demonstrate the inability of preservatives used within the commercial juice/cider industry to consistently control these pathogens (CDC. coli O157:H7 from the edible portion of spinach or cabbage plants which were inoculated during germination. 2002b. shredded lettuce. two outbreaks of E.. any degree of pathogen internalization is alarming. 1997). coli O157:H7 infections (Brooks et al. generated considerable alarm among consumers and health officials in response to the outbreaks (CDC. Although control measures have since been implemented. While Wachtel et al. coli O157:H7. continues to be recovered from retail-level sprout samples (Samadpour et al. Also in 1996. coli-contaminated water (Riordan et al. it has been shown that E. 1997)... coli O157:H7 infections. In recent years. and thus. (2002) and Warriner et al.gov/ola/2006/ foodsafety1115. 2001b). a total of 168 . html). alfalfa. ETEC O169:H41 has emerged as the primary serotype involved in outbreaks of food-borne ETEC infections. United States (http://www. Initial contamination was most likely introduced at the farm level..fda. In 1993. Warriner et al.78 BEAUCHAMP AND SOFOS specific examples. lettuce and mixed leafy green salads were associated with 14 domestic outbreaks of E. coli O157:H7 (http://www. (2005) did not recover internalized E. an unrelated outbreak of illnesses stemming from consumption of commercial unpasteurized apple juice was implicated in the death of a young girl in Colorado. coli O157:H7 infections (http://www. The low infectious dose associated with clinical cases. As with sprouts. E.html). 2003). (2002a. Also in 2006. and 0. coli O157:H7 may be internalized into the edible portion of lettuce plants.fda. although the pathogen was most likely introduced during cross-contamination events at the food preparation level (http://www.fda.fda.html). 2003).cspinet. Even so. 2001. children (Յ5 years old). 2001b).html).org/foodsafety/outbreak/pathogen. 1997). 204 illnesses.

.fda. the FDA issued the Guide to Minimize Microbial Contamination of Fresh Fruits and Vegetables. However. Sofos. 2005. 2003). 2004). 1997). Measures to control the colonization of cattle by E. the FDA released another guidance document entitled Guide to Minimize Microbial Food Safety Hazards of Fresh-Cut Fruits and Vegetables. behavioral management. hide contamination and/or carcass contamination events during hide removal and evisceration may be minimized. COLI CONTROL MEASURES DURING PRODUCTION AND PROCESSING Safer Supply Chains The volume and international breadth of human illnesses associated with alfalfa and radish sprouts justified extensive modifications to pathogen control programs within the sprout industry.gov/~comm/ift3-4a. human to food product. Unfortunately. quaternary ammonium compounds. fda. Diez-Gonzalez. coli O157 and Salmonella (http://www. 2003). successive rinsing and sanitizing steps may be more effective against surface contamination. which used inadequately chlorinated water during parsley processing and packing operations (Naimi et al. Minimizing Contamination Events during Processing Contamination may be introduced by many different routes (e. 1997. The carrots served at both venues were grown in the same state. 1998. coli (http://www. Although their application is unlikely to be counterproductive.. although a single source was never identified. Sofos et al.to 200-ppm sodium hypochlorite (bleach) solutions during processing (Warriner et al. Code of Federal Regulations.. In 1998... By reducing the number of animals shedding the pathogen. and processing of sprouts. Callaway et al. This route of contamination warrants significant attention. Seed decontamination technologies include the application of acidified sodium chlorite. animal to environment. and improved hide removal technologies (Gill. 2005. 2005. all of which are capable of reducing levels of E. In February of 2008. including recommendations on worker health and hygiene and sanitation operations and for the preparation and maintenance of processing water (http://www.. which outlines similar microbiological control measures. as it was also implicated in multiple outbreaks of E. the most significant advances in process control almost certainly include those which address worker education and hygiene (9 CFR 416.cfsan. It was later determined that the parsley was grown at a single farm in Baja. coli O157:H7 include the feeding of probiotics. and feedlot pen maintenance (Brashears et al.gov/~dms/prodguid. Proposed strategies include transport and lairage management (cleaning holding areas and separating “clean” versus heavily soiled lots of cattle). two outbreaks stemming from food served in Minnesota restaurants which contained fresh chopped parsley resulted in a total of 77 illnesses (Naimi et al. vaccination programs.g.gov/~dms/prodguid. vast improvements have been made to the conventional germination. 1999c). ozone gas. 2005). the lot numbers on individual packages/bags of certified seed can now be traced back to the original premises at which the seeds were grown. As a result...cfsan. hide decontamination.gov/~dms/prodgui4.cfsan.cfsan. Hadley et al. .html). lasting results when applied under realistic parameters. most likely in response to the massive outbreak associated with fresh-cut bagged spinach in 2006. 1994. Bari et al.html). ADVANCES IN DIARRHEAGENIC E. sodium hypochlorite is only moderately effective in reducing pathogen counts (Beuchat.html). In 1998.. when used under typical processing conditions. 1997) because of its rapid inactivation in the presence of organic material (Lillard. 2003). 2004).. 2006).fda. cultivation. 2005. coli O157:H7 contamination (Warriner et al.. the use of backlighting equipment and air curtains. 1980. 2001). prebiotics or other feed additives/ingredients. Surface contamination can also be transferred into previously sterile tissue through cuts/ punctures in the skin of fruits and vegetables (Warriner et al. water chlorination. In addition. 1997. and processing equipment to food product) (Bell.fda. Other efforts include increased pest control and sanitation programs at the processing level. such programs have yet to provide repeatable. food product to food product.html). Among the most significant of the improvements was the screening of water used during hydroponic cultivation for E.5. 2003). coli O157:H7 infections associated with unpasteurized apple juice and cider (CDC.CHAPTER 5 • DIARRHEAGENIC ESCHERICHIA COLI 79 hotel and airline patrons in Rhode Island and New Hampshire became ill after eating shredded carrots contaminated with ETEC serotype O169:H41 (http:// www. 2006). and irradiation. The majority of fresh produce is washed in 100. and final product testing for E.. 2005. The level of hide contamination has been correlated to the subsequent proportion of contaminated carcasses (Woerner et al. Stopforth and Sofos. Mexico. Thus. and technologies which minimize hide contamination are under extensive investigation. Beuchat and Ryu. Russell and Axtell. harvesting. adoption of hazard analysis and critical control point (HACCP) concepts.

on surfaces of seeds. In-plant contamination issues may be reduced by simply reviewing the basic fundamentals related to personal hygiene and hand washing. In lieu of pasteurization. Sapers et al. In an effort to reduce the incidence of cross-contamination events between carcasses. knives are rotated between consecutive carcasses (one knife is applied to a carcass. it is not surprising that the majority of fresh-meat-related outbreaks and recalls involve ground meat (Rangel et al. The outer surfaces/trimmings from carcasses are generally intended for ground meat production. product and equipment handling. 2005). rinds. and milk contaminants from the surface of nonintact beef (FSIS. 1998. and fresh or processed meat products (Beuchat.. raw produce with surface defects are rejected. and the levels of contamination on outer surfaces are minimized before inner tissues are exposed. 1997. coli and also reduce the number of carcasses which fall from overhead rails and onto the ground and must then be diverted to areas reserved for trimming and additional FSIS inspection. 1999b. and protein compounds (Acuff. 2005). Gill.. 1999. 1999. 1998. Cords et al. 2005). 2005. as well as the incidence of hide and carcass defects (Scanga. Knives are invaluable food processing tools but may also be contaminated with pathogens (Bell. 2005). Sofos et al. has also reduced cross-contamination events during processing (Scanga. microorganisms can contaminate previously sterile inner tissue through cuts or tears in outer skin. 2001) describing sanitary operations. supervisors must provide access to and/or enforce the use of appropriate restroom and eating facilities and good hygiene practices. or hides (CDC. even though the outer surface typically encounters a higher level of contamination than any other carcass region (Gill. at which time contamination may be transferred from the hide to the carcass (Sofos. 2005).. peroxides and peroxyacid solutions. According to the section of the Code of Federal Regulations (9 CFR 415. Inappropriate terminology aside. 1999). Carcasses are sterile before hide removal. ingesta. and appropriate behavior during illness (FSIS. coli. Employee education and training courses are typically included in the prerequisite program portion of a HACCP system and can be customized to fit the needs of an individual operation. such treatments may be used to decontaminate the outer surfaces of fruits destined for juice production and only when accompanied by rigid microbiological “hold and test” programs. organic acids.. Therefore. which requires the removal of all visible soil. feces. Knife trimming has been implemented to address the “zero tolerance” policy. 1997). Thus. Available decontamination fluids include chlorine and chlorine derivatives. 1999c). improper cooking is very likely to occur and should be anticipated. unpasteurized juice must be labeled as such (21 CFR 120. iodophors. spot decontamination efforts effectively reduce microbial counts and limit the spread of particulate matter over carcass surfaces during subsequent carcass washing steps (Sofos et al. especially with the chain speeds used in many domestic commercial facilities. 2005. Inactivation Decontamination with scientifically validated decontamination fluids may be valuable in minimizing the incidence of diarrheagenic E. The advent of mechanical devices as processing aids. ozone and electrolyzed oxidizing water. 1999b). Other carcass-spot decontamination technologies include steam pasteurization or steam vacuum units. 2005). Code of Federal Regulations. nuts. Sofos. Sofos. Although cooking (72ЊC or 160ЊF) raw ground beef before consumption should inactivate pathogens of concern. Once proper behaviors are introduced and improper practices have been corrected. specifically STEC contamination. 1999a.. . attempts to remove contamination from the entire surface of every carcass using spot decontamination strategies is not feasible. 2005). In addition.4. 1998. Beuchat. 1999b). “all [direct and indirect] food-contact surfaces including…utensils and equipment. Warriner et al. Sofos et al. the number of potential harbors for pathogenic contamination may be reduced by covering tears in the subcutaneous fat layer with plastic film prior to washing carcasses (Simpson et al.80 BEAUCHAMP AND SOFOS as workers can serve as original vectors of enterically derived contamination or transfer existing contamination to unsoiled areas or products. Sofos et al. When present. 2006. Sofos et al. 1993). While zero tolerance implies that all such matter is removed. The evisceration process also has the potential to generate high levels/ incidence of contamination on carcasses (Gill. 2006). produce.” The dual-knife system has been a great asset to the meat packing industry. 2005. which limit worker contact with food products. 2005. Advances in mechanical hide removal should reduce the incidence of hide-to-carcass transfer of pathogenic E. 1999a.. 2002).24. 1999a. 2005). while the other knife is submerged in sanitizer) (Scanga. it is imperative that injurious events to protective outer surfaces are minimized. must be sanitized as frequently as necessary to prevent the creation of insanitary conditions and the adulteration of product. which are used to remove visible contamination or to systematically treat the area most likely to become contaminated during hide removal (Sofos and Smith.. eggs. Sapers et al... 1994. 1994.. Code of Federal Regulations. trisodium phosphate.

and pulsed ultraviolet light or electric field treatments (Guan and Hoover. coli is limited.cfsan. dose. and time. coli (http://www. 2005). 1997. In 1998..gc.. alterations in flavor profiles... Leistner and Gould. Dry fermented sausages are the exception. can influence . and depth of penetration. acids. pH. including diarrheagenic E. Other nonthermal processes include high hydrostatic pressure. packaging conditions. Murano et al. texture. Low doses of irradiation are highly lethal for most microorganisms.wisc.htm). Code of Federal Regulations. Appropriate cooking and handling recommendations are available for many raw or partially cooked food products. It is extremely important that these solutions be applied under appropriate conditions. such as heat (Bacon et al. seeds.g. and a thermal pasteurization guide for milk. 2003. Alkaline phosphatase is an LT enzyme naturally found in raw milk and is inactivated at temperatures just above those required to inactivate most milk-borne microorganisms. 2000. Russell and Axtell. edu/fri/briefs/foodirrd..24). 1995. Foley et al.hc-sc. is available at http://www. 2006). 2004. as well the components (e. specifically E. 2005. which produce inhibitory compounds (e. with little impact on the flavor. 2003. The rate and extent of product chilling. spices.edu/fri/ briefs/foodirrd. Lacroix et al. alkaline phosphatase inactivation is used to verify the successful application of pasteurization treatments (15 min at 71. sprouts.. 2006).CHAPTER 5 • DIARRHEAGENIC ESCHERICHIA COLI 81 Warriner et al. Jay. Kamat et al. 2006).fda..cfsan. 1998. Lipids. air flow. the administration of Ն3 kGy successfully inactivates the pathogen.html. many decontamination treatments tend to be more effective when used sequentially or when combined with other antimicrobial factors. water hardness. temperature. pulp and fat content).gov/~ebam/bam-27. minimally processed.. 1999b. 2002. 2002). other organic compounds. and rapid oxidation (Kuby.gov/~ebam/bam-27. coli (Davidson and Harrison. Pordesimo et al. 2003... 1999a. Antimicrobial agents within processed food product formulations and sprays applied to final products or in packaging material may be used to control postlethal pathogen growth. and tools are available to inhibit pathogen growth in both processed and unprocessed products. 2006). 2005. cfsan. 2002. 1998. 1999.fda.html). The successful pasteurization of milk and other dairy products can be determined by screening for the presence of postpasteurization phosphatase activity (http://www.fda. and meat products (Bari et al. 1997. as affected by packaging. ready to eat). Sofos et al. proteins. Sofos et al.. 1997. 1998). and level of target microorganisms associated with a product (NACMCF. fruit juice manufacturers may adopt HACCP programs which include alternative scientifically validated processes which are capable of reducing Ն5 log cycles of E. it became mandatory for all noncitrus juices entering interstate commerce to be pasteurized or be clearly labeled as an unpasteurized product. bacteriocins... 2004. stocking density. NACMCF. 2005. 2001. diacetyl. and adherence to such recommendations should eliminate the risk associated with raw foods. and many improvements to irradiation technologies have been developed in recent years (http://www. 2006).gov/~ear/pmo01toc.8ЊC) (http://www. coli.ca/fn-an/alt_formats/hpfbdgpsa/pdf/res-rech/mfo25-eng.. the efficacy of pasteurization treatments depends on the process. relative humidity. Hardin et al.. shock waves..pdf). Grade “A” Pasteurized Milk Ordinance. Duffy et al. outbreaks of E. Sofos et al. 1999. Nonmeat ingredients also appear to increase overall efficacy of irradiation treatments (Bricher. or color of treated ground beef samples (Arthur et al. 2002. as sublethal exposures may only enhance the stress tolerance of diarrheagenic E.24. 1982).wisc. although all surfaces of a product may not be exposed or adequately decontaminated (Warriner et al. With adherence to the guidelines for optimization of temperature.7 or 30 min at 62.g. However. ultrasonication. Samelis and Sofos. 2004. The generation of free radicals is also responsible for the generation of hydrogen peroxide and quality defects in irradiated produce. Irradiation programs have been scientifically validated and are available to inactivate E. which include mushiness.. and pressure during application influence the killing power of many decontamination fluids (Arrit et al..html). 2005). or as an alternative to pasteurization.. and the use of antimicrobial ingredients to control diarrheagenic E. possible pathogen contamination must be addressed during processing of products intended for immediate consumption (i. 2002). Inhibition Postlethality contamination events pose a significant risk to human health. 1998). 1998. http://www. Pasteurization is recommended (21 CFR 120. Murano et al. For these reasons. In general. Cutter et al.e. Sofos and Smith. However. coli O157:H7 associated with juices.. Lewis et al. Sofos and Smith. 2004. Smith and Pillai. 2002.. In general. coli infections are still associated with fresh. the use of various lactic acid bacteria cultures.htm).. Spray applications may be more convenient than immersion treatments (Beuchat. Kang and Fung. and ethanol) are added to raw sausage formulations to ensure proper fermentation and products of acceptable quality and safety (Lahti et al. 2002). and/or readyto-eat food products. NACMCF. coli O157:H7 (21 CFR 120. contact time. Vickers and Wang.

. 1997). modified atmosphere packaging is used for microbial inhibition. Simpson et al. as well as in the recovery of injured cells (Hepburn et al. Instead. COLI The FDA and USDA conduct routine sampling and testing of food products. Product quality and safety are almost certainly compromised after encounters of abusive temperatures for sporadic/extended periods of time during holding. Sorbitol is added to MAC (SMAC) to differentiate E. and vancomycin (Hussein et al. and gram-negative or E. or quantify the presence of specific microorganisms. and the expected level of contamination. potassium tellurite. ETEC. Temperature monitoring/recording systems. repeatable. coldshortening in rapidly chilled beef. 1993. Sanderson et al. 2006). Chilling food products too rapidly or storing them at Ͼ4ЊC may result in quality defects (e. Additional compounds added to selective media to suppress the growth of background flora include antimicrobials to which E. microbiological analysis of raw materials and finished food products should not be used in place of a comprehensive pathogen control program or to “confirm” product safety. 2008). 2005). As oxygen and other gases in the environment affect microbial growth. To date. Enrichment and Isolation Enrichment protocols usually precede most conventional and rapid detection methods. coli. coli O157:H7 in food products further compromises the integrity of microbiological testing programs (Buchanan and Doyle.. it is important to employ the most appropriate method regarding the type of information needed. Popular solid media used to isolate generic biotype I E. or distribution (Koutsoumanis and Taoukis.. Although results of microbiological analyses may be delayed as a result. and pork or freeze-damaged fresh produce) (Lee et al. Diarrheagenic E. but it is capable of growth at temperatures that barely exceed (7 to 9ЊC or 45ЊF) the recommendations (ICMSF. coli O157:H7 under modified atmospheres is not well understood and may be similar to its performance under aerobic conditions (Hao and Brackett.cfsan. Levine’s eosine methylene blue agar. 1995). Smith et al. enrichment protocols are critical when attempting to isolate very low levels of pathogenic E.. IDENTIFICATION. coli. lamb. (2008) found that brain heart infusion supplemented with potassium tellurite. 2000). Hussein et al. coli O157 from non-O157 strains. 2004). coli enrichment broths include brain heart infusion. sample composition. or interactions between food components and reagents used in detection protocols (Gill. and numerous selective and differential media.. 1996). fda. 1989). The performance of E. conventional culture methods remain the gold standard in pathogen detection. Practical detection methods must be sensitive. and CO2) and include common vacuum packaging (Phillips. and cefixime was more successful than other selective enrichment broths in the suppression of background flora during enrichment of STEC recovered from cattle feces. MAC and Hektoen enteric agar are most useful when isolating EIEC (Doyle and Padhye. cefsulodin. routine microbiological testing should be incorporated as part of a multifaceted program to validate/verify the efficacy of antimicrobial interventions.gov/~ebam/bam-a1. DETECTION. Microbiological tests can be used to detect. AND CONFIRMATION OF DIARRHEAGENIC E. to effectively inhibit the proliferation of these pathogens during cold storage of produce and meat products. although the need for such programs remains a topic of debate. vancomycin.. specific. are available (Zadik et al. tryptic soy broth.cfsan. In any respect. coli from samples with or without excessive levels of background flora (fresh produce and fermented foods).. which METHODS FOR ISOLATION. gov/~ebam/bam-a1. coli broth supplemented with novobiocin (Hajna. novobiocin. 1996). and economical and also generate results expressed in practical units in a timely manner. 1993. temperature must be lowered as quickly as possible and maintained for the duration of product holding (Hao and Brackett. a nonhomogeneous distribution of cells throughout the product/sample. 1993). Such technologies manipulate individual levels of the main components of air (O2. and EAEC include violet red bile agar. EPEC. and 3M PetriFilm rehydratable films (http://www. N2.html). identify. 2005. detection of microorganisms in food product samples can be problematic due to low levels of contamination. MacConkey agar (MAC). can be used to effectively maintain the correct temperature during each phase of cold storage. Selective E.fda. Hussein et al. many tests can be used for more than one of these objectives. which require as little as 18 h for the detection of diarrheagenic E. 1955. In general. coli are particularly resistant. such as cefixime. http://www. transport. While .82 BEAUCHAMP AND SOFOS the fate of microorganisms during storage. and processors are also encouraged to adopt scientifically validated microbiological testing programs. The low incidence of E. equipped with audible default alarms.. coli can survive but will not grow at recommended refrigeration temperatures (4ЊC or 40ЊF).html)..g. 2008). 2002. Thus.

stIa/stIb and lt (ST and LT enterotoxins) for ETEC.gov/~ebam/bam-a1.. antibody-coated magnetic beads are added to samples during (pre-) enrichment steps and interact with the antigens present on the surfaces of target cells. beads are gathered together using a magnet. hemorrhagic colitis agar.. the most popular combinations of detection and confirmation methods are those which employ conventional culturing and enumeration and PCR technologies (Lazcka et al.. A similar single-reaction protocol was developed by Moyo et al. coli O157:H7 and O157 or customized combinations of E.fda. Higuchi et al. which then allows for the removal of unbound sample components (Karch et al. and other proprietary chromogenic media (Huang et al.cfsan. Paton et al. 1986. Fratamico et al. http:// www3. that some strains of E.cfsan.html) and TaqMan (Applied Biosytems. 2005). http://www. 2007. http://www. com/Qualicon/en_US/products/BAX_System/bax_ ecolimp.. include rfbEO157.fda. and stx2 (Gannon et al.gov/~ebam/bam-4a. 2007). or Southern blot assays using labeled probes can be used to confirm the identity of a product (Fratamico et al. Moreover.gov/~comm/ift3-4a. 2002. 1991. PCR products are visualized via electrophoresis on agarose gels and enzyme-linked hybridizations.. rainbow agar. DNA hybridization.fda. enzymebinding proteins. however. stx1 and stx2 (Shiga toxins) for STEC/ EHEC. 1990).. other media used to differentiate E.cfsan. coli O157:NM and O157:H7 from non-O157 E. coli (Meng et al. hylA. are not designed to indicate the presence of other classes of diarrheagenic E. coli O157 strains typically do not (Schmidt et al. and food components tend to antagonize the results of some types of rapid tests (http://www. Real-time PCR utilizes the fluorescent signal of a reporter dye which increases in intensity as the target sequence is amplified (Fratamico et al. levels of the target organism can be quantified by comparing these curves to standardized models (Fratamico et al.com/cms/groups/applied_ markets_marketing/documents/generaldocuments/ cms_039421. 1993..html).cfsan. The most commonly used DNA-based detection technologies include genetic probes. including aggR (transcriptional activator of AAF I expression) for EAEC (Nataro et al... fliCH7. 1996). 2001). http:// www. eaeA. specific to STEC and EHEC...gov/~ebam/bam-4a. Hybridized probes labeled with fluorescent substrates. Other differential substrates include rhamnose. (2008) have developed a real-time fluorescence-based multiplex PCR protocol capable of detecting all six classes of diarrheagenic E. 1992. 1997. 1992.. coli O157 include BAX (DuPont Qualicon. In general. 1993. and bacteriophages (http://www. specifically designed primer sets are used to amplify eight different virulence genes in a single PCR reaction.gov/~ebam/bam-a1.cfsan.. It should be noted. 1990).CHAPTER 5 • DIARRHEAGENIC ESCHERICHIA COLI 83 readily ferment sorbitol.cfsan.. or the coupling of specific probes with target cell DNA. 2005.dupont. coli O157 and O157:H7 strains (Karch et al. Software is available which generates a curve of intensity based on initial fluorescence and level of fluorescence at each ensuing cycle. and 4-methylum-belliferyl-␤-d-glucoronide (MUG) (Chapman et al.. coli. EPEC.html).. and then visualized (Entis et al. Immunomagnetic separation is commonly used to concentrate target cells which are present in low numbers within large samples and can select both sorbitol-positive and -negative E. 1993. presumptive positive samples identified by rapid technologies must be confirmed by conventional culture methods (http://www. or radioactive isotopes (less common) are adsorbed onto a thin membrane (solid-phase hybridization) or captured from liquid-phase assays by a solid support. coli O157:H7. Guion et al. ipaH for EIEC. March and Ratnam. http://www2.pdf) systems. 1999). Currently. In addition to SMAC. http:// www. Livak et al. Wang et al.fda. coli virulence genes or target sequences.. 2001a). . eaeA (intimin) for EPEC and STEC/EHEC. To do so. and daaD for DAEC. The optimal sensitivity and specificity of a test are observed when detecting pathogens in broth systems. Popular automated real-time multiplex PCR systems used to detect E. 1997. 1994). coli are sorbitol negative and can induce false-positive results (Borczyk et al. PCR is the most extensively used rapid technology for detection of STEC and involves the extraction of genetic material. the majority of rapid detection methods.. like biotin-streptavidin.. Zadik et al. 2007).html).. 2005). Rapid detection methods have become increasingly popular in the food industry for the detection of specific pathogens and toxins (Lazcka et al.appliedbiosystems. or more specifically identify a microorganism. coli O157 can ferment sorbitol. 1996). specifically E.. and therefore. Multiplex-PCR protocols are capable of detecting multiple target sequences and can be used to screen samples for multiple pathogens. Available methods are not as specific or sensitive as conventional methods...cfsan. 2000). (2007) for use in the detection of EAEC. Okrend et al. PCR.fda. coli include SMAC supplemented with cefixime and potassium tellurite. Thompson et al.html).gov/~ ebam/bam-a1.html)..html. The target sequences in many multiplex-PCR protocols used to detect STEC strains.fda. In brief. and some species other than E. and many commercial kits are available and approved for use to detect E. and the amplification of the target sequence (Lazcka et al. 1995. while E.gov/~ebam/bam-a1. 5-bromo-5chloro-3-indoxyl-␤-d-glucoronide (␤-galactosidase).fda.html). stx1.

during which target antigens interact with two types of antibodies. and O145. In general. (2008) recently described a series of plating media used to differentiate and confirm the identity of STEC serotypes O26. coli by agglutination with O. and fully automated versions of standardized biochemical tests which identify the standard biochemical characteristics of E.. the efficacy of any PCR protocol directly depends on the proper enrichment and purification of food samples. coli and E. http://www.gov/~ebam/bam-4a. and the second type is attached to an enzyme-labeled “indicator” compound (Fratamico et al. the resulting fragments are then separated by electrophoresis on an agarose gel. the incidence of non-O157 STEC infections among health districts which use an enzyme immunoassay for Shiga toxin to screen clinical stool samples is disproportionately higher than that among districts which use culture-based screening methods (CDC.. non-O157 serotypes were differentiated by general appearance and color of colonies following spread plating and incubation on MAC Confirmation or Identification Phenotypic and genotypic characteristics can be used to confirm the identity of or classify a microorganism. 2001). Manual. O157:H7 Ϫ ϩ ϩ ϩ Ϫ Ϫ ϩ Ϫ ϩ Ϫ ϩ Ϫ Ϫ Ϫ Ϫ Data derived from Meng et al. 2001a). Table 4. 2001). 2005). 2005). O103. Phenotypic indicators of E. as many substances can antagonize PCR reagents (Entis et al.cdc. while O157:H7 has historically been the most common serotype (80%) associated with STEC infections (http://www.fda.gov/~ ebam/bam-a1. Agglutination assays utilize antibodycoated particles which interact with target antigens on cell surfaces or with soluble toxin antigens (Hajra et al. O111. as well as sorbitol-positive and -negative O157. coli ϩ ϩ ϩ ϩ Ϫ Ϫ ϩ Ϫ ϩ Ϫ ϩ ϩ ϩ ϩ Ϫ E.gov/foodborneoutbreaks/ecoli/ STEC2002. coli O157/O157:H7 Phenotypic indicator MUG ONPG Lysine decarboxylase Ornithine decarboxylase H2S Citrate utilization Methyl red Voges-Proskauer Indole Urease Arabinose Sucrose Sorbitol Rhamnose Cellobiose a a Generic biotype I E. and phage typing (Hiett. Correspondingly.pdf). 2005).. and particles that do not interact with the antibodies are washed away. the genetic material of a pure cell culture is cut apart by enzymes which target very specific DNA sequences..fda. 2005. compare. Phenotyping methods include the screening of known biochemical characteristics.html).html. The use of enzyme immunoassays in clinical diagnostic laboratories to screen for STEC is becoming increasingly common. Pulsed-field gel electrophoresis (PFGE) is the DNA-based molecular typing assay used by PulseNet and FoodNet. and track human clinical cases of food-borne illness and human pathogens recovered from food products (Swaminathan et al. EIEC.. and EHEC but not DAEC.cfsan.. coli and E. Recent Advances in Analytical Methods Possé et al. coli O157. serotyping.cfsan.84 BEAUCHAMP AND SOFOS ETEC. the first type is isolated by precipitation because it is covalently bound or adsorbed to a solid surface. Briefly. The use of multiple enzymes increases the specificity of digestive enzyme-based typing assays (Hiett. 2007). The resulting pattern of DNA fragments can then be compared to the DNA patterns of other isolates. and genetic homology can be measured (Farber.and H-group antisera is also used to confirm the identity of pure cultures (Meng et al. Enzyme-linked immunosorbent assays and immunoprecipitation are both “sandwich” assays. Briefly. These assays are unable to differentiate viable from nonviable cells (Fratamico et al. rapid. 2007). two branches of the CDC which investigate. . Serotyping of diarrheagenic E. (2001b) and http://www. coli O157:H7 are available (Table 4). 1996).

gov/ecoli/ reportingtimeline. 2007). or to an established outbreak (http://www. Additional drawbacks include bacteriophages with a very broad/narrow target host range and the eventual development of resistance by target host cells (Greer and Dilts. (2007) described the use of a recombinant phage specific for E. including those for E. Bioreporter cells carrying the Vibrio fischeri lux operon were added to the reaction and emitted a bioluminescent signal in response to OHHL production by infected cells after as few as 4 h (Brigati et al. Mead et al. Rangel et al. 1999). 2003). 2007). IPTG. http://www. While the sensitivity and specificity of some biosensor assays rival or exceed those of conventional detection methods. 2007. Bacteriophages are species-specific viruses that. maximum fluorescence was observed after 3 h (Oda et al. or d-arabinose.. and potassium tellurite. html). coli O157:H7 and/or Shiga toxins. coli O157:H7.to 3-week interval between consumption of contaminated food and being classified as part of an E.. although antibody-based assays are the most popular (Nakamura and Karube. and (iii) the documentation of epidemiologically and genetically related illnesses and/or contaminated food products may not be available and/or correct (CDC. are stabilized by avidin-biotin systems. Cieslack et al. 3. coli O157:H7 was examined. Swaminathan et al.. l-rhamnose. coli-specific bacteriophages infect and replicate within viable cells and can indicate the presence of extremely low levels of E. 3. 2008). refractive. coli O157:H7 in as little as 5 min (Radke and Alocilja... Brigati et al. 2003. Although most require at least 1 to 3 h. coli in food samples (Goodridge et al. the CDC estimates a 2. Ruan et al. 2007. coli outbreak (http://www.. coli within 1 to 2 h (Goodridge et al.. 1999. (ii) an accurate recollection of all activities and foods eaten before the onset of symptoms is nearly impossible. 2005. which initiates the production of N-(3oxohexanoyl)-l-homosterine lactone (OHHL) in infected cells.. the identity of suspect sorbitolpositive colonies (purple) was confirmed using a phenol red base plus l-rhamnose (Possé et al. isopropyl-␤-d-thiogalactopyranoside (IPTG). many infections go unreported... 2004). overall sensitivity and specificity in the presence of food components and background flora remain unproven. X-Gal. by adsorption to gold. Labeled E. 2006. 2002.. diagnostic testing by state health authorities. Biosensors are devices associated or integrated with cellular transduction systems and include cell components and biologically or synthetically engineered compounds (Lazcka et al.cdc. to a contaminated food product. nucleic acid-. or electrochemical impedance indices are then used as indicators (Bergwerff and Knapen. and thus. or by forming self-assembled monolayers. bile salts no.. coli O157:H7. When attribution is possible. some biosensor assays may detect E. This interval includes incubation time.cdc. PFGE. Radke and Alocilja.htm). Summers. Extended enrichment protocols may also be necessary when using bacteriophages to detect E. and using an epifluorescence microscope. Briefly.cfsan. the molecular typing assay used to link human clinical and/or food isolates.htm).. novobiocin. 1999. In one study. when labeled with an indicator compound (i. Loessner and Busse. 2001). 2002). is conducted by trained laboratory technicians with standardized equipment and protocols and generates distinctive DNA fragment patterns or genetic “fingerprints. Sorbitol-positive and -negative O157 strains were plated onto differential media consisting of MAC supplemented with sorbitol. 2001). a much more thorough examination of each method is required before it will be accepted as a viable option for the detection of STEC/EHEC in foods (Lazcka et al. 2004). or antibody-based biosensor assays have been developed for use in the detection of pathogens. Tanji et al.. McGrath et al. 2003). pathogen-specific antibodies.” Once generated. While these and other phage assays demonstrate potential for use in the rapid detection of E. 1990. and completion of the analyses required to link a patient to other isolated cases of illness. PFGE profiles are made available to other regional laboratories with access to the national . Following incubation. the ability of green fluorescent protein-labeled PP01 bacteriophage to detect E. can be used to detect target organisms among a diverse background of unrelated microflora. d-raffinose. and potassium tellurite. SURVEILLANCE AND TRACE-BACK PROGRAMS Locating the food product responsible for human illness can be rather difficult for a number of reasons: (i) only a small proportion of the human population is at risk for severe infection which requires medical attention.CHAPTER 5 • DIARRHEAGENIC ESCHERICHIA COLI 85 supplemented with sucrose. onset and progression of symptoms which require medical attention. Lazcka et al. A multitude of enzyme-.e. fluorescent protein). 1997..fda.gov/~ebam/bam-4a. and deviations in optical. novobiocin. adsorption of green fluorescent protein-PP01 to cells was observed after only 10 min (4ЊC). fluorescent. 1990. 5-bromo-4-chloro-3-indolyl-␤-d-galactopyranoside (X-Gal). A second medium was used to confirm the identity of suspect colonies and was composed of a phenol red base supplemented with dulcitol.gov/ecoli/ reportingtimeline.. sorbose. 2006. bile salts no.

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old. Muriana. because this provides an opportunity for the pathogen to survive in a variety of niches at various points within food processing operations and ultimately contaminate product contact surfaces and finished products (Tompkin. 2002). The optimum growth temperature is 30 to 37ЊC. Stillwater. and John B. Eastern Regional Research Center. non-spore-forming rod that exhibits tumbling end-over-end motility at 22ЊC. 4c.911 (Nolan et al. 4d. and severe systemic infections in the unborn or neonates. with about 2. monocytogenes can also survive and/or grow at water activity (aw) levels that are usually too low for other bacteria to survive. Between 1998 and 2002. 95 . L.. Peter M. 4e. DC Chapter 6 Listeria monocytogenes Anna C. monocytogenes increases as the temperature decreases.. U. 1992). 4b. Porto-Fett. pregnant. New information indicates that once the pathogen has been ingested and disseminated to the maternal organs. 4a.900 cases and about 260 deaths (mortality rate of ca. and 7) (Khelef et al. Cargill.. England. Juneja and J. 2000). Sofos © 2010 ASM Press. that being aw values of Յ0. Department of Agriculture. Biochemically. and it hydrolyzes esculin and exhibits slight ␤-hemolysis on blood agar (Ryser and Marth. it is able to migrate to the placenta. 1/2c. what is now known as Listeria monocytogenes was first documented by E. 1999). 2004). of which about 500 are fatal. Symptoms range from an asymptomatic infection to flu-like symptoms. In general. S. Timothy A. but it can grow at Ϫ0. Although healthy adults and children can also suffer from listeriosis. and immune-compromised individuals (“YOPIs”) are particularly susceptible (CDC. 3c. The ability to survive and/or grow over a wide range of temperatures. peripheral monocytosis. 1/2b. Until recently. Of the estimated 2. Corporate Food Safety and Regulatory Affairs. about one-third involve pregnant women (Mead et al. 4ab.6 (Pearson and Marth. 2006). Freier. in laboratory rabbits. pH. Because the pathogen is opportunistic and is not necessarily adapted to humans. intrauterine infections in pregnant women. wherein it is protected from the maternal immune system (Bakardjiev et al.S. it was thought that pregnant women were more susceptible because their immune system was changed and/or compromised during pregnancy. 9. MN 55440-9300.Pathogens and Toxins in Foods: Challenges and Interventions Edited by V. Jeffrey E. A few years later Nyfeldt (1929) reported the first case of illness in humans that was attributed to this bacterium.. 1989. OK 74078. 1996). Luchansky • Microbial Food Safety Research Unit.7% of bacterial food-borne illnesses over this 5-year Jeffrey E. Muriana • Department of and Agricultural Products Research and Technology Center. Peter M. Freier • LISTERIOSIS There is still much that is not known about how this disease is manifested in the human body (McLauchlin. Luchansky TYPE OF ILLNESS AND CHARACTERISTICS OF THE ORGANISM In 1924 in Cambridge. Minneapolis. the pH that will support growth of L. N. D.4 and 9.. Service. S. K. 3a. such as fever and muscle aches in healthy individuals.0%) (McLauchlin et al. it is catalase positive and oxidase negative. 1999). it probably has multiple routes of infection and types of symptoms. but growth has been observed between pH 4. Murray and colleagues (1926) as the causative agent of a septic illness. Porto-Fett. Call. Wyndmoor. The optimum pH range for growth is pH 6 to 8. Call. PA 19038. Oklahoma State University. L. monocytogenes is a gram-positive.500 food-borne cases per year in the United States. 1998). Anna C. G.. 1999). although listeriosis represented only 0. the principal route of infection for foodborne listeriosis begins with the ingestion of contaminated food. Food Timothy A. Washington.4 and 45ЊC and can survive freezing for prolonged periods (Rowan and Anderson. and John B. 3b. depending on how it enters the body. Inc. Ryser and Marth. there were 27 outbreaks of food-borne listeriosis reported worldwide. Between 1976 and 2002. and aw values is of great concern to the food industry. There are currently 13 serotypes (1/2a. Agricultural Research Animal Science. however. 2006). young. to septicemia and meningitis in immune-compromised patients.

it was responsible for 54% of all deaths (CDC. the pathogen is usually present at levels of ca. 4 for cooling. It is accessed by ca. 1997).82% (577 of 31. . and the Predictive Microbiology Informational Portal are worth mentioning. 2001). ComBase. and animals raised as food (CFSAN.. levels are typically low. Paoli et al. Such progress has also been accelerated by the availability and comparison of the whole genomes of select serotype 4b and 1/2a strains (Nelson et al.0 log10 CFU/g but sometimes can be found at levels as high as 7. namely. The PMP is a stand-alone online application used by ca. 3 for heat inactivation. More detailed information on molecular characterization of the determinants involved in invasion of host cells. when found in various meat and dairy products. 2002. Gombas et al. primarily because the pathogen can grow at low temperatures.0 log10 CFU per package/pound. such as glycine betaine and carnitine (Chattopadhyay. soil. As summarized by Farber and Peterkin (1991). as well as for structure stabilization of macromolecules.0 to 3. L. monocytogenes in foods. ComBase is a relational database that contains over 40.800 samples) among 12 brands of commercial frankfurters and levels of 1. monocytogenes growth/survival at low temperatures requires maintenance of membrane fluidity for appropriate enzymatic activity and transport of solutes across the membrane. 2006. whereas competitors cannot. Wallace et al. including 23 models for growth. The pathogen has also been recovered from a variety of raw and ready-to-eat (RTE) foods (Meng and Doyle. monocytogenes may exhibit a thermotolerance response when exposed to temperatures above its optimum growth temperature. and guidelines pertaining to L. being associated with plants. INTRINSIC AND EXTRINSIC FACTORS THAT AFFECT SURVIVAL AND GROWTH IN FOOD PRODUCTS AND CONTRIBUTE TO OUTBREAKS The psychrotrophic nature of L. 1998). Efforts to fill research voids needed to conduct risk assessments are aided by the ability to predict the fate of the pathogen on various foods using the tools of predictive microbiology. Further expansion of our knowledge of the mechanism(s) of pathogenesis at the molecular level will ultimately result in more direct and more effective interventions to better manage the occurrence. In this regard. Glaser et al. SOURCES AND INCIDENCE OF L. period. and Latin-style cheese as foods of higher risk (CFSAN. and/ or the uptake or synthesis of compatible cryoprotectant solutes.000 online users in some 35 countries worldwide each year to predict the fate of pathogens and spoilage microbes in a variety of foods.0 to 5.. the Pathogen Modeling Program (PMP). RTE foods that were consumed without reheating. 2003). It has also been recovered from stool samples of an estimated 1 to 10% of healthy humans (Farber and Peterkin. research data. 30% of the food industry for hazard analysis of critical control point validation. 2003). A Joint Agency risk assessment prioritizing the relative risk of listeriosis among 20 food categories identified deli meats. feed. and numbers of this pathogen in our food supply. survival. may be consumed without any reheating and/or further preparation. monocytogenes is of particular concern. monocytogenes and other food-borne pathogens under diverse environments relevant to food processing operations.6% (532 of 32. The PMP contains some 40 models. such as Latin-style cheese and deli meats. The Predictive Microbiology Informational Portal is a comprehensive website that presently is comprised of predictive models. 2006). 4 for survival. and 3% in seafoods. 11% in fresh vegetables. 1991). 2005). For example. 5. monocytogenes. 2007) and primarily because RTE foods. (2003) reported a prevalence of 1.96 LUCHANSKY ET AL. undercooked chicken. heat shock proteins (Rowan and Anderson.arserrc.5 5 ϫ 10 CFU/g in six categories of RTE foods. intracellular motility. monocytogenes virulent. MONOCYTOGENES IN THE ENVIRONMENT AND FOODS L. (2003) reported a prevalence of 1. The bacterium is found in numerous foods with a prevalence ranging from 13% in raw meats to 3% in dairy products. monocytogenes and RTE meat and poultry products (http://portal.705 samples) and levels of Ͻ0.3 most probable numbers (MPN)/g to 1. When found in RTE foods. water. persistence. and 6 for irradiation. given that these temperatures may trigger physiological responses that induce synthesis of polypeptides. The majority of cases linked to foods are associated with refrigerated. such as ribosomes. RTE foods provide an ideal environment for L. sewage. monocytogenes is widespread in nature. 2004.000 data sets/records of the growth. Likewise. The use of genomic and proteomic tools has generated appreciable insight into the genes and proteins that make L. 2007).0 log10 CFU/g.gov). and cell-to-cell spread is beyond the scope of this chapter but can be found elsewhere (Kathariou. L. 1. relevant regulatory policies. given that refrigeration is one of the most common interventions used to ensure safety and extend shelf-life (Gandhi and Chikindas. Many factors affect thermotolerance of L. Gandhi and Chikindas. Refrigerated.. and/or inactivation of L.

monocytogenes to accumulate these intracellular solutes allows it to grow at NaCl concentrations of Յ12% (Razavilar and Genigeorgis. Parish and Higgins (1989) reported that L. regardless of the acid used to adjust the pH. environmental monitoring. when at near-optimum temperatures. Ingham et al.87 did not support growth of L.5) cells of L. sanitary design of equipment and facilities. (2006) evaluated the survival of L. monocytogenes growth in foods is pH 4. The lowering of aw in foods is another strategy widely used for controlling food-borne pathogens. and the microbiological media and/or incubation conditions used to recover injured cells (Doyle et al. monocytogenes. The authors reported that jerky products with an aw of Յ0. monocytogenes strain EGD-e also developed an acid tolerance response at 20ЊC when cells were acid adapted (pH 5. the glutamate decarboxylase system. 2000). 2001). According to Garner et al. SPREAD. The adaptation of L. sanitation. monocytogenes to environmental conditions present in RTE meats. 1998). 2002. (2006).6 with HCl. and dairy products. monocytogenes accumulated higher levels of glycine betaine and carnitine when inoculated onto the surfaces of bologna. Acidic stress may also increase the ability of the pathogen to survive in foods and/or may facilitate expression of its virulence genes (Ryser and Marth. such as osmotic and acidic shocks. by accumulation of solutes. monocytogenes control interventions. 2000). monocytogenes is one of the most difficult challenges faced by manufacturers and handlers of RTE meat. heat treatment or other listericidal steps. such as glycine betaine and carnitine (Bayles and Wilkinson. comprised of lisR and lisK (Shen et al. environmental stresses. 2000. monocytogenes to a low-aw environment is induced. poultry. exposure of L. differences in the strains used or their physiological state. 2007). thus challenging control of this pathogen in salted RTE foods. frankfurters..5) compared to those that were not acid adapted (pH 7. Gandhi and Chikindas.. presence of deleterious chemicals in the growth media. B sigma factor (␴ ). Their results showed thatL. O’Driscoll et al.5) with either acetic acid or lactic acid prior to exposure to a lethal pH (pH 3.. (2003) reported that cells of L. . in the acid conditions within the gastrointestinal tract.0 to 5. Angelidis and Smith.. and cold chain management. monocytogenes (Bayles and Wilkinson. monocytogenes by exposure to sublethal pH. The stress response of L. such as fermented meat and dairy products. Cotter and Hill. 2003).0). and/or by binding of water to a range of macromolecular components (Stecchini et al. wieners. Smith (1996) evaluated the adaptation of osmotically stressed cells of L. segregation of raw product areas from cooked/pasteurized product areas. seafood. Other important control considerations. FOOD PROCESSING OPERATIONS THAT INFLUENCE THE NUMBERS. and a two-component regulatory system. and in the phagosomes of macrophages (O’Driscoll et al. 1996. monocytogenes survived for 21 and 5 days at 4 and 30ЊC. For example. Greenacre et al. 1996). acid-adapted (pH 5.4. its growth at low pH is dependent on the medium or food composition. such as the addition of NaCl and organic acid salts. respectively. 1999). by removal of water using physical methods. history/adaptation of the cells prior to inoculation. such as the intrinsic and extrinsic factors of the food and specific L. As one example. monocytogenes to low-pH environments is the result of the activation of stress adaptation mechanisms.. The minimum pH requirement reported for L.0). As another example. in orange serum samples adjusted to pH 3. may enhance its pathogenicity and consequently the association of listeriosis with these foods. are addressed elsewhere in this chapter.47. monocytogenes strain LO28 showed greater tolerance toward lethal pH (pH 3. Smith. monocytogenes on processed meat surfaces. Overcoming this challenge requires the flawless execution of numerous food safety-related systems and operations. Studies have shown that glycine betaine and carnitine are the most effective solutes for osmotic adaptation of L. The intracellular solutes that are synthesized or that are taken up from the food or natural environment compensate for the external osmotic strength without affecting the macromolecular structure of the cell (Duché et al. at least in part.. (1996) reported that. and bratwurst at refrigerated temperatures than when inoculated in liquid media supplemented with either of the osmolytes. The aw of foods can be altered by addition of osmolitic compounds. 2003). OR CHARACTERISTICS Control of L. monocytogenes on vacuum-packaged beef jerky and related products stored at 21ЊC for up 28 days. such as induction of proteins. 2006. and the preincubation temperature (Phan-Thanh et al. Its ability to adapt to acidic environments is of particular concern because the pathogen encounters these environments in low-pH foods. the strain and its physiological state. However. 2004). ham. Tolerance of acidic environments can be induced in L. such as soft cheese and deli meats. including proper sourcing and storage of ingredients. pH homeostasis.CHAPTER 6 • LISTERIA MONOCYTOGENES 97 such as the proximate composition of the food. The ability of L. but the organism survived in jerky at aw values Յ0.

maintenance. Air balances should be repeated whenever new equipment is added or whenever the facility is modified in ways that could impact the air balance. Ingredients added after cooking. public data do not exist for many meat and poultry ingredients. so worst-case estimations of the levels potentially present before the kill step should be conducted to assess the adequacy of this control point. For example. segregation of employees that work in raw versus treated areas can best be attained by having dedicated employees for each area. soap. This kill capacity is not unlimited. These systems must also be designed to quickly reduce the humidity generated during sanitation and maintain low humidity levels during operation. Modern systems that treat the air supply with heat. Carefully planned procedures then become critical for limiting L. nor do they exist for most other ingredients. Segregation of Raw and Treated Areas Because L. dry spice rubs. paper towel. but limiting the occurrence of L. 1999). and equipment for production. and/or UV radiation are available. reworked product. monocytogenes in the posttreatment environment to the minimum level possible will greatly protect the treated/finished product (Tompkin. such as spice blends. and heatsensitive nutrient supplements. The RTE industry has used numerous practices to attain this goal. and other processing aids will enter the treated product area. packaging materials. If there is a regulatory or public health-determined “zero tolerance” for L. such as sauces. For many operations. are even more critical. While most other microbial food-borne pathogens can be controlled by a well-validated cooking or pasteurization step. monocytogenes. monocytogenes detected in several types of raw meat and poultry (Table 1). and restrooms) to avoid cross-contamination. monocytogenes is not able to multiply within the system or be disseminated in the air stream. 2002). and sanitation should be maintained for the pretreatment and posttreatment areas. monocytogenes thrives in the cool. sanitation equipment and supplies. monocytogenes. A well-designed hand wash station incorporating such things as touchless water. utensils. monocytogenes. as well as water and air.98 LUCHANSKY ET AL. monocytogeneskill-step product. monocytogenes in the posttreatment environment. fork lifts/hand trucks. Processing aids. becoming more negative in the raw areas. A critical limit for the level of kill necessary to produce a safe product needs to be determined based . Manufacturers should also consider how ingredients. when the generation of microaerosols could spread L. especially when the product is directly exposed to that environment. monocytogenes is not attainable. Ingredients in which L. monocytogenes during the cooking or pasteurization step has been determined for many products and processes (CDC. cafeterias. quantitative testing should be conducted to determine maximum levels. monocytogenes growth is key to protecting public health. monocytogenes is capable of multiplication should be stored at temperatures and for times that will not allow for an increase in numbers of the pathogen that might subsequently overwhelm the final kill step. Air pressure should be highest in the most critical high-care area. 1988. especially including the sanitation shift. it is important to minimize the presence and levels of L. Thus. monocytogenes is ubiquitous in the environment and is often present on raw ingredients. garbage/inedible containers. Sourcing and Storage of Ingredients A consideration of all inputs to the process or product is a key step in controlling L. Complete exclusion of L. and brine chill solutions. pallets. Many facilities even provide separate employee welfare areas (locker rooms. employing two complete sets of workers is impractical. Unfortunately. should also be carefully considered if they will be contacting the post-L. Juneja and Eblen. monocytogenes in the final product. damp environment typical of the postcook or postpasteurization environments of RTE manufacturing facilities. monocytogenes and from exposure to conditions conducive to L. Facility air chilling systems should be designed so that L. HEPA filtration. packaging materials. Air balances should be conducted to ensure that these pressures are maintained during all phases of operation. lubricants. and final hand sanitizer dispenser controls and with comfortable water temperature and adequate sink space (so employees are not rushed) is just one of the recommended requisites. L. Kill Step The capacity to kill L. protection of cooked or pasteurized RTE products from recontamination by L. ingredients added postcook must also be free of any viable L. quality assurance supplies. One of the most important considerations is provision of footwear and outer clothing that are worn only in the high-care areas and that are adequately cleaned and sanitized on a routine basis. monocytogenes cross-contamination when employees transition from raw to treated areas. Separate sets of tools. and even home refrigerators. monocytogenes. If any ingredients added prior to cooking are suspected of having occasional high levels of L. Baseline studies conducted by the USDA/FSIS indicated the highest levels of L. retail slicing areas.

CHAPTER 6 • LISTERIA MONOCYTOGENES 99 Table 1. bolt/screw/rivet penetrations.fsis. then protein residues can be baked onto the equipment. then fats and oils will not be melted and removed. Excellent information on best practices for the sanitary design of equipment and facilities has been compiled by the American Meat Institute and is available at www. sweeping. and high-pressure spray) and/or do not receive adequate exposure to cleaning and sanitizing chemicals.). where clogs and backups can occur. Cleaning is the critical step that removes food residues and allows the chemical sanitizer to function optimally. electrical panels. The first step is dry cleaning. monocytogenes growth temperature range. condensation. or adapted from existing published data (Table 1). monocytogenes in the overall facility environment is to strive for clean. monocytogenes found in USDA baseline sampling Type of animal product (sample type) Market hogs (carcass swab) Broiler chickens (carcass rinse) Cows and bulls (carcass swab) Young turkeys (carcass rinse) a a No. pumps. conveyor rollers.221 Highest level (MPN/cm ) 46 51 43 0. The third step is applying the appropriate detergent. most probable numbers. the appropriate sanitizer can be applied. leaking steam and water valves. although it can sometimes come from other sources. as it will not rinse properly if allowed to dry. removing as much product residue as possible by scraping. monocytogenes are typically the floor and drain systems. and time within the L. etc. where it is critical that all remaining food and detergent residue be completely rinsed from the equipment. etc. the potential exists for an “L. unbroken floors that remain dry during food production shifts. including juices from the product. cracks. because if it is too cool. monocytogenes control points. and if too hot. is one of the most important L. It is important that the foam not be allowed to dry on the equipment. bearings. A four-step cleaning process is recommended. Examples of growth niches are hollow equipment elements that are not hermetically sealed (support structures. Once validated. This detergent should be matched to the application by a cleaning chemical expert.meatami. and shoveling. equipment controls. monocytogenes that may be present in raw milk. such as dust from a cardboard box. It is generally accepted that the heating times and temperatures necessary to eliminate Salmonella in cooked meat and poultry products are also adequate to eliminate L. monocytogenes that could potentially be in the product before the kill step. on a worst-case estimation of the load of L. This temperature is important. discharged chilling water from packaging equipment. monocytogenes. When the equipment and room are clean. if moisture can infiltrate the junction.usda. The most important .). sealed. Detergent is commonly applied as a foam solution.asp. MPN.gov/Science/Baseline_Data/index. leading to a high-risk L. and cooling coils. monocytogenes situation. monocytogenes is from a growth niche that exists in or near product contact surfaces on equipment in the exposed posttreatment product area. water softness. however. Sanitary Design of Equipment and Facilities The most typical cause of the contamination of RTE product with L. foaming. gaskets. the kill step must undergo routine verifications to ensure its continuing efficacy.org (accessed 15 April 2008). etc. residual water left from the cleaning/sanitation process. compressed air lines. Levels of L. sampled 2. Sanitation Sanitation. Anytime two solid pieces of material are combined. especially in critical high-care areas. Care should be taken to pick up and dispose of this residue and not rinse it down the drainage system. Traditional milk pasteurization times/temperatures are also considered generally adequate to eliminate the typically low levels of L. The second step is the rough rinse with hot water (approximately 140ºF). These data can be generated by conducting quantitative testing of the uncooked product or ingredients. rubber floor mats. and even poorly managed antimicrobial foot baths or floor foaming systems. motors. monocytogenes sandwich. water. monocytogenes growth depends on temperature and other extrinsic conditions. turbulent flow of clean-in-place systems. The most effective way to limit the levels of L. The final cleaning step is the final rinse.3 2 Source: http://www. Accessed 15 April 2008.297 2. The most problematic areas of equipment that can become growth niches for L.” The most common harborage sites for L. residues to be removed. The time necessary for L.112 1. monocytogenes are the areas that do not receive physical disruption (scrubbing. drive chains. scrubbing. A growth niche is an area that contains the essential ingredients of food. Water can be supplied from numerous sources. as it needs to match the cleaning needs (clean in place. The food source is typically residue from the product being manufactured.112 1.

Many facilities double and even triple sanitize to ensure for the most complete kill of L. Environmental Monitoring Areas capable of harboring L. consideration for sanitizer application is coverage. or home use. RECENT ADVANCES IN BIOLOGICAL. the final sanitizer should be applied at a level that can be left on equipment without a final rinse.. monocytogenes in the central. distribution. monocytogenes in food products will also kill L. Dry heat has much less killing capacity. If the product is temperature abused at any step of manufacture. Equipment can be covered with a tarp and steamed. sampling large areas. monocytogenes. Despite advances that reduced by about 40% both the prevalence of the pathogen in certain . most heat-protected areas of the equipment. Most sanitizers should be applied as a mixture with cool water. the times and temperatures necessary to kill L. If regulations allow. 2003). Environmental monitoring gives manufacturers the ability to find and eliminate L. and sampling areas that are the most difficult to reach and clean.04 cell/g) (Shank et al. retail. Flood sanitizing (low-pressure and high-volume) application of the sanitizer is most effective. so higher temperatures and longer application times are typically required. the potential for L. storage. In addition. monocytogenes harborage sites and growth niches. single-ply tissues. 1996). thus. and the frequency and magnitude of product recalls have resulted in the “zero tolerance” policy in effect at present in the United States. monocytogenes growth niches by doing routine monitoring several hours after the start of processing operations or at the end of production but before sanitation. monocytogenes to grow to infectious numbers increases. One recommendation for aggressive searching is to reward employees for finding positive environmental samples. A neutralizing buffer should be used to moisten the sampling device. as pointed out by Buchanan and colleagues (1997). monocytogenes growth conditions does not end when the product leaves the manufacturing facility. in contrast to looking specifically for L. However.. is used first and is followed by a quaternary ammonium solution that is slower acting but has residual killing activity. such as Listeria spp. most sensitive methods possible. Others include using the fastest. Heat sanitizing processes should be validated for the ability to kill L.. CHEMICAL. such as Germany (about 4 or 5 cases per million inhabitants). Using a broader indicator group. 2003). except in special circumstances. Sampling devices should be optimized to pick up the target group from the equipment so that it can be transferred into the detection system. Many manufacturers occasionally send temperature monitoring devices with the product during transit and storage and then review the monitoring information to ensure that the cold chain is routinely being maintained. the key to minimizing listeriosis is to prevent L. heat can be effectively used to kill L. it is noteworthy that the relative frequency of listeriosis in countries that practice zero tolerance. monocytogenes multiplication (Chen et al. or immersed in hot water. monocytogenes and other microorganisms. monocytogenes.100 LUCHANSKY ET AL. For very difficult-to-sanitize equipment. a fast-acting oxidizing sanitizer. highlighting the importance of the cold chain in minimizing this disease (CFSAN. 2004). When using wet heat. The RTE meat and poultry industry has found the greatest success in finding L. monocytogenes in or on equipment. is about the same as that in countries that do not. Other methods (fogging and garden sprayer) may not give 100% coverage and should be avoided. the nature and number of the most susceptible members of our society. The sanitizing method should be carefully evaluated for employee safety and degradation of heat-sensitive machine components. or Listeria-like organisms. The two biggest keys to success are aggressive searching and diligent corrective action. monocytogenes is arguably the food-borne pathogen that has consistently garnered the most regulatory attention over the past 25 years. Cold Chain Management Protection from L. Microcellulose sponges. Often. placed in an oven. monocytogenes are not always visually apparent. Clean-out-of-place tanks can be very useful for cleaning and heat sanitizing smaller parts. A joint risk assessment conducted by the FDA and the USDA indicated that if all home refrigerators could be maintained at or below the recommended 41ºF. retailers and display case manufacturers have made improvements in maintaining cold temperatures during retail display. and gauze all work well and pick up the target group more efficiently than cotton-tipped swabs or direct-contact plates (Vorst et al. cases of listeriosis could be cut by Ͼ98%. enhances the ability to find growth niches. such as the United States. The severity and mortality of the disease. such as a mixed peracid. that being an allowable limit of Յ1 cell/25 g food (0. Samples should be kept refrigerated and be processed in a timely manner to prevent overgrowth by competitors or die-off of the target group. AND PHYSICAL INTERVENTIONS TO GUARD AGAINST THE PATHOGEN L.

1996). This ruling provides manufacturers with three options for the plant/product which ultimately determine the frequency of regulatory testing: use of both a postprocess lethality step and an antimicrobial to control outgrowth (lowest testing frequency) (alternative 1). In efforts to expand the application of nisin as an antimicrobial.. water activity. pressure. 2006).to 3. and their inhibitory activity against various food-borne pathogens (Muriana. for which it has not yet been approved. One virulent lytic bacteriophage. The only bacteriocin that has been granted such approval is nisin. Carlton et al. or use of appropriate sanitation alone (most testing) (alternative 3). Baltimore.and postprocessing.9-log10-CFU/cm reduction of L.. Salt Lake City. OmniLytics. efforts have intensified to develop and implement biological. EBI Food Safety. which is allowed for use in low-moisture/lowsalt pasteurized processed cheese. Bacteriocins Bacteriocins are inhibitory peptides and proteins produced by bacteria. The Netherlands).5-log10 reduction 9 with higher phage titers (3 ϫ 10 PFU/ml). 2006). pH.5 ϫ 10 PFU/ml phage titer). 2001). and the composition of foodstuffs (Hugas et al. MD. Neetoo et al. monocytogenes (1. When the bacteriophage was applied to soft cheese. The level of inactivation by HPP depends on the type of microorganism. (2008) have shown a 2 3.0. and the fact that not all organisms are equally affected. Other bacteriocins have been used in this regard by employing bacteriocinproducing lactic acid bacteria for production of cultured milk or whey preparations that are used directly as ingredients. that has been granted GRAS (generally recognized as safe) status by the FDA on a petition submitted by Intralytics and that has been granted approval as a food additive for use on RTE meat and poultry products (FDA. monocytogenes relative to growth in controls at the highest level of 2 nisin used (2. chemical. treatment time. 2002). Listex-P100 (EBI). (2005) observed a 2. which improved to a 3. a change in membrane structure is believed to be the main cause of inactivation (Lado and Yousef. phage P100. displayed a host range against 95% of the 250 Listeria isolates tested (Carlton et al. additional guidelines are in place for producers of red meat and poultry products (Anonymous. This has been developed into a product. 2005). the cost of scale-up to handle commercial-sized quantities of product.asp). use of either a postprocessing lethality step or an antimicrobial to control outgrowth (moderate testing frequency) (alternative 2).fsis. .CHAPTER 6 • LISTERIA MONOCYTOGENES 101 foods and the occurrence of listeriosis (CDC. Wageningen. and inhibition of enzyme activity.000 IU/cm ). fatty acid composition. Use of purified bacteriocins would constitute a direct food additive and require FDA approval. Inc. that are prone to quality changes using typical thermal processes. UT. Cellular functions sensitive to pressure include modification of membrane permeability. researchers have been examining its use in a variety of different food applications. also known as cold pasteurization. In addition. Using nisincoated plastic films for vacuum-packaged coldsmoked salmon.gov/Regulations_&_ Policies/New_Technologies/index. The FDA/WHO Codex High-Pressure Processing High-pressure processing (HPP) represents a promising nonthermal process for the preservation of sensitive products. their generalized GRAS Irradiation Irradiation.usda. Several disadvantages of HPP are batch-wise processing. however. status for use in foods. cell and membrane morphology. and/or physical interventions to control the pathogen both pre. temperature. Pressures of 100 to 600 MPa are being used to control microbial growth at low or moderate temperatures. Due to this “incentivized” offering by USDA/FSIS. along with a description of the antimicrobial process (http://www. USDA/FSIS also provides a venue for companies to implement antimicrobial approaches via plant trials to provide efficacy data for USDA acceptance that can be listed on the USDA’s New Technologies web page. without affecting organoleptic properties. monocytogenes to nisin and other bacteriocins that may occur during repetitive selective pressure in food applications.. 2002). and minimally processed meat products (Molins et al.0-log10 decrease in viable counts 8 of inoculated L. such as sliced deli meats. 2003). Concern has also surfaced for the resistance of L. cooked. PHYSICAL INTERVENTIONS BIOLOGICAL INTERVENTIONS Bacteriophage Several companies have proposed utilizing bacteriophage as a biological anti-listerial intervention for food and other applications (Intralytics. protein denaturation. is an effective control measure in maintaining the quality of raw. Those produced by lactic acid bacteria have long been proposed as food “biopreservatives” because of the historical use of these organisms in food fermentations..

25% of the total formulation (Anonymous. Thermal Surface Pasteurization Listeria contamination on RTE products often represents postprocess surface contamination. More recently. UV rays are restricted to the treatment of food or equipment surfaces and eradication of airborne contaminants and. which reduces the oxidizing capacity of the solution.0.0-log10 reduction of L. 2004).8-log10 reduction) in less time (60 to 75 s). 2002). ultrasound. can be of some practical use in reducing L.to 4. monocytogenes (1. its effectiveness is also limited by the degree of organic material. monocytogenes in food production and storage areas. Radiant heat prepackage surface pasteurization has also achieved similar lethalities of L. more of the undissociated form of an organic acid can enter cells and dissociate to generate the toxic anion. Because of their poor penetration power. When foods are treated directly. Either method alone provides an alternative 2 process or in combination with chemical antimicrobials could provide for an alternative 1 process. Prepackage pasteurization requires the product to be packaged immediately upon exit from the oven to eliminate the possibility of recontamination. 2004. Zhu et al. monocytogenes. 8 countries that permit its use in meat. while examining electron beam irradiation of RTE products in the presence of various acidulants.0. Other Physical Methods As reviewed elsewhere in detail (NACMCF. which requires that it be used quickly upon generation. 2000). In 2001. 2001). including meat and poultry (Anonymous. Additional research demonstrated that this combination of acidulants had appreciable inhibitory activity for suppressing the growth of L.to 2. Although flavor changes of raw meats induced by irradiation may be masked during cooking. Stekelenburg. it shows more consistency in application on surfaces with minimal organic background. monocytogenes in raw and uncooked meat. Submersed-water. The use of ozone is limited by its short half-life. for which treatment of the entire mass of product may not be necessary. including 12 countries that allow irradiation for control of pathogens in poultry. which may be absorbed and neutralized with time by the food itself. and therefore. Such chemicals are more effective when used at a pH below the pKa of the acid. Oxidizing Solutions Ozone is a strong oxidizing agent approved with GRAS status for the sanitization of bottled water in 1982.0-log10 o decrease after 2 to 10 min of exposure at 91 to 96 C (Muriana et al. flavor and quality challenges still exist for its application to RTE meat products. respectively.. gamma radiation. (2005) demonstrated a 2. however. nonthermal plasma.to 4. and even filtration. 2001). pulsed electric fields. additional methods that could be used as microbial interventions may also include microwave processing.102 LUCHANSKY ET AL. 2003). with reduced purge loss or need for special pasteurization bags and reduced chilling requirements (Gande and Muriana.0 to 2. Wade et al. and 13 that allow its use for fish and seafood (Molins et al. thus. The combination of the sodium/potassium salts of lactate and diacetate has become the standard ingredient for the processed meat industry ever since the USDA/FSIS increased the allowable level of lactate to 4.. and environmental surface sanitation. a combination of the two processes eliminates this aspect while providing a 3. oscillating magnetic fields. 40 countries permitted the use of irradiation in different types of foods. Among the different forms of irradiation.0. . postpackage pasteurization has been used commercially as a postprocess lethality step for surface pasteurization of RTE deli products capable of providing a 2. UV.0.5 kGy. including L.... The problem with many foods in which this form of inhibition takes place is that direct application of the acidulant may create a temporary acidified film around the product. As a general oxidant. Under such conditions.48 log10 CFU/g) on alfalfa sprouts and seeds that were continuously sparged for 5 to 20 min with ozonated water than that on those treated with water alone.8% and of diacetate to 0. monocytogenes (Muriana et al. 2006). vegetables. it attacks chemical/organic constituents of the cell wall/ membrane of bacteria and other microbes. (2003) found a greater efficacy of the reduction of L. monocytogenes on hams at 1. 2001. such as fruits. and electron beam (generated by electricity) are considered to be bactericidal. thus inhibiting the organism. 2003). monocytogenes in formulated/processed RTE meats (Barmpalia et al. and studies have shown their effectiveness under various conditions and in different foodstuffs. CHEMICAL INTERVENTIONS Organic Acids Many organic acids have GRAS status as food ingredients.to 5. ohmic heating.0-log10 reduction of L. monocytogenes (2. Bedie et al. Alimentarious Commission considers irradiation a safe technology for controlling L. it was approved as an antimicrobial agent on food.

serotype 4b strains are responsible for all major outbreaks and about 50% of sporadic cases. 2007a. including carbonyl compounds. Instead.. Although all methods provide useful information.. 2000. Moreover. have met with varying levels of success at reducing the prevalence of this pathogen. monocytogenes (Faith et al. food treated via SLIC tastes better because there is less impact on flavor. 2000. Thus. and the compact SLIC system fits directly on existing production lines at a nominal. Subtyping has also been of great value in identifying sources of contamination in food processing plants (Berrang et al. de Valk et al. 1/2b. It became quite apparent by the late 1980s that the overwhelming number of food-borne listeriosis cases were caused by only three serotypes... research on the implementation and optimization of typing strategies. resulting in cost savings of $0. most studies evaluated the delivery of food-grade chemicals as an ingredient and/or a bath. relative to typeability.to 5. and the majority of the strains involved in food-borne outbreaks comprise two distinct genomic divisions that correlate with flagellar antigens (Rocourt. and/or discriminatory capability (Arbeit. (1992) demonstrated the ability of liquid smoke to inactivate L. and/or that were applied into or onto the packaging material. 1995). 2000.0-log10 reduction of L. 2007b).3-log10 reduction when combined with heat pasteurization (Gedela et al. some methods are better than others. however. monocytogenes using only phenotypic methods: between 20 to 40% of strains are non-phagetypeable. Regarding the latter. a major phenolic component of liquid smoke. Another .5 to $2 million per year for a small. have also demonstrated effective antilisterial properties on RTE meats and have suppressed growth of L. monocytogenes within 24 hours at refrigeration temperatures. Rørvik et al. and strains that are indistinguishable should be shown as such. Additionally.CHAPTER 6 • LISTERIA MONOCYTOGENES 103 Smoke-Derived Flavorings and Extracts Traditional smoking of commercial food products has been replaced largely by “liquid smoke” treatment because of the quick and easy application. monocytogenes when inoculated at low levels or provided as much as a 5.. 1997). The more often a particular method is practiced. Sim et al. dip. or spray for the finished product. Liquid smoke is known to possess a variety of phenolic compounds that have demonstrated inhibitory properties toward pathogens and spoilage organisms. isoeugenol. 2006). one-time cost of $5.000 to $10. liquid smoke is used mostly for flavor attributes and does not adequately provide the preservation characteristics of traditional smoked products. the results obtained with a given typing method must also be epidemiologically relevant (Tenover et al. 1992).. As summarized elsewhere (Ryser and Marth. Regardless. Relative to other interventions that use food-grade chemicals. reproducibility. it is often difficult to differentiate isolates of L. The literature is replete with various methods for typing and/or tracking listeriae (Sauders et al. Depending on the product. the sprayed lethality in container (SLIC) method was developed to introduce an antimicrobial solution into the packaging container just prior to when the finished meat or poultry product is placed within the package and then rely on the vacuum-packaging step to evenly distribute the antimicrobial purge. serotyping has limited value for epidemiologic applications. Thus. Other components derived from liquid smoke.. whereas strains that are nonidentical should be shown as different. 2002). has intensified to establish unequivocal relationships among strains for molecular subtyping studies. the same results should be obtained each time by all persons using a standardized method.or medium-sized frankfurter processing plant. DISCRIMINATIVE DETECTION METHODS FOR CONFIRMATION AND TRACE-BACK OF CONTAMINATED PRODUCTS Subtyping offers an approach for investigating the relatedness of isolates and identifying and tracing the sources of epidemics (Lyytikäinen et al. the easier and less costly it becomes... 1999).. and 4b. this system was effective at delivering about a 2. such that total coverage of both product and package is achieved (Luchansky et al. serotypes 1/2a. was the only phenol shown to appreciably inhibit L.000 for the equipment. namely.002 per pound. monocytogenes in hot dog exudates. such as the use of postprocess thermal treatments and/or application of biological and food-grade chemicals. Regarding scientific criteria. and discriminatory power) considerations for selecting a suitable method for subtyping. using this system would result in significantly shorter processing times (2 or 3 s). 2003). For example. Lauric Arginate Current practices. the same or similar O antigens are found in several species/strains. there are several practical (cost and ease-of-use) and scientific (typeability. Faith et al. 2005). all strains should be unambiguously typeable by the method(s) used.0. In further testing with 11 different phenols. 1994). used alone or in combination. reproducibility. production costs are appreciably reduced to about $0. despite a general usefulness in studies of food-borne illness.

1994). we used pulsed-field gel electrophoresis to identify possible sources of contamination in a dairy processing plant producing Minas frescal cheese (MFC). and/or with foods. Նaw 0. ՆpH 4. monocytogenes on farms. we must be ever vigilant in following appropriate good manufacturing practices. approach that was used to type L. especially in databases containing gels of DNA fragments produced by scientists in different labs. Յ100 cells/g) in lower risk foods that do not support its growth. on the potential threat of listeriosis.g.. There are numerous examples already published describing the use of molecular methods to subtype and source L. whereby specific genetic loci are identified. samples from sites that previously tested positive for the pathogen were collected from the processing environment and MFC on multiple visits. In the mid-1990s the techniques of pulsed-field gel electrophoresis and ribotyping confirmed that L. and analyzed in comparison to similar loci from other strains by multiple sequence alignment analyses (Maiden et al. and DNA sequences are readily analyzed by portable computer software for simultaneous comparisons of various clones.e. monocytogenes grouped into these same two distinct groups (Brosh et al. monocytogenes with raw and pasteurized milk. One of the main advantages of multilocus sequence typing over DNA fragment-based typing schemes is the elimination of the ambiguity in fragment migration. 1989). Minas Gerais. isolates from sporadic cases of listeriosis and animal isolates could not be grouped into distinctive subsets based on multilocus enzyme electrophoresis types. As such.4. monocytogenes. Based on the association of L. it may be time to revisit the zero tolerance policy and consider allowing minimal levels of L. and plant F environmental samples were serotype 1/2a and displayed the same AscI or ApaI fingerprints.g. good agricultural practices. the threat to public health. monocytogenes. standard operating protocols. given the advancements in PCR amplification and sequencing technologies. monocytogenes from our food supply.92. and hazard analysis and critical control point programs. absence of inhibitory substances. to eliminate harborage points within a food processing facility. several sites within the processing plant and MFC samples tested positive. Interestingly. however. sequenced. One common method is multilocus sequence typing.. All 344 isolates recovered from retail MFC. I (4b. “YOPIs”) are at greater risk due to their age and/or health status. we will illustrate the approach using a study that we recently completed (Brito et al. isolates were grouped into one of two types (based on their enzyme locus profiles). nucleic acid sequencebased typing schemes have flourished. Automated DNA sequencers with laser fluorescence detection using a single gel lane can provide upwards of 500 bp of DNA sequence for as little as US$10. where. all tested negative for L. Our results validated that systematic culturing and analyses of products and processing facilities can identify areas that harbor L. amplified. in practice it may be more a question of better managing efforts to identify harborage points and applying interventions to lessen the prevalence and levels of this pathogen and.. This would allow food safety professionals to focus available resources on foods that present a greater threat to human health and to ostensibly reduce the occurrence and severity of listeriosis worldwide. Samples from 9 of 10 MFC brands from retail sites located in Juiz de Fora. in plants. 6 of 10 from “brand F” of MFC tested positive. and 3b) and II (1/2a and 1/2c). the farm/plant that produced brand F MFC was sampled. tested negative for L. 1998). in turn. 2008). given its ubiquity. in turn. In addition. which also coincided with groupings based on flagellar antigens a and c and on b (Piffaretti et al. CONCLUDING REMARKS Although in theory and from a policy perspective one can talk about eliminating L.00 (core facility charge). monocytogenes. We must also be pragmatic with regard to when. .. Over the past decade.104 LUCHANSKY ET AL.. so as to minimize the load and occurrence of the pathogen. monocytogenes (e. 1/2b.. placed together as artificially contiguous DNA sequences. monocytogenes was multilocus enzyme electrophoresis. Following renovation. plant F MFC. with appropriate interventions. we also demonstrated that it is possible. persistence. and pathogenicity. These results helped establish that the storage coolers served as the contamination source of the MFC. and/or combinations thereof) and that some people (i. dairy farms. A wealth of information collected from the numerous studies conducted thus far confirms that some foods present a greater risk for listeriosis due to their ability to support survival/growth of the pathogen (e. Brazil. monocytogenes will not reach the consumer’s plate. Automated DNA sequencing is more accurate than ever before. To this end. Ն4ЊC. and concomitantly continue efforts to develop and implement effective interventions to ensure that an infectious dose of L. cheese processing plants. Thus. and Latin-style cheese and. and how much of our resources should be directed toward developing policies and hiring inspectors versus directed toward filling research voids. Due to space constraints..

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a researcher under Daniel E. chronic conditions. Cosby Clinical pathologists in France in the early 19th century first identified a typhoid bacillus that was the causative agent for typhoid fever. Inc. was the first American to identify Salmonella as a separate strain or genus. However. The elderly. and ankylosing spondylitis. such as reactive arthritis. However. such as S. Salmon’s name as administrator was listed first on the research paper. Salmonella spp. GA 30605. Salmon and Smith (1885) first isolated Bacillus cholerae-suis. infants. Russell Research Center. the Salmonella infection may spread from the intestines to the blood stream and then to other body sites and can cause death. Athens.. The majority of Salmonella spp. In the United States. 2004). TYPE OF ILLNESS AND CHARACTERISTICS OF THE ORGANISM Salmonellosis is a human infection caused by Salmonella bacteria. Although Smith actually identified the bacteria. fever. L. enterica serovar Gallinarum. Nelson A. N. Jason Richardson. Voetsch et al. Juneja and J. in some persons the diarrhea may be so severe that the patient needs to be hospitalized. and those with impaired immune systems are more likely to have a severe illness. and discriminative detection methods are also discussed. Salmonella spp. Hazelwood. GA 30605.4 million cases of illness have been estimated to occur in the United States annually. 2007. the presence of outer membrane lipopolysaccharides. 1993).Pathogens and Toxins in Foods: Challenges and Interventions Edited by V. Russell Research Center. The establishment of a human Salmonella infection rests on the ability of the organism to attach (colonization) and enter (invasion) intestinal columnar epithelial cells (enterocytes) and specialized M cells overlying Peyer’s patches (Ponka et al. (2004) determined that the average confirmed annual human illness in the United States caused by nontyphoidal Salmonella infections was approximately 15. use of interventions. now known as Salmonella cholerasuis.. enterica serovar Pullorum and S. are facultatively anaerobic gramnegative non-spore-forming rods belonging to the family Enterobacteriaceae. Jason Richardson and Nelson A. 1990.000 hospitalizations and 400 deaths. and a propensity to invade host cells (Hakalehto et al. have resulted from Salmonella infections in patients. Sofos © 2010 ASM Press. In this chapter. The majority of persons infected with Salmonella develop diarrhea. so the new bacterium was named for Salmon. along with the sources and incidence of Salmonella in the environment and food commodities. Salmonella spp. L. from swine with “hog cholera” (Le Minor. Bacterial prerequisites for the onset of these chronic diseases include the ability of the bacterial strain to infect mucosal surfaces. Factors affecting survival in food processing operations. In rare instances. Salmon in the USDA’s Bureau of Animal Industry. This agent was eventually identified as Salmonella. approximately 1. Cox. Burslem et al. In these patients. and nonmotile Salmonella strains resulting from dysfunctional flagella do occur. 1981). Cosby • USDA-ARS-Bacterial Epidemiology and Antimicrobial Resistant Research Unit.. 1995). 108 . Sockett et al. and most persons recover without treatment. Cox • USDA-ARS-Poultry Microbiological Safety Research Unit.. MI 63042. Theobald Smith. The annual cost associated with salmonellosis has been estimated to be several billion dollars (Voetsch et al.. The illness usually lasts 4 to 7 days. However.. have the ability to metabolize nutrients by Stan Bailey • Biomerieux. are capable of adapting to extreme environmental conditions. are motile by peritrichous flagella. and Douglas E. Reiter’s syndrome. and abdominal cramps 12 to 72 hours after infection. unless the person is treated promptly with antibiotics. the sections are intended to give the reader a basic understanding of the characteristics and illness. Washington. DC Chapter 7 Salmonella Stan Bailey. Athens. K. Douglas E. nonflagellated variants.

4. . Table 1. . .1 1. . . enterica serovar Newport are the most frequently isolated Salmonella serovars from human salmonellosis cases. . IV. swine. . . Percent positive Salmonella tests in FSIS HACCP a verification samples—2006 Product % Positive 11. enterica subsp. and VI. .6 2. S. . . . . . . . The two species are S. . . Because chicken is the meat product with the highest frequency of Salmonella-positive samples. . . 2000). . Salmonella has been thought of as primarily a pathogen of poultry meat. . .4 2. . and ground chicken (45. houtenae. and other farm animals. . .12:i:Muenchen Saintpaul Braenderup % 19. S. arizonae. . . . . . .5. .4 4. . . . enterica subspecies are considered significant etiological agents for human food-borne-related illnesses. enterica serovar Enteritidis. .[5]. dependent on the Salmonella strain and the type of food matrix. These sources usually become exposed to Salmonella by either direct or indirect contact with contaminated fecal matter. . .3 7. . enterica group (D’Aoust. enterica serovar Kentucky. . . . . . and DNA homology. . . . . . . .5 1. . . . . . . . . . . . S. . . . .CHAPTER 7 • SALMONELLA 109 the respiratory and fermentative pathways. a Source: www. . . . Turkeys . . . . . . Ground beef . . However. . However. S. . . enterica serovar Typhimurium. . As seen in the 2004 data (Table 2). The Centers for Disease Control and Prevention (CDC) has adopted as its official nomenclature the scheme in which the genus Salmonella contains two species.3 5. . 2004 Rank 1 2 3 4 5 6 7 8 9 10 a SOURCES AND INCIDENCE IN THE ENVIRONMENT AND FOODS The natural habitat of Salmonella is the gastrointestinal tract of animals. . . . . S. . . . . . . Historically though. .gov/Science/Progress_Report_Salmonella_Testing_ Tables/index. . .4. each of which contains multiple serotypes (Brenner et al. . . S. it is ubiquitous and has been isolated from numerous other sources. .1 Broilers. eggs. Ground turkey . . it is important to remember that public health agencies in the United States consider all species of Salmonella important to public health. . . . serovar Enteritidis associated with raw or lightly cooked shell eggs. . Salmonella enterica serotypes in humans versus a chickens (slaughter). S. enterica subsp. . . enterica subsp. . . . . . The biggest change in poultry isolations in recent years is the rise in the incidence of S. with the largest number of these in the S. . . . . . .9 Chicken (slaughter) Serovar Kentucky Typhimurium Heidelberg Enteritidis Schwarzengrund Montevideo Thompson Mbandaka Infantis 1. . . Therefore. enterica is divided into six subspecies. . . IIIb. indica). .9 2.5 and 7. . Table 2. . . . . . The nomenclature of Salmonella has changed significantly over time and has progressed through a succession of taxonomical schemes based on biochemical and serological characteristics. . . bongori. . . . The optimal temperature of growth is between 35 and 40ЊC. .3%). . . Broiler chickens (11. . . . enterica subspecies are differentiated biochemically and through genomic relatedness. . enterica serovar Heidelberg are the serotypes most frequently associated with poultry. IIIa. . many people assume that the serotypes most frequently associated with human illness would be the serotypes most commonly found on poultry. . . . . enterica and S.[5]. . Furthermore. S.1 9. . . USDA. . . .12:i:% 44. . .3 1. S. serovar Typhimurium. . . enterica subsp. . . each referred to by a Roman numeral and a name (I. . 2000). . . . . . . . . Of particular concern since the mid-1990s has been a pandemic associated with S. enterica. . . . . enterica subsp. principles of numerical taxonomy.4 13. . . . . serovar Enteritidis in broiler chickens.0 4. . Salmonella strains vary in their pathogenic abilities. . . fruits. . . .9 1. .fsis. isolation procedures should recover all serotypes of Salmonella. .usda.2 14. . II. However.7 1. . . . . turkeys (20. . .8 0.1 2. . . . Food Safety and Inspection Service (FSIS) hazard analysis critical control point (HACCP) samplings confirm this association of Salmonella with raw meat and egg products (Table 1).. . . . The Salmonella incidence data from the CDC for humans and from the National Antimicrobial Resistance Monitoring System for farm animals are relatively consistent from year to year. . salamae.8 2. . and S. . . . . . . .0 45. . . and S. Cows/bulls . . . . . . . S. . . . . . . . . . . serovar Kentucky and the increase in the incidence of S. . . . . . enterica subsp. . . . . . . . . . and vegetables. . . . S. .3 2. incidence among the FSIS-regulated meat products in 2006.5 1. .1 6. . .4 13. .450 serotypes have been identified. . . Salmonella strains have an optimum pH for sustained growth between 6. . . . . diarizonae. However. . . Steers/heifers . .4 Values are percentages of total isolates positive for the serotypes. . enterica subsp. . .0 0. . .0%) had the highest Human Serovar Typhimurium Enteritidis Newport Javiana Heidelberg Montevideo 1.asp. . . S. .4%). . A total of approximately 2. Ground chicken . . S. . . . . such as seafood. . . . . the range of growth can occur between 2 and 54ЊC.0 20. . . Market hogs . .

serovar Typhimurium at pH 5.005% (Ebel and Schlosser.. whether the products are from animal origin or another source. 1997. Meat and poultry have a pH range of 5.5 to 7. the recent and numerous outbreaks associated with other vectors. pH.5 M NaCl) ascribed to the induced synthesis of the Omp C outer membrane proteins. Like most other paratyphoid (non-host-adapted) Salmonella serotypes. and an increased resistance to the antibacterial lactoperoxidase system and surface-active agents. The overall incidence of serovar Enteritidis contamination of eggs from commercial flocks in the United States has been estimated at around 0. 1990. leading to an increased thermal resistance at 50ЊC. reveals that only 5 outbreaks were definitively linked to poultry meat or eggs. 1993).7.5 to 7. The growth of Salmonella in acidic environments has been demonstrated to be significantly more robust if the bacterium has been preconditioned by previous exposure to lower pH conditions (Huhtanen. 1993).4. 2006. Heat is used widely in food manufacturing processes to control ..com. This significantly contributes to outbreaks occurring from contaminated food products.0.99 and 1.. an enhanced tolerance to high osmotic stress (2.. competitive microflora. primarily produce.110 BAILEY ET AL.93. Salmonella growth is inhibited in the presence of 3 to 4% NaCl. Most fresh fruits have a pH range of 1. Salmonella can readily adapt to extreme environmental conditions. and vegetables have a pH range of 3. serovar Enteritidis appears to gain access to the contents of eggs (Miyamoto et al. The remaining 18 outbreaks were associated with nonmeat products.3.5 to 9. and their effectiveness in influencing the epidemiology of serovar Enteritidis in the United States is documented by Mumma et al. High salt concentrations have long been recognized for their ability to extend the shelf-life of foods by inhibiting the growth of endogenous microflora (Pohl et al. the propensity of these pathogens to persist in hostile environments further heightens public health concerns and the need to control this pathogen in the food chain (Humphrey.. studies have also shown that several other serotypes can also be recovered from internal organs and tissues (Bailey et al. Intrinsic factors that affect survival and growth of Salmonella in food products include. salt tolerance increases with increasing temperature in the range of 10 to 30ЊC (D’Aoust. nutrient content. Of particular concern is that after invasion through mucosal epithelial cells serovar Enteritidis can be disseminated systemically to a wide array of internal organs..5. The prevalence of serovar Enteritidis in egg layers and the incidence of serovar Enteritidis in human illnesses peaked in the late 1990s in the United States. including multiple outbreaks associated with tomatoes. acidity. regulatory.8.about-salmonella. and food production industries must be aware of the almost ubiquitous presence of Salmonella. 1991b). By colonizing the ovary (the site of yolk maturation and release) and the oviduct (the site of albumen secretion around the descending yolk).5. Extrinsic factors that affect survival and growth of Salmonella in food products include.5) and promote the survival of Salmonella within the digestive system of humans (D’Aoust. 2004). temperature. such as crystal violet and polymyxin B (Leyer and Johnson.00 and in eggs. a greater surface hydrophobicity. and fish ranges between 0. 2007).gov). but are not limited to. Acid stress can also trigger enhanced bacterial resistance to other adverse environmental conditions. serovar Enteritidis is usually introduced to chickens via the gastrointestinal tract.1 to 6. and the federal government. The presence of Salmonella spp. Salmonella has been shown to proliferate at pH values ranging from 4. De Buck et al. but are not limited to. and natural/added antimicrobials in the food. Humphrey et al. However. 2000). fresh fruits. Fish and shellfish have a pH range of 5. 1991a). and raw milk (http://www. as demonstrated by the growth of S. natural cheeses. The largest outbreaks in 2006 were associated with tomatoes and peanut butter and in 2007 with pot pies (http:// cdc. This bacteriostatic effect results from a dramatic decrease in water activity (aw). an analysis of the 23 Salmonella outbreaks in which the etiology of the causative food was known. and packaging/atmosphere. 2007a). INTRINSIC AND EXTRINSIC FACTORS THAT AFFECT SURVIVAL AND GROWTH IN FOOD PRODUCTS AND CONTRIBUTE TO OUTBREAKS Although the potential growth of food-borne Salmonella is of primary importance in safety assessments.00.95 to 1. that have increased acid tolerances in foods heightens the level of public health hazard because this could minimize the antimicrobial action of gastric acidity (pH 2. While meat and poultry must always be considered potential sources of Salmonella. 1975). The aw in fresh meat. and vegetables from 0. Egg quality assurance programs have been sponsored by the industry. Cox et al. poultry. Salmonella will not grow in foods with an aw value of Ͻ0.8 to 7. 2004). suggest that public health. Despite the widespread perception of meats and eggs as the predominate source of Salmonella. however. storage conditions.8 to 6. moisture content. cantaloupe. including reproductive tissues (Gast and Beard. 2005. states. even though the optimum pH for growth is 6. (2004). 1993).

Salmonella can actively grow within a wide temperature range and has also exhibited psychrotrophic properties. This in turn could possibly influence the ability . Hinton et al. The emergence of antimicrobial resistance in Salmonella which can be traced to food-producing animals has generated much controversy and attention. However. but traditionally not considered to harbor the organism. the kinetics of the freezing process. reducing/eliminating the organism at the processing plant becomes a challenge. Immersion chilling can be a source of cross-contamination if no antimicrobials are used. Smith and Berrang. Once at the processing facility. SPREAD. 2000. In addition. D’Aoust. the physiological state of Salmonella. With regard to meat products.. The prevalence rate of Salmonella in preharvest poultry is higher than any of the other meat commodities. it is important to point out that. the feed is withdrawn to allow clearance of the gastrointestinal tract. without promoting the growth of Salmonella spp. The occurrence of Salmonella in food products which are capable of harboring the organism. Increases in geographic distribution have also caused changes in the handling and storage practices for these commodities. as previously discussed. the birds enter the killing stage. 2003). the behavior of Salmonella within other types of commodities. This adaptive response uncovers potentially serious implications for the safety of thermal processes that expose or maintain food products at marginally lethal temperatures. The increase of the pH and decrease in lactic acid within the crop may actually be the cause of Salmonella increase within the crop or a combination of the two (Corrier et al. such as fruits and vegetables. The meat animal and aquaculture industries are believed to contribute to increased Salmonella-resistant bacteria (Threlfall. or animals directly or indirectly in the fields. specifically poultry. the problem with eliminating Salmonella is complicated by the fact that these organisms are natural commensal microflora of the digestive tract and usually present in very high numbers. The rapid adaptation of the organism to rising temperatures in the microenvironment with a level of enhanced thermotolerance is quite distinct from that described in conventional time-temperature curves of thermal lethality. 1994. However. Gaseous mixtures consisting of 60 to 80% (vol/vol) CO2 with varying proportions of N2 and/or O2 have been found to inhibit the growth of aerobic spoilage microorganisms. crosscontamination from transport coops can occur. exposing cells to low temperatures can increase the ability of some Salmonella spp. 1991b).CHAPTER 7 • SALMONELLA 111 the bacterial quality and safety of end products. Widespread refrigerated storage of foods packaged under vacuum or modified atmosphere to prolong shelf-life has also been utilized.. 2002. such as Pseudomonas spp. Angulo et al.. and cross-contamination from the neck-cutting knife can occur (Mead et al. as is seen in immersion chilling. as reflected in the ability to grow in foods stored at 2ЊC to 4ЊC (D’Aoust. Air chilling is also utilized and is effective in reducing Salmonella cross-contamination but is less effective in reducing total bacterial load because there is no washing effect.. is not fully elucidated (Beuchet. untreated sewage or irrigation water. to grow and survive at refrigeration temperatures (Alpuche-Aranda et al. The use of antimicrobial agents in production and processing facilities has contributed to the emergence and persistence of resistant strains. 1994). 1999. This could be of particular concern if the Salmonella spp. which could provide protection from interventions utilized in the chill tanks. rapid exposure to decreased temperatures can increase the ability of some Salmonella spp. therefore. presents serious problems to food safety. Once the birds reach market age. A more in-depth review of stress responses and the ability of Salmonella to survive in foods can be found in the work of Humphrey (2004). This has raised concerns for the efficacy of chill temperatures in ensuring food safety. 1976). Fruit and vegetable contamination can come from the use of noncomposted animal manure. 1994). It has been shown that the composition of the freezing menstruum.. 2002). defeathering. but with the addition of chlorine or other antimicrobials. Furthermore.. OR CHARACTERISTICS The ecology and behavior of Salmonella in foods of animal origin have been studied extensively. Scalding. FOOD PROCESSING OPERATIONS THAT INFLUENCE THE NUMBERS. Salmonella has the ability to acquire greater heat resistance following exposure to sublethal temperatures. 1994). the chiller has been shown to greatly reduce Salmonella and other microorganisms on the carcass. become entrapped and/or attached within feather follicles. and the serovar-specific responses to extreme temperatures determine the fate of Salmonella during freezer storage of foods (Corry. During transportation to the processing plant. to grow and survive at refrigeration temperatures.. 2006). (D’Aoust. 2000). This process may increase Salmonella incidence in the crop due to consumption of litter and fecal matter. prior to catching. and evisceration also cause cross-contamination between carcasses. utilization of antibiotics at subtherapeutic levels as growth promoters could also be contributing to resistance (Antunes et al.

S. hatch cabinet disinfection. sanitation standard operating procedures. the environment.S. alternative methods of achieving similar results may be possible. but now the programs are paid for through industry checkoffs. In other cases. but Salmonella-positive grow-out broilers are processed separately and can be sold to the consumer. Evidence of the role that on-farm interventions can play in effectively helping to control Salmonella can be seen in Sweden and Denmark. serovar Kentucky. In Denmark. There are several technologies that are currently being researched and in some cases used by the U. The size and competitive nature of the U. they must be killed and properly disposed of. In 2007. not from the ingredients themselves.112 BAILEY ET AL. the killed vaccines are a collection of autogenous strains associated with a particular company or production complex. The European experience has confirmed that the best way to control Salmonella in food systems is to control the pathogen on the farm and to prevent it from ever entering the processing plant. respectively. 1984). it is important to remember that Salmonella and other pathogens could potentially contaminate the products. therefore. unless there is a concomitant decision by the entire industry to implement the technology. CHEMICAL. the initial phases of the program are as in Sweden. Interventions are usually not aimed against Salmonella in these types of processing facilities. and extensive biosecurity in breeder and broiler operations should yield beneficial results. 2000. Live vaccines are often deletion mutant strains of S. poultry producers suggest that as much as 50% of the industry is now using killed-cell vaccination or a combination of killedcell and live vaccines to help reduce Salmonella in broiler breeders and their progeny. or packaging steps within the processing chain. Therefore. if any Salmonella-positive flocks are identified. Interviews with U. 2008. AND PHYSICAL INTERVENTIONS TO GUARD AGAINST THE PATHOGEN Probably more work has been done to control Salmonella in chicken production and processing than in any other food. from a processing view. serovar Heidelberg or S. Sweden’s and Denmark’s programs were initiated in about 1990 and 1995. which has never been considered a product in which Salmonella would be of concern. serovar Enteritidis and some other strain. such as S.S. serovar Typhimurium and S. No Salmonella-positive feed was permitted. pathogenic organisms from adulterating the food product. and processed chickens was conducted. Crosscontamination of the products could occur during the collection. the Salmonella presence increases the likelihood of the resulting product being contaminated and a possible illness or outbreak occurring from consumption of the product. consisting of strains of S. Takeuchi and Frank. good agricultural practices. 1996). The more troubling concern is that Salmonella has the ability to survive for many years in the finished product when stored at ambient temperature (D’Aoust. no matter the product being processed. Therefore. Initially the cost of implementing the programs in both countries was paid for by the government. washing. it is hoped. Salmonella can survive and proliferate on the product (Mahmoud and Linton. but it will likely not be economically feasible to implement this same program in the United States. In Sweden. Killed vaccines can be more universal in nature. . RECENT ADVANCES IN BIOLOGICAL. Final economic analysis for a similar program in the United States is ongoing. An example is Salmonella in dry-roasted cocoa beans which are utilized to produce chocolate products. adequate standard operating procedures. HACCP. competitive exclusion treatments in breeders and broilers. Salmonella was isolated from peanut butter. without the extensive costs of eradication programs. extensive testing of feed. Thermal inactivation of Salmonella in molten chocolate is difficult because the time-temperature conditions required to effectively eliminate the pathogen in this product would likely result in an organoleptically unacceptable product. A comprehensive review of these efforts is discussed below. or other practices should be in place to prevent. Research and anecdotal evidence suggest that the use of live and killed-cell vaccines in breeders. where on-farm control programs have significantly suppressed Salmonella in broiler chicken production. and all breeder birds that tested positive for Salmonella were eradicated. therefore. However. of Salmonella to survive in these products. Sanitizers utilized to decontaminate the surface of these products lack the ability to infiltrate the cracks and intercellular spaces. It was found that the contamination of the product was due to cross-contamination from environmental sources. poultry industry which offer the opportunity to significantly reduce Salmonella. Salmonella has been recovered from numerous other types of products. serovar Typhiumurium and are used to boost immune response and will not persist in the birds or the environment for long periods of time. Zhuang and Beuchat. poultry industry make implementation of new Salmonella intervention technologies that would significantly increase costs of production a challenge. In both programs.

such as company policy. the level of Salmonella can be reduced by 20 to 50% from prevaccination levels. Second. As few as 1 to 10 cells can colonize a day-of-hatch chick compared to the more than 1. 2000). Several factors. Preempt. At the broiler grow-out level. Preempt is no longer commercially available. which was a continuous-flow defined culture comprised of 21 specific strains of bacteria. a process in which wood pine (or some other product) shavings are put down on top of the old bedding material between every flock. Rodent control has been shown to be critical for control of S. At the broiler breeder level. and control of movement of people and their vehicles (anything entering the property or facilities) are linked under this category. These products are used in many countries around the world today. however. 1990a). where all of the houses are built on concrete floors and out of materials designed to handle the harsh winters. Many people have made “probiotic” or defined CE products (in which all the component bacteria are known). Several commercial products were eventually made using the concepts initiated by Nurmi. companies have a policy on rodent control.000 and 40. which gained FDA approval and was used briefly by some companies in the United States in the late 1990s or early 2000s.. In the integrated poultry production operations. Most U. Help in controlling the movement of bacterial pathogens associated with food safety is a side benefit of these biosecurity programs. Therefore. For the purposes of this discussion. entry into and exit from the facilities always involve foot baths and can also include shower-in for entry into the facility.” which has made getting approval more problematic. the houses are cleaned down to the concrete and disinfected with chemical washes by a contract cleaner between every flock. much more resistant to becoming colonized with Salmonella than chicks of the same age with no added microflora. but implementation of the program is left up to the individual grower.S. the hatchery may be the most critical area for Salmonella control for two reasons. Cleaning and disinfection programs vary widely. The FDA has taken jurisdiction over these types of products and handles them as they do all “drugs. in a very short time. flock health. In 1972. but many have dirt floors. Most biosecurity programs are implemented primarily to control and prevent movement of viruses and other potential disease-causing organisms which will affect the health and production of the animals. some houses have concrete floors. The other area of “on-farm” pathogen control falls under the general category of biosecurity. the development and use of undefined competitive exclusion (CE) cultures probably have been the most effective interventions (Bailey et al. but not in the United States (at least not legally with pathogen claims) because they were comprised of many strains of bacteria (undefined culture). rodent control. Growers within companies will either clean out the houses between flocks or rear broilers on what is called “built-up” litter. Biosecurity varies widely in the poultry industry. even if only one or a few eggs are contaminated with Salmonella.000 cells needed to colonize an older chicken (Cox et al. such as quaternary ammonium compounds or iodine compounds. There was one product. the newly hatched chick is more susceptible to colonization by Salmonella than older birds. This process can often continue for 1 or 2 years (5 to 10 flocks) before cleanout. serovar Enteritidis in egglaying flocks and likely plays a role in the control of all types of Salmonella in broiler grow-out. Preempt never proved to be particularly effective in reducing Salmonella. In Scandinavia. anecdotal responses from many in the poultry industry suggest that after 1 or 2 years. and most are not built to be completely washed and cleaned between flocks. and effective disposal of the litter. biosecurity generally involves restricting access to the facilities to those authorized and the use of dedicated or disposable footwear and footbaths with disinfectants. A few companies also have installed disinfection stations that will spray the tires and undercarriages of trucks and other vehicles entering and leaving the grow-out facilities. Outside of preventing Salmonella from ever entering the breeder or grow-out farms and being an effective method for preventing Salmonella in processed broilers. These chicks can then become colonized with Salmonella and subsequently spread the Salmonella to other chicks during . First. Esko Nurmi and coworkers were the first to publish on the use of CE cultures to help control Salmonella in broiler chickens. cleaning and disinfection. but to this point no products have been demonstrated to be as effective as the undefined CE products. and there were production problems in maintaining the balance of organisms in the continuous flow. availability of bedding material..000 hatching eggs in a single hatch cabinet. Effective programs are available for those that choose to follow them. making them. when these eggs hatch they can spread Salmonella to many other chicks in the hatch cabinet. some of which were not or could not be identified. because there are often between 10. In the United States.CHAPTER 7 • SALMONELLA 113 There is a paucity of peer-reviewed research concerning the long-term effectiveness of these programs when implemented in large-scale commercial operations. Nurmi took intestinal material from healthy adult birds and fed the material back to newly hatched chicks. play into this decision.

1. chiller pH control [acidified]. solution can be applied by spraying or dipping chilled or prechilled carcasses for up to 15 s Peroxyacetic acid Trisodium phosphate Disruption of cell membrane causing leakage of intracellular fluid. propylene glycol concentration 1.114 BAILEY ET AL. 1991. 1-diphosphoric acid 8–12% solution in conjunction with a water spray containing 20 ppm chlorine.3–2. just before the product goes to the consumer. can effectively prevent the spread of Salmonella during the actual hatch period (last 2 or 3 days in the hatch cabinet). “Food ingredients and sources of radiation listed or approved for use in the production of meat and poultry products. prechiller or chiller solution: 50–150 ppm sodium chlorite and any GRAS acid to achieve pH 2. On 23 December 1999. Several chemicals.. leading to cell death Oxidation of cell components.fsis. the primary means of attempting to control Salmonella in poultry has been the use of chemical disinfection in the processing plant. 1997. Since the implementation of the HACCP pathogen reduction plan in 1996 (http://www.3 g/lb of poultry. chiller treatments. including hydrogen peroxide or properly controlled formalin.8–3. Chemical applications could be applied by spray with or without scrubber brushes or as dips. resulting in the leakage of the cellular components. Chemicals used in poultry processing to eliminate or reduce Salmonella are listed in Table 3.. 1994.2 Not to exceed 0. 110 ppm hydrogen peroxide. online reprocessing [OLR]. An association with Salmonella serotypes recovered from the hatchery can be found on the final processed carcass (Bailey et al. transport or in the grow-out house. Except in certain circumstances. The primary reason for this approach is to try to emulate the pasteurization of milk. 2007b). details of the antimicrobial mechanism have not been completely elucidated . oxidation of cell membrane Strong oxidation of cell membrane and other cell components. also used in processing water at 10–30 ppm Not to exceed 3 ppm in poultry process water contacting whole fresh carcass Suggested mode of action Broad-spectrum germicides: oxchlorous compounds act by breaking bonds on cell membrane surfaces Cetylpyridinium chloride Hydrophilic portion reacts with the cell membrane. FDA and FSIS entered into a memorandum of understanding that outlines the procedures that are followed by FDA and FSIS regarding the joint review of requests for the use of food ingredients and sources of radiation in meat and poultry products. Commercial antimicrobials commonly used during poultry processing Antimicrobial Acidified sodium chlorite Use Spray of dip solution: 500–1. used in gaseous or aqueous phase Maximum concentration of 220 ppm peroxyacetic acid.gov/ OPPDE/rdad/FSISDirectives/7120.” In January 2000..usda. a survey of intervention treatments at five processing plant locations (prescalder brushes. in which a terminal treatment for pathogens is applied at the end of processing. the Food Safety and Inspection Service (FSIS) published in the Federal Register a final rule. Many studies have shown the need to properly clean eggs and to disinfect between hatch groups (Bailey et al. FDA will now list in its regulations (21 CFR) food additives and sources of radiation that are safe and suitable for use in the production of meat or poultry products. gov/oa/background/finalrul. resulting in cell death Oxidation of the cellular membrane and cellular constituents at high concentrations. it breaks the cell wall Direct molecular reaction and indirect reactions involving free radicals.200 ppm sodium chlorite and any GRAS acid to achieve pH 2. In 2006. Approved chemicals with application for addition to meat and poultry production are listed (http://www. Cox et al.fsis. and 13 ppm 1-hydroxyethylidene-1.htm).9. and postchill treatments) was responded to by eight integrated companies with a total of 100 processing plants.3(o)(2).usda.htm). resulting in cell death Chlorine-sodium hypochlorite Chlorine dioxide Ozone Antimicrobial agent as stated in 21 CFR 170. 1990b. 2002). More intervention efforts were Table 3.5 times that of cetylpyridium chloride 20–50 ppm in chill water.

. Traditional cultural isolation from samples involves an overnight preenrichment step utilizing a nonselective enrichment. the various sources from which Salmonella can be isolated. generate hydrogen sulfide gas in TSI and LI media. Phenotyping and genotyping methods exist in order to evaluate Salmonella strains in research and from a public health standpoint. such as buffered peptone water or lactose broth. and modifications to conventional tests) are utilized to detect the presence of Salmonella in samples (McMeekin. chromogenic plating media can be utilized (Schonenbrucher et al.e. and incubated for an additional 24 hours. xylose lysine deoxycholate. biotyping.CHAPTER 7 • SALMONELLA 115 made in the chiller treatments (93%) and in OLR (86%) than with the scalder (18%) or postchill dips (12%). Typical Salmonella isolates will produce acid and gas from glucose in TSI agar medium and will not utilize lactose or sucrose in TSI or in differential plating media. Rappaport-Vassiliadis. However. To reduce the screening time. chemical treatments in the processing plant are the principal method that companies are currently using to control Salmonella and indirectly Campylobacter in chicken production and processing. Serotyping. The 2-day limitation is due mainly to the samples having to be enriched prior to testing to increase sensitivity and specificity (Feng. 1997) and McMeekin (2003). The enormous number of Salmonella serotypes. The first would be direct health concerns that some of the chemicals may complex with the proteins in the meat and potentially be a human health concern. 2008). The key to effective chlorine treatments in the chiller is to maintain chiller water at or near pH 7. and Hektoen enteric agars. and 8 of these used sodium chlorite. and R typing are examples of phenotyping methods. Many of the traits listed above traditionally have formed the basis for the presumptive biochemical identification of Salmonella isolates (Cox and Mercuri. rapid methods (i. flagellum (H) antigens on the peritrichous flagella.. and the capsular (Vi) antigen. such as brilliant green. antibody/ DNA-based tests. The biochemical classification of food-borne and clinical Salmonella isolates is generally coupled with serological confirmation evaluating the somatic (O) lipopolysaccharides on the bacterial outer membrane. xylose lysine desoxycholate. XLT4. phage typing. After preenrichment. There are potentially some concerns that may arise from this extensive use of chemicals during processing. and fail to hydrolyze urea. which takes 4 to 6 days to complete. With most of the rapid methods commercially available. 1976). 1996).0. generally produce hydrogen sulfide and decarboxylate lysine. 2003). and Hektoen enteric agars. As seen in this recent industry survey. Depending on the area of the country where the plant is located or whether trisodium phosphate is being used in the plant. modified lysine iron. Sodium chlorite (33%) and trisodium phosphate (24%) were used most frequently. DISCRIMINATIVE DETECTION METHODS FOR CONFIRMATION AND TRACE-BACK OF CONTAMINATED PRODUCTS Isolation and identification of Salmonella by traditional cultural methods requires a series of steps for isolation and identification. the samples are then plated onto selective agar plates. After incubation. bismuth sulfite. and selenite cystine broths. Salmonella spp. screening time for a Salmonella presence in the sample can be performed in as few as 2 days. Chemical treatments used for OLR were far more diversified. such as tetrathionate. presumptive positive samples are then subjected to biochemical screening on triple sugar iron (TSI) and lysine iron agars. and incubated for 24 h. and genetic differences that occur within a single serotype mean that it is not sufficient to carry out only serotyping in order to accurately determine the source of infection . a positive result through utilization of rapid methods still needs to be confirmed by traditional cultural methods (Feng. The second is the perception of many consumers that the use of chemicals is bad for them (note the rapid interest in all things organic). Only 12 plants used a postchill treatment. The significant number of illnesses due to Salmonella has made epidemiological trace-back to the cause of infection a very valuable tool to correct problems and reduce the incidence of food-borne outbreaks. such as brilliant green sulfa. After enrichment. Salmonella spp. are oxidase and catalase negative. the chill water may need to be acidified. Chiller treatments were overwhelmingly chlorine (hypochlorous acid) followed by peracetic acid and chlorine dioxide. 1997). Ninety plants used carbon dioxide to control pH in the chill tank compared to three plants that used citric acid. produce an alkaline reaction from the decarboxylation of lysine to cadaverine in lysine ion (LI) agar. the sample is then transferred to selective enrichments. miniaturized biochemical kits. In addition. there are export issues with many countries that do not allow the use of any chemicals during processing. and do not hydrolyze urea. and finally. which involves another day of incubation. A more detailed review of rapid methods can be found in the work of Feng (1996. Food safety managers at the processing plants felt strongly that multiple hurdles will have to be used for successful reduction of Salmonella and that none of the interventions will work without attention to the entire process. Briefly.

. and N. S. N. However. Cosby. and pulsed-field gel electrophoresis (PFGE). J. E. 2000.. Pestana. A. and the monitoring and accessing of changes in gene expressions. Miller. PFGE is utilized by PulseNet. F.. J.. Antibioticresistant typing has been beneficial in evaluating the evolving increase in antibiotic-restraint strains of Salmonella. that may no longer be the case. I. J.. S. Craven. a combination of the discussed methods should increase obtainable information. D. and another situation for a totally different method. the method should be combined with other epidemiology tracking methods (De Oliveira et al. J. A. All the methods discussed in this section have been developed and utilized for a number of reasons. 2003. Poult.. K. Multilocus sequence typing is an example of an RNA-based method. Bailey J. Food Prot. J. A. Cox. 1995). and N. efforts to control Salmonella will continue to be a significant issue well into the future as new products are continually introduced into the food arena. Food Prot. 68:2698– 2701.116 BAILEY ET AL. Berrang. peanut butter. N. Advances in methods to genetically characterize Salmonella have greatly assisted epidemiologists in their ability to accurately determine the source and spread of Salmonella. and M. 2007).. S. 65:742–745. L. R. Bailey J. and a dramatic reduction of Salmonella in certain food products. 2005). S. whether for control programs or in trace-back studies. Cox. Bailey J. 2001). evaluation of evolutionary changes. Reu. 179:601–608. such as ribotyping and PFGE (Nair et al. Swanson. Cox. Salmonella stimulate macrophage macropinocytosis and persist within spacious phagosomes. E. L. Numerous studies have suggested that antimicrobial resistance among bacteria is on the rise. 2002). C. Significance and sources of antimicrobial-resistant nontyphoidal Salmonella infections in the United States. Cosby. with the increase in globalization. 1993). E. E. 2000. but they also lack some of the discriminative power to identify strain differences (Rankin and Platt. Cox. cantaloupes. Food Microbiol. Johnson. 2002. . J. Microb. It was reported that multilocus sequence typing was more discriminatory than PFGE (Kotetishvili et al. Salmonella has been thought of as a problem associated almost exclusively with meat and poultry products. 63:867–870. PFGE is still considered the “gold standard” for the subtyping of Salmonella strains and has been highly effective in epidemiological investigations tracing back sources of outbreaks (Swaminathan et al. A. Int.. J. N. Exp. 6:77–83. 1-day standardized protocols. certain circumstances may call for one method. 2000. 1994. 2005. S. from an epidemiological standpoint (Reeves. J. and other produce suggest that vigilance and interventions from the farm all the way through food processing must be maintained at all times in order to ensure the production of safe and wholesome foods without Salmonella. Genotyping is usually performed by extrachromosomal analysis (plasmid profiling and restriction digestion of plasmids) or through chromosomal analysis. J. Racoussin. and S. Food Prot. Bailey. On the practical side. Sci. These include but are not limited to conventional isolation and monitoring. Serotype tracking of Salmonella through integrated broiler chicken operations. however. due mainly to the fact that the method does not have the discriminatory power needed to accurately access problems from an epidemiological standpoint. C. Ross and Heuzenroeder. and M. Peixe. C. V. L. A few examples of DNA-based molecular methods are PCR. Angulo. Tauxe. It is imperative to remember that when considering a method. A. It is clear that the problem of Salmonella in the global food chain and its current and projected repercussions for human health are causes for concern. REFERENCES Alpuche-Aranda. Incidence of Salmonella from poultry products and their susceptibility to antimicrobial agents. Stern.. J. which is the national surveillance network for bacterial foodborne disease in the United States.. Hatcheryacquired salmonellae in broiler chicks. Recent epidemiological studies have clearly demonstrated that if it were ever true that meat and poultry were the primary sources of outbreaks. E. Cohen. N. Richardson. amplification of the discriminatory ability to detect differences between strains. Outbreaks associated with tomatoes. ribotyping. and L. due to the reproducibility between laboratories. Amplified fragment length polymorphism has been shown to be more discriminatory than other DNA-based methods. Increased understanding of Salmonella at the molecular level will possibly lead to better intervention strategies. Sousa. J. 82:97– 103. Movement and persistence of Salmonella in broiler chickens following oral or intracloacal inoculation. and the simplicity of analysis of strain relatedness. P. Phage typing and biotyping have been utilized in epidemiological and surveillance studies. 1994. Examples of PCR-based methods include random amplified polymorphic DNA and amplified fragment length polymorphism (AFLP). While a few of the molecular methods are gaining attention due to their discrimination power. Commercial field trial evaluation of mucosal starter culture to reduce Salmonella incidence in processed broiler carcasses. and this has led to changes in control and treatment strategies. CONCLUDING REMARKS Historically. Drug Res. Med. and D. Antunes. M. 73:1153– 1157. real-time screening methods.. PFGE has gained much recognition since being implemented around 1998.

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and acid from aerobic mannose tests (De Paulis et al. Although the true morbidity and incidence of SFP are unknown. is not an infection but is a foodborne intoxication caused by one or more staphylococcal enterotoxins (SEs). ornithine decarboxylase. Additional biochemical tests can provide further speciation and confirmation. on the basis of coagulase production or the presence of a coagulase gene. 2003). Coagulase-positive staphylococci include S. Methods used for phenotypic identification of staphylococci include a conventional scheme (Kloos et al. 119 . 1991) and commercial kits. Juneja and J. also known as staphylococcal gastroenteritis. DC Chapter 8 Staphylococcal Food Poisoning Keun Seok Seo and Gregory A. Bohach • Department of Microbiology.. such as mannitol-salt agar. hyicus. aureus is implicated in a variety of illnesses. aureus (Denys. Due to the complexity and prolonged time of conventional methods. catalase... Crass and Bergdoll. aureus. also known informally as staphylococci. 1914). caused by a transposon or transcriptional and posttranscriptional defects of the coagulase gene. including the coagulase tube test alone or in combination with Gram-staining. and osteomyelitis. 1971. and Planococcus. a coagulase-deficient phenotype. Moscow. K. Other media. pyrrolidonyl arylamidase. S. members of the genus Staphylococcus. which includes the genera Micrococcus. In 1914. Speciation by phenotypic properties can lead to misidentification. 1986). 53 Staphylococcus species and subspecies are recognized (Kloos et al. 1986). The name Staphylococcus is derived from the Greek words “staphyle” and “kokkos. another toxin-mediated illness. the etiological agent of staphylococcal food poisoning (SFP). Staphylococci are often divided into coagulasepositive staphylococci and coagulase-negative staphylococci (CNS). Washington.5 billion. The International Organization for Standardization suggests BairdParker agar (ISO6888-1) and rabbit plasma fibrinogen agar (ISO6888-2) to enumerate coagulase-positive staphylococci from food and animal feeding stuffs (De Buyser et al. including skin infections. schleiferi. respectively.” which mean grapes and cocci. 1894). University of Idaho. Molecular Biology. S. has been Keun Seok Seo and Gregory A. has been extensively studied and is known to produce a cadre of extracellular pathogenic factors. 2000). CHARACTERISTICS OF THE ORGANISM Taxonomy According to Bergey’s Manual of Systematic Bacteriology (Schleifer. endocarditis. Barber purposely drank stored milk from a cow showing signs of mastitis and provided the first evidence that a soluble toxin was responsible (Barber. such as toxic shock syndrome.S. including some CNS. N. 2003). S. It appears to have been initially reported in 1894 as a family outbreak due to undercooked meat from a sick cow that became contaminated with S. describing their Gramstained appearance under the microscope (Schleifer. aureus. and colony morphology.Pathogens and Toxins in Foods: Challenges and Interventions Edited by V. delphini. 1986). intermedius. This chapter focuses predominantly on SFP. Although several species. and Biochemistry. at a cost of more than $1. clearly most cases are attributable to S.750 hospitalizations. CNS comprise a heterogeneous group of many species. cases annually exceeds 185. with 1. S. catalase-positive and nonmotile cocci occurring singly. plus several toxin-mediated diseases. urease. aureus. an estimate is that the number of U. In addition to SFP. Bohach Staphylococcus aureus. are located within the family Micrococcaceae.000. including novobiocin susceptibility. Sofos © 2010 ASM Press. For example. Staphylococcus members are gram-positive. thermostable nuclease production. Currently. ID 83844. 1991).5% of food-borne illnesses in the European Union (WHO Surveillance Programme. SFP. or in irregular clusters. clinical laboratories may use key tests for presumptive identification of S. SFP also constitutes an estimated 4. in pairs. and S.. have been implicated in SFP (Breckinridge and Bergdoll. Stomatococcus. Staphylococcus. are useful.

4% of staphylococci (Jousson et al. Test kits that distinguish species by biochemical reactions are frequently used. The gap gene. most commercial kits are not designed to identify veterinary or food-borne CNS. 2002). S. It also contains species-specific variable regions. such as PCR-based DNA fingerprinting. schleiferi. identification at the species level. S. cohnii. sucrose. rapid bacterial identification by PCR-restriction fragment length polymorphism (RFLP) uses species-specific and ubiquitous DNA as a target. 2004). S. Some drawbacks of molecular genetics-based species-level identification are associated with the developing databases and include faulty. or S. hyicus. S. 1994).1%) had a coagulase-deficient phenotype (Luczak-Kadlubowska et al.. 1971). One study reported that 54 of 110 S.. several manufacturers supply kits. S. although 0.. Growth is inhibited in the presence of 0. which is equivalent to 3. other relatively rapid and inexpensive molecular methods.83. advances in taxonomy have been accomplished through techniques based on genotypic analysis. the optimum growth pH is 7.3). sucrose.5 M NaCl (Townsend and Wilkinson. 2004) because the 16S rRNA gene contains regions well conserved in all organisms that are ideal for PCR and sequencing. 2005).. and acid tolerance is dramatically decreased in the presence of potassium sorbate.. epidermidis. Its growth can be delayed in the presence of 80% CO2 (Genigeorgis et al. The molecular mechanisms of staphylococcal osmotolerance are based on intracellular levels of osmoprotectants.. S. Universal pathway and function genes. carnosus. its resistance to common food preservative materials is generally unremarkable. 1961). such as the sodA (Poyart et al. At the subspecies level. In another study. .. aureus is able to grow at a lower water activity (aW) than other commonly encountered mesophilic bacteria... sequencing is relatively expensive for routine identification in many clinical diagnostic settings... glycerol. Currently. The upper limit of growth can be extended to Ͼ44ЊC by addition of NaCl and monosodium glutamate (Jay et al. is a well-conserved gene in Staphylococcus spp. However. It has been reported to be capable of growing at an aW as low as 0. and glycine betaine which increase corresponding with the level of NaCl in the environment (Townsend and Wilkinson. aureus has an aW as low as 0. aureus. Survival and resistance Although S. In recent decades. have been developed and used successfully for Staphylococcus sp. 2005). Although the precise pH extremes tolerated are dependent on several factors. and/or redundant sequence entries..83 in the presence of NaCl. The accuracy of these systems is usually adequate to identify S. sequencing of the 5Ј 16S rRNA gene fragment could discriminate among subspecies of S.0 to 7. aureus is facultative. Vitek-2 (bioMérieux).120 SEO AND BOHACH reported (Fox et al. Thus. For example. such as API (bioMérieux). real-time PCR of 16S rRNA genes (Skow et al. In general. Genetic methods to identify Staphylococcus spp. BD Phoenix (Becton Dickinson).. and it has been suggested that a gap gene PCRRFLP assay may be a useful tool for differentiating clinical isolates of 24 staphylococcal species (Yugueros et al. 2005). 1990). In a study by Becker et al. except for osmotolerance.8 with 5% NaCl..86 is generally accepted as the minimum (Marshall et al. 2000). It has been reported that. Walk/Away (Dade MicroScan). hominis. 1992). and the development of species-specific PCR amplification primers targeting 16S rRNA genes (Yamashita et al. simulans with the systems above (Matthews et al. partial gene sequencing is widely used. succinus. 1996. or glycerol humectants. are being used more frequently as PCR targets.. aureus and most common CNS clinical isolates. S. 2007). In particular. outdated nomenclature. and S. aureus is known for acquiring genetic elements conferring resistance to heavy metals and antimicrobial agents. aureus may grow at Ͻ10ЊC and that conventional refrigeration may not always ensure the safety of foods (Angelotti et al. S. Additionally...5 (range of 4. PCR amplification of the 16S to 23S rRNA gene intergenic spacer (Martin et al. Since the 5Ј end of the 16S rRNA gene contains enough information to identify most Staphylococcus spp.. Physiology Growth The optimum growth temperature for S. 1969). (2004). incomplete. aureus is 37ЊC (range of 6 to 48ЊC). chromogenes was misidentified as S. a similar technique unambiguously identified 97. 16S rRNA gene sequence analysis has emerged as one preferred technique (Clarridge. Vandenesch et al. and NaCl (Stewart et al. encoding glyceraldehyde-3-phosphate dehydrogenase. aureus human clinical isolates (49. and poor quality assurance. 2005) or the other genes. under some conditions. 2004). 2001) and tuf genes (Heikens et al. 49 species and subspecies of staphylococci were identified by partial 16S rRNA gene sequencing. and Replianalyzer (Oxoid). Staphylococcal osmotolerance is problematic for food safety since blocked growth of competing bacteria can result in a more likely overgrowth of S.1% acetic acid (pH 5. S. whose nucleotide sequences are fairly homologous among bacteria.1) or at a pH of 4. proline. offer increased speed and accuracy and include 16S rDNA sequencing (Becker et al..2 to 9. 2006).

intracellular K levels are high in unstressed S.9 27 28. Many animal species are often heavily colonized and can directly contaminate food during processing of domestic animals and their products. and sugar to develop flavor and color. S. S. no detectable S. tuna. Prevalence of S. and aging them. In Iberian dry cured hams. equorum and S. Curing is a traditional means of meat preservation using NaCl. and micrococci were dominant. (1993) NA.332 616 214 % Positive for S. Reservoirs Staphylococci are ubiquitous in air.3 4.6 7.650 3.. CNS. At 30ЊC. aureus. xylosus. of S. In low-salt bacon (5 to 7% NaCl) held at 20ЊC. CNS became dominant.CHAPTER 8 • STAPHYLOCOCCAL FOOD POISONING 121 1992). whereas. the micrococci became dominant. (2007) Moon et al. Cavett (1962) and Tonge et al.8 No. cold smoking. humans. aureus. 1992). cream pies. sewage.. the two predominant organisms in the ripening process are S. milk.1 30 Reference Atanassova et al. aureus CFU/g NA NA NA Ͻ100 Ͼ10 Ͼ3 Ͼ3 Ͻ10 Ͻ10 Ͻ10 NA NA NA a % Positive for SE 34. (1964) found that when high-salt vacuum-packed bacon was held at 20ЊC for 22 days. aureus in several foods Product Raw pork Raw meat Raw milk Ground beef Ground turkey Frozen prawns Shrimp Beef carcasses Boneless beef Pork carcasses Ready-to-eat fast food Vegetables Bakery products a No. S. sciuri. potato. such as custard. not available.8 NA NA 26 NA NA NA NA 47 73.8 7. suggesting that the transport systems are constitutively active (Townsend and Wilkinson. Foods that require considerable handling during preparation and that are left at room temperature without subsequent cooking or adequate heating are usually involved. However. aureus 57. milk products. xylosus. (2007) Summer et al. of samples tested 135 139 714 1.7 20. While colonized food handlers are the main source of dissemination of staphylococci to others and in outbreaks.9 30 6 23. and S. In contrast. followed by S. whereas the low-affinity system has broader substrate specificity. and animals. In a study of Italian dry fermented sausages. Osmoprotection systems in other bacteria require ϩ activation of K transportation systems and a subsequent synthesis of osmoprotectant transporters under ϩ osmotic stress. aureus may survive when drying is extended and at temperatures over 60ЊC (Holley. xylosus appears to be the most frequently isolated from several Italian dry sausages. ice cream. (2006) Yeh et al. 1996). sandwich fillings.8 31. problems with food-borne pathogens are minimized. and soft cheeses. the high-affinity system is stimulated by a lower concenϩ tration of Na and is proline specific. xylosus. saprophyticus. Survival during food processing is an important area of applied research. High. and chocolate éclairs.and low-affinity transport systems operϩ ate in S. bakery products.155 1. egg. S. environmental surfaces. After 4 months of aging. and pasta salads. water.6 7. 2002). aureus. over 97% of the isolates were CNS. This high Table 1. dust. . and both are believed to contribute to product flavor (Gardini et al. including S. shellfish in a mayonnaiselike dressing. (1977) Sanjeev et al.468 1. aureus was found (Portocarrero et al. (1987) Swartzentruber et al. and many foods and on food equipment. equorum. aureus in country-cured hams was investigated by spraying fresh hams with S.86 is conducted within 3 hours. milk. saprophyticus. Food products with high protein contents are particularly good growth substrates for S. poultry and eggs. Humans and animals are the primary reservoirs. (2006) Phillips et al.7 2. Both depend on Na for activity. In a study of Iberian drycured hams. equipment and environmental surfaces can also be implicated. aureus. 1985). aureus is efficiently carried in the nares of at least 20 to 30% of the general population.9 9.082 1. (2005) Oh et al. and S. Jerky is a dried stable product made from lightly salted and spiced meat.8 8. the most frequently isolated staphylococcus was S. Enterococcus faecalis. nitrate.. aureus and then curing.or cream-filled pastries. 2003). (1980) Phillips et al. When drying to reduce the aW below 0. S. Frequently implicated foods include raw and processed meat. cohnii (Rodriguez et al. catalase-positive cocci dominated. The survival of S.090 50 46 1. (2001) Moon et al. chicken. at 30ЊC. Some commonly contaminated foods are summarized in Table 1. (2007) Moon et al. S. (2007) USDA (1996) Guthertz et al.

1987). 2006) and S.. Nv. Staphylococci. Dinges et al. Basic Biophysical Characteristics SEs and SEls are moderately sized (~25 KDa) structurally stable proteins. aureus is a potential public health concern due to the high prevalence of SE genes in mastitis isolates (Fitzgerald et al. Iv. A related toxin. and SEC-caprine). 1994). 2004). 1972). Except for SEA.. 1989). Therefore. They may be denatured in high concentrations of urea or guanidine hydrochloride. SEA.. chromogenes. 1982). 2000).. and SEC3. but sensitive to pepsin at pH 2 (Balaban and Rasooly. 1967. except SEA (Tremaine et al. SEC. SEC2. This effect is largely due to the staphylococcal quorum sensing Agr system which regulates staphylococcal virulence factors in response to the density of an autoinducing peptide (AIP).. be designated SE-like (SEl) until their enterotoxigenic activity can be confirmed (Lina et al. Although all proteins in this family share significant homology. simulans are commonly isolated from dairy samples (Taponen et al. 1996). haemolyticus are associated with infections in hospitalized patients (Crass and Bergdoll. and papain. for example S. which is produced relatively early. SEA is thermally inactivated by heating at 121ЊC for 8 min (Denny et al. Since the improvement of genomic analysis. SEC. Pasteurization has minimal effect on SE biological activity. and S. SEC-ovine. 2000. Inactivation of Agr results in decreased expression of SEs. and osmotic conditions and is also regulated at the molecular level. xylosus. which has facilitated sequencing capabilities. and therefore. Cleavage of SEs by some proteases does not always result in the loss of biological activity. SEs are expressed minimally during early growth but rapidly increase during the late-exponential or stationary growth phases. and Uv) have been identified. The heat resistance of SEs has been well documented and complicates approaches to prevent SFP since heating may kill staphylococci but not necessarily inactivate the toxins already expressed.. and four additional toxins (types Gv. Production occurs maximally in the absence of NaCl and is nearly abrogated in Ͼ6% NaCl.. and bologna at as low as 10ЊC and as high as 46ЊC (Vandenbosch et al. 1967). SEB activity is not altered by heating for 16 h at 60ЊC in pH 7. some have been shown to lack emetic properties or have not yet been tested. 1973). one serious problem for the dairy industry is mastitis. Mastitis caused by S. pH. SEB. was originally designated SEF but was subsequently shown to lack emetic properties.122 SEO AND BOHACH prevalence of staphylococci in raw foods of animal origin emphasizes the need for effective processing to ensure safety (Genigeorgis.. SEs may regain biological activity (Spero et al. Another study showed that thermal processing of canned infant formula alters SEA and SED serological activity. In the presence of excessive glucose. Major features of SEs and SEls are summarized in Table 2. Bergdoll. and SEE) were recognized. SEB production in brain heart infusion medium is inversely related to the NaCl concentration (Pereira et al. 2000).3 (Schantz et al. rennin. but not confirmed to exhibit emetic activity in the monkey feeding assay. a reduced pH decreases . The optimum temperature for SEB and SEC production was 40ЊC in protein hydrolysate medium. AIP levels and SE expression occur maximally at neutral pH. SEs are partially resistant to gastrointestinal and other proteases. five classical and antigenically distinct SEs (SEA. minor differences in the antigenicity of SEC resulted in its differentiation into subtypes. toxic shock syndrome toxin 1 (TSST-1). S. the International Nomenclature Committee for Staphylococcal Superantigens (INCSS) recommends that proteins related to the SEs. intermedius is commonly isolated from canine samples (Khambaty et al. SE Environmental Regulation SE production is affected by temperature. which regulates AIP levels. and SED production was detected in ground beef. three novel variants of SEC (SEC-bovine. PROPERTIES OF SEs AND RELATED PROTEIN TOXINS SE Classification For many years. Crude toxin preparations are more resistant than purified toxins. the SEF designation is no longer appropriate. chymotrypsin. aureus. In addition. other than S. Recently. sciuri. SEB.. S. 1986). 1976). epidermidis and S. once the denaturing conditions are removed. SED. but not their biological activity (Bennett and Berry... SEC1. evidence has been mounting to indicate that SEs are expressed by other species and that their risk for SFP is underestimated (see below). suggesting that other organic molecules help stabilize the SE structure. 1993). suggesting that their antigenic determinant confirmation is not directly related to their biological properties. However. SEC retains its serological activity after heating for 30 min at 60ЊC (Borja and Bergdoll. SEC expression is also linked to the level of pH in the culture. ham. 13 novel SEs and related proteins (types G through R and U). S. such as trypsin. are often considered potential pathogens and are common inhabitants of the skin. For example.

1. 8. defined as gene cluster encoding pathogenic or virulence factors. 13.1.. 6. 7. (1997) Deringer et al.7.1 28. 6.2. the SEC3 crystal structure is shown in Figure 1 as a representative. B The alternative sigma factor (␴ ) responds to an environmental stimulus.4 27. 12. h SaPI (designation of S. 20 3. 22 6. (1989) Couch et al. 5. 12. 17. However. Some genomic islands are similar to SaPI but have key features missing.3 pIB485. MHC II binding site.2. SaPIbov Type II υSa3. 12.1.0 Emesis Yes Yes Yes f ND ND ND ND ND Yes Yes Yes Yes ND Yes ND Weak ND No ND No b Genetic location φSa3mu. 15. (2001) Orwin et al. 18. 5. egc SaPI3 h d Type II υSa3. 20 ND 3.3 2. egc Type I υSa␤.8 24. high levels of NaCl. 22. 14.9 26.3.2.2. 8. (1997) Deringer et al.2. 17. SaPI is a mobile pathogenicity island encoding SE genes and other virulence factors..15. sen.1.8 25. 5. 20 5.4.3.4.2. (1998) Omoe et al. φSa3mw pIB485 e d d Human TCR interaction(s) c Reference(s) Betley and Mekalanos (1988) Bayles and Iandolo (1989). 21. suggesting that expression of SEB is not directly regulated by B ␴ (Schmidt et al. 13. (1998) Orwin et al. 7. 1997). (1998) Omoe et al. 12 5. 16. 7. (2003) Ranelli et al.6.2. The proteins are ellipsoidal and folded into two domains.7 5. 6. 21. 3. 5.3 9 5. 12. alkaline pH.9. not determined. SaPI3 Type II υSa3. 9. Toxins lacking one or both cysteine residues are generally nonemetic or weakly .1. 16. SE Structural Characteristics The three-dimensional topologies of the SEs and SEls are highly conserved. such as high temperature. SaPI3 h e Nomenclature in this table follows INCSS guidelines (Lina.1.4. 6.CHAPTER 8 • STAPHYLOCOCCAL FOOD POISONING 123 Table 2. also affects expression of SEB by regulating Agr and Rot regulation (McNamara et al. The oligonucleotideoligosaccharide-binding fold containing a ␤-barrel structure capped with an ␣-helix comprises much of one domain and is present in other toxins including Figure 1.6 26. egc pIB485. f ND. 23 5. 8. 16.3. 14 3. pF5 d φSa3n d g Type I υSa␤. or catabolites (glucose. 3. 14.3. sem. 13. 6. enterotoxin gene cluster harboring the sei. except for SElH.1. 18.9 30. 5. 6. 15. 11. 21. 6. Emesis as determined by primate feeding assay. 2004).3.6 ND 5.8 31. 17..1 26. (1988) Zhang et al. d Genomic island.8 25. by recognizing its target promoter. galactose. 7. 5.0 26. Biochemical and functional properties of SEs and SEls (grouped according to sequence and functional similarities) SE or SEl SEA SED SEE SElJ SElP SElN SElO SElH SEB SEC1 SEC2 SEC3 SElU SEG SElR SEI SElK SElL SElM SElQ a b c a MW 27. 8. pF5 Type I υSa␤. 17. Ribbon diagram of SEC3.5 26.9. and Shiga toxin. e Plasmids encoding SEs.2. SaPIbov Type I υSa␤. 6. 20 2. and cysteine loop are indicated. 5. SaPI1.4. (1985) Deringer et al.9.3.2 27. 2004). 13.2. egc Type I υSa␤.4. 18. 5. Major features including the TCR-cell receptor binding site. Numbers indicate human TCR V␤ elements. (2002) 1. the Sar locus. 1991). Most SEs contain two cysteines forming a variable length flexible cysteine loop. 13.0 27. (2004) Munson et al. Kappler et al. 5. and seo genes with or without seg. g egc. 6.3. (1997) Letertre et al. 22 Vα10 1. connected by a central ␣-helix. 6. 13. such as drug resistance or toxins. 12. 14. AIP levels and concurrently SEC expression (Regassa et al. and maltose). pertussis toxin.1. (2003) Jarraud et al.1.4. The figure is oriented with domain 1 containing the oligonucleotide-oligosaccharide-binding fold on the right and the N and C termini on the left. 6.1. Another global regulator. 12. 6. 21. glycerol. (2001) Petersson et al. aureus pathogenicity island). 13. 6. 18. 2000). 13.6 27. (2005b) Jarraud et al. cholera toxin. 21.0 24. (2001) Orwin et al. 23 1. (2001) Jarraud et al. 9. the promoter of B seb differs from the target promoter for ␴ . egc h φSa3mw. 14 1.1 26. GTTT(N14–17)GGGTAT (Kullik and Giachino. SEB expression is B negatively regulated by ␴ . 8.2 27. (2003b) Munson et al. SaPIbov Type I υSa␤.1 26.1. SaPIbov Type II υSa3. sucrose. egc φSa3mw.

house musk shrews. headache. SFP is usually a self-limiting illness. and esophagus. 2000. must be interpreted with caution. including contraction of abdominal muscles. SEB. FOOD-BORNE OUTBREAKS SFP Characteristics SFP is caused by ingestion of food containing SE preformed by metabolically active staphylococci. Emesis Emesis involves coordinated gastrointestinal activity. In 4 milk. 2007). and SED produced detectable levels of SEB 6 and SED (1 ng/ml) at a density of 6 ϫ 10 CFU/ml. The minimal number of S. The severity depends on an individual’s susceptibility to the SE. particularly those that require systemic administration rather than oral feeding. in an outbreak in the United States in which students consumed 2% chocolate milk contaminated with SEA. forceful contractions of the stomach pylorus. Victims experience severe nausea and characteristically projectile vomiting (Balaban and Rasooly. ultimately stimulating the central vomiting center in the fourth ventricle (Sugiyama and Hayama. A strain producing SEA. 1982). Recent studies demonstrated that long-term ϩ stimulation by SAgs induces development of CD4 ϩ CD25 regulatory T cells in humans. 1988). SE Potency In Vivo Several studies assessed the amount of SE required to initiate SFP symptoms.. SEA. SEC3.124 SEO AND BOHACH emetic (Bergdoll. and the general health of the victim. 1969).. and then SEA (4 ng/ml) was detected at a cell density 7 of 3 ϫ 10 CFU/ml (Noleto and Bergdoll... SEA and SED were detected as low as 10 and 7 10 CFU/ml. the amount of toxin in the food ingested.. respectively. 1988). Considering the variability among strains. and relaxation of the fundus. resulting in an acute uncontrolled release of proinflammatory cytokines and immunomodulatory cytokines in a chronic response (McCormick et al.. Binding activates APCs and extensive proliferation of T cells. SAgs stimulate a high percentage of T cells. A recent study using the house musk shrew demonstrated that SEA induces a release of 5-hydroxytrptamine (5-HT) in the intestine and that the 5-HT3 receptors on vagal afferent neurons are essential for SEA-induced emesis (Hu et al. the amount of contaminated food eaten. SEC2. cardiac sphincter. aureus required to produce detectable and toxic levels of SE in food has been investigated. little is known about the molecular and cellular mechanisms by which SEs induce emesis. the relevance of this information for SFP in general must be cautiously considered.4 ␮g/ kg of body weight) is required to cause emesis (Raj and Bergdoll. 2001). SED. SEC1. piglets.. However. This suggests that immunomodulation induced by SAgs has an important role in staphylococcal diseases. Le Loir et al. pain. 2003). 2007). Results with these models. and bovines. often abrupt and severe in onset. 2004) (see below). and muscle ache.. A massive outbreak in Japan caused by lowfat milk contaminated with SEA showed that the total intake of SEA per individual was estimated at approximately 20 to 100 ng (Asao et al. Dinges et al. Most bind to the outside of the peptide binding groove of MHC II molecules on antigen-presenting cells (APCs) and to T-cell receptors (TCRs) bearing specific beta chain V␤ sequences on T cells. The monkey feeding assay described by Bergdoll (1988) is considered to be the standard method. SAg Activity SEs and SEls belong to a family of toxins produced by S. abdominal cramps. An early study in which human volunteers ingested partially purified toxin demonstrated that 20 to 25 ␮g of SEB (~0. diarrhea. Studies with primates demonstrated that SEs stimulate neural receptors in the abdomen. aureus and Streptococcus pyogenes. with a short incubation period (1 to 8 h). SEE. which share several properties. zinc binding can occur at the outer surface or in a classical motif (H-E-X-X-H) in the groove between two domains (Baker and Acharya. Depending on the toxin. capable of suppressing the responses of responder T-cell proliferation (Seo et al. have been used (Hu et al. The cysteine loop is not directly associated with emesis and is believed to provide proper orientation of critical residues located near the disulfide linkage (Warren et al. 2000. 1974). since they may mimic toxic shock syndrome rather than SFP. rodents. and SElH bind zinc which is required by some toxins to bind major histocompatibility complex class II (MHC II) (Baker and Acharya. 2007). and the possible interference by food in SE detection.. the average dose of SEA in the carton was 144 Ϯ 50 ng (Evenson et al. 1965). variable environmental conditions in food. Other animal models. This gap in knowledge is due to a lack of a convenient and inexpensive animal model confirmed to mimic human SFP following oral administration. including kittens. 2004). The illness may be accompanied by subnormal temperature and transient changes in blood . However. including superantigen (SAg) activity. 2003). Other symptoms include convulsive retching. which transmit impulses through the vagus and sympathetic nerves. and ferrets.

Deaths are rare. 2008. 2002). of cases 197 Reference State of Alaska (1975) 1983 1986 1990 1992 2000 2004 Caribbean cruise ship United States Thailand United States Japan Brazil 215 ca. intermedius. the SE most frequently produced by S. attempts to isolate the causative staphylococci from patients are not warranted. In the United States. prostration. S. SFP Incidence SFP occurs as isolated cases or outbreaks.525 outbreaks were reported.. Although there is considerable variability among strains from different geographic regions. SFP accounted for an average of 4. Kwon et al. despite intravenous electrolyte and fluid replacement. seccanine. In addition.. Typically. 1998).. suggesting that SE genes might be transferred horizontally. Due to the lack of a unified surveillance system for the European Union..000 485 1. and SEC (Chiang et al. and shock can occur in severe cases which may require hospitalization. 1999). Outbreaks typically receive considerable publicity. tst. namely.413 cases occurred between 1993 and 1997. Recent studies demonstrated that SaPI is mobilized by staphylococcal bacteriophages and endogenous prophages (Lindsay et al. 2004. as well as genes for Table 3. It is estimated that ~1.000 CDC (1983) Evenson et al. Dehydration. the most commonly observed SEs are those harbored on the enterotoxin gene cluster (egc). SEI. Although it has likely afflicted humankind for centuries. with fatality rates ranging from 0. (1995) USFDA (1992) Asao et al. and sel) (Fitzgerald et al.CHAPTER 8 • STAPHYLOCOCCAL FOOD POISONING 125 pressure and pulse rate. or chronically ill patients. There has been one report of a U. 2000). sem. Some examples of recent outbreaks are summarized in Table 3. prophages. followed by SED. definitive diagnoses of isolated cases are not usually accomplished. Although there is a national surveillance system for food-borne disease in the United States through FoodNet (CDC. 2. 8. Kerouanton et al.364 14. 2000). those encoded by the sei. or affected individuals may not seek treatment. the elderly. It has been estimated that the mean annual number of cases from 1982 through 1997 could have been 185. and the cost of SFP in the United States is approximately $1. Numerous studies have assessed SE and SEl profiles of S.. 1999). AL Cream-filled pastries Low-fat chocolate milk Éclairs Chicken salad Low-fat milk Ordination meal No. 2006).5 billion annually (Harvey and Gilmour... Another coagulase-positive species. (1988) Thaikruea et al. 2002). 1998). accounting for 1. In Japan.S. since it is not usually life-threatening. followed by those on the genetic element named bovine staphylococcal pathogenicity island (SaPI bov) (sec. aureus in food which may have been discarded. (2003) Do Carmo et al. Fatal cases are usually associated with fluid and metabolic abnormalities.. Distribution of SEs among Staphylococci Many SE and SEl genes are associated with genomic islands and mobile genetic elements such as the staphylococcal pathogenicity island (designated SaPI)..6% of total food-borne disease cases (Olsen et al. Large outbreaks of SFP Yr 1975 Site or country Commercial airline Product Omelets and ham for airplane meal in Anchorage. the exact incidence is unknown for several reasons.964 persons between 1980 and 1999 (Shimizu et al. aureus implicated in SFP is SEA. and seo genes with or without the seg gene. 2007). or plasmids (Baba et al.870 ca.. 47 outbreaks of SFP involving 3..750 hospitalizations and two deaths typically occur each year from SFP in the United States (Mead et al. (2004) . 2000). Since SFP is not an infection. In contrast. the duration of SFP varies from several hours to 1 day. which is known to frequently express SEs (Jones et al.5% of food-borne disease cases (926 outbreaks) between 1993 and 1998 in 15 European Union countries (WHO Surveillance Programme. sen. SFP is not officially reportable. the reported prevalence of SFP varies greatly from country to country... aureus from various sources. aureus. Confirmatory diagnosis is not always practical since it usually requires identification of SE-producing S.181 cases were reported between 1983 and 1987 and 42 reported outbreaks involving 1.03% for the general public to 4. 2000). 1. is a well-known SE producer that possesses a variant SEC gene. Srinivasan et al.4% for the very young. 2007. involving 59. outbreak by shredded pork and coleslaw contaminated with a community-acquired methicillin-resistant S. The known locations of SE and SEl genes are summarized in Table 2. Lawrynowicz-Paciorek et al.060 (Mead et al. 2000.

SEC. aureus strains for SE-encoding genes. aureus. CNS also harbor SE genes. 2000). In fact. schleiferi. 2001). 1996). Valle et al. CONCLUDING REMARKS SFP has been a major concern of the food industry and medical profession for many decades. intermedius (Khambaty et al. with a physicochemical transducer to convert and amplify minute signals from the biological component into a measurable signal. SE Detection Methods There is considerable interest in detecting SEs or their genes for epidemiological purposes. Recently the ability of biosensors to overcome these shortcomings has been investigated. and SEI (Omoe et al. cohnii from goat milk and cheese produce SEE (Vernozy-Rozand et al. Olsvik et al. in a single reaction. staphylococci other than S. Letertre et al. Enzymatic bio-nanotransduction systems use specific antibody conjugated to nano-signal-producing DNA templates. Hoover et al. Others extended this method and developed PCR techniques for larger numbers of SE genes in multiple reactions (Omoe et al. Biomolecular interaction analysis-mass spectometry uses surface plasmon resonance to detect SEB binding to antibody immobilized on a gold electrode. 2005b). haemolyticus. PCR is an extremely powerful tool to rapidly screen S. and S. TransiaTube (Diffchamb AB). suggesting that emesis is not necessarily a feature of the toxins that provides a selective advantage to staphylococci. such as an antibody.. and sec3 have been described (Chen et al. 2002). Multiplex PCR methods that subtype the sec gene into its variants sec1. SFP produces considerable morbidity and economic impact. (2004) described an assay which involves PCR amplification of SE genes using degenerate primers. 1988. treatment. hominis. S. Sergeev et al. Currently. S. SED. Some of these related proteins are confirmed to lack emetic activity. in addition to the five classical SEs. 2004). These systems typically link a biological component. xylosus and S.. and great advances have been made in regard to its prevention. and etiology. It is more likely that enterotoxicity is a secondary effect of SEinduced immunomodulation and activity as SAgs. S. Several multiplex PCR methods have been developed. Although it is not usually life-threatening. SElH. such as SEG. Breckinridge and Bergdoll. S. S. (2003b) developed a real-time PCR to detect the sea to sej genes. Monday and Bohach (1999) developed an assay which.126 SEO AND BOHACH other SEs (SEA. detects nine SE genes. Biomolecular interaction analysis mass spectrometry was developed and applied to detect SEB and TSST-1. followed by matrix-assisted laser desorption ionization– time-of-flight to identify and differentiate the bound toxins. major advances were made in understanding the complicated epidemiology of SFP. S. These are usually based on passive agglutination or the enzyme-linked immunosorbent assay and include the SET-RPLA kit (Denka Seiken). hyicus subspecies hyicus.. 1994). Recent application of this method detected SEB at a level of 0. It is unclear whether these species have the capacity to frequently cause SFP since one study reported that CNS produce very small amounts of SE (Ͻ10 ng/ml) (Bergdoll... se-int (Futagawa-Saito et al. A 1991 outbreak of SFP in the United States involving 265 persons was conclusively linked to an SEA-producing strain of S. T-cell mitogenicity has been used to detect novel SEs based on their SAg activity (Holtfreter et al. This method detected and differentiated SEB and TSST-1 at levels of 1 ng/ml in milk and mushrooms (Nedelkov et al.. Some laboratories have raised antibodies for the detection of certain SEs. 1990). commercial kits are available to detect the classical SEs (SEA to SEE) in food. 1982. 1996). lentus. their performance time and compromised accuracy due to food matrices limit their effectiveness. BioDetect Test Strip (Alexeter Technologies). gallinarum. and S. S. xylosus from various sources reportedly produce SEs (Bautista et al.. sec2. 1983. the visual immunoassay (Tecra). Udo et al. equorum.. S. 1995). cohnii. we have only recently learned that. S.11 ng/ml (Branen et al. For example. SEB. saprophyticus.. and Ridascreen (rBiopharma). S. 2007). since fewer reagents are available. S. Detection of more recently identified SEs is problematic. Therefore. particularly antisera. 2004). albeit less frequently than S.. . aureus should not be neglected in regard to risk of SFP.. and several past dogmas are now considered obsolete. 1971. cohnii from dry cured ham produce SEC and SED (Rodriguez. many additional molecular variants and recently identified SEs and SEls exist. capitis. capitis.. S. An understanding of the exact impact in the United States would require more stringent and uniform reporting requirements. diagnosis. This assay has the advantage that it can detect previously unidentified SE genes. followed by characterization of the amplicons by microchip hybridization with oligonucleotide probes specific for SE genes. S. 1999. Signals may be amplified by in vitro transcription of DNA templates bound to target molecules producing RNA nano-signals specific for multiple targets in the sample. Despite the usefulness of methods described above. and SEE) and a novel SE-related gene. In recent years.. warneri..

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which is the only Shigella sp. Also. on a yearly basis worldwide range from 164. Shigella flexneri. including bacterial. Etiological agents comprise a diverse group of pathogens. Common to some bacterial pathogens. the severity of illness is dependent upon which Shigella spp. Consequently. by a series of sophisticated interactions between the host immune system and their own genetic factory. As with other enteric pathogens. and. or simply the transfer of Shigella via the fecaloral route. to carry the genetic information for the Shiga toxins. boydii is commonly isolated in the Indian subcontinent. regions of either chromosomal or plasmid DNA that encode for most of the genes responsible for virulence. viral. Washington. S. College Park. 1999). 131 . follows a cascade of events modulated by intrinsic factors provided by the host. Most of the 163. flexneri and S.Pathogens and Toxins in Foods: Challenges and Interventions Edited by V. 1999). are the most susceptible and have the highest mortality rates. and parasitic agents. Shigella spp. The numbers of isolated Shigella varieties are nearly reversed in industrialized countries where 16% and 77% of infections are caused by S. the pathogenesis of Shigella spp. particularly those who are malnourished. excellent review of the key components of the “cross-talk” between the host and bacterial pathogen and the role of host cell proinflammatory responses is provided elsewhere (Sansonetti. the infected host is unable to retain fluids. establish an infection by intracellular multiplication and intercellular spread. DC Chapter 9 Shigella Keith A. with nearly 1. flexneri is the most common Shigella spp.2 million people affected live in developing countries. in this case colonic epithelial cells. followed by S. S. they remain a scourge throughout the developing world and continue to be a significant health concern.. have not drawn as much attention as other human microbial pathogens. respectively. is a concern primarily in developing countries. Estimates of disease caused by Shigella spp. Although Shigella spp. Children under the age of 5 years. and Shigella sonnei. MD 20740. leading to diarrheal episodes (Sansonetti et al. The genus Shigella comprises four serotypes based on the antigenicity of each lipopolysaccharide (somatic O antigens): Shigella dysenteriae.7 to 200 million people infected. Lampel • Food and Drug Administration.1 million deaths attributed to this pathogen (Kotloff et al. Destruction of the intestinal epithelial cells and mucosal inflammation is a consequence of the host’s polymorphonuclear leukocytes and a subsequent recruitment influx of chemokines and cytokines at the sites of Shigella invasion. Juneja and J. invade selected target host cells. and some cases have been reported recently from Europe and the United States. notably type 1. K. HFS-710. dysenteriae. Sofos © 2010 ASM Press. As with other gram-negative pathogens. A short. Several factors are responsible for differences in the morbidity and mortality rates between developing countries and developed (industrialized) countries and include socioeconomic trends and nutrition. Shigella spp. the host-pathogen interactions are mediated by bacterial pathogen effector molecules that utilize the type III secretion system to effectively deliver these proteins to specific sites within the host cell. After consumption of contaminated foods or water. this pathogen is able to survive and transit the stomach milieu to the intestine. 1997). 5100 Paint Branch Parkway. particularly in developed countries where the mortality rate due to these pathogens is quite low.. 2006). S. Shigella boydii. The number of deaths associated with this type of illness is estimated to be in the millions and is considered the fourth leading cause of mortality in the world (Murray and Lopez. In developing countries. is the causative agent of disease. owe their pathogenesis to genetic determinants that reside on pathogenicity islands. isolated. sonnei. Keith A. Lampel Diarrheal diseases afflict a significant number of the world’s population each year. N. Briefly. sonnei isolates.

do not produce gas from glucose utilization (S. coli (Lan and Reeves. coli and Shigella chromosome was provided by data generated from the sequence of the S. positive strains of S. The four species of Shigella are divided into 55 serotypes and subserotypes. coli (Maurelli et al.. flexneri (group B) 8 serotypes and 11 subserotypes. 1998). coli bacteria are that they are nonmotile and are nonlactose fermenters. This is based on DNA-DNA reassociation studies and transconjugants between Shigella and E.. the more selective ecological niche that shigellae occupy–for instance. Shigellae are short (1 to 3 µm). Further corroborative data to place shigellae and E. S. Species S. nonencapsulated. Asia. coli pathogens to attach to the host cell surface prior to invasion. flexneri 2a genome (Jin et al.e.. such as the malate dehydrogenase (mdh) gene. flexneri. do not produce hydrogen sulfide.. high mortality rate if untreated Elicits less severe dysentery than S. coli in the same phylogenetic group come from sequence analysis of the 16S ribosomal RNA genes (Cilia et al.. S. flexneri 6 are positive. They are facultative anaerobes. distinguished by serology Produces mildest form of shigellosis S. are. 2002. Falkow et al. boydii serotypes 13 and 14 produce gas). 1985).. boydii (group C) 20 serotypes. nonmotile. Further evidence of the relatedness of the E. One of the major differences between Shigella spp. dysenteriae Biochemically identical to S. . boydii 13 and 14 have been noted [NB: S. Table 1. 2002). rod-shaped cells. The evolutionary relatedness of EIEC to Shigella has been discussed by Lan et al. ribotyping. dysenteriae Serogroup A Serotypes and subtypes 15 serotypes Geographic distribution Indian subcontinent. S. Wei et al. 11 subtypes 20 serotypes S. gram-negative. contains a homologous virulence plasmid of shigellae) and biochemical properties similar to those of Shigella. the identity of “black holes. Genetically.. coli strains and are placed within ECOR groups A or B1 (Johnson. Recent analysis using multilocus enzyme electrophoresis. Central America Most common isolate in developing countries Indian subcontinent. (2004). sonnei a D 1 serotype a Forms I and II are serotypically distinguishable. Characteristics of Shigella spp. Some serotypes of EIEC. provided additional support that Shigella spp. tribe Escherichiaeae. 1969). coli–may also support the notion that Shigella spp. boydii B C 8 serotypes. coli strains. In addition. EIEC has pathogenic factors (i. sonnei is positive [Edwards and Ewing. it has been noted (Strockbine and Maurelli. are independent from E. rarely isolated in developed countries Most common isolate in developed countries Distinguishing characteristics Type I produces Shiga toxin. lactose negative (some biotypes of S. 1972]).. non-spore-forming. such as EIEC 0124. and pathogenic E. distinguishing itself from other pathogenic E. Shigella spp. Shigella spp. coli and are considered to be clones of this species. Accordingly. and their conclusion is that these two pathogens comprise a pathovar of E. In addition. 1963. are closely related evolutionarily to E. Africa. Proponents of reclassifying Shigella spp. boydii 13 is the exception to many genetic biochemical characteristics. do not have any adherence factors such as those that enable E. 1999). Brenner et al. Shigella species are grouped as E. Enteroinvasive Escherichia coli (EIEC) is capable of causing dysentery. dysenteriae (group A) contains 15 serotypes. within E.” deletions of genetic regions of the Shigella chromosome compared to the genetic map of E. coli. they are host adapted to humans and higher primates in contrast to E. boydii serotype 13 is positive). These bacteria are members of the family Enterobacteriaceae. also share identical O antigens with Shigella (Sansonetti et al. coli posit that shigellae are clones of Escherichia spp. Important physiological/biochemical traits follow. lysine decarboxylase negative (S. 1957. coli (Luria and Burrous. causes most severe dysentery. 2005) that this organism may not be Shigella]). Other attributes that distinguish EIEC from other E.132 LAMPEL TYPE OF ILLNESS AND CHARACTERISTICS OF THE ORGANISM Bacillary dysentery or shigellosis is caused by all four members of the Shigella genus. in essence. flexneri and S. strains of E. and S. 2003). coli strains relates to the physical mechanism of entering host cells. 1996). sonnei (group D) one serotype (Table 1). and DNA sequencing of select genetic loci. flexneri S. coli. suggests that these two bacteria are genetically drifting apart from each other. and are ornithine decarboxylase negative (S.

unsanitary conditions exist (Kotloff et al. antibiotic therapy with ampicillin. S.who. manifesting in diarrhea (Rout et al. 1989).. particularly at zoos and primate centers (Line et al. 1993)... reactive arthritis. 1993). Kinsey et al. Preferred treatment is hydration. dysenteriae type 1. Further examination showed that S. and bloody. and particularly in human-made and natural disasters. particularly children under the age of 5 and the elderly. Later in infection. Overall. are rarely found in infants under the age of 6 months. showed that Shigella was isolated from stool samples collected from asymptomatic children under the age of 5 years. Children ages 1 to 5 are most susceptible to infections due to shigellae. This may be a reflection of the anatomical events of bacterial destruction of the colonic mucosa and the inability of the host to reabsorb fluids. this augments the likelihood of symptomatic and asymptomatic carriers coming into contact with uninfected and susceptible populations. Banish et al. coli O157:H7. S. For Shigella species. relapse. and mortality rates. This may account for its ability to cause the most severe and prolonged form of dysentery with the highest frequency of fatality. with summer months showing the highest number of incidences. is a rare postinfection sequela of Shigella. the bacteria spread from cell to cell.CHAPTER 9 • SHIGELLA 133 Shigellosis is a highly communicable disease due in part to the rapid spread of the pathogen within certain populations. HIV (human immunodeficiency virus)-infected individuals can experience both chronic diarrhea and dysentery from a Shigella infection and usually manifest more severe clinical symptoms (http://www. Asymptomatic carriers of Shigella may exacerbate the spread and maintenance of this pathogen in developing countries. Shiga toxin. where poverty and crowded.. 1973. which is endemic in developed countries. Simon et al. the dissemination of the bacteria from person to person can be extremely swift and can be responsible for the high secondary attack rate when introduced within environs such as crowded and institutionalized populations... Shiga toxin. the principal natural reservoir is humans. and copious amounts of watery diarrhea are accompanied by large numbers of leukocytes. 1988). In warmer months. Two studies. as evident by the presence of blood. Many other human bacterial pathogens may have multiple animal or environmental niches. and the sole etiological species is S. dysenteriae 1 (Raghupathy et al. Shigella spp. due to the low infectious dose of shigellae. 1989). 1991). as with Shiga-like toxin of E. nalidixic acid. Higher primates have also been known to harbor Shigella.. dehydration. coli O157:H7. 1992.. producing more destruction and sloughing of the colonic mucosal cells.. Studies with volunteers have shown that ingestion of only 100 virulent cells is sufficient to elicit clinical symptoms (DuPont et al. 30% of the isolates are S. Fever is common. dysenteriae is isolated from 6% of patients with bacillary dysentery in developing countries. dysenteriae type 1 carries the genetic information for a potent enterotoxin. Another contributing factor is the low infectious dose of Shigella that is required to cause disease.html). Clinical presentations range from mild diarrhea to severe dysenteric syndrome. and hemolytic uremic syndrome (HUS) (Bennish. reactive arthritis is associated with individuals of the HLA-B27 histocompatibility tissue type (Brewerton et al. mucoid stools.. tenesmus. flexneri 1b was isolated from stool samples of these employees and that four monkeys were shedding the identical . It accounts for deadly epidemics of bacillary dysentery and frequent pandemics in Central America. The illness can persist in a patient for 1 to 2 weeks. intestinal perforation. flexneri and S. Of these. The onset of shigellosis usually occurs within 2 days after ingestion and is usually a self-limiting disease that resolves itself within 5 to 7 days. shigellosis outbreaks are seasonal. and causes the most severe form of the disease. trimethoprim/sulfamethoxazole. sub-Saharan Africa. Infections caused by S. people’s interactions increase. seizures.. one in Bangladesh (Hossain et al. 1978). pus. Further complications of shigellosis have been identified as toxic megacolon. or ciprofloxacin may be implemented. In many geographical areas.. the latter consisting of abdominal pain. 1985.. Lopez et al.. and mucus in stools.int/vaccine_research/diseases/ diarrhoeal/en/index6. sonnei. HUS. 1981. In one instance (Kennedy et al. Bunning et al. As with other gram-negative bacteria. and for patients with severe symptoms.. 1994) and another in Mexico (Guerrero et al. parts of south Asia. more commonly associated with E. These children face an increase in attack. 1976). although cases have been reported. 1994). three animal caretakers at a monkey house presented with diarrheal symptoms. boydii can range from mild to severe. 1999). may be involved with HUS by damaging the vascular endothelial cells of the kidney (Karmali et al. 1975. Septicemia is not a usual complication from shigellosis. The disease can be fatal in immunocompromised individuals and malnourished people. This phenomenon is compounded by pediatric malnutrition in developing nations. The mildest form of the disease is associated with S. particularly in crowded communities and/or in environments with poor sanitary conditions. The presence of high numbers of leukocytes and shigellae in stool specimens occurs in the early stages of the disease.

In other cases. infected patients. shredded lettuce from Spain Cruise ship Multistate. a relatively new category.134 LAMPEL serotype. such as cholera and dysentery. refugees fleeing from Rwanda into Zaire in 1994). (1990) Lee et al. dysenteriae 1 (Goma Epidemiology Group. In the United States. troops Europe. 2001). Increased risk also extends to family contacts of day care attendees (Weissman et al. have been added (Islam et al. (2006) Terajima et al. (2000).. flexneri S. From carriers. Many of the outbreaks listed in Table 2 represent this type of Table 2. this pathogen is spread by “the four F’s”: food. An increase in human exposure may have contributed to zoonotic transmission of these Shigella spp. food handlers Commercial airline. boydii were isolated from preadult to adult gorillas. 1990). (1995) CDC (1994) CDC (1999). flexneri S. (2004) Reller et al. sources of contaminated foods implicated in Shigella outbreaks varied extensively. and food obtained from unsafe sources (Bean and Griffin. (2003) CDC (2000).. Most cases of shigellosis are caused by the ingestion of fecal-contaminated food or water.g. which are inanimate objects (e. foods are considered factors only when contaminated by human waste. contaminated equipment. as more single. food preparer Operation Restore Hope. such as when natural or human-made disasters destroy the sanitary waste treatment and water purification infrastructure.. Michigan. (1995) Kapperud et al. 1995). and massive population displacement. flexneri. (Nizeyi et al. sonnei S. Improper storage of contaminated foods is the second most common factor accounting for food-borne outbreaks due to Shigella (Smith. fresh parsley Multistate. 1986. Other contributing factors are inadequate cooking. tomato Japan. 2001). The disease was spread by direct contact of the caretakers with excrement from the infected monkeys. U. 1994). and the low infectious dose for causing disease increases the risk for shigellosis. fingers. 1974). Examples include the war in Bosnia-Herzegovina. and S. sonnei S. S. Transmission of Shigella in this population is very efficient. Shigellosis can be endemic in other institutional settings. In a recent study of free-ranging mountain gorillas in two national parks in Uganda. and flies. the major contributing factor for contamination is the poor personal hygiene of food handlers. Flies can transmit the bacteria from fecal matter to foods. food handlers Outdoor music festival. Wetherington et al.. oysters Isolate S. Kimura et al. (1991) Hedberg et al. sonnei S. moose soup. These conditions place a population at risk for diarrheal diseases. sonnei Reference(s) Wharton et al. sonnei. (1992) Gessner and Beller (1994) Sharp et al. (e.g.. S. Naimi et al. where crowding and/or insufficient hygiene create an environment for direct fecal-oral contamination. Levine and Levine. food preparers are a common means for transmitting Shigella from an infected host to susceptible populations. cold sandwiches Alaska. fomites. developed countries assume the conditions of developing countries. As evident in Table 2. As such. sonnei S. 1987). particularly in day care centers. Outbreaks of shigellosis Yr 1987 1988 1988 1991 1992–1993 1994 1994 1998 2000 2001 2001 Location. utensils and cutting surfaces). and nursing homes. which led to explosive epidemics of diarrheal disease caused by Vibrio cholerae and S.S. bean dip New York. are monitored until stool samples are negative for Shigella. famine and political upheaval in Somalia. Somalia. sonnei Shigella spp. mental hospitals. In the case of foods. of late. feces. source of contamination Rainbow Family gathering. sonnei S. S. Since humans are basically the sole source of Shigella. such as prisons. sonnei S. (2004) . outbreaks of shigellosis and other diarrheal diseases in day care centers are increasing.. SOURCES AND INCIDENCES IN THE ENVIRONMENT AND FOODS Foods The primary means of human-to-human transmission of Shigella is by the fecal-oral route.and two-parent working families turn to these facilities to care for their children (Pickering et al. To reduce the spread of shigellosis.

all cases were due to S.003). the numbers dropped to 6% for the years 1971 to 1990 and subsequently to 4% for 1991 to 2002. http://www.gov/~dms/ prodsur7. In later reporting periods. 44 samples (4. strawberries.gov/ ~dms/prodsur6. the amount of foods being transported from one country to another has increased.5%) were found to be contaminated with either Shigella (1. cfsan.S. For instance. Shigella. 9/1. . samples were collected specifically from high-volume imported fresh produce from 21 countries. causing additional cases. FDA domestic and imported surveys can be found at http://www.fda. http://www.html (2000 domestic interim results).gov/OC/pages/programs/impstatR. During this time span. Environment In addition to analyzing food directly for microbial pathogens. celery. and the Council of State and Territorial Epidemiologists have collaborated to collect data in reference to the sources of waterborne disease outbreaks (http://www. cilantro. Surveillance of food-borne illnesses caused by Shigella spp. FDA included broccoli. the prospect of microbial food contamination still persists.. continues in many countries. Environmental Protection Agency (EPA). and E. cfsan. this raises the issue of how to ensure that imported foods are safe for the consumers.CHAPTER 9 • SHIGELLA 135 passage.cdc. who contaminated the food and infected 25 other people. in which 327 samples were analyzed. AL.gov/ nheerl/articles/2006/waterborne_disease/waterborne_outbreaks. not only within the immediate population but also within a wider population.5%. 7 were positive for Salmonella and 1 was positive for Shigella. cfsan. In the context of food-borne illnesses. 35/1.pdf). Out of the 1.fda.html) and has continued this program to the present. thus elevating the secondary attack rate. Data from the 2005 import assignment. From 2003 to 2004. produce commodities have been a major route of dissemination in developed countries. with many reporting to a central repository at the World Health Organization. coli O157:H7 (http://intranet. whereas 23 cases were reported for untreated lake water.S.gov/~dms/prodsur9. In the United States. with increasing globalization of commerce.fda. Craun et al. shigellosis was reported for 56 cases from treated recreational (fountain) water. are found in water polluted with human feces. In the first survey.html (2001 assignment) and http://www. parsley.fda. Although a wide range of food sources have been implicated as the source of Shigella. sonnei (CDC. looseleaf lettuce.cfsan.cfsan.6% of the total cases of gastrointestinal illnesses.S.003) or Salmonella (3. as well as produce grown in the United States.epa. In one case. 2006a. One effort was the initiation of a surveillance program to monitor imported and domestically grown produce. coli O157:H7. Shigella spp. of the 486 samples that were tested. no E. representing 9. FDA issued a Domestic and Imported Produce Assignment for sampling specified produce commodities starting in 1999 (FDA survey of imported fresh produce. none were found for E. from 1995 to 2004 there were 136 outbreaks attributed to Shigella. Most of the commodities collected were analyzed for Salmonella. This has become a much more acute issue as food has become an integral part of the global economy. Contamination most likely occurred by an influx of water from a river in close proximity to the wells. and unless each shipment of food imported into or exported from one country to another is sampled. monitor food supplies through several different mechanisms.fda. the United States does monitor recreational water. whether treated or nontreated. CDC. and testing is done usually if a food is suspected of being contaminated. FY 1999 field assignment. Shigella was one of the two most common etiological agents for waterborne disease outbreaks.003 imported samples that were collected. coli O157:H7 was isolated from any sample. Standards for food safety seem to vary greatly around the globe. showed that 8 samples were found to be positive with either Salmonella (5 samples) or Shigella (3 samples). Furthermore. Not uncommonly. Foods are not routinely tested for the presence of Shigella.S.gov/ mmwr/preview/mmwrhtml/ss5512a1. an investigation of a farm in which Shigella was found in parsley revealed that the well water used for cooling and washing the produce was contaminated with this pathogen. Subsequent findings of the U. As reported in 2006 from a CDC surveillance report.0%. Produce that were of interest to the U. In this report. One of the striking features about food-borne outbreaks caused by shigellae is that often contamination of foods may not have originated at the processing plant but rather from either a food handler or feces-containing water used for irrigation or used at other points in food processing. In the latest posted results for 2006 (http://intranet. scallions (green onions). since 1971. cantaloupe.pdf. shigellosis accounted for 16% of all illnesses associated with waterborne human pathogens from 1961 to 1970. and tomatoes. the U. the U. In addition.pdf). regulatory agencies. 2006). In some cases. rapid dissemination of the pathogen occurred.gov/OC/pages/programs/impsumry. such as the Food and Drug Administration. as exemplified by moose soup prepared by a recovering individual in Galena.htm?s_ cid=ss5512a1_e).

moh. In August 1994.5 and 9. and hazard analytical critical control point (HACCP).pdf). shigellae are unable to grow and do not survive well in an acidic environment. 2002a). One of the lessons learned from a recent E. In two incidents. initially in the country where the food was originally prepared and second. in the processing plants. Partially. 2002a). 1994). Shigella spp. Other countries have followed suit and recognized the importance of identifying environmental conditions that may influence the spread of pathogens such as Shigella. Shigella was listed as one of the principal microbial pathogens responsible for most of the cases of waterborne gastroenteritis in New Zealand (http://www. one in October 1989 and the other in August 1994. Although no growth is observed. over the past few years (CDC. A multiple-antibiotic-resistant strain of S. Salt (NaCl) concentration and its effect on the growth and survival of S. flexneri 2a was isolated from several ill passengers and crew members in 1989. Under laboratory conditions. In media with a pH of 4.. and processing stages.. attention to details in all the growing and processing steps is required for reducing the advent of a food-related outbreak. such as formic or acetic acid.000 to 34. Koutsotoli et al. 1991). flexneri and S. between 4. With brain heart infusion medium. sonnei are greatly influenced by temperature and pH. 14% of the passengers and 3% of the crew members from the 1989 outbreak reported having diarrhea (Lew et al.8 to 5. transporting.. Yet. 2006b) was to identify potential sources of contamination for food commodities that are presented to the consumer in their raw state. respectively (Bagamboula et al. are considered more acid tolerant than some other enteric bacteria. Besides good manufacturing practices. are obviously not confined to the United States. this can be attributed to better vigilance on the part of the regulatory agencies.50 and 4. flexneri 2a was isolated from a patient.75.000 cases occur each year. the availability of chlorinated water may be scarce. Epidemiological data collected from several outbreaks in Greece indicate the widespread nature of this pathogen. and the suspected source of contamination was spring onions (http://www. In two reports covering the recent outbreaks over the past 3 decades.. only by a member of the galley crew on the cruise ship. 2004). flexneri at 7ЊC and up to 48ЊC. flexneri in broth cultures at different pH values with different organic acids (Zaika. with one death occurring (CDC. and the source of this outbreak was identified as German potato salad. lower temperature appears to support the survival time of S. passengers and crew members reported having gastrointestinal symptoms. 2006).doc). to either grow or survive under a myriad of laboratory conditions in broth cultures is obviously influenced by temperature and pH. growth for S.govt. sonnei can grow at temperatures as low as 6ЊC and S. as well as an increased scrutiny by the produce growers and the food industry overall. A report from New Zealand indicates that 18. survival of shigellae can be extended.. In addition.nsf/pagesmh/5821/$File/water-bornedisease-burden-prelim-report-feb07. sonnei and S. S.and food-borne diseases due to Shigella spp. INTRINSIC AND EXTRINSIC FACTORS THAT AFFECT SURVIVAL AND GROWTH IN FOOD PRODUCTS AND CONTRIBUTE TO OUTBREAKS Laboratory Conditions The ability of Shigella spp. five outbreaks were reported in 2004.2% NaCl). This includes following GAP and good manufacturing practices (GMP) during the growth. and 37% of the passengers and 4% of the crew from the 1994 outbreak reported having diarrhea. S. no growth of shigellae is observed.publichealthreports. Water. no growth is observed and survival of the bacteria declines. in broth medium is observed over a range of temperatures. . or nitrite (300 to 700 ppm). 2007b). 2000. sonnei and S. salts (3. S. flexneri was observed at a minimum pH of 4.org/userfiles/ 119_4/119435. S. nz/moh. depending on the temperature. flexneri grow in culture media with nearly the same pH values. Under certain culture conditions with media supplemented with organic compounds. and this characteristic may enable these pathogens to transit through the acidic conditions of the human digestive system. sonnei was responsible for all of the outbreaks reported (Alamanos et al.136 LAMPEL Waterborne outbreaks due to Shigella spp. Various studies have addressed the impact of these environmental concerns on how storage conditions of foods can affect the growth or survival of the pathogen. harvesting. coli O157:H7 outbreak related to consumption of spinach (CDC. In regions of Greece and other Mediterranean countries. Growth of Shigella spp. Contamination was introduced by infected food handlers. the latter also dependent on the type of acid supplemented in the medium. under some laboratory conditions and in fecal samples. HACCP principles can be applied. Data from the CDC on reported food-borne cases in the United States show a decline in the number of illnesses due to Shigella spp.3. good agricultural practices (GAP). have also been reported on cruise ships (Rooney et al.

fresh fruit salad. Recently. do not grow in broth medium when the pH is less than 4. data on the recovery of S. variable recovery of shigellae was obtained after 1 to 6 days (ICMSF. 1996). 1997. sonnei was recovered from both commodities. such as butter or margarine. Shigella survived for 13 to 92 days. radish. 1996. Shigella spp.5% to 7%. and a reduction of 10% at 3 kGy is observed.. sonnei and S. and lack of chemical and biological inhibitors. 28ЊC. hdeA and gadC. growth was observed. 19ЊC. and raw ground beef. sonnei is from 4 h to 10 days (ICMSF. Shigella survived for more than 10 days at refrigerated temperatures (4 to 10ЊC). flexneri and S. . nondeleterious pH values. ICMSF. In foods. onion. ice cream. incubation conditions. it was speculated that these bacteria lowered the pH of the environmental milieu and may have produced conditions unfavorable for both growth and survival of Shigella.8 to 5. 2002b). Acid resistance seems to be modulated by a sigma factor encoded by the rpoS (katF) gene. in 300 to 700 mg/liter NaNO2 and in 0. 2002b).8 to 5. with specific attention to produce/vegetables. A recent paper (Warren et al. and wine. Few studies have examined the survival and growth of Shigella in foods commonly associated with food-borne outbreaks.47) and fresh fruit salad (pH 3. and broths (beef. lactic acid bacteria were identified as part of the microbial flora.73) and incubated at 4ЊC for 4 to 48 h varied.0. S. In neutral pH foods. such as cheese. whereas with the other two commodities. In acidic foods.5 mg/liter of sodium hypochlorite (NaClO) in water at 4ЊC. S. celery. chicken.5 to 1. S. Under conditions favoring growth. initial growth did occur and was followed by a significant decline in the number of recovered cells (Bagamboula et al. whereas S. sonnei and S. and minced pork meat) stored frozen.. In some cases.. while at least two other genes. Growth and survival rates of Shigella are impeded in the presence of 3. and mayonnaise. such as citrus juices and carbonated soft drinks. some data were reported that indicate how these pathogens may survive or grow in foods. potato salad. and in some foods (e. as will be exemplified below. and 37ЊC.. inoculation numbers. Rafii and Lunsford.2% NaCl at pH 4. For example. respectively (ICMSF. S. broccoli. flexneri or S. flexneri survived well in broth with pH values higher than 4. and lemon). pH 3. or tomato juice. sonnei colony-forming units recovered was observed compared to the initial inoculation level. In a more detailed analysis. Under normal storage conditions for tomatoes and refrigerated temperatures (2 to 8ЊC) for potato salad and ground beef. carbonated beverages. strawberry. flexneri can survive at 4ЊC for 21 days in foods commonly implicated in outbreaks. a variety of produce was tested with regard to their effect on the growth and survival of Shigella (Rafii et al. sonnei on three different foods: tomato (smooth surface). Survival of shigellae was longer in foods stored frozen or at refrigeration temperatures than at room temperature. and room temperature and with a brief exposure to 80ЊC.1 (Bagamboula et al. 1996. survived up to 170 hours and 2 to 28 hours. Although different strains. 1996). Data from Zaika (2002b) indicated that S. Shigella can survive a temperature range of Ϫ20ЊC to room temperature. Other examples include citric juices (orange. Shigella spp. Zaika and Scullen. In order to address these concerns. 1995. Shigella sp. Survival of shigellae in foods under acidic conditions depends on temperature and type of acid. no S. green pepper. In foods. cauliflower..3 to 3. flexneri was isolated only from fruit salad and not strawberries. and at temperatures of 12ЊC. including temperatures above suggested proper storage settings. and recovery/enumeration parameters were reported in these studies. pH 3. are involved (Waterman and Small. shigellae can be recovered after 100 days when stored frozen or at 6ЊC. such as salads with mayonnaise and some cheese products. and vegetable). no significant decline in the number of S. These organisms can survive on dried surfaces for an extended period of time and in foods (e. tested in orange juice at 4ЊC and grape juice at 20ЊC. S.. 2002a). 2007) examined survival of S. flexneri did not grow at 22ЊC in either apple juice.9 to 4. shrimp. In most cases. Growth of Shigella at elevated temperatures (12 to 37ЊC) was mixed. 4ЊC. As noted above.. Composition of the food matrix and resident microbial flora had a pronounced effect on survival. grape. carrots). can survive in a broad milieu of food matrices. such as carrot.. In most vegetables and other types of foods tested. the survival time for S. Growth can occur in foods under the right environmental conditions.5.g. can survive in media with a pH range of 2 to 3 for several hours. They are sensitive to ionizing radiation. dysenteriae. Bagamboula et al. For instance. potato salad. with NaCl concentrations from 0. cabbage. 1996). flexneri and S.4. sonnei can survive for the longest period of time at room temperature. 1996). survival time is quite different at Ϫ20ЊC.CHAPTER 9 • SHIGELLA 137 Survival and Growth in Foods Smith (1987) summarized which foods have commonly been associated with food-borne outbreaks caused by Shigella spp. efforts have been directed to determine how well this pathogen grows and survives in foods. sonnei.g. seeded onto strawberries (pH 3. sonnei was recovered from tomatoes after 2 days. Depending upon the in vitro conditions. pea.

2007a). 1994).fda. but the primary reason was the lack of effectiveness in sampling and analysis of the final product. Produce commodities have been a common vehicle for food-borne diseases caused by Shigella spp. coli O157:H7 and Salmonella spp. 2007a). the concept of actually implementing such a process becomes a complex issue. salt concentration. in foods include not only pH. respectively). The food industry and regulatory agencies must grapple with whatever system is developed to reach a satisfactory approach that meets the demands of all concerned parties. . CDC. worldwide (Smith. current bacteriological methods are oftentimes consuming and laborious. can methods be developed that would eradicate pathogens from foods yet retain the quality of that particular commodity? As simple an idea as this is. 1999). this may occur at the manufacturing site but more likely happens at a point between the processing plant and the consumer. can be contaminated at the site of collection and shipped directly to market. including the health and safety of the consumer. considering that the infectious dose is 10 to 200 organisms and the number of shigellae on the foods may greatly exceed this figure. In addition. FOOD PROCESSING OPERATIONS THAT INFLUENCE THE NUMBERS. in some cases of shigellosis caused by the ingestion of contaminated produce. This has been noted both with a broad stroke with other microbial pathogens (Tyrrel et al. In addition.cfsan.. 1987. lettuce and parsley).g.736 were attributed to Shigella. 1989). but also the chemical components of the food matrix and the indigenous microbial flora.gov/~lrd/haccp. Salmonella and Campylobacter were the leading causes of food-borne illnesses reported by FoodNet (CDC. For example. a CCP could be directed at preventing ill workers from entering the workplace or instituting proper sanitation. In contrast. This approach would be instituted as a preventive program. present in food during short or prolonged storage.g. Therefore. OR CHARACTERISTICS Over the past few years. recovery may require special enrichment procedures for successful isolation of Shigella (Andrews. such as vegetables (e. Several drawbacks to this approach have been elucidated (Hall. A monitoring system such as HACCP may be well suited for pathogenic bacteria. SPREAD. An alternative to end product testing is the HACCP (http://vm. chemical. 2007b). that are known to be associated with specific foods (e. has consistently marked this pathogen as the third leading cause of bacterial food-borne diseases in the United States (http://www. can provide more in-depth insight into the effect that these environmental parameters have on the number of Shigella spp.. For Shigella. comprised of seven principles. knowing the potential for contamination is important to prevent contaminated foods from reaching the consumer. as indicated by the CDC data detailing the low number of confirmed sources of food-borne outbreaks (CDC. From epidemiological data. One of the traditional approaches to address the problem of microbial contaminated foods in the processing plant is to inspect the final product.000 population. representing 1. such as E. Of the 17. water activity. such as a food handler with poor personal hygiene. coli O157:H7 (Wachtel et al. Another factor is that foods.. 2. 2002). with less reliance on end product testing. either as individual or collective components. This pathogen is usually introduced into the food supply by an infected person. and temperature.gov/mmwr/ preview/mmwrhtml/mm5614a4.. 2006) and with specific bacteria. 1995. and physical hazards. However.91 incidences per 100.138 LAMPEL Factors that affect growth and survival of Shigella spp. the physiological state of the pathogen may be a significant factor in why shigellae are not likely to be isolated from foods.. the number of laboratory-confirmed cases of food-borne illnesses caused by Shigella spp. meats and egg products. Understanding the dynamics of the aforementioned factors. Kapperud et al. Recovery of shigellae is a daunting process. such as E. Whether these conditions lead to either an increase in the total count of the pathogen or the maintenance of sufficient numbers of infectious agents.html) system. CDC. including the responsibility of the food industry to identify certain points of the processing system that may be most vulnerable to microbial. establishing specific critical control points (CCP) for preventing Shigella contamination of foods is not always suitable under the HACCP concept.htm. foods are rendered unsanitary at the site of production/harvest by fecal-laden irrigated water or poor sanitary hygienic practices by those responsible for harvest or by a food handler postharvest. it becomes obvious how one contaminated commodity can transverse the commercial food process maze and affect a great number of unsuspecting consumers. it is apparent that Shigella spp. From several studies.252 cases identified through FoodNet surveillance (CDC Foodborne Diseases Active Surveillance Network). as Shigella may be present in low numbers and/ or stressed/injured in the suspected food samples. In some cases. survive well in most foods.cdc. the ability to increase in numbers may not be a significant point.

cdc. www. acceptance is moving more slowly. such as the FDA (FDA’s Center for Food Safety and Applied Nutrition.html). The FDA published a GAP document and the FDA’s Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits and Vegetables in 2007 (http://www. such as Salmonella and E. including (i) preventing contamination of fresh produce with pathogens.foodproductiondaily. the CDC estimated that there were approximately 448. and spices (21 CFR 179.fda.000 cases of shigellosis in the United States.S.gov/~dms/prodpla2.CHAPTER 9 • SHIGELLA 139 Although HACCP is a method for controlling food safety and preventing food-borne outbreaks. all four Shigella spp. A point of emphasis is that foods are not routinely examined for the presence of Shigella unless epidemiological data suggest that it may be the source of an outbreak. Vegetables and salads are commonly contaminated sources (Smith. simple methods to reduce or eliminate the pathogen load in raw vegetables.S. coli O157:H7. (iii) improving communication with producers.26. shigellosis continues to be a major public health concern.cfsan. 2006). FDA’s action plan identifies steps that could contribute to the achievement of the objective (http://www. Produce Safety from Production to Consumption: 2004 Action Plan to Minimize Food-borne Illness Associated with Fresh Produce Consumption (http://www. including irrigation. preparers. “Food Irradiation: a Safe Measure. and other samples.com/news/print NewsBis.html). CHEMICAL. dysenteriae. vegetables.asp?id=77092). which included reported cases (from 1983 to 1992) and the number of cases from passive (1992 to 1997) and active surveillance via FoodNet (http:// www. AND PHYSICAL INTERVENTIONS TO GUARD AGAINST THE PATHOGEN Introduction of shigellae into foods. For each objective. gov/ncidod/dbmd/diseaseinfo/foodirradiation.htm). 2005). unpasteurized orange juice.cfsan. Mexico. government ..foodsafety. In the United States and other countries (e. Debate over whether there is an adverse effect on the “taste” of the raw commodity after irradiation or over the cost of implementing irradiation on a larger scale may lessen the appeal of this process to reduce the number of shigellae in foods. were isolated from all types of samples (6 to 17%).. which are not indigenous to. where control over all CCPs may be more problematic. particularly raw vegetables/produce. government agencies. Total aerobic and E. Means to control microbial pathogen contamination may involve chemical treatment or irradiation.html) and the CDC (http://www. 2000). 2003b. the application of food irradiation is slowly being accepted by consumers. 1987). Irradiation of foods is supported by U.html). Although the CDC has reported that food-borne outbreaks of shigellosis in the United States have declined recently. (ii) minimizing the public health impact when contamination of fresh produce occurs.net). 2007). Although public attention has been drawn to the control of other microbial pathogens.” http://vm. Canada [http://www. In compiling data from 1982 to 1997. sold by either street vendors or small grocery stores.. as demonstrated with cut parsley artificially inoculated with S. RECENT ADVANCES IN BIOLOGICAL.com /news/ ng.gov/~dms/prodpla2. in which freshly squeezed. and at the international level by the Codex Alimentarius Commission (Codex.. in Europe. FDA has approved the use of irradiation for meats. and consumers about fresh produce. and (iv) facilitating and supporting research relevant to fresh produce. the third leading cause of food-borne outbreaks by bacterial pathogens (CDC.html). Briefly.gov/ncidod/dbmd/foodnet). http://www.gov/ ~dms/opa-bckg.gov/opacom/catalog/irradbro.gov/~dms/prodguid. Overall. pathogens such as Shigella. harvesting.fda. fresh fruits.cdc. coli counts indicated that the samples were contaminated with fecal matter.asp?id=28788-canada-extends-food]).fda. Codexalimentarius. but only 10 European Union members have approved facilities for food processing (http://www. foods.cfsan. Another approach is chemical applications with acetic acid or chlorinated water. U. were analyzed for the presence of Shigella (Castillo et al. fda. An example of this problem was recently exemplified in a survey conducted in Guadalajara. the FDA further committed to addressing the problem by releasing an action plan to reduce produce-related illness. These have been shown to be effective. The U. 2003a. including S. A recent consumer report on the application of irradiation of fresh produce covers many pertinent topics ranging from food safety to the question of whether there are any adverse effects of irradiation of foods (Groth. foods potentially contaminated with Shigella could also be successfully treated with ionizing radiation. such as wiping cloths. and hand packaging. In June 2004. In contrast.g. sonnei (Wu et al. but rather introduced into.S.foodproductiondaily. HACCP would work at the processing plant but would be more difficult to implement at the site of production. most likely occurs during processing. poultry. The plan had several objectives. Irradiating foods is an accepted process in 40 countries. are most likely to be undetected.

is used to screen for lactose-negative colonies (Shigella are lactose negative). produce alkaline (red) slants. acetate. A survey of how extensive this problem is and how the U. 2001. notably E. is catalase negative and has ornithine decarboxylase activity. and biosensors. with the exception of S. and salicin. ... raffinose. Media containing chromogenic or fluorogenic indicators have been applied to isolating and detection regimens for a number of pathogens. from feces). S. are catalase positive. Most methods entail growth in broth medium which is followed by plating on selective agars. flexneri serotype 6. coli and Shigella spp. e. However.. Some Shigella spp. with slight modifications. boydii serotypes 13 and 14..140 LAMPEL regulatory departments and agencies have implemented programs to test imported and domestic produce for the presence of select bacterial agents as a means to obtain baseline data and to take regulatory action when warranted. malonate. Similar to other enteric bacteria. gov/~ebam/bam-6. except for S. Suspected colonies that are gram-negative. www. Although most Shigella spp. acetate. the authors detail one method to isolate shigellae from foods. One Shigella sp. boydii. and S. which reflect a much smaller number of matrices than foods. are oxidase negative. rhamnose. 2005). from each other. dysenteriae type I. Other biochemical tests are used to identify the serotypes of Shigella.g.gov/~dms/ prodact. coli O157:H7 and Salmonella spp. dulcitol. xylose.g. do not utilize citrate.gc. 2002. potassium cyanide. such as S.html) uses a very similar scheme. In the Bacteriological Analytical Manual (Andrews and Jacobsen. sonnei and S. acid (yellow) butt. sonnei (Warren et al. These biochemical tests and those presented in several laboratory protocols aim to differentiate Shigella spp. Growth on Christensen citrate. are unable to grow on highly selective Salmonella-Shigella medium. there is not one definitive method that has the robustness. detect. rapidity. S. A protocol from the International Organization of Standards (ISO 21567:2004. dysenteriae type 1. however. Two or three different selective media should be used to increase the chance of recovering Shigella. from E. S. some species.html). Highly selective media include SalmonellaShigella and Hektoen agars. Shigellae are negative for the Voges-Proskauer test (S. The ability to utilize mannitol.ca/fn-an/res-rech/analy-meth/ microbio/index_e.. pose a challenge to laboratory personnel isolating Shigella (e. no enrichment medium exists to selectively grow Shigella from either food or fecal samples. dysenteriae 3). www. Confirmation is usually performed using biochemical tests. Further biochemical characterizations show that Shigella spp. one medium containing a chromogenic indicator has been tested and should be commercially available in the near future. Currently. Eosin methylene blue and Tergitol-7 agar are alternative low-selectivity agars. and identify Shigella spp.fda. from foods can be divided into three major categories: conventional bacteriological. Shigella spp. coli and also to distinguish Shigella spp. and efficacy to be effective in isolating Shigella from foods. either automated or commercially available biochemical strips. Desoxycholate and xylose-lysine-desoxycholate agars are intermediate selective media and are preferred media to isolate Shigella spp. and no gas.) offers another method.S. on these agars. and. are negative for H2S production. boydii and S. boydii serotype 13 are positive).html). adonitol. Presently. no H2S.cfsan. Discriminative Detection Methods for Confirmation and Trace-Back of Contaminated Produce Methods used to isolate. dysenteriae. sonnei may after a long period of incubation). Growth of Shigella on MacConkey agar.fda. Clinical samples. or acetate agar is one characteristic that discriminates between E. nucleic acid based. glycerol. This severely hampers the ability of laboratories to isolate Shigella from foods. do not ferment xylose. A range of selective agar media is recommended to plate cultures after growth overnight in broths. Microbiology of food and animal feeding stuffs— Horizontal method for the detection of Shigella spp. Health Canada (http://www. sodium mucate.. this medium fares poorly with foods having a high number of indigenous microbial populations. inositol. FDA is addressing this issue has been published (Beru and Salsbury. As for Shigella. phenylalanine deaminase. Shigella spp. In that study chromogenic agar was used with tomatoes artificially contaminated with S. a low-selectivity medium. and indole and the presence of ornithine decarboxylase and o-nitrophenyl-␤-d-galactopyranosidase have been used to physiologically discriminate between Shigella spp. or mucate as a sole carbon source.cfsan. and sucrose and lactose fermentation (although S. particularly when this pathogen competes with the indigenous flora present in foods. shigellae are unable to utilize citrate. ferment glucose. all shigellae are methyl red positive and are unable to produce acid from glucose and other carbohydrates (acid and gas production occurs with S. have variable reactions and may be missed. nonmotile rods are inoculated onto lysine iron or Kliger iron agar and incubated 18 to 24 hours at 37ЊC.hc-sc. and lack lysine decarboxylase. flexneri serotype 6.

Another aspect of identifying causative agents of food-borne outbreaks of diarrheal disease is the ability to link the people affected in one region to other geographical locales and ultimately to the source of contamination. One alternative to enrichment uses a filter that lyses the bacterial cell and sequesters the DNA in the membrane and can be used directly as a template in the reaction (Lampel et al. are essential for accuracy. and S. namely. the amplification of PCR products can be monitored in real time. To ensure that negative results are truly that.cdc. such as using a probe within the reaction. There are several different platforms that are available. Form (or phase) I is virulent and has a smooth colony texture. Unlike other methods that may take several days to draw a conclusion regarding the presence of Shigella in foods. much of this type of analysis is automated. Also. each with their own strengths. The CDC has established several surveillance programs to monitor food-borne outbreaks in the United States. templates in sufficient quantity and free of PCR inhibitors prepared from all types of food matrices are critical. Although S. Therefore. Most of these protocols require enrichment in broth for 6 to 18 h..who. Other technologies An alternative to agarose gel electrophoresis is the application of an enzyme-linked immunosorbent assay at the conclusion of a PCR run to detect the ipaH gene of Shigella spp. In each system. 2003). Two approaches are being entertained. PCR assays can yield a result in less than 1 day.. Also.cdc. the same problems exist. therefore. One potential problem for PCR-based assays is the presence of inhibitors of the reaction deriving from the food matrix. sonnei isolates. 2001). at http://www.gov/ and.gov/ncidod/dbmd/phlis data/shigella. these particular primers do target copies of the ipaH gene in the chromosome. ipaH (Wang et al.CHAPTER 9 • SHIGELLA 141 Serological testing using polyvalent antiserum is used to identify the Shigella groups A to D.. has been used to establish a database to type and identify Shigella strains (Coimbra et al. 1973). can increase the specificity of the reaction in a very short period of time. These include PulseNet (http://www. As for using a PCR-based assay to detect Shigella in foods. in foods. Some O-antigen structures of EIEC strains share homology with O-antigen structures of some Shigella serotypes (Tulloch et al. and in some cases. bacillary dysentery. as do the shigellae.html). The advantage of these primers is that the ipaH gene is present in multiple copies in the virulence plasmid (five copies) and chromosome (seven genes/homologues. cognates) of Shigella (Jin et al. http://www. is a much more sensitive means of detecting the presence of microbial pathogens than conventional bacteriology and the use of DNA probes. an extra step.gov/foodnet/). viable but nonculturable cells may persist in the population and may not be resuscitated by enrichment. The World Health Organization (WHO. false-negative reactions may not be truly indicative of contamination. antiserum targeting both forms should be applied to identify immunologically S. 2002. ensuring an opportunity for a successful reaction. Ribotyping. require enrichment in broth. another means of subtyping microbial pathogens that uses gel electrophoresis patterns of PCR-amplified RNA genes. DNA-based assays Several types of DNA-based assays have been applied to detect Shigella spp. in instances when the large virulence plasmid is lost. Realtime PCR has the advantage of reducing the time for analysis. Both forms carry distinct antigens.htm. meaning less chance of mistakes and reduced labor. In this regard.. control reactions using washes or homogenates with target DNA added.gov/pulsenet/) and FoodNet (http://www. whether or not to include an enrichment step prior to PCR. from stool specimens (Set- . DNA probes and PCR were developed to detect Shigella from clinical samples. This added step may be sufficient for some pathogens. Initially.cdc. Injured. coli O antigens of the Alkalescens-Dispar bioserogroup or EIEC. 1997). Therefore. boydii have reciprocal cross-reactivity with E. if not all. sonnei has only one serotype.who. the 5 to 12 target sequences increase the sensitivity of the assay compared to primers targeting just one copy of a gene. As with conventional PCR. two forms of this pathogen exist on agar plates. weak. PCR. primers are selected that target one specific virulence gene. others can be found at http://www. 2000). pulse-field gel electrophoresis surpasses most other nucleic acidbased typing methods. subsequently digested with a specific endonuclease. an in vitro amplification system. flexneri.. Wei et al. but shigellae pose a more difficult task.cdc. Hence. EIEC causes the same disease. Several serotypes of S.. template preparation is one of the key steps in ensuring the correct result in analyzing foods by PCR. specifically for Shigella. S. Several manufacturers provide kits. A note of caution should be taken.int/emc/) also monitors outbreaks and publishes some of their findings in the Weekly Epidemiological Record (http://www. whereas form II is irreversibly avirulent and has a rough colony phenotype on agar surfaces. and most. int/wer/index. dysenteriae.

2000). Epidemiol. have poor sanitation. Clearance of Shigella flexneri carriers in a zoologic collection of primates.. Castillo. Vet. 2002. Bagamboula. Gaithersburg. Beru. Bacteriol. 19:529–536. Immunomagnetic separation technology. Salsbury.. Uyttendaele. et al. and trends. In one report. J. Children under the age of 5 years often have the highest mortality rate in developing countries. 98:637–650.1 million deaths worldwide each year. laboratories.int/vaccine_research/diseases/shigella/en/). http://www. A. and present an appropriate milieu for facilitating rapid transmission of shigellae via the fecal-oral route. N. A community waterborne outbreak of gastro-enteritis attributed to Shigella sonnei. Maipa. MD. J. Villarruel-Lopez. and D. Although considered a self-limiting disease. The sensitivity of these instruments is approaching the level of molecularbased methods. One limiting step is the incorporation of an antibody or antibodies that target each Shigella serotype and subserotypes.. International AOAC. p.. Growth and survival of Shigella sonnei and S. W. Polynucleotide sequence relationships among members of the Enterobacteriaceae. and J. Foods present a different scenario. J. M. can be routinely grown in laboratory conditions. Microbiol.. http://www. J. F. However. Andrews. WHO and public health researchers around the globe have recognized the necessity for a vaccine against Shigella (WHO. Ray (ed. Food Protect.pdf. usually for the aforementioned conditions. J. 1988. G. Bean. J. J.01–6.who. M. Even in fecal samples in which ample numbers of shigellae are shed in infected individuals. Bunning. 6. H. 55–113. S. Walters. Johnson. Assoc. Torres-Vitela. FL. Sack. S. Archer. Montali. 2000. whether in developed or developing countries. p. Water.):87S–107S. R. K. A. D. Citarella. Shigella. Caffrey. N. 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1996). 1999). Although cholera epidemics have been essentially eliminated in the United States. Other Vibrio species that can cause human disease include V. 2002). Wright and Keith R.. damsela. which estimates that these figures probably reflect only 1% of the actual disease incidence (Zuckerman et al. K. Metabolically they are moderate halophiles and facultative anaerobes that use glucose as a sole carbon source. Skin infections from Vibrio species are also common and are associated with exposure of wounds to seawater or by handling of seafood during processing. Vibrio species are responsible for both sporadic and epidemic disease. metschnikovii. and V. alginolyticus. gastrointestinal symptoms range from mild watery diarrhea and vomiting to the severe. and the capacity for global spread.. but they all secrete chitinase. Schneider The primary pathogens in the genus Vibrio are Vibrio parahaemolyticus. furnissii. As summarized in Table 1. however. and V. but reported cases are either relatively rare or do not involve food-borne transmission. Food Science and Human Nutrition Department. N. The global incidence of this disease remains devastatingly high: approximately 100. DC Chapter 10 Pathogenic Vibrios in Seafood Anita C. V. hollisae. V. Nonepidemic V. 2007). V. V. Furthermore. 1984). Kaneko and Colwell (1975a) postulated that the distribution of vibrios in marine environments is determined by their adsorption to and utilization of the chitinous exoskeletons of planktonic organisms. and susceptible persons have predisposing conditions. Many species produce rapidly lethal septicemia. V. 2000). vulnificus. mimicus. V.000 cases are reported annually worldwide to the WHO. All species appear as curve-shaped rods with one or two single-polar flagella in standard culture (Baumann and Schubert. and symptoms vary greatly among the different pathogenic species. cincinnatiensis. Washington. FL 32611.000 to 300. Juneja and J. PATHOGENS: TYPE OF ILLNESS AND CHARACTERISTICS OF THE ORGANISMS V. Although most bacterial foodborne infections have declined in recent years. cholerae This species was first described as the causative agent of epidemic cholera disease by Pacini in 1854 and later by Koch in 1883. cholerae is also responsible for occasional seafood-borne disease in the United States and can be distinguished from epidemic disease by the severity of symptoms. purging diarrhea associated with cholera epidemics. Figure 1 shows attachment of V. Gainesville.. 2007). the initiation of vibrio microcolonies on these animals is thought to be critical for carbon cycling in the world’s oceans (Keyhani and Roseman. aquatic bacteria that commonly inhabit coastal ecosystems throughout the world. V. Sofos © 2010 ASM Press. V. cholerae. • estimated annual incidence of vibrio disease in the United States actually increased by 78% in 2006 compared to 1996 to 1998 (CDC. Wright and Keith R. the disease is still endemic in parts of Asia and Africa and more recently South and Central America. usually related to hepatic or immune systems. Vibrios are gram-negative. related diseases are generally transmitted to humans via consumption of contaminated seafood or drinking water. and several species are also pathogens of fish and shellfish. fluvialis. vulnificus cells to a planktonic diatom in culture. carchariae (Hlady and Klontz. and it remains one of the deadliest bacterial disease agents worldwide (Lipp et al. Because vibrios are indigenous to aquatic environments.Pathogens and Toxins in Foods: Challenges and Interventions Edited by V. 359 FSHN Bldg. Vibrios account for about 75% of seafood-borne bacterial illness in the United States (Feldhusen. demonstrating the close association of these two marine species. Schneider Newell Drive. the Anita C. 146 . The various species demonstrate a range of fermented sugars and enzymatic profiles. University of Florida. the serogroups of associated strains. systemic disease seldom occurs in healthy individuals.

After docking to the intestinal cells. and it serves as a model for the study of other bacterial toxins with similar structures (Sandvig and van Deurs. vulnificus Symptoms Gastroenteritis Less common Common Common Less common Severe diarrhea Common No No No Septicemia No Rare Rare Common Wounds Rare Rare Less common Common ... Zuckerman et al. Other possible virulence factors include outer membrane proteins or porins (Provenzano and Klose. 1987b). Interestingly. Ford et al.. the toxin transfers ADP ribose to G proteins that are involved in this process. The disease requires a relatively large infectious dose. 1995).. 2003).CHAPTER 10 • PATHOGENIC VIBRIOS IN SEAFOOD 147 Figure 1. and chemotaxis is important for colonization and induction of CT expression in vivo (Lee et al. and an inner membrane protein ToxR that is produced by all Vibrio species and coordinately regulates the other virulence factors (Miller and Mekalanos. (Figure provided by Maria Chatzidaki-Livanis. cholerae bacteria lacking CT are not able to cause epidemic disease (Morris. leads to frequently fatal dehydration. potentially epidemic. The activity of CT has been studied extensively. and susceptibility to the disease varies widely among healthy adults (Sack et al. this pilus also serves as the receptor for a lysogenic filamentous bacteriophage (CTX␸) that carries the genetic material for the production of CT (Waldor and Mekalanos. 1991). Nontoxigenic V. Specifically.. Treatment of cholera includes oral or intravenous rehydration and antibiotic therapy. 2007. 1969).. 2001). estimated to be about 1 million bacteria. especially tetracyclines. which produces rapid fluid loss that. ToxT (DiRita et al. but multiply drug-resistant strains have been reported (Morris. As shown in Figure 2. cholerae (nonepidemic) V. if untreated.. releasing the A subunit into the cytoplasm.. V. Genetically Table 1. This rapid loss of large fluid volumes accounts for the severity and high mortality associated with cholera. Activity of the A subunit blocks the host machinery for recycling the intracellular signaling molecule. Vaccines offer protection against disease but are expensive to produce and deliver. the toxin enters the cell and transverses across the cytoplasm. and these proteins combine to make the active holotoxin (Kaper et al. The net result is the permanent activation of host adenylate cyclase. cholerae is sensitive to a variety of antibiotics. 2000). leading to secretion of massive amounts of chloride ions into the lumen of the intestine. Motility is also required for virulence (Richardson. The single A subunit has the enzymatic activity responsible for symptoms and is surrounded by five B subunits that provide the binding capacity of the toxin. Following ingestion. resulting in characteristic voluminous diarrhea.. The primary virulence factor is the cholera toxin (CT) that is secreted from the bacterium during infection and is responsible for the characteristic diarrhea (Finkelstein and LoSpalluto. cyclic AMP.) Epidemic cholera is characterized by massive watery diarrhea. 2003). which causes significant increases in cyclic AMP levels. Lyerly et al. cholerae (epidemic) V. Typical symptoms associated with different Vibrio species Vibrio species (type) V.. parahaemolyticus V. vulnificus bacteria that are attached to diatom are indicated by arrow. Subsequent osmotic imbalance induces dramatic water loss from the cells and tissues. 1984). strains following infection by this phage. 1972). Genes for TCP and CTX are both regulated by the same transcriptional activator. 1982). cholerae strains are converted to toxigenic. CT is comprised of two protein subunits designated A and B. 1991). 1987a). 2002. 1988. 2007). the bacterium multiplies in the lumen of the small intestine and uses pili to anchor to enterocytes in the gut (Taylor et al. 1996). Other virulence factors include the toxin coregulated pilus (TCP) that is expressed during colonization of the small intestine (Taylor et al. V. V. and the immunity provided may be limited (Taylor et al.

and typical symptoms are dramatically different from those of other pathogenic vibrios. (1976). vulnificus V. Disease incidence is relatively rare compared to the . parahaemolyticus was first described as the causative agent of outbreaks of gastrointestinal illness in Japan (Fujino. parahaemolyticus involves rehydration. V.. but fatalities are rare and generally follow septicemia (Su and Liu. (Adapted from Kaper et al.. 1953). parahaemolyticus. leading to fluid accumulation (Baffone et al. Some clinical isolates produce a genetically similar toxin called the TDH-related hemolysin (TRH). followed by endocytosis and release of the A subunit (black circle). V.. 1995).. parahaemolyticus V. whose activity is characterized by the Kanagawa phenomenon that is based on lysis of human red blood cells on Wagatsuma agar (Miyamoto et al. which ribosylates G proteins to activate adenylate cyclase. 2007). Experimental genetic deletion of the gene (tdh) encoding TDH corresponds to loss of symptoms in animal models (Nishibuchi et al. and septicemia are also caused by V. A vaccine is not available for V. Diarrhea may result from toxin-mediated 2ϩ induction of Ca -activated chloride channels. Treatment for V. Occasional dysentery. vulnificus was first described by Hollis et al.) engineered. parahaemolyticus. and other strains produce both TDH and TRH (Honda et al. Disease can result either as a consequence of seafood consumption or from exposure of wounds to seawater or through the handling of seafood (Blake et al. parahaemolyticus disease and better understanding of virulence mechanisms may lead to improved prevention strategies. 2005). V. and massive diarrhea. 1995.. However. 1969). live attenuated oral vaccines hold great promise and generally involve deletion of the gene (ctxA) encoding the enzymatic A subunit of CT (Kaper et al. and infected persons may also benefit from antibiotic therapy with tetracyclines or quinolones (Morris. Activity of CT. and vaccine development seems unlikely due to the typically mild nature of symptoms. CT B subunits bind host intestinal cells. 1992). Unfortunately. necessitating the construction of new vaccines. wound infections. 1988)... The subsequent increase in cyclic AMP (cAMP) results in protein phosphorylation. these vaccine strains still retain some reactogenicity. Symptoms associated with these infections contrast greatly with epidemic cholera and generally present as mild gastroenteritis with watery diarrhea that resolves without treatment. and emerging strains can be antigenically different from vaccine strains.. parahaemolyticus produces several hemolytic toxins. especially for persistent diarrhea. 2003). recent increases in the global spread of epidemic V. 2007). The first described toxin was the thermal stable direct hemolysin or TDH. extrusion of chloride ions. Toxic molecules related to type three secretion systems have also been implicated in disease and are under investigation (Liverman et al.148 WRIGHT AND SCHNEIDER Figure 2. 1979).

2006a. Howard and Lieb. V. In compromised hosts. Another hemolysin. vulnificus septicemia. and only 30 to 60 cases are reported annually in the United States (Feldhusen. 1991). and persons who are at risk for this disease generally exhibit some type of underlying condition that includes alcoholic cirrhosis.. 1984). A CPS operon was recently described for V. For example. and the early administration of antibiotics is critical to the outcome of disease (Klontz et al. In these persons. 2007).. A recent survey in Japan detected six cases of V. 1985. vulnificus also causes gastrointestinal illness with mild diarrhea and vomiting.. and these infections may result from exposure of breached skin surface to seawater or contaminated seafood handling (Shapiro et al. (2005).. CDC. V.. intravascular coagulation. Acapsular variants show deletion mutations in this operon and are locked in the translucent phase. Paranjpye et al. this single species is responsible for nearly all of the deaths related to bacterial contamination of seafood in the United States. (1981) found that exogenously administered iron in mice dramatically reduced the 50% lethal dose from about 10. The mortality rate is exceptionally high (Ͼ50%). vulnificus are rarely seen in healthy adults or children..CHAPTER 10 • PATHOGENIC VIBRIOS IN SEAFOOD 149 other vibrio pathogens. while strains with partial CPS expression retain an intact operon and reversible phase variation. 2006. Chatzidaki-Livanis et al.. 1985. 1990).) whose relevance to virulence is unclear. vulnificus disease. where increased exposure of consumers to raw seafood and lower immune status is more common (Park et al. the V. primary septicemia can occur within hours of exposure and produce symptoms that are similar to other types of gram-negative toxic shock. and risk of disease may be greater in countries outside the United States. Furthermore. 2000).. The different colony types are shown in Figure 4 and have been observed with other Vibrio species. parahaemolyticus but shows genetic similarity to those found in V.000 to Ͻ10 bacteria.. 1987). Yoshida et al. DNases. Amako et al.. 2001). and quorum sensing (Kim et al. vulnificus to cause systemic disease by providing protection from the lytic activity of serum and from phagocytosis during systemic disease (Tamplin et al. Combinations of antimicrobials may be more therapeutic and . However. cholerae and other Vibrio species (Yamamoto et al. Experimental evidence supports epidemiological data indicating that host iron status contributes to Figure 3. Encapsulation by polysaccharide is a primary virulence factor and contributes to the ability of V.. Multiple types of translucent-phase variants have been identified with differing degrees of encapsulation and variation in the genetic organization of the CPS operon.. has been implicated in disease in animal models (Lee et al. vulnificus and showed genetic similarity to the Esherichia coli group 1 capsule (Chatzidaki-Livanis et al. Encapsulation is marked by formation of opaque colonies.. pili (Paranjpye and Strom. vulnificus septicemia is rapidly fatal. including rapid drop in blood pressure. V. as shown in Figure 3. 1988). 2007). which appears more translucent and has reduced capsular polysaccharide (CPS) expression and thus decreased virulence in animal models (Yoshida et al. 1996. however. 2003). Wright et al. as well as numerous secreted molecules (proteases. a fulminating... Unlike the other vibrio pathogens. vulnificus hemolysin is unrelated to the hemolysins of V. hepatitis C. Bollous lesions associated with V.. A distinguishing characteristic of V. 1985. 1998. 2003).. Wright et al. 2001). Fan et al. phospholipases. 2005. This organism is the most common cause of serious wound infections associated with Vibrio species. vulnificus sepsis is the appearance of bollous lesions on the extremities.. etc. diabetes. 2005). hemochromatosis (iron overload). this phenotype may undergo spontaneous phase variation to an alternate colony type. 1988) (reviewed by Oliver [2005]). illnesses caused by V. and multiple organ failure.. 1996). as reviewed by Gulig et al. 2001). Simpson et al. and immune system dysfunction (Hlady and Klontz. vulnificus hemolysin is unlikely to be a virulence factor because inactivation of the vvhA gene encoding the hemolysin does not reduce virulence in mice (Wright and Morris. Production of bacterial iron-sequestering siderophores also contributes significantly to virulence (Litwin et al. referred to as repeats in toxin or RTX. V. vulnificus septicemia during a 20-day period (Ono. excess physiological iron as a consequence of hereditary hemochromatosis increases susceptibility to disease (Blake et al. Kim and Rhee. 1991. 2006b. This species is very much an opportunistic pathogen. Other reported virulence factors include flagella (Lee et al. 1979). wound infections may also lead to septic shock and death.

cholerae Since 1817 there have been at least seven pandemics of V. they commonly appear to coexist without damage to the host. algal blooms. V. It should also be noted that combined parameters of temperature. Motes et al. pH. the distribution of vibrios in the environment is likely to be determined by adaptations that evolved to allow coexistence with these hosts in marine ecosystems. Motes et al. fischeri. cholerae (Kaneko and Colwell. more recent epidemics in South America clearly implicate raw or undercooked seafood and shellfish. cholerae that have spread throughout the world (Lipp et al. cholerae is transmitted primarily through contaminated drinking water... thereby providing camouflage for the adult squid (Ruby. Presumably. ENVIRONMENTAL SOURCES: INCIDENCE IN THE ENVIRONMENT AND FOODS Vibrio species are found naturally in aquatic environments and are particularly evident in estuaries with temperate climates that support their preference for moderate salinity.. and salinity have interactive effects on growth and survival and vary with depth. 1982. and availability of invertebrate or vertebrate hosts. Kelly. Furthermore. Ruby and Lee. mollusks. competition with other bacterial flora. and neutral to alkaline pH (Tantillo et al. 1977). 1996. and crustaceans. larvae of bobtail squid (Euprymna scolopes) are colonized by V. but tolerances to extremes in temperature and salinity vary with species. ultraviolet irradiation.150 WRIGHT AND SCHNEIDER Figure 4. vulnificus showing opaque (right) and translucent (left) phenotypes associated with virulence and capsule expression.000 bacteria/g) that generally exceed their distribution in the water column (Tamplin et al. however. 1995).. the relationship of bacteria and squid is beneficial for both organisms. 1982).. Additional biological factors that are likely to influence the prevalence of these species in the environment include fluctuations in nutrient availability. In cholera-endemic regions of the world.. Thus. V. For example.. some pathogenic Vibrio species are recovered from oysters in high numbers (Ͼ1. 1973. more saline environments that generally do not support survival of V. responses to these parameters vary somewhat among species. 1998. Kelly.. Both natural and human-made disasters exacerbate outbreaks of cholera due to a breakdown of infrastructure for sewage disposal and water treatment and to delay of medical treatment. vulnificus or V. 1988. and minocycline combined with cefotzxime or fluoroquinolone is recommended (Chuang et al. warmer water temperature. 2004). parahaemolyticus can be recovered from colder. Tauxe et al. Improved understanding of the biology of Vibrio species supports the essential link of this bacterium to the aquatic environment as the primary reservoir of disease (Colwell et al. 1991. These bacteria grow optimally at temperatures between 22 and 42ЊC within a pH range of 5 to 11 for salinities ranging from 1 to 8% (Kaspar and Tamplin. vulnificus is not available. 1982). Although Vibrio species can be pathogens of fish. Wright et al. Kelly and Stroh. 1994). Sporadic cases reported in New Jersey and Florida were attributed to imported blue crab (CDC. Specific physical parameters greatly influence the environmental distribution of the different pathogenic Vibrio species. 1993. laying the foundations for the field of epidemiology and establishing principles of modern public health. 1998). V.. 1998). 2002). In general. 1996. Although cultural taboos often prohibit the consumption of shellfish in regions where cholera has been endemic historically. increase survival.. extremes in temperature and salinity may limit distribution of a particular Vibrio species in shellfish. and the development of a light organ in this squid is dependent on colonization by these bioluminescent bacteria. A vaccine against V. 1988. cholerae has a relatively low salinity requirement compared to . the numbers of all Vibrio species peak in warmer months and decline after extended exposure to cold temperatures. Thus. and even higher levels are recovered from the intestinal tracts of fish that feed upon shellfish (DePaola et al. Colony morphology of V. 1998). They also form intimate associations with marine plankton and mollusks through adaptations that enhance their environmental survival. V. Kelly and Stroh. In 1849 John Snow famously traced the source of a cholera outbreak in London to a tainted well.

1994). O139. 2007. cholerae serotype. (Reprinted from Nair et al. originated in the Bay of Bengal and spread to the Western Hemisphere (Nair et al. non-O139 are now observed. cholerae serotype. Epidemics generally follow the monsoon cycles that facilitate disease by extensive flooding that alters the local estuarine environment. 2002). most outbreaks occurred only in Japan and were generally associated with uncooked fish products (Fujino et al. However. cases were infrequent and often transmitted to humans via contaminated blue crabs (Molenda et al. cholerae. parahaemolyticus emerged in India and Indonesia in 1992. Bangladesh. In 1992 a newly emergent epidemic V. this Figure 5.. Genetic analyses indicated that this strain evolved from existing seventh pandemic O1 strains via acquisition of genes for encapsulation. and epidemic strains that are non-O1. V. This strain also produces a unique polysaccharide capsule (Waldor et al. 2007). and most environmental strains also lack genes for this receptor. its distribution includes freshwater ponds and river basins.. Global distribution of “pandemic” V.. However. Strains continue to evolve. The acquisition of the capsule probably facilitated environmental distribution and survival against host immune defenses. 1972). U. This emergent type was genetically and biologically distinct from strains recovered in prior epidemics. all epidemic cholera was caused by a single O1 V. Prior to 1992. most environmental isolates of V. parahaemolyticus strain designated serotype O3:K6 (Nair et al. cholerae may be facilitated during human passage as a consequence of the close proximity of bacteria and phage in the human intestinal tract.S. and therefore. Gene transfer requires that the bacteria express TCP as the receptor for phage.. as well as in South and Central American countries. parahaemolyticus also do not cause disease. parahaemolyticus disease. As shown in Figure 5. this profile changed as a “pandemic” strain of V. cholerae (Lipp et al. 1996). Strains isolated during these earlier outbreaks showed very diverse serotypes. Prior to 1992. and it appeared to result from clonal expansion of a single V. parahaemolyticus Similar to the epidemiology of V..CHAPTER 10 • PATHOGENIC VIBRIOS IN SEAFOOD 151 other pathogenic species. environmental isolates were serologically heterogenic and were collectively referred to as non-O1 or nonagglutinating strains. Genes encoding CT are carried by bacteriophage viruses that infect the bacterium and transfer the toxin genes to the bacterial genome to convert nontoxigenic strains to toxigenic variants (Waldor and Mekalanos. cholerae commonly lack the toxin genes (ctxA and ctxB) associated with epidemic disease and therefore are relatively avirulent. 1994). parahaemolyticus have been rapidly changing.) . and Indonesia.. This strain is now pandemic in India.. because they lack the genes for TDH and TRH toxins. 1953). the epidemiology and environmental distribution of V. and environmental conditions that enhance uptake of DNA are also likely to increase the prevalence of pathogenic V. Transformation to toxigenic V. Most environmental isolates of V.

Furthermore. Genetic studies confirm biotype 1 was more likely to be derived from human infection via oyster consumption. 1987. 1982). genetic analyses showed clinical strains are associated with specific genotypes that have relatively infrequent distribution in most environment isolates (Chatzidaki-Livanis et al. Preventative measures.. A survey of Galveston Bay found that “clinical” genotypes were seasonal and had wider distribution in late summer months. Unlike other pathogenic vibrios. Clams.. and Alabama. 2007. and strains now exhibit multiple serotypes due to genetic mutations affecting lipopolysaccharide and CPS genes.. Recent examination with a mouse model also showed that strains with the “clinical” genotype were significantly more associated with systemic disease compared to those with “environmental” types (P. 1982. After 2004. 1999).. 2004). As the “pandemic” strain spread throughout the world. parahaemolyticus was first observed in the Chesapeake Bay by Kaneko and Colwell (1973). when the number of cases is generally higher (Lin et al. it is unclear why this strain is so successful in its dissemination. 1998. Recent outbreaks of V. while 95% of Gulf Coast and 51% of mid-Atlantic samples were positive (Cook et al. Novotny et al.. and fish are rarely implicated in disease. vulnificus As with other vibrios. Tilton and Ryan. C. 2003).. Rosche et al. Investigation of the environmental distribution of this species is hampered by the relative scarcity of virulent phenotypes compared to avirulent phenotypes. as well as in Canada.. parahaemolyticus isolates were TDH positive. 1990). and these numbers are used to determine regulatory policy as to the acceptable levels of V.000. are being developed to ensure the survival of these animals and prevent transmission of disease to humans (Ravi et al. vulnificus is seasonal and related to water temperature. V. These data support the hypothesis that this “clinical” genotype may have increased virulence. However.. V. A survey of Mobile Bay. as these products are more likely to be cooked prior to consumption. vulnificus are almost always associated with raw oyster consumption and particularly oysters harvested from the Gulf Coast (Blake.. Genetic analysis of biotype 3 indicated that these strains were a genetic hybrid derived from recombination of the fish and oyster . the O3:K6 serotype subsequently diverged. Wright. 2005). V. 2003a). and Connecticut.152 WRIGHT AND SCHNEIDER disease now has much wider distribution in the Western Hemisphere and is largely associated with raw oyster consumption. Outbreaks have now occurred along the Pacific coast from Alaska to Chile. as levels in the midAtlantic oysters can be similar to those from the Gulf Coast (Wright et al. 2004. West Coast were negative for this bacterium. However. however. including probiotics and vaccines. This differential geographic distribution is likely to reflect lower water temperatures in Pacific and North Atlantic shellfish harvesting waters relative to the Gulf of Mexico. 1979). and most isolates recovered from oysters are virulent in animal models (DePaola et al. 2003b). Seafood-borne infections related to V. Risk assessment analyses estimated that the ratio of virulent to avirulent phenotypes was about 1:10.. 2005. while biotype 2 was specific for fish infections.. Nilsson et al. 2006a. V. parahaemolyticus in regions previously unaffected by this disease has followed global warming trends. Coincidental changes in the genetic background of clinical isolates may also contribute to greater capacity for environmental survival and/or enhanced ability to cause disease. Esteve-Gassent et al. distribution of V. biotype 3 was pathogenic in humans and transmitted by fish (Bisharat et al. 1994). outbreaks were also reported in California.. and their prevalence was inversely correlated with water temperature (DePaola et al. vulnificus in the Gulf Coast compared to that of Atlantic oysters is not entirely clear. Motes et al.. New Jersey. the reason for the greater disease incidence of V. with large outbreaks reported in the United States beginning in 1998 in Texas.. parahaemolyticus in commercially processed oysters. 2003). V. mussels. and the number of total V. Seasonal distribution of V. vulnificus is also a pathogen in marine fish and invertebrates and is a significant problem for aquacultured fish.08% of total V. New York. A. Fish pathogens are generally distinct from most human disease isolates and can be differentiated by biotype (Tison et al. vulnificus does not express a toxin marker equivalent to CT or TDH that is indicative of virulence. 2002).. showed that only 0.. 1996). vulnificus is generally not recovered from shellfish at temperatures below 18ЊC (Tamplin et al. but these strains still appear to be genetically related. Most (90%) oyster samples from the U. Cook.S. parahaemolyticus was isolated in samples from the Pacific Northwest only when temperatures exceeded 15ЊC (Kaysner et al. Gulig and A. parahaemolyticus generally showed linear increase with temperature. parahaemolyticus in Alaska also correlated with water temperatures exceeding 15ЊC (McLaughlin et al. AL. The emergence of epidemic V. unpublished data). Oregon.. and geographic disparity in distribution of more virulent strains could account for the predilection of disease coming from Gulf Coast oysters.

1999). 1982). Seasonal fluctuations in the environmental distribution of vibrios are likely to be aggravated by the progression of global warming. the oyster parasite Perkinsus marinus produces an immunosuppressant protease that was shown to decrease in vitro clearance of V.. 2002). Vibrios absorb to these chitinaceous organisms. and standard monitoring methods for fecal pollution do not prevent or predict vibrio contamination of seafood (Tamplin et al. 1996). oysters and clams are consumed frequently without processing or cooking. resulting in increased bacterial populations. Limiting growth is particularly critical for V. 2005). Reported cases of V.. 2005. vulnificus in oyster hemocytes (Tall et al.. in which intact muscle tissue is sterile. . Increased incidence of disease also corresponds historically to increased summer harvesting of oysters. Disease incidence dramatically increases when shellfish are harvested from water at higher temperatures. Both factors may work synergistically to increase vibrio distribution (Lipp et al. Oliver. (Data provided by Roberta Hammond. 1982). Furthermore.) genotypes (Bisharat et al. Unlike fish. A survey of Florida cases over recent years supports this trend toward increases in wound infections (Figure 6). which rose from 8% in 1970 to 30% in 1994 (Altekruse et al. Therefore. and 86% of vibrio-induced septicemia occurs from April until October (Hlady and Klontz. 2005) and during exposures resulting from Hurricane Katrina (CDC. molluscan shellfish are filter-feeding organisms. Vibrio species are ubiquitous in shellfish harvesting waters in summer months. Parasitic disease may debilitate oyster immunity. and algal blooms may influence the density of the vibrio load in oysters.. 1993.CHAPTER 10 • PATHOGENIC VIBRIOS IN SEAFOOD 153 Figure 6. Recent reports show increases in the number of cases associated with wounds compared to other sources. the most important intrinsic factor for vibrio-associated diseases is the initial load of bacteria in the shellfish.. they generally do not appear to have deleterious effects on oyster health. Finally.000 CFU/g in summer months. 1982). as populations may double within 8 to 9 minutes under optimum conditions (Joseph et al. Epidemiological data indicate that V.. oyster health may contribute to the vibrio burden of oysters. parahaemolyticus. Kelly. vulnificus in Florida from 1990 to 2005. 1997). For example. Other factors related to vibrio contamination of shellfish are algae or zooplankton that serve as the primary diet for mollusks. Recent trends suggest increased incidence of wound infections in Texas and Florida (Oliver. which not only increases water temperatures but also decreases salinities along coastlines. 2005). and their bacterial flora reflects the content of their environment. Optimum growth for pathogenic vibrios in oysters is between 30 and 37ºC (Kaspar and Tamplin. Extrinsic Factors Temperature is the primary parameter for controlling growth of Vibrio species in seafood products during storage and processing. as they are ingested with the plankton. parahaemolyticus disease incidence increases as temperatures approach 15ЊC. It should be noted that although vibrios are maintained at levels ranging from 100 to 10. INTRINSIC AND EXTRINSIC FACTORS: ROLE IN SURVIVAL AND GROWTH IN FOOD PRODUCTS Intrinsic Factors Most food-borne vibrio infections are caused by consumption of raw oysters.

may not thoroughly heat the entire oyster and result in survival of vibrios postcooking. 1960). Vibrio pathogens die or become nonculturable at refrigeration temperatures. 1991). with upper limits for growth temperature in V.5% NaCl (Kelly. Survival and growth of Vibrio species in seafood are also related to salinity. cholerae will grow in culture medium without additional salt. Thus. and numerous studies have shown that it is effective in inactivating bacteria (Cabelli and Haffernau. 1973). 2005). parahaemolyticus grows well at 8% NaCl. For example. and postharvest treatments (PHP) to ensure product safety.. 1961. The oyster industry responded to these guidelines by implementing pre. parahaemolyticus. UV light allows continuous dosage. vulnificus in oysters. and infections are very rarely associated with properly cooked seafood. 1991) and led to the establishment of the National Shellfish Sanitation Program (NSSP). but heat resistance increased at higher inocula and with addition of 7% NaCl. Survival of V.5% and growth is inhibited at 6. FOOD PROCESSING AND RECENT ADVANCES IN BIOLOGICAL. enteric bacteria. 1995).1% NaCl. Furthermore. vulnificus does not survive at 8. respectively). Implementation of these methods requires validation and verification that these treatments meet standards for reduction of vibrios in . 1965). 1991). the depuration process does not effectively remove Vibrio species from shellfish (Vasconcelos and Lee. viable but nonculturable bacteria retain virulence in animal models but with increased infectious dose (Oliver and Bockian.1 minutes. Some food processing. 1982. Tripp. Comparison of heat resistances for pathogenic vibrios showed that D55 values varied among species (Johnston and Brown. harvest restrictions. alginolyticus is differentiated from the other pathogens by the ability to grow in 10% NaCl. which has been practiced throughout the world for approximately 100 years (Canzonier. standard pasteurization conditions (70ЊC for 2 min) produced a Ͼ7-log reduction in all three species. The primary enrichment medium (alkaline peptone water) uses pH 8 to achieve selective growth of these species. V. However. Decimal reduction time was reduced significantly at 53ЊC for pH 5 to 6 compared to pH 7. strict time and temperature regulations have been established to limit exposure of seafood to elevated temperatures that will promote the growth of these species (NSSP. 1991) or by UV light (Tamplin and Capers. respectively. V. Depuration is the preferred method for removal of fecal coliforms. However.. parahaemolyticus and V. such as mild steaming. Unfortunately. vibrios appear to be firmly attached to the shellfish tissues and are not effectively removed by depuration. as vibrios persisted in shellfish for up to 96 hours of treatment at concentrations that were similar to those in untreated shellfish (Rodrick and Schneider. and viral pathogens in many countries and has a distinct advantage over other treatments in that it does not kill mollusks (Richards. Entry into a dormant “viable but nonculturable” state has been postulated (Xu et al. 1991). Kawabata and Harada. V. 1992) during recirculation into wet storage tanks (Furfari. vulnificus are both moderate halophiles and will not grow below 0.5 s. and V. while V. one of the oldest processing practices for molluscan shellfish sanitation was depuration. vulnificus at 44 and 42ЊC. 1971. The guidance document focused on consumer education.154 WRIGHT AND SCHNEIDER Consequently. Vibrios are fairly tolerant of high pH but will not grow below pH 6 (Beuchat. 2002). particularly purine and pyrimidines found in DNA. Cortelyou et al. Vibrios are also sensitive to elevated temperatures. parahaemolyticus (D55 of 1. 1972. parahaemolyticus and V. V. 1973). Kelly. but species differ greatly in their responses to salinity. Experimental evidence indicates that Vibrio species grown at refrigeration temperatures remain viable but cannot be recovered by standard culture methods. and lethal changes are directly related to the depth of penetration and amount of particulate matter (Huff et al. Colwell and Liston. In 2003 the FDA and the NSSP set forth guidelines intended to minimize consumer risk from naturally occurring V. Control methods have been evaluated and adjusted in an effort to reduce annual vibrio infections. parahaemolyticus at lower temperatures can increase with addition of NaCl or by adjusting the pH of growth medium (Beuchat.. these bacteria die at even mildly elevated temperatures (45 to 55ЊC). However. This application was driven by a series of typhoid fever outbreaks during the 1920s that were associated with the consumption of raw oysters (Canzonier. 1970). 1982). 1991). 1954. vulnificus and V.and postharvest controls aimed at reducing levels of V. 1990). 1969. 1960. The process involves the removal of potential pathogens by placement of shellfish in sanitized seawater that is usually treated either by ozonation (Schneider et al. All vibrios require some salt for growth. vulnificus and V.. CHEMICAL. Oliver et al... In contrast. Beuchat (1973) reported thermal resistance at 47ЊC (D47 values) ranging from 0. The bactericidal activity disrupts unsaturated bonds.75 min) was considerably more resistant compared to V. AND PHYSICAL INTERVENTIONS Historically. 1959) and viruses (Hill et al. cholerae (12 and 22.8 to 65. UV light irradiation is currently the most common depuration method in the United States and the United Kingdom.

Cook. Unfortunately. HPP Recent progress in the high-pressure processing (HPP) of foods in general. In shellfish areas with confirmed association with a vibrio outbreak. 2007). and survival at low temperature is also enhanced by more gradual rates of cooling (Bryan et al. Refrigeration of oysters is required within 10 hours of harvest in summer months. All Vibrio species die rapidly at temperatures exceeding 55ЊC (Johnston and Brown. or oysters are transferred to deeper. Thus. this study also found that ice immersion could increase the number of fecal coliforms in oysters.. Cook. Temperature controls are even more relevant for intertidal oysters. and/or freezing. harvesting during intertidal exposure is avoided. 2003). and oysters in particular. 1999).. therefore. and liquid nitrogen treatment was recently validated as an oyster PHP for reduction of V. Furthermore. refrigeration. 2000). even with an extended exposure of 10 to 15 days (Kaspar and Tamplin. Recent studies have shown the ability of freezing coupled with frozen storage as a means of reducing the number of recoverable . the allowable time between harvest and refrigeration is reduced. After prolonged exposure to low temperature storage.. suggests that this method can be applied to shellfish. vibrios can also become nondetectable on standard media but still be viable and in virulence (Baffone et al.. suggesting that this method should be used with caution. 1992). Heat treatment of oysters also improves shucking yields compared to untreated oysters and benefits both the consumer and the distributor. Treatment of oysters at 400 MPa for 10 minutes resulted in an about 5-log-unit reduction of total viable count. ultra-low temperature treatment (ϽϪ70ЊC) has been shown to effectively reduce vibrios when it was followed by extended frozen storage at Ϫ20ºC for 1 to 2 weeks.. Approved and validated treatments include high hydrostatic pressure. The use of HPP at 310 MPa caused the opening of the shell with 100% efficiency as the adductor muscle of the oyster was detached in the process (He et al. and produces a “selfshucking” product. These treatments are generally used in combination with approved transport and storage practices involving icing. but vibrios are not eliminated in oysters. Ultra-Low Temperature Treatment Recently. 1993. even following rapid decreases in temperature. Refrigeration Seafood held at ambient temperatures can support rapid growth of Vibrio species (Gooch et al. Ultra-low freezing can be achieved by immersion of shellstock in liquid nitrogen or CO2. 2002). Furthermore. Heat Treatment Heating is also an effective treatment for elimination of Vibrio species. growth was observed with food products at 9. H2S-producing organisms. vulnificus in oysters (Quevedo et al. pasteurization (heat shock). 2000). 2002. product with a significant bacterial load at harvest will still present increased risk to the consumer. and individual quick freezing. Immersion of oysters in ice prior to refrigeration results in greater reduction compared to simple refrigeration but also does not completely eliminate V. Maintenance of refrigerated or freezing temperatures during transport and storage is also regulated. 2002). Therefore. Vibrio content in oysters following nitrogen treatment and frozen storage was significantly reduced to levels that were below those specified by the NSSP (Wright et al.. cooler seawater prior to harvest.. lactic acid bacteria. both warm and cold temperature processing have been used in controlling and killing pathogenic Vibrio species in molluscan shellstock. 2007). Murphy and Oliver. 2005). Traditional pasteurization is also done at 75ЊC for 8 min and can reduce vibrio counts to safe levels for the consumer but produces some biochemical and sensory changes in the meats (Cruz-Romero et al. refrigerated storage results in only moderate bacterial reductions and is not appropriate for PHP that is aimed at risk reduction.5 to 10ЊC. as evidenced by the lack of disease association with cooked seafood products. Application of elevated pressure dramatically reduces the numbers of Vibrio species. 1994..CHAPTER 10 • PATHOGENIC VIBRIOS IN SEAFOOD 155 oysters. This treatment has the advantage of producing a high-quality product with extended shelf life. Heat shock treatment involves immersion in hot water (50ºC for 5 to 10 minutes) combined with frozen storage and achieves the desired reduction within 2 weeks (Andrews et al. and coliforms (Lopez-Caballero et al. as these oysters are exposed to ambient air temperatures and may warm to levels that promote growth of Vibrio species. depending on the process. 1994). Bacterial levels decline somewhat with refrigeration. vulnificus. The simplest and most common processing practice for all fish and shellfish handling is the immediate transfer of the product to ice or refrigerated storage. however. and lack of compliance with these recommendations may account for a significant proportion of disease. The rate of decline is inoculum dependent.. the time-temperature matrix varies with the ambient air temperature and infection risk.

length and method of relay.. These “beneficial” bacteria can outcompete pathogens. Irradiation The United States has lagged behind other countries in the application of food irradiation technologies partially due to the U. problems with seafood safety have been exacerbated by recent outbreaks of V. Increased incidence of V. vulnificus bacteria in Gulf Coast oysters (Parker et al. raw oysters.com). and Cosmetic Act of 1958. foodnavigator. California also requires all oysters harvested from Gulf Coast states to be treated during certain months. Applications are primarily against gram-positive bacteria (Lipinska. but this type of analysis is ineffective in preventing naturally occurring contamination of vibrios in shellfish. spices and vegetable seasoning. polluted waters to areas approved for harvesting and permitting the process of natural biological purification. In the 1980s.. the FDA approved irradiation as a safe and effective method of decreasing or eliminating harmful bacteria in herbs. vulnificus diseases (NSSP. parahaemolyticus. Various food additives have also been investigated but were not effective in live oysters (Sun and Oliver. parahaemolyticus disease necessitated expanding PHP programs to include Pacific Coast oysters. depuration and relaying to higher salinities present logistical and regulatory problems and could increase contamination by other more halophilic Vibrio species. Unfortunately. Unfortunately. Problems associated with relaying are that it is a labor-intensive process and that up to 50% of the original harvest can be lost (Furfari. parahaemolyticus cultures were less sensitive to irradiation compared to V. The rate of purging depends on the type of contaminant. a substantial number of shellfish consumers still prefer live. presumably because refrigerated temperature guidelines limit growth but do not eliminate vibrios in oysters. including oysters. vegetables. Unfortunately. 1994.000 bacteria/g.156 WRIGHT AND SCHNEIDER V. poultry. 2003). V. lack of activity against higher organisms. hemochromatosis (iron overload). these strategies did not completely eliminate disease mortality from V. Furthermore. and demonstration of effectiveness toward Listeria monocytogenes (Farber. which classified irradiation as a food additive. 1987). diabetes. or be lethal to potential pathogens.. Relaying The basic concept of relaying involves transferring shellfish from restricted. Furthermore. and a small number of cases are still reported annually. and India (http://www. Relaying also includes transporting oysters to areas with higher salinity in order to reduce levels of V. and various environmental factors. fruits. Food. Drug. Birkenhauer and Oliver. and grains (Morrison. the European Union. as normal shelf-life is maintained with no increase in mortality compared with untreated control oysters (Mallett et al. and new guidelines provide for monitoring oysters with recommended closures when V. water quality. or immune system dysfunction. limit their growth. 1993). and the bacteriocin nisin is now permitted in over 40 countries worldwide.S. but an exception to this rule is a class of antimicrobial molecules. PHP of oysters to reduce the numbers of vibrios has been mandated for Gulf Coast oysters. Filter-feeding shellfish can purge themselves of certain abiotic and biotic substances when transferred or “relayed” to relatively clean seawater. . PHP is now implemented for Ͼ25% of oyster shellstock from the Gulf Coast and requires a minimum reduction of 3. termed bacteriocins. Erdman and Tennant (1956) showed an average 85% reduction in the number of coliform bacteria in the soft-shell clam (Mya arenaria). Antibiotics are generally not added to foods. including the United Kingdom. Acceptance of these molecules as biological controls is a consequence of their natural presence in milk products. Probiotics or biological controls use microbiological products or microorganisms as food additives to reduce pathogen load. were not approved by the FDA until 2006. species of shellfish. 1986). However. vulnificus. 1979). such as cirrhosis. parahaemolyticus levels exceeded 10. and other foods. Oysters appear tolerant to irradiation processing at levels of a Ͻ2. Government Regulation Standard mitigation and monitoring strategies for shellfish were originally based on fecal coliform content. 2003). Meat. Russia.5-kGy absorbed dose. 1977). fresh. vulnificus (Motes et al.52 log most probable numbers (MPN)/g reduction in vibrio content in order to validate a treatment for commercial processing. 1998). higher levels of irradiation increased mortality and resulted in a yellow exudate and an unpalatable product. Food Additives and Biological Controls Although consumer markets for the treated seafood have been growing. 1994). currently accepted bacteriocins are not effective against gram-negative bacteria and thus will not clear pathogenic vibrios in oysters. Warning labels on oyster products and extensive educational programs targeted at-risk individuals with underlying diseases. that are produced by bacteria and are specifically lethal to other bacteria.

. and aeoromaonads are common isolates on TCBS (Cleland. 2005. Tarr et al. such as V. Unfortunately. also ferment sucrose and are common isolates on this agar. vulnificus are based upon a cytolysin gene. colistin. molecular methods are used for species identification because they improve specificity and sensitivity of detection. 2006). However. 1993).. all of these isolation media lack adequate specificity to reliably identify species. Therefore. 2004).gov/~ebam/bam-9.. TCBS is still used to isolate this species. 1998).cfsan. vulnificus. Additionally. Probes for V.0 with 1% peptone and 1 to 2% NaCl. parahaemolyticus and V. vulnificus on the basis of sucrose fermentation (Kelly. 1982. 1991) and cellobiose colistin agar (Hoi et al. in practice. a new chromogenic agar medium (CHROMagar Vibrio CV. A number of PCR methods are also used to amplify species. alginolyticus. 2003. Lee et al. In one study. fda. which are not useful for species identification. Clinical strains V. while removing inhibitors (Stevens and Jaykus. This medium differentiates V. Improved separation technologies may function to concentrate and increase sample size. 1986). usually alkaline peptone water at pH 8.or virulence-specific DNA (Brauns et al. V. Enumeration of vibrios generally begins with homogenization of fish or shellfish tissue . parahaemolyticus targets the gene for a thermal-labile hemolysin. TCBS can also inhibit recovery of Vibrio species from the environment compared to recovery on nonselective agars (Wright et al. cholerae from stool samples and utilizes alkaline pH (8. 2004. 1992). Therefore. and more recent methods include realtime detection and quantitative PCR (Panicker et al. 2004).. vulnificus do not ferment sucrose and therefore cannot be discriminated from each other on this medium. serotype-. Increasingly. parahaemolyticus and appears to be more sensitive and accurate than TCBS for primary isolation of this species (Hara-Kudo et al. is the FDA-specified method for vibrio identification (Kaysner and DePaola.. Paris. parahaemolyticus include modified cellobiose polymyxin B colistin agar (mCPC) (Tamplin et al. cholerae from V. Multiplex PCR analyses for simultaneous identification of multiple food-borne pathogens... however. vulnificus and V. Thiosulphate citrate bile salts sucrose (TCBS) agar was originally developed for isolation of pathogenic V. which yields typical yellow colonies surrounded by a yellow halo on this medium. and species-specific identification of V. 2004). 2003. Recently. including vibrios. 2004).. and cellobiose fermentation is used for differentiation of V. vulnificus and V.html) specifies two basic approaches for enumeration of vibrios: (i) MPN and (ii) colony counts determined by direct spread plating using DNA probe identification of colonies (Kaysner and DePaola. cholerae.. Unfortunately. have been reported (Wang et al. cholerae recognize genes for CT (Wright et al. V. while potentially pathogenic isolates are discriminated by probes detecting genes for TDH and TRH virulence-associated hemolysins.. Unfortunately other Vibrio species. 1991). 1998). 2001). and NaCl (1%) to suppress the growth of nontarget bacteria (Kobayashi et al. 1997.. ox bile. using alkaline phosphatase-labeled oligonucleotides. Species Identification Commercial formats. Campbell and Wright. growth in enrichment broth..CHAPTER 10 • PATHOGENIC VIBRIOS IN SEAFOOD 157 DETECTION METHODS FOR CONFIRMATION AND TRACE-BACK OF CONTAMINATED PRODUCTS Selective Differential Agar Media Several media are available to isolate Vibrio species. species identification cannot be determined upon primary isolation and requires additional testing for confirmation. and the phenotypic plasticity of these species frequently complicates their application. these assays are costly. Lyon. parahaemolyticus and V.. parahaemolyticus show more heterogeneous serotypes. 1985). and toxin-specific identification are also available but rely upon expressed antigen and may require cell processing for detection. 2003. Media recommended by the FDA to differentiate V. 49% of the bacteria isolated on TCBS were reported as not vibrio. are available for biochemical identification of vibrios.. Panicker and Bej. 1993). vvhA (Wright et al. CHROMagar Microbiology. France) was described for more effective recovery of V. Lipopolysaccharide antibody or “O” antigen has been used traditionally to identify and classify epidemic V. Blackstone et al. 2007. Peptide antibiotics.6). such as the API 20E and Biolog systems. Enumeration of Pathogenic Vibrios The Food and Drug Administration (FDA) Bacteriological Analytical Manual (http://www.. 2001). Karunasagar et al. parahaemolyticus generally is not recovered on these media. is often employed prior to plating (Kaysner and DePaola. Incubation at 40ЊC further inhibits nontarget bacteria and is important for maintaining the selectivity of mCPC. DNA probes for V. and/or polymyxin are used as selective agents. and confirmation by additional assays using antibodies or molecular probes is required. A nonradioactive DNA probe format. Brasher et al. Antibodies for species-. described above (Nordstrom et al. PCR reactions are often inhibited by food matrices and generally require strain isolation and some purification of the DNA template for optimum sensitivity.. 1963).

. Species-specific growth is indicated by isolation of selective agars (usually TCBS. Cohen. A. Interacting effects of pH. and S. offer more rapid analysis and increase assay sensitivity and specificity necessary to accurately detect low numbers of V. Krieg and J. and salt concentration on growth and survival of Vibrio parahaemolyticus. However. and G. Citterio. K. vulnificus organisms.. cholerae. B. Biosensors that recognize specific cellular targets or products. Evidence for the presence of a capsule in Vibrio vulnificus. 17:787–791. colistin agar. Pierfelici. as only viable cells were detected. Monitoring of this organism in shellfish is now common practice on the Pacific Coast. 516–550. shellfish monitoring should discriminate pathogenic vibrios in hours rather than days. Molecular techniques. Vibrionaceae. such as fecal coliform analysis. Amako.. bacterial. L. and that virulent strains are difficult to detect in the environment. 2005. and G. Several studies have shown that PCR. L. G. Emerging technologies for detection of Vibrio species include DNA microarrays. R. Low temperature pasteurization to reduce the risk of vibrio infections from raw shell-stock oysters. Quantitative or real-time PCR (QPCR) assays provide quantitative analysis while reducing the need for postassay processing. E. MPN is based on titration of homogenates into multiple enrichment cultures.. Wright et al. J. W. Okada. Int.. and spectroscopic methods. L. 1997. W. colony counts are derived from dilutions of homogenate that are spread plated on nonselective media. of Food Microbiol. D. Donelli. CONCLUSIONS Vibrios are potentially lethal food-borne pathogens that naturally inhabit estuaries and river basins. S. Recent side-by-side comparison of the standard FDA MPN analysis versus direct QPCR confirmation of positive MPN enrichment samples demonstrated that the QPCR-MPN assay had excellent correlation with the standard method and enhanced the sensitivity of the assay (Wright et al. Emerg. 130:2741–2743. Ideally. H. Donelli. Microbiol. CDC (2005) surveillance spanning from 1997 to 2004 documented a 78% increase in disease related to Vibrio species. monitoring is complicated by the facts that their presence does not correlate with conventional measurements of seafood safety. 2000. Alternatively.. 3:285–293. the formation rate of the PCR products is detected by increased fluorescence signal that corresponds directly to the DNA target concentration. However. ‘In vivo’ studies on the pathophysiological mechanism of Vibrio parahaemolyticus TDHϩ-induced secretion. P. Baumann. parahaemolyticus (CDC. Contam. have been employed to detect pathogens such as E. J. Casaroli.158 WRIGHT AND SCHNEIDER samples in phosphate-buffered saline. can reduce the limit of detection to 1 bacterium/g (Panicker and Bej. 2007). L. The perceived threat of diseases associated with these organisms has impacted tremendously the seafood industry. 1973. usually in alkaline peptone water. 2007). such as cell wall proteins. P. Emerging foodborne diseases. Baltimore. Bergey’s Manual of Systematic Bacteriology. L.. recovery of cells and subsequent culture are often preferable for confirmation or additional assessment (Tims and Lim. Swerdlow. Parks. These recent increases of vibrio infections in the United States are primarily a consequence of the number of cases related to outbreaks of V. The number of tubes with positive growth for each dilution is used to calculate a statistically valid assessment of the MPN in the sample. Greater understanding of the role of these bacteria in estuarine ecosystems and the risks associated with environmental. 89:31–39. 2004) and the genetic basis of endemic versus pandemic disease (Dziejman et al. and host factors is crucial for control and safety of seafood products. S. temperature. Andrews. 25:844–846. Blackstone et al. Casaroli.. and R. 2003). L. and Y. 1984. M. Vittoria. with confirmation by biochemical.. but molecular technologies can be combined with standard enrichment protocols to increase both sensitivity and sample size. coli O157:H7 (DeMarco et al. 38:133–137. In N. B. Evaluation of postharvest processed oysters disputed the possibility that dead cells may be enumerated by QPCR-MPN. REFERENCES Altekruse.. Citterio. Dis. 2007). R. Falzano. Microbiol. Infect. K. Microb. described above. when used in combination with MPN methodology. Appl. F. Gen. cholerae (Carter et al. In these assays.). Food Addit. MD. 1984. Campana. Both assays can be complicated by the overgrowth of competing nontarget organisms. W. Schubert. Miake. p. 1999) and V.. R. Williams & Wilkins. 2005. 2003. Sensitivity of QPCR assays is limited by the small sample volume size. Vittoria. or molecular identification of species.. R. Chen. Pathog. Campana. and D. biosensors. or mCPC). Family II. 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Juneja and J. Finland.O. Germany. however. 164 . 1999. 1999).. liver. 1989. Y. K. pseudotuberculosis is transmitted by the fecal-oral route. enterocolitica (Wren. Sofos © 2010 ASM Press. and outbreaks are uncommon. depending on the age of the person infected. pseudotuberculosis initially travel to the small intestine and bind to the intestinal epithelium of the terminal ileum. however. the specialized cells of the follicle-associated epithelia that function in antigen uptake.533 confirmed cases in the European Union. Ludwig-Maximilians University Munich.. especially in infants (Jones et al. Smego et al. pseudotuberculosis infection. Jalava et al. 2003).6. seroprevalence of anti-Yop antibodies among 750 healthy Austrians was shown to be 29. 2006). which indicates that the majority of infections are either subclinical or mild (Tomaso et al. 2004). pseudotuberculosis can cause a variety of symptoms. Of the 9. 00014 Helsinki University. These bacteria penetrate the intestinal mucosa through M cells. 1999).. The highest incidences of 14. Miia Lindström. Common symptoms are fever. often in schoolchildren. After penetration of the intestinal epithelium. enterocolitica. pseudotuberculosis than for Y. 1999). which is more typical for Y.. 2004. respectively. 2003). Enteropathogenic Yersinia can also disseminate from the intestinal tract to the major lymphatic organs. Due to several outbreaks. enteropathogenic Yersinia replicates within the local lymphoid follicles known as Peyer’s patches. Most human illness worldwide is caused by Y. Maria Fredriksson-Ahomaa • Institute of Hygiene and Technologie of Food of Animal Origin. the majority was found to be Y.7%. however. although diarrhea may persist for several weeks. DC Chapter 11 Yersinia enterocolitica and Yersinia pseudotuberculosis Maria Fredriksson-Ahomaa. 7. and diarrhea (Bottone. P. The linking of unnecessary appendectomies can help to identify a Yersinia outbreak (Nuorti et al. enterocolitica and Y. pseudotuberculosis has emerged as an outbreakassociated organism (Table 1). Yersiniosis caused by Y. 2004. D-85764 Oberschleissheim. mandatory notifications of yersiniosis were reported to have taken place in most of the member states of the European Union. pseudotuberculosis infection (Wren. In older children.Pathogens and Toxins in Foods: Challenges and Interventions Edited by V. Most reported cases of yersiniosis are sporadic. After ingestion. Sometimes diarrhea may be bloody and fever may be high. the incidence was only 1.. most infections are localized and self-limiting because the host’s inflammatory response is usually able to eliminate the invaders (Bottone. 2003). Schoenleutnerstrasse 8. Smego et al.2. Miia Lindström and Hannu Korkeala • Department of Food and Environmental Hygiene. Infections are acquired primarily through the gastrointestinal tract as a result of contaminated foods or water (Bottone. and lungs. Washington.. In recent years. 2006). University of Helsinki. Symptoms typically develop 4 to 7 days after exposure and may last 5 to 14 days. Diarrhea is a more dominant symptom of Y. 1999. enterocolitica and Y.6. abdominal pain. Infection with Y.. have occurred mainly in Finland and Japan (Tsubokura et al.000 inhabitants were observed in Lithuania. and Hannu Korkeala TYPE OF ILLNESS AND CHARACTERISTICS OF THE ORGANISM Yersiniosis Yersiniosis is an infectious disease caused by Yersinia. N. Sweden. food-borne yersiniosis being due to Yersinia enterocolitica or Yersinia pseudotuberculosis. Occasionally. like the spleen.7. enterocolitica and Y. In 2004. enterocolitica infections. while abdominal pain and fever are more frequent in Y. and 6. The bacteria can then spread to mesenteric lymph nodes. Unnecessary appendectomies have also been observed with yersiniosis patients. Box 66. and Germany. Outbreaks of Y. The minimal infective dose for humans has not been determined. only a few countries conduct national surveillance for Y.8 cases per 100. pseudotuberculosis is high in Finland (Figure 1). However. 1999). the incidence of Y. Y. In Austria. resulting in mesenteric lymphadenitis.. enterocolitica. pseudotuberculosis (EFSA. Nuorti et al. 12. Finland.. right-sided abdominal pain and fever can be the predominant symptoms and may be confused with appendicitis (Smego et al. 2006).

1999. Y. pseudotuberculosis and Y. like Y. Multilocus sequence analysis and DNA-DNA hybridization studies suggest that Y. Y. 2005). Y. and Y. chloramphenicol. 2003). pestis is transmitted by fleas or in aerosols. Y. (2003). 2003). kristensenii (Sprague and Neubauer. pseudotuberculosis. 1999. (2006) typically among adults. rohdei. 2005). (1989) Hallanvuo et al. aldovae.. Y. pseudotuberculosis diverged within the last 200 million years and that Y. like systemic infection and bacteremia. co-trimoxazole. aleksiciae. Nuorti et al. enterocolitica. enterocolitica. Y. enterocolitica (Wren. In more severe infections. A number of antimicrobial agents are active in vitro against enteropathogenic Yersinia strains isolated from human and nonhuman sources. PSEUDOTUBERCULOSIS 165 Table 1.000 years (Achtman et al. the third-generation cephalosporins.7 Mb) (Thomson et al. (1989) Tsubokura et al.. Reported Yersinia pseudotuberculosis outbreaks Yr 1977 1981 1981 1982 1984 1984 1984 1985–86 1987 1997 1998 1998−2000 2001 2003 Country Japan Finland Japan Japan Japan Japan Japan Japan Finland Finland Canada Finland Finland Finland Location Kindergarten School School Mountain area School Mountain area Restaurant School School School Household School.ktl. and 3-4/O:3. Number of yersiniosis cases in Finland from 1995 to 2006 (http://www3. Y.. The reactive arthritis is associated with the presence of the HLA-B27 antigen. O:3 O:1 No. Y.6. and fluoroquinolones (Stock and Wiedemann. tetracyclines. pseudotuberculosis is an enteric pathogen that causes gastroenteritis through oral infection. aleksiciae. pestis. non-spore-forming rods or coccobacilli. Sprague and Neubauer. The genome of these pathogens is very similar in size (4. antimicrobials may be useful. 12 species: Y. Y. 1989. enterocolitica and Y. respectively. 2003). 1999. (2004) Jalava et al. ENTEROCOLITICA AND Y. The newest species. pestis. oxidase-negative. most commonly in the knees. pestis is 47. The G+C content of Y. Y.3 and 47.CHAPTER 11 • Y. pseudotuberculosis. (1989) Tsubokura et al. Y. has recently been separated from Y. complications such as joint pain (reactive arthritis) and skin rash (erythema nodosus) may occur.500 to 20. Genome sequence data confirm that Y. (1989) Nakano et al. 2006). catalase-positive. cafeteria School School Serotype O:1b O:3 O:5a O:4b O:3 O:4b O:5a O:4b O:1a O:3 O:1b O:3 O:1. mollaretii. 2-3/O:9. Hannu et al. Y. Y. enterocolitica and of Y. 2-3/O:5. ruckeri (Bottone. enterocolitica is heterogeneous in its biochemical and antigenic properties. The genus Yersinia is a group of gram-negative. pseudotuberculosis are closely related. with DNA sequence homology of nearly 97%. which are facultative anaerobes and have the ability to grow at refrigeration temperatures. which is lower than that of most members of the family Enterobacteriaceae. while Y. The vast majority of clinical isolates belong to bioserotypes 1B/O:8. The joint pains. (1989) Tertti et al. pseudotuberculosis that has emerged within the last 1. These include aminoglycosides. (1984) Tsubokura et al. frederiksenii.6 to 4. pestis and Y. Strains of bioserotype 1B/O:8 have been found mostly in the United States and . Characteristics of Y. These bioserotypes have been shown to have different geographical distributions (Table 2). Baumgartner et al. (1999) Hallanvuo et al. kristensenii. ankles. Y. Uncomplicated cases of diarrhea usually resolve on their own without antimicrobial treatment. bercovieri. (1989) Tsubokura et al. enterocolitica and Y. pestis is a clone of Y.27. Three species.. are pathogenic for humans. However. (2003) Nowgesic et al. (1989) Tertti et al. 2006). or wrists. (2004) Jalava et al.fi/stat/). intermedia. Y. of cases 82 19 188 140 63 11 39 609 34 36 74 122 89 76 Source Water? Unknown Vegetable? Water Unknown Water Barbecue? Unknown Unknown Vegetables? Milk Iceberg lettuce Iceberg lettuce Carrot Reference(s) Tsubokura et al. and Y. (1989) Tsubokura et al. enterocolitica. Wren.. usually develop within 1 week to 1 month after the initial infection and generally resolve after 1 to 6 months (Granfors et al. pseudotuberculosis The genus Yersinia is a member of the family Enterobacteriaceae and is composed of at least Figure 1. whereas both share only about 50% homology with Y.

serum resistance Attachment. monkey Hare. Ecological and geographical distribution of most common serotypes of Yersinia enterocolitica and Yersinia pseudotuberculosis Species Y. 1990).. pseudotuberculosis are serotypes O:1 to O:5. YadA (Yersinia adhesin A). Ail. 2001). This pYV codes for an outer membrane protein. wild animals Geographical distribution North America. . pseudotuberculosis (Table 3). and citrate. Australia Canada. invasion Serum resistance Fluid secretion in intestine Stimulation of T cells Iron uptake Y. With Ysc. At least two chromosomal genes. Japan. enterocolitica belonging to biotype 1B and Table 3. pseudotuberculosis Serotype(s) O:8 O:5. Italy. rabbit Human.3 O:1–O:3 O:4 and O:5 Hosts Human. but not of Y. enterocolitica and Y. however. pig. Strains that are largely responsible for human yersiniosis in Europe. Virulence factors of Yersinia enterocolitica and Yersinia pseudotuberculosis Genomic origin Plasmid Chromosome Gene ysc-yop yadA inv ail ystA ypm irp a Determinant Yops YadA Invasin Ail YstA YPM HPI Function Resistance to phagocytosis Attachment. The inv gene. pseudotuberculosis strains are considered to be pathogenic. Germany. Among Y. Table 2. a set of secreted proteins called Yops (Yersinia outer membrane proteins). pig. which is homologous for Y. pig. The yst gene in the chromosome of Y. Japan Australia. enterocolitica. 1997). sheep. Yops protect Yersinia from the macrophage by destroying the phagocytic and signaling capacities of the macrophage and finally by inducing apoptosis. The plasmid alone is not sufficient for the full expression of virulence in Yersinia.166 FREDRIKSSON-AHOMAA ET AL. pig. 1995). pseudotuberculosis strains there is little variation in biochemical reactions. Virulence Factors of Y. cat. rat. Yersiniabactin is a siderophore. pseudotuberculosis Several virulence factors have been identified that are common to the pathogenic Y. Far East Far East Canada and only seldom in Japan and Europe. goat. invasion. Canada. pig. cattle. that is essential for bacterial replication in host tissue (Cornelis et al. The ail gene. France. Strains belonging to biotype 1A are considered to be nonpathogenic. The most common serotypes of Y. sheep. dog. enterocolitica and Y. 2005). rat Chinchilla Human. pseudotuberculosis. 1999). dog. which is highly correlated with virulence. which can be used to divide the species into four biotypes (Tsubokura and Aleksic. Europe. goat. are needed for mammalian cell invasion. which promotes serum resistance in Y. wild animals Human. pseudotuberculosis. encodes a heat-stable enterotoxin. Strains of bioserotype 3/O:3 have been recovered mostly in Japan and China (Fukushima et al. except with the sugars melibiose and raffinose. which differ in their geographical distribution (Table 2). 2001). Japan Europe. 1995). enterocolitica Biotype(s) 1B 2 and 3 2 and 3 3 3 and 4 5 Y. and their secretion apparatus called Ysc (Yop secretion). Yst (Yersinia stable toxin) (Delor et al. dog. codes for a surface protein. enterocolitica + + + + + + − a + Y.27 O:9 O:1. an outer membrane protein. Europe.. which appears to play a vital role in promoting entry into epithelial cells of the ileum during the initial stage of infection (Isberg and Barnes. pseudotuberculosis (Miller et al. Australia. enterocolitica and Y. Y.. and the United States belong to bioserotype 4/O:3 (Bottone. extracellularly located Yersinia cells that are in close contact with the eukaryotic cell deliver Yops into the cytosol of the target cell. pseudotuberculosis strains can be classified into 21 serotypes (Tsubokura and Aleksic. enterocolitica and Y. which is synthesized by Y. United States All continents Europe. 1998). sheep Human. strains of this biotype have constituted quite a large fraction of strains from patients with gastroenteritis and may act as opportunistic pathogens (Pujol and Bliska. pets.3 O:3 O:2. All pathogenic Yersinia species harbor an approximately 70-kb plasmid. invasion Attachment. All Y. North America Europe. cat. pig. pets. Japan.2. termed pYV (plasmid for Yersinia virulence). The virF gene is an important activator of the production of Yops. Canada. codes for Inv. pseudotuberculosis + + + − + − + + Strains belonging to biotype 1B.. inv and ail. rodent Human.

domestic pets. Serotypes O:3 and O:8 have sporadically been isolated from captive monkeys (Iwata et al. High-pathogenicity-islandpositive Y. enterocolitica O:9 has frequently been isolated from stools of cattle and goats in France (Gourdon et al. Several studies have been conducted to isolate Y. and nonpathogenic Yersinia enterocolitica strains belonging to the most common bioserotypes. 1999).. and experimental animals to wild and captive animals (Table 4). In Japan.. 1985. Fredriksson-Ahomaa et al. serotypes O:5. pseudotuberculosis can also synthesize a superantigenic toxin. Niskanen et al. These bacteria have been recovered from diverse animal sources. The highest prevalence of pathogenic Y. such as cold and humid weather or starvation. 2004). Most animals are healthy carriers. In contrast. serotypes O:3. The frequency of YPM-producing strains has been shown to be higher in the Far East than in Europe. pseudotuberculosis-derived mitogen). there was a trend of increasing prevalence as pigs mature. where systemic symptoms in infected patients were less common than those in the Far East (Fukushima et al.. enterocolitica and Y. Bowman et al. 2001). SOURCES AND PREVALENCE IN THE ENVIRONMENT AND FOODS Animal Reservoirs of Y. ranging from farm animals. enterocolitica and Y. and serotypes O:3 and O:5. In the United Kingdom. pseudotuberculosis and. and sows. 1995). 2008). 2007. 2006). 2006). sources of human infections. hence.CHAPTER 11 • Y. Pathogenic Y. enterocolitica has been observed with pig tonsils. O:8. pseudotuberculosis infection among captive monkeys have been reported in Japan (Kageyama et al. pseudotuberculosis at the farm level for pigs (Gürtler et al. Jerret et al. This serotype has a worldwide distribution in the pig population and also has been shown to be the most common serotype in the United States (Bhaduri and Wesley. .27 and O:9 are common in pigs. weaned pigs. Some studies have been conducted to identify the primary reservoir of Y. 2005. (1990) reported that Y.. enterocolitica bacteria have frequently been isolated only from slaughtered pigs. Y. pets... Y. low-. with serotype O:3 being the most common (Fredriksson-Ahomaa and Korkeala. Y. but they may become ill and excrete the bacteria under stress. PSEUDOTUBERCULOSIS 167 Figure 2. pseudotuberculosis Animals have long been suspected of being reservoirs for pathogenic Y. and serotype O:5. pseudotuberculosis is one of the most common infectious causes of death among farmed deer in Australia. The highest prevalence was observed among fattening pigs.. and domestic animals (especially pigs and ruminants). 2002).27 in cattle and sheep (McNally et al. pseudotuberculosis from farm animals. enterocolitica and Y. pseudotuberculosis only (Figure 2). YPM (Y. and O:9 have been isolated from wild rodents. FredrikssonAhomaa et al. 2003a). the lowest prevalence was found mostly among suckling pigs. 2007.. enterocolitica strains are also called highpathogenic strains because they can cause lethal infections in mice. the seroprevalence of anti-Yop antibodies has been shown to be high in cattle (76%) and goats (70%) in Germany (Tomaso et al. but the principal reservoir hosts are believed to be rodents. wild birds.... and wild animals (Table 5). Several outbreaks of Y. In all studies.. The host range is broad. 2007b). However. 2001c).. ENTEROCOLITICA AND Y. 1999). especially from field mice (Hayashidani et al. Nesbakken et al. 2005. The yersiniabactin genes are clustered within a region of the chromosome referred to as a high-pathogenicity island (Carniel.. Distribution of different virulence determinants among high-.27 from dogs and cats (Fukushima et al.

Fredriksson-Ahomaa et al. enterocolitica positive using PCR but none with culturing..27 O:1. enterocolitica in pigs (Fredriksson-Ahomaa et al.. 2000a. the detection rate was shown to be clearly higher with PCR than with culture methods. (1988) Slee and Skilbeck (1992) Fukushima et al. The tonsils remained positive for Y. which explains the high prevalence of this pathogen in the tonsils of slaughter pigs. O:5. sheep. (1985) Fukushima et al. lungs.. (2001) found 40% of the mesenteric lymph nodes of pigs Y. enterocolitica strains belonging to pathogenic bioserotypes have rarely been isolated from the environment. diaphragm. esophagus.200 210 2. 2004. enterocolitica and Y. The Environment Contaminated with Y. and no antibodies against Y. (2003) Jerret et al. Table 4. of positive samples 52 8 185 21 16 4 2 3 29 8 2 4 9 4 4 % Positive 4 4 7 5 6 1 1 1 18 4 1 3 8 3 2 Country Japan Finland Australia Australia Japan Japan Japan Sweden Australia Japan Japan China China China Japan Reference Fukushima et al. O:8 O:3. (1990) Fukushima and Gomyoda (1991) Fukushima and Gomyoda (1991) Zheng et al. Bhaduri et al. 2000b. Additionally. Pathogenic Y. rabbit Rodent Monkey Y.. (1989) Niskanen et al. This pathogen was detected on the brisket saw. O:5. (1985) Fukushima and Gomyoda (1991) Niskanen et al.3 Y.2. 2004). O:8. 2007a). (2007) found 9% of the gestating sows positive but none of the farrowing sows. Nesbakken et al. enterocolitica was isolated from the air in the Table 5. Boyapalle et al. enterocolitica could be isolated from only 35% of the tonsils (Fredriksson-Ahomaa et al. strains of bioserotype 4/O:3 have occasionally been isolated from the environment in slaughterhouses and butcher shops (Nesbakken. PCR has been applied in only a few studies investigating the prevalence of pathogenic Y. O:9 O:3. (1995) Zheng et al.. pseudotuberculosis Y. Animal reservoirs for Yersinia enterocolitica and Yersinia pseudotuberculosis Reservoir type Farm animals Pets Wild animals Animal source Pig Cattle. Bowman et al. enterocolitica in 12% of fecal samples in the United States using PCR compared to 4% using a culture method. goat Dog. cat Chinchilla Bird Deer Hare. O:9 O:5. O:2 O:1–O:3 O. Prevalence of Yersinia pseudotuberculosis in animal sources Source Pig Cattle Sheep Dog Cat Bird Deer Hare Rabbit Mouse Rat No. trachea.. 2001.1–O:5 O:1–O:5 O:1–O:3 Zoo animals O:2.639 449 252 318 259 468 153 215 139 148 107 148 237 No. and the handle of the coffee maker used by slaughterhouse workers. (1995) Kageyama et al. and kidneys) hangs. pathogenic Y. (2005) detected pathogenic Y. aprons used by trimming workers. (1995) Zheng et al.27. enterocolitica has been detected by PCR on a variety of environmental sources in a Finnish slaughterhouse (Fredriksson-Ahomaa et al. 1988. However. liver. enterocolitica can be measured. Bhaduri et al.. Korte et al. enterocolitica during the last weeks of the lives of fattening pigs. heart. O:8.168 FREDRIKSSON-AHOMAA ET AL. (2002) Slee et al. enterocolitica negative in both the tonsils and feces. the computer keyboard used in the meat inspection area. (2007) have shown that piglets under 2 months of age are Y. 2007a. O:9 O:3. Boyapalle et al. 2005). 2000b). Most (88%) of the pig tonsils were shown to be positive with PCR when pathogenic Y.3. The young pigs became carriers in the tonsils and feces when they were about 60 to 80 days old and seropositive shortly thereafter. (2002) . evisceration knife.27. the hook from which the pluck set (tongue. In these studies. pseudotuberculosis O:1–O:4 O:1–O:3 O:1–O:5 O:3 O:1. enterocolitica O:3. O:8 however. of samples 1.

at a considerably high rate (Tsubokura et al. Germany. Fredriksson-Ahomaa et al. but Lee et al. Transmission is likely to occur more often via cross-contamination of cooked pork or foods not normally harboring Y. Ostroff et al. In epidemiological studies. Consumption of raw pork may play an important role in countries like Belgium.. including pig tongues. with some epidemiological studies linking consumption of uncooked or undercooked pork (Tauxe et al. etc.27 (Falcao et al.. Jalava et al. Lambertz et al. Vishnubhatla et al. Jones et al. Y. Y. enterocolitica in ground beef.. 1994). Y. pseudotuberculosis. Foods Contaminated with Y.. pseudotuberculosis has been isolated from fresh water. Satterthwaite et al. hearts. vegetables. enterocolitica in fermented pork sausages by use of PCR. (1990) reported Y. Direct person-to-person contact has not been demonstrated. Lee et al. In Korea.. The detection rate of pathogenic Y. enterocolitica O:8 strains have been isolated from stream water (Iwata et al. 1997).27 in Japan in pork (Logue et al. Pathogenic Y. although pathogenic isolates have seldom been recovered from food samples. 2003b).. where it can survive for a long time. pork and pork products have been implicated as the major source of human Y. Ostroff et al.27 and 2/O:9 have sporadically been found in the United Kingdom. 2005). and 3/O:3 and 3/O:5. Sandery et al. enterocolitica and Y. but this pathogen has been detected in 3% of lettuce samples with PCR (Fredriksson-Ahomaa and Korkeala. 1987. pathogenic strains have only sporadically been isolated from beef. No pathogenic Y. and birds (Fukushima et al.. pseudotuberculosis is widely spread in the environment (soil. enterocolitica O:3 infections have been associated with eating raw or undercooked pork within 2 weeks before onset (Tauxe et al. 2006). The environment can be contaminated by the feces of infected animals... 2000. such as river. In the United States. untreated drinking water has been reported to be a risk factor for sporadic Y. where raw minced pork with pepper and onion is a delicacy that can be purchased in ready-to-eat form from butcher shops. Another transmission route may be from person to person... FredrikssonAhomaa and Korkeala. enterocolitica can frequently be detected in environmental waters. 2006b). 1989). PSEUDOTUBERCULOSIS 169 bleeding area. 2001b. natural water and sewage have shown to be sporadically contaminated with pathogenic Y. enterocolitica O:3 infections in infants who were probably exposed to infection by their caretakers. enterocolitica has been detected in fish and chicken. The only pathogenic bioserotype found in northern Europe and Germany is 4/O:3 (Johannessen et al. enterocolitica and Y. however. enterocolitica O:3 infections have been associated with the household preparation of chitterlings (intestines of pigs. 1996. enterocolitica 4/O:3 has also been isolated occasionally from water (Thompson and Gravel.. pseudotuberculosis to Humans The primary transmission route of human yersiniosis is proposed to be fecal-oral via contaminated food (Figure 3). Bioserotypes 2/O:5. Y. 1986).. pseudotuberculosis. mainly wild animals.. which are a traditional dish in the southern United States) (Lee et al. and The Netherlands. (2001) detected a high prevalence of yst-positive Y. 1998). Y. Y. Transmission of Y.). and from chitterlings (Table 6). (2004) isolated one ail-positive Y. enterocolitica strain of bioserotype 3/O:3 from 673 ready-to-eat vegetables. In particular. Fukushima et al.. Indirect person-to-person transmission has apparently occurred in several instances by transfusion . Raw pork products have been investigated widely due to the association between Y. 1999. 1990. enterocolitica in foods has been shown to be clearly higher by PCR than by culturing (Fredriksson-Ahomaa and Korkeala. and kidneys.. enterocolitica belonging to serotype O:5. This pathogen has sporadically been isolated from pork in Japan (Tsubokura et al. 2005). 2003).CHAPTER 11 • Y. (2007) reported recently a relatively high prevalence (11%) of pathogenic Y. enterocolitica and Y. 2006). 1997). enterocolitica. PCR-positive samples were also obtained from the floor in the eviscerating and weighing areas and from the table in the meat-cutting area.. livers. 2003a). Y.. 2003b. which can be direct or indirect. water. (1996) have shown with PCR that pathogenic strains of Y. enterocolitica infection. and mountain stream water. well. the epidemic strain was also found on the soil and on the production line (washing and peeling equipment) at the farm of origin (Jalava et al. FredrikssonAhomaa et al.. pseudotuberculosis has been isolated from iceberg lettuce and raw carrots implicated in food-borne outbreaks in Finland (Nuorti et al.. 1987. In Japan. 1994. rodents. 2004). This may happen when basic hygiene and hand-washing habits are inadequate. 2001a. enterocolitica infections in Norway (Ostroff et al. pseudotuberculosis Food has often been suggested to be the main source of Y. Fukushima et al. The highest detection rate has been obtained from pig offal.. 2004.. 1994). In a large point source outbreak caused by raw carrots contaminated by Y. In Brazil. ENTEROCOLITICA AND Y. Thisted Lambertz and Danielsson-Tham. 1989. like deer. pseudotuberculosis has very rarely been isolated from foods. In a case-control study. enterocolitica and pigs.

however. (2001) Boyapalle et al. Y. washing and peeling equipment was shown to be contaminated with the outbreak strain. enterocolitica may be transmitted to humans indirectly from pork and offal via dogs and cats (Fredriksson-Ahomaa et al. of culture-positive a samples (%) 7 (47) 79 (80) 38 (35) 8 (2) 0 32 (32) 23 (23) 4 (2) 5 (5) 6 (2) 6 (7) 0 0 0 0 0 0 No. (2000) Thisted Lambertz and Danielsson-Tham (2005) Fredriksson-Ahomaa et al.170 FREDRIKSSON-AHOMAA ET AL. In these cases. (2007a) Lambertz et al. pseudotuberculosis is also a food-borne pathogen. (2001) Vishnubhatla et al. however. Elevated serum antibody concentrations have been found among people involved in swine breeding or pork production (Seuri and Granfors. Liver. of PCR-positive samples (%) 10 (67) 82 (83) 77 (70) 278 (79) 133 (38) 47 (47) 31 (31) 63 (25) 35 (35) 50 (17) 9 (10) 9 (6) 11 (11) 0 0 3 (3) 6 (12) Reference Vishnubhatla et al. a common risk for pig farmers and slaughterhouse workers. the transmission routes are unclear. (2001) Chicken Fish Lettuce Tofu a b c Pathogenicity of isolates confirmed. 2001c). (2001) Fredriksson-Ahomaa and Korkeala (2003b) Fredriksson-Ahomaa and Korkeala (2003b) Boyapalle et al.. 2006). Except pig tongues and offal. Jalava et al. (2001) Vishnubhatla et al. fresh produce and untreated surface water have been possible infection sources. (2007) Fredriksson-Ahomaa and Korkeala (2003b) Fredriksson-Ahomaa and Korkeala (2003b) Fredriksson-Ahomaa and Korkeala (2003b) Vishnubhatla et al. and kidney. of contaminated blood products (Bottone. Pathogenic Y. heart. In the reported outbreaks. (2007) Johannessen et al. may also be a transmission route. the Figure 3. but this pathogen has rarely been isolated from foods implicated in the illness. Y. of samples 15 99 110 350 350 100 100 255 100 300 91 150 97 43 200 101 50 No. Some recent epidemiologic investigations in Finland have linked outbreaks of Y. the most likely source of Yersinia has been blood donors with subclinical bacteremia. especially young children. In the outbreak linked to carrots. pseudotuberculosis occurs in water and in the environment and has been isolated from various animals. 1999). Transmission of Yersinia enterocolitica and Yersinia pseudotuberculosis to humans.. . Table 6.. (2001) Fredriksson-Ahomaa and Korkeala (2003b) Lambertz et al. pseudotuberculosis to domestically grown iceberg lettuce and carrots (Nuorti et al. Prevalence of pathogenic Yersinia enterocolitica in foods with PCR and culture methods Sample Pig tongues Pig offal Chitterling Ground pork Ground beef Minced pork Pork c b No. 1992). 2004. Direct contact with pigs. Pet animals have also been suspected as being sources of human yersiniosis through close contact with humans.

enterocolitica on pork can reach 10 CFU per cm after 5 days at 10°C (Nissen et al. the bacterium is able to grow in 5% NaCl.58 obtained with a concentration greater than 0. 2006). 1998). 2002. However. induced by any temperature downshift of 10°C or more. Extrinsic Factors The most important extrinsic factors include temperature and gas atmosphere. In some Finnish Y. thus having the ability to grow at refrigerator temperatures. a nitrite concentration of only 80 mg/kg has been reported to inhibit the growth of Y. Y. While 7% or more of NaCl is bacteriostatic (RobinsBrowne.. enterocolitica to low temperature is dominated by mechanisms involved in biodegradative metabolism (Bresolin et al. With citric acid. pseudotuberculosis could have been transmitted from infected animals to the lettuce and irrigation water by feces at the farm before distribution (Nuorti et al. even after repeated freezing and thawing (Toora et al.16% (vol/vol) (Karapinar and Gonul.. pseudotuberculosis outbreaks detected in spring. Bae et al. A combination of direct contact with wildlife feces during storage and cross-contamination of the equipment was the most likely contributing factor. 1992). ENTEROCOLITICA AND Y. 2000). 2000). Although Yersinia can grow at temperatures as low as 0°C. Unchlorinated drinking water from wells. iceberg lettuce was probably contaminated with irrigation water. 2006). The early and mid-exponential growth of Y. the organism grows much more slowly as temperatures drop below 5°C (Goverde et al. Goverde et al. 2006). growth was detected even at a pH of 3. 2006). Y. at the levels most commonly present in foods. enterocolitica at low temperature involves the activation of genes encoding environmental sensors and regulators related to signal transduction (Bresolin et al.31% (vol/vol). An epidemiological study has shown evidence that Y. salt alone will not prevent growth. 1992). pseudotuberculosis was transmitted from infected pets to humans in a familial outbreak in Japan (Fukushima et al. enterocolitica in fermented sausages (Asplund et al. Membrane rigidity and fluidity are maintained by changing the membrane composition.81 obtained with a concentration of 0. As in most bacteria.. At the acclimation phase. while acetic acid inhibited the growth at pH 5. 1994. mRNA decay. In Korea. Thus. the acute cold shock response. This bacterium is also able to multiply over a wide pH range from approximately pH 4 to 10. pseudotuberculosis infection (Han et al. with an optimum pH of around 7. as only few foods have an alkaline pH. Nutritionally. 1997) for Yersinia. Chattopadhyay. 2006) are induced. INTRINSIC AND EXTRINSIC FACTORS THAT AFFECT SURVIVAL AND GROWTH IN FOOD PRODUCTS AND CONTRIBUTE TO OUTBREAKS Intrinsic Factors The most important intrinsic factors are nutrition. 1992). springs. It has been shown that the number of 9 2 Y. .. Yersinia is generally considered to be sensitive to low pH conditions. the tolerance of a high pH is relatively unimportant. 1994). and water activity. 2004).. PSEUDOTUBERCULOSIS 171 exact mechanism of contamination of carrots on the farm could not be clarified (Jalava et al. unsaturated fatty acids and phospholipids predominate.. catabolic events... The late exponential and early stationary phase of adaptation of Y. the cold shock response in yersiniae is featured by the expression of cold shock proteins (Csp). 1993).CHAPTER 11 • Y. and other preservative hurdles are required. However. Yersinia can withstand freezing and can survive in frozen foods for extended periods.. In another outbreak. The doubling time at the optimum growth temperature (approximately 28 to 30°C) is around 34 min (Schiemann... 1994.6. Yersinia is unusual among pathogenic enterobacteria in being psychrotrophic. At low temperature.. utilization of endogenous resources. At the cellular level. 1989). Citric acid is less inhibitory than acetic acid. Yersinia is not a fastidious organism.. enterocolitica can tolerate both sodium nitrate and nitrite of up to 20 mg/ml for 48 h in vitro (De Giusti and De Vito.. while saturated and cyclic fatty acid contents become a majority upon a rising temperature (Nagamachi et al. flagellar synthesis and chemotaxis (Bresolin et al. and longer-term adaptation to low temperature involve different mechanisms (Bresolin et al. untreated mountain spring water could be linked to Y.. Harrison et al. and termination of transcription (Ermolenko and Makhatadze. and defense against oxidative stress. a well-known phenomenon in many pathogens exposed to temperatures outside of the mammalian host. Csp also bind mRNA and regulate ribosomal translation. Csp bind single-stranded nucleic acids and might thus play a role as RNA chaperones and transcription antiterminators (Jones and Inouye. Also. the production of Csp declines... 2001). 2003). 2006). 1991. 1998). pH. and streams contaminated with feces of wild animals is considered an important transmission route in mountainous areas in Japan (Fukushima et al. Salt tolerance of Yersinia is moderate and strongly dependent on storage temperature. the vehicles have been vegetables of the crop stored at chilled temperature from a previous year.

and kidneys) (Fredriksson-Ahomaa et al. Bacterial virulence functions are often temperature-regulated due to the high energy cost of unnecessary expression of virulence genes (Hurme and Rhen. Furthermore. ↓. shipping. D55. 1998). 2000b. the head meat and tongue should be pasteurized before delivery. 1999. in which contamination of carcass and offal can occur. FOOD PROCESSING OPERATIONS THAT INFLUENCE THE NUMBER. especially with Y. Preventing the access of wild animals to irrigation water and fields could reduce the risk of contamination of surface water and soil. diaphragm. the spread of pathogenic Yersinia from tonsils to pluck set and other parts of the carcass by knives and hands is unavoidable because the tonsils are usually removed together with the pluck set (tongue. Effect of temperature on activity of virulence-related b genes in Y. Furthermore. 2004. like evisceration and incision of the submaxillary lymph nodes. even in the presence of high numbers of other bacteria (Barakat and Harris.. serum resistance a b Genes/function 37°C ↓ ↓ ↓ ↓ ↑ ↑ ↑ ↑ DISCRIMINATIVE DETECTION METHODS FOR CONFIRMATION AND TRACE-BACK OF CONTAMINATED PRODUCTS Several difficulties have been associated with isolating pathogenic Yersinia from food and environmental samples. respectively.. and tongue should occur in a separate room with separate equipment. the ability of bacteria. 1995. 2000).. Thus. tonsils.172 FREDRIKSSON-AHOMAA ET AL. 2006). 2001a). The inspection of head. By removing the head together with the tonsils and tongue prior to evisceration. lack of identification between species. Yersinia can grow in anaerobic conditions. esophagus. 2008). serum resistance ysc. heart. such as long incubation steps taking up to 4 weeks. The maximum growth temperature of Yersinia is between 42 and 44°C. 2000). Modified from the work of Straley and Perry (1995). OR CHARACTERISTICS OF ENTEROPATHOGENIC YERSINIA Slaughter pigs often carry pathogenic Y. harvesting. and D60 of 1 min. but with higher levels of CO2 the length of the lag phase will increase and growth will be slower (Pin et al. particularly in tonsils and feces (Fredriksson-Ahomaa and Korkeala. Yersinia is relatively sensitive to heat.. Table 7. upregulation. Y. Bredholt et al. 2003a). many of the Yersinia virulence factors are expressed at 26°C but not at 37°C (Table 7). have been reported for vacuumpackaged minced beef (Bolton et al. 1994). 1998). However. SPREAD. . strict slaughter hygiene remains important in reducing contamination in slaughterhouses.. Additionally. There are several meat inspection procedures. lungs. it is impossible to reject asymptomatic pigs contaminated with pathogenic Yersinia at postmortem meat inspection. As a facultatively anaerobic bacterium. the access by rodents and birds to storage facilities should be prevented. 2000). the use of a mechanized bung cutter in connection with enclosing the anus and rectum in a plastic bag to minimize fecal contamination has been shown to reduce feces contamination of carcasses (Nesbakken et al.. yop/secretion of Yops ail/attachment. Niskanen et al. including Y. during irrigation. enterocolitica and Y.. at 50°C (D50).55 min. During evisceration. It is also destroyed by pasteurization at 72°C for 15 to 20 s. It can also grow well in modified atmospheres (Harrison et al. D values.. enterocolitica.. with thermal treatments employed in food processing destroying it readily. enterocolitica has been shown to grow well on meat when packaged by vacuum or in modified atmosphere and stored at 5°C (Doherty et al. Bodnaruk and Draughon. enterocolitica Effect of temp on gene a expression 26°C inv/invasion irp/iron uptake rfb/lipopolysaccharide yst/enterotoxin production virF/transcriptional activator yadA/adhesion to animal cells. In addition. 1 min. Jalava et al. trachea. to persist in samples in a viable but noncultivable state can ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↑. pseudotuberculosis in the oral cavity. and the cleaning of processing equipment should be adequate (Nuorti et al. liver. Furthermore. packing. 2003b. and processing. 2002. Yersinia contamination in the later stages of the food chain has not been studied sufficiently. downregulation.. Conventional culture-dependent methods have several limitations. pseudotuberculosis. 1999). and 0. the contamination of pathogenic Yersinia could probably be reduced. The presence of symptomless carriers together with the widespread occurrence of pathogenic Yersinia in herds renders the control of this bacterium at farm level difficult. and lack of discrimination between pathogenic and nonpathogenic strains (FredrikssonAhomaa and Korkeala. or thermal resistance. Fresh produce may become contaminated with pathogenic Yersinia.

which can be used prior to PCR to increase the sensitivity and decrease the risk of falsepositive results due to detection of dead cells. 1996. investigations have been undertaken to develop rapid and reliable methods. 1998). they are not necessarily the most effective methods to remove inhibitors from complex matrices (Kaneko et al. real-time monitoring. Many of these methods use primers targeted to the virF or yadA gene located on pYV (Table 8). (2002) have designed a selective enrichment medium. Some of the PCR methods have been created to detect more than one gene at the same time. pseudotuberculosis Using PCR Several methods have been developed to detect Y.. 2004).CHAPTER 11 • Y. Wolffs et al. The ail gene located in the chromosome of pathogenic Y. pathogenic Yersinia organisms are often dominant bacteria and Table 8. enterocolitica. Sandery et al. while method B uses a two-step PCR with primers targeted to yadA on the pYV. Fredriksson-Ahomaa et al. Knutsson et al. (1996) NCFA (1998) Jourdan et al. agarose gel electrophoresis. ail a ϫ Kaneko et al. DNA extraction procedures using silica particles or chelex resin have commonly been used.. Jourdan et al. enterocolitica and Y.. PCR methods targeted to chromosomal virulence genes have also been established.. pseudotuberculosis in clinical specimens of infected individuals than to find them in asymptomatic carriers or in foods and environmental samples. (2007a) Özbas et al. enterocolitica. 16S rRNA yadA. (1999) Boyapalle et al. such as PCR. The method most frequently used to detect PCR products has been electrophoresis in an agarose gel. Fukushima et al. (2001) AGE. Method A is based on a one-step PCR with primers targeted to the ail gene in the chromosome.. with some variations. Fredriksson-Ahomaa et al. Numerous commercial DNA purification kits are available. inv virF. 2000. (2000). especially Y.. ENTEROCOLITICA AND Y. for Y. Vishnubhatla et al. 2001. enterocolitica in food samples (Table 8).. Thus. 2001. Boyapalle et al. enterocolitica in foods. and some of these kits have also been used in PCR assays designed for Y. Real time. These two methods have been published by the Nordic Committee on Food Analysis (NCFA. ail. enterocolitica and Y. enterocolitica strains is the most frequently used target. Detection of Y. Because of possible plasmid loss during culturing. 2001. 2004. Detection of Yersinia enterocolitica and Yersinia pseudotuberculosis in food samples using PCR Gene region(s) or gene(s) Plasmid virF yadA Chromosome ail Type of PCR Nested Nested Single Semi-nested Nested Single Single Single Multiplex Multiplex Multiplex Multiplex Detection AGE AGE Real time AGE AGE Real time AGE Real time AGE AGE AGE AGE a Y. There are. (1998) Lantz et al.. two standardized conventional PCR methods for detection of pathogenic Y. pseudotuberculosis Using Culture Methods It is generally easier to find Y. enterocolitica.. Buoyant density centrifugation... however. enterocolitica and Y. (1995) Nilsson et al. During acute gastroenteritis or with organ abscesses. Yersinia PCR-compatible enrichment medium. 2003). 2000.. 2007a) or SYBR green dye (Fukushima et al. however. 2007). 2000.. (2005) Sandery et al. 1995. (2001) yst Plasmid and chromosome virF. for detecting pathogenic Yersinia. . PSEUDOTUBERCULOSIS 173 be a problem (Alexandrino et al. pseudotuberculosis Reference(s) NCFA (1998) NCFA (1998) Wolffs et al. Boyapalle et al. enterocolitica in food and environmental samples using PCR (Fredriksson-Ahomaa and Korkeala. enterocolitica from food samples to remove inhibitors and to prevent false-positive results due to DNA originating from dead cells (Thisted Lambertz et al. enterocolitica ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ Y. as they are rapid and simple to use. so far. The Yersinia methods are based on duallabeled probes (Jourdan et al. has been used to separate Y. Fredriksson-Ahomaa et al. (2000) Vishnubhatla et al.. Detection of Y. 2007a). There are already some realtime PCR methods designed for qualitative detection of pathogenic Y. the use of real-time monitoring by fluorescence techniques has substantially increased. in food samples. ail yadA. 2003a).

is seldom successful (Fredriksson-Ahomaa and Korkeala. Many different selective plating media developed for enteropathogens have been used for isolation of Yersinia (Fredriksson-Ahomaa and Korkeala. two oligonucleotide probes are available from the FDA’s (U. ITC. (1988). differentiation of Yersinia from competing organisms. like API 20E. 24 h) CIN (32°C. 24 h) SSDC (30°C. and Micronaute E. API Rapid 32 IDE. according to Schiemann (1979). enterocolitica. which has been shown to be efficient for recovery of strains of bioserotype 4/O:3. the most widely used is MacConkey (MAC) agar. pseudotuberculosis. Several entirely new media for isolation of Yersinia have been designed to improve selectivity. The methods are based on specific segments of the virulence plasmid or the chromosomal DNA with known virulence functions. This kit has high positive identification rates of 93% and 90% for Y. 2003a). direct isolation. enterocolitica from food and environmental samples (Table 9). Y. 18 h). 1 day) CIN (30°C. 2003). irgasan-ticarcillin-potassium chlorate broth. PSB. 48 h) CIN (32°C. 2001).. 14–21 days) Slightly selective PSB (10°C. The CIN agar is most commonly used for both Y. 10 days) PSB (25°C. Cefsulodin-irgasan-novobiocin (CIN) agar. pseudotuberculosis from foods. Citrobacter. Because of the high number of other bacteria and the low number of pathogenic strains of Yersinia in food and environmental samples. tripticase soy broth. 2003. TSB. Y. MAC agar has been used alongside CIN and is generally considered to be useful for the isolation of Y. phosphate-buffered saline broth with sorbitol and bile salts. Food and Drug Administration) Center for Food Safety and Applied Nutrition for identification of pathogenic Y. pseudotuberculosis is usually also able to grow on them. Most of the media have been designed for Y. modified Rappaport broth. SSDC. enterocolitica and Y. widely used for identification of presumptive Yersinia isolates. Salmonella-Shigella-sodium deoxycholate-calcium chloride agar plate. 18 h). are widely used for bioserotype 4/O:3 due to their high selectivity and commercial availability (ISO. pseudotuberculosis Reference CIN (32°C. Enterobacter. even on selective media. Niskanen et al. 2003a). 1 day) PBS (4°C.50% KOH for 20 s has widely been used for isolation of Yersinia in foods (FDA. enterocolitica and Y. Morganella. 2001. 2–3 days) a Isolation medium/media (conditions) Y. phosphate-buffered saline. supplemented with 1% mannitol and 0. enrichment in liquid media prior to isolation on solid media is required. Klebsiella. and Serratia. microarray chip technology has shown increased importance and potential in rapidly determining a complete gene profile in pathogenic microorganisms. such as Aeromonas. Currently. pseudotuberculosis. 2003. This can be done rapidly and with high specificity with DNA-based methods. can easily be isolated by direct plating on conventional enteric media. enterocolitica and Y. To increase the number of Yersinia strains in these samples. However. 2003a). enterocolitica Y. enterocolitica isolates. NCFA. The API 20E system. enterocolitica and Y. 2003). pseudotuberculosis. 48 h) CIN and MAC (30°C. Alkali treatment with 0. despite its low selectivity. 18 h) ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ APHA (1992) APHA (1992) FDA (2001) ISO (2003) ISO (2003) APHA (1992) ϫ PBS. Proteus. 1998). enterocolitica (FDA. NCFA. pseudotuberculosis. and SalmonellaShigella deoxycholate calcium chloride agar. Far less information is available on reliable methods for recovery of Y. The most widely used selective enrichment broth is irgasan-ticarcillinpotassium chlorate broth. Isolation methods of Yersinia enterocolitica and Yersinia pseudotuberculosis most commonly used for food samples Enrichment medium (conditions) Not selective TSB (22–25°C. Of the traditional enteric media. MAC (22–25°C. can be difficult.15% bile salts. Several PCR and DNA colony hybridization assays have been designed to verify the pathogenicity of Y.174 FREDRIKSSON-AHOMAA ET AL. 1–3 days) Selective ITC (25°C. . it is important to assess the pathogenicity of isolates (Fredriksson-Ahomaa and Korkeala. (2002) have shown that cold enrichment for 2 weeks in phosphate-buffered saline broth. These probes are specific for the ail gene in the chromosome and the virF on the pYV. especially followed by alkali treatment. thus. enterocolitica isolates recovered from nonhuman sources are considered nonpathogenic. pseudotuberculosis can be identified with commercial rapid identification kits. but its growth is mostly poor and delayed. respectively. according to Wauters et al. MRB.S. has been shown to be accurate in identifying Y. pseudotuberculosis. Myers et al. Several different methods are available for isolation of Y. however. (2006) have recently developed a microarray chip combined a Table 9. The majority of Y. In addition. 2 days) MRB (22–25°C. MAC (22–25°C.25 to 0. is productive for isolation of Y. ISO. when incubated at a temperature between 25°C and 30°C instead of 37°C for 18 to 24 h (Neubauer et al.

Falcao et al. 1998) have demonstrated differences in the geographical distribution among Y. which is simple and quick to perform but may possess a low reproducibility. Jalava et al. Kageyama et al. Amplified fragment length polymorphism. 2005). food-borne yersiniosis being due to Yersinia enterocolitica or Yersinia pseudotuberculosis. thus. (2005). pseudotuberculosis into subtypes is necessary to identify contamination sources and routes. important virulence genes of Y. have occurred in Finland and Japan. enterocolitica. pseudotuberculosis Reference(s) Fearnley et al. (2002) Fredriksson-Ahomaa et al. (1994). The primary transmission route of human yersiniosis is proposed to be fecal-oral via contaminated food. (2005). between strains belonging to the same bioserotype. this method is a commonly used technique in epidemiological studies of Y. 2006a). (2004). (2003) Fredriksson-Ahomaa et al. . It allows discrimination between strains belonging to different bioserotypes and also. Fredriksson-Ahomaa et al. 2002. 1998). (1999. (2006a) Voskressenskaya et al. pseudotuberculosis strains at the subserotype level. Kuehni-Boghenbor et al. often in schoolchildren. (1994) and Kageyama et al. random amplification of polymorphic DNA (RAPD) assay. 2006b. The reported cases of yersiniosis are mainly sporadic and outbreaks are uncommon.. 2003. ENTEROCOLITICA AND Y. (1994. pseudotuberculosis (Table 10). enterocolitica. a recently adopted PCR-based typing method. Both techniques have been used to investigate the relationship between genotypes and host sources (Fredriksson-Ahomaa et al. and restriction endonuclease analysis of the plasmid (REAP) have also frequently been applied to characterize enteropathogenic Yersinia strains (Table 10). enterocolitica and Y.CHAPTER 11 • Y. Ribotyping. enterocolitica infection with consumption of uncooked or undercooked pork. enterocolitica has frequently been recovered only from slaughter pigs. (2007) Niskanen et al.. Molecular techniques represent valuable alternatives for subtyping of Yersinia. differentiation of Y. Some epidemiological studies have linked Y. Nuorti et al. However. Molecular Characterization In epidemiological studies. and yst genes. enterocolitica ϫ ϫ ϫ ϫ ϫ Y. amplified fragment length polymorphism.. 2005). Pathogenic Y. (2005) Favier et al. pseudotuberculosis. 2008). also allows differentiation of Y. The RAPD assay. By removing the head together with tonsils and tongue prior to evisceration and by using a mechanized bung cutter in connection with enclosing the anus and rectum in a plastic bag. In the United States. especially to characterize Y. The low isolation rate Table 10. enterocolitica and Y. PSEUDOTUBERCULOSIS 175 with multiplex PCR for detection and characterization of four virulence genes in Y. (1994. (2006a) Makino et al. (2006a) Fukushima et al. Pulsed-field gel electrophoresis (PFGE) has proved to be highly discriminatory for molecular typing of Yersinia. Fukushima et al. household preparation of chitterlings (traditional dish made of intestines of pigs in the southern United States) has often been associated with Y.. (2004. (2002). enterocolitica and Y.. these bacteria have seldom been isolated from foods. has been used in several studies to characterize Y. Multiplex PCR was used to amplify the virF. 2006) ϫ ϫ ϫ ϫ a AFLP. enterocolitica strains within serotype-related clusters (Fearnley et al. Most human illness worldwide is caused by Y. 2006a). Frequently used methods for molecular subtyping of Yersinia enterocolitica and Yersinia pseudotuberculosis Typing method AFLP RAPD REAP Ribotyping PFGE a Y. pseudotuberculosis strains (Voskressenskaya et al. RAPD is also able to distinguish Y. thus. (2006). PFGE allows subtyping of Yersinia strains belonging to the same serotype (Niskanen et al. Lambertz et al. outbreaks of Y. pseudotuberculosis strains belonging to the same serotype using REAP. CONCLUDING REMARKS Yersiniosis is an infectious disease caused by Yersinia. 2007b). Fredriksson-Ahomaa et al. pseudotuberculosis are important food-borne pathogens.. enterocolitica and Y. enterocolitica strains (Fredriksson-Ahomaa et al. 2006a. 2006). ail. 2006b. Han et al. Despite the fact that Y. Kuehni-Boghenbor et al. (2006) Fredriksson-Ahomaa et al. Several DNAbased methods have been used in molecular typing of Y. it has been used in two outbreak studies by Makino et al. in some cases. Using this technique. (2002. Ribotyping has been shown to be a useful tool for molecular typing. pseudotuberculosis infection. enterocolitica could simultaneously be detected directly from pasteurized whole milk. the contamination of pathogenic Yersinia could probably be reduced.. enterocolitica O:3 infections.

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Additional species have been described more recently. 1968). trota. Chronic diarrheal disease caused by this genus occurs most commonly in children. O:16. hydrophila bacteria in drinking water. A. but the role of lesser-known food-borne pathogens in the causation of illness cannot be discounted. jandaei. and Aeromonas hydrophila is the type species. • Department of Food Science and Technology.Pathogens and Toxins in Foods: Challenges and Interventions Edited by V. Characteristics of Organisms and Type of Illness These gram-negative bacteria are of the order Aeromonadales and family Aeromonadaceae. and the immunocompromised (von Graevenitz and Mensch. Arcobacter. globally occurring aquatic. and food safety concerns involving each pathogen. Sofos © 2010 ASM Press. hydrophila is listed in the Environmental Protection Agency’s (EPA) first and second Contaminant Candidate List as a “potential waterborne pathogen. especially in crowded. veronii biovar veronii. GA 30602. Helicobacter. oxidasepositive facultative anaerobes. Harrison The occurrence of food-borne illness may be dominated in the media and public awareness by outbreaks related to a small group of microorganisms. and O:34 were found to predominate in clinical species. wound infections. with rare short chains. A. Athens. A. hydrophila (48%). DC Chapter 12 Other Bacterial Pathogens: Aeromonas. AEROMONAS SPP. and A. 16S ribosomal DNA and gyrB sequence studies have been used to establish taxonomic species relatedness. Serotypes O:11. Strains may be motile with single. nosocomial infections. GA 30602. Bergey’s Manual of Systematic Bacteriology (Brenner et al. thorough overview of the significance. Aeromonads also cause disease in fish. septicemia. 2005) lists 17 DNA-DNA hybridization groups and 14 phenospecies of this genus. and bile tract infections. • Mark A. Harrison 181 . K. veronii biovar sobria. hydrophila. Carbohydrates are utilized both oxidatively and fermentatively. A. Aeromonas species are indicated as important human pathogens causing gastrointestinal and other infections in healthy and immunocompromised hosts. aquaculture Elaine M. A. They are coccobacilli or bacilli. Aeromonas species (aeromonads) are ubiquitous. though serotyping may be useful as a rapid method for identification of aeromonads to the genus level. Juneja and J. and community-acquired infections. Conditions caused by Aeromonas in adults include diarrhea. including domestic animals and birds. endocarditis. sobria and A. To a lesser extent. The University of Georgia. A. meningitis. There is evidence that some species are pathogenic for humans and warm-blooded and coldblooded animals. Plesiomonas. and Streptococcus Elaine M. Washington. with an additional 52 provisional groups. characteristics. the ground. A. the elderly. caviae. D’Sa • Department of Foods and Nutrition. and marine water bodies. polar flagella or lateral or peritrichous flagella.” An EPA method (method 1605) exists for the detection and enumeration of A. The University of Georgia. D’Sa and Mark A. The most implicated clinical species is A. Environments in which they have been detected include drinking and wastewater.. In accordance with the Safe Drinking Water Act. Serotyping studies determined the existence of 44 serogroups based on O antigen studies. 2006). caviae (about 25% each). Athens. A. This chapter covers some of these bacterial pathogens and will attempt to give a concise. The serological heterogeneity of several strains precludes its usefulness for taxonomic studies. occurring singly or in pairs. surfaces. salmonicida is a significant pathogen of fish (USEPA. N. schubertii have been associated with human infections. followed by A. Mycobacterium. Aeromonads have been implicated as a cause of traveler’s diarrhea.

Virulence in Aeromonas species may be due to cellassociated or extracellular factors. though no outbreaks due to the presence of these organisms in water biofilms have been reported. by their resistance to 150 µg O/129 and variable reaction to ornithine decarboxylase. A seasonal association is observed between aeromonads present in drinking water supplies and those identified in human cases of gastrointestinal disease. 21st edition (APHA. capsules.5% NaCl. The optimum pH range is from 5.5 to 9. carbenicillin. tetracyclines. 1997). proteolytic cleavage of a C-terminal peptide from this produces an active fragment that is approximately 40 amino acids long.. “S-layers” are cell surface proteins that are associated with LPS. 2005). in addition to cell-associated flagella. trota and 30% of A. ampicillin. water reuse. they are unable to grow on thiosulfate-citrate-bile saltssucrose agar. associated with the Tap gene cluster (Tap A).and third-generation cephalosporins. Two important families of type IV pili produced in gastroenteritis-associated Aeromonas species are the Tap (short. adhesins. 2006). but their role in human infections has not been established (USEPA. They are part of the normal microflora of healthy animals and humans and are found at high levels in sewage (USEPA. and treated drinking water. 1993). They are susceptible to second. hemolysins.0 is tolerated. Some species (including most A. but most such host-microbe interactions do not result in disease. 2006). A high infectious dose 10 (>10 ) has been reported. and nuclease have also been identified. 2006). and lipopolysaccharide (LPS). rigid) pili. while environmental samples are handled according to methods in APHA’s Standard Methods for Examination of Water and Wastewater. Strains that produce these are believed to be more pathogenic for fish. and cytolytic activity. which may explain why no outbreaks associated with treated drinking water have been described (Rusin et al. One or more broth enrichment procedures (with or without antibiotics) may be needed to recover aeromonads where low numbers of organisms are expected. Aeromonas spp. and have a negative string test. outer membrane proteins. and intensive care units. chloramphenicols. which are susceptible to ampicillin). Both the polar flagella and multiple lateral flagella facilitate adhesion and thus are thought to be important virulence factors and significant in the formation of biofilms that are resistant to disinfection (Kirov et al. Enzymes produced include toxins. aminoglycosides. Sources and Incidence in the Environment and Foods Aeromonas species have been detected in a variety of foods. with an extended temperature range of 0 to 45°C for some. salmonicida) do not grow at 35°C or above (Mateos et al. ready-to-eat meats and dairy products. and a lipase that digests host cell plasma membranes is also produced. Presence and adherence of pili are essential in virulence. Food specimens are transported and handled according to the procedures in the FDA’s Bacteriological Analytical Manual (Andrews and Hammack. carbapenems. The optimum growth temperature for Aeromonas spp. 2004). lipases.0. and thus have resulted in notable losses to the aquaculture industry (Moyer. liquefy gelatin. Other enzymes like amylase. most are able to ferment d-mannitol and sucrose (USEPA. and ticarcillin (with the exception of A. 2006). do not ferment i-inositol.. 1997).. proteases. Another aerolysin is a dimer which produces a pore in the outer membrane protein. caviae. with . Lack of personal hygiene. and consumption of untreated water containing large numbers of aeromonads are risk factors for the spread of disease. Serine proteases and metalloproteases function in toxin activation. leakage of cell contents.5 to 9. pili.. seafood. Phenotypically. Tap pili attach to enterocytes. Increased transmission rates occur in day care environments. is 22 to 35°C. 1998). nursing homes. lecithinase. “Aerolysins” are toxins produced by some Aeromonas species that have hemolytic. Intrinsic and Extrinsic Factors That Affect Survival and Growth in Food Products and Contribute to Outbreaks Aeromonas species occur in biofilms that resist disinfection. chitinase. elastase. One type is produced from cells as “proaerolysin”. and agglutinins. Aeromonas may be ingested from water and food through the environment. while Bfp pili are thought to be important in attachment to human erythrocytes and in intestinal colonization. Most motile aeromonads are resistant to penicillin. and cell destruction (USEPA. No single culture medium is considered optimal for recovery of all aeromonads. associated with the tapABCD gene cluster (Barnett et al. enterotoxic. wavy pili. Numbers of aeromonads in water are reported to be higher in summer months (Janda and Abbott. 2006). a wider range of 4. facilitating altered cell permeability. and trimethoprimsulfamethoxazole. Optimum NaCl concentration is from 0 to 4%. and Bfp (bundle-forming) long. including raw produce. quinolones.182 D’SA AND HARRISON environments. raw meats. 2005). Aeromonads are unable to grow in 6. are differentiated from Vibrio and Plesiomonas spp.

and multiple tube tests commonly used. Aeromonas levels do not correlate well with commonly used indicator organisms for fecal pollution of drinking water. Chemical. they are placed in the family Campylobacteraceae. Maintenance of sanitary food handling and distribution practices lowers the threat from this genus in the causation of food-borne illness. Discriminative Detection Methods for Confirmation and Trace-Back of Contaminated Products Several methods have been used for the detection. due to the heterogeneity observed. 2003) was carried out and is recommended for identification. cryaerophilus hybridization groups 1A and 1B. The tendency of the cells to form biofilms makes accurate determination of their numbers in water systems a difficult task (USEPA. 2005). Concluding Remarks The importance of aeromonads as pathogens of food-borne origin dates back to the 1950s. 2006). were designated species of a distinct genus in 1991 (Vandamme et al. tube. with a view to identifying pathogenic strains. Recent Advances in Biological. colistin resistance can be used as a phenotypic marker. even in the presence of 5 to 6 log10 background flora. Concentrations of 10 to 30 mg/liter ampicillin have been used in ADA. or Characteristics Aeromonads are inactivated by commonly used disinfectants used in water treatment and by routinely used food processing and preparation methods. officially separating them from the closely related species of the genus Campylobacter. A. membrane filtration. Commercial identification systems have been found to give inaccurate results. and illness manifestations in humans and animals. Starch ampicillin agar and commercially available Aeromonas medium (Ryan’s medium) have also been used. Their presence in water sources and raw and ready-to-eat foods lends authenticity to their capacity to cause outbreaks of food-borne disease under the right circumstances.CHAPTER 12 • OTHER BACTERIAL PATHOGENS 183 combinations of plating. typical food-borne pathogen intervention methods are effective in inactivating Aeromonas spp. Phenotypic diversity makes identification based on biochemical tests challenging. Spread. Pulsed-field gel electrophoresis has been found to be increasingly useful and is replacing ribotyping as a typing method. Within the group. since the level of aeromonads in water is not close to the infectious dose for humans. Molecular detection methods used for clinical or environmental samples have a detection limit greater than 3 to 4 log10 CFU/ml and generally need an enrichment step. followed by screening and identification. with which they share a relatively common background of sources.2 mg/liter). Screening of colonies may be done by the oxidase test. as mentioned above. Together with Campylobacter spp. Additionally. Hence. Molecular methods for detection of aeromonads have been studied. Detection of aeromonads in environmental samples is influenced by temperature (should be greater than 15°C) and residual chlorine levels (should be less than 0. and identification by commercial kits or biochemical methods. 2000). 2006). cefsulodin irgasan novobiocin or blood ampicillin agar may be used. Modified bile salts irgasan brilliant green agar has been used for recovery of 2 to 7 log10 aeromonads from foods. Microarray technology is useful for the study of populations in biofilms (USEPA. A. first observed in veterinary specimens. characteristics. MacConkey agar. A. ARCOBACTER SPP. following their isolation from humans.. and Physical Interventions To Guard against the Pathogen With little evidence of Aeromonas causing foodborne illness. and plate tests (Abbott et al. Food Processing Operations That Influence the Numbers. hydrophila from drinking water (USEPA. This is not considered a significant problem though. nitrofigilis (Houf et al. the membrane filtration method using ADA has been included in the APHA’s Standard Methods for Examination of Water and Wastewater (APHA. 2005) and EPA method 1605 (ADA with vancomycin). A. Some of these methods target virulence genes... enumeration. butzleri (thought to be the primary human pathogen). 1991).. The EPA method 1605. Typing methods are useful in epidemiological studies. Antigen-antibody methods are not commonly used for identification of aeromonads. For recovery of aeromonads from clinical specimens. cibarius (Houf et al. in foods. which is a membrane filtration method using ampicillin dextrin agar (ADA) with vancomycin has been validated for isolation of A. the genus is not considered to be a public health threat in water. 2006). Characteristics of Organisms and Type of Illness These “arc-shaped” bacteria. Seven species have been identified: A. halophilus (Donachie .. A. Among these. and isolation of aeromonads. skirrowii. The characterization of 193 strains using carbohydrate.

using genus. unhygienic food preparation practices and environmental conditions. tests used in their identification include those used to identify Campylobacter spp. In vivo studies have suggested the potential virulence of A. Wolinella. beef. and A. and person-to-person contact. and A. Definitive biochemical tests used to identify Arcobacter and distinguish between the species are limited and somewhat unreliable. ducks. and turkeys]. and cadmium chloride susceptibility (Wesley. They are also a part of the indigenous flora of healthy animals. One study found the distribution of Arcobacter spp. 1997). 2005). growth in the presence of glycine or sodium chloride. hydrolysis of indoxyl acetate. the organisms have been isolated from water (surface and drinking water). 1992). Campylobacter. Arcobacter strains are negative for all three tests (Burnens and Nicolet. (Stampi et al. fatty acid analysis.. sewage. catalase activity. Wesley et al.6%). (Bolton et al. in seafood and raw milk remains unknown. Sources and Incidence in the Environment and Foods Limited studies have been conducted globally on the prevalence of these organisms. butzleri (Wesley et al. Susceptibility to antimicrobial agents has also been used to characterize this genus (Kiehlbauch et al. and postchilling steps of a commercial poultry operation. nitrate reduction. in porcine abortions in the United States to be A..184 D’SA AND HARRISON et al. ready-to-eat poultry. along with biochemical tests and antimicrobial resistance patterns. Helicobacter. are gram-negative. 1998).. raw foods (including poultry [chickens. prechilling. Since these organisms are relatively metabolically inert. 1977). and 33 (23..7%) to nalidixic acid. Molecular and nucleic acid-based techniques. 114 (82%) to azithromycin. mastitis.. cryaerophilus 1A at 16%.9% of Arcobacter spp.3%). in 10. This. 1997). 1997). A. growth on MacConkey agar. Isolation rates of up to 43% from porcine abortions have been reported. makes the determination of the accurate prevalence of the genus a study in statistical extrapolation. Plasmid analysis (with a plasmid detection rate of 33% for Arcobacter spp. Infections with these organisms cause economic losses in livestock herds and compromise human health. A total of 125 (89. Analysis of farm management practices revealed that feeding of alfalfa and use of individual waterers protected the cattle from infection with .6%) and A. (2007) sampled 325 broiler carcasses during a three-month period in 2004. cryaerophilus 1B (18. 1994). and Arcobacter spp. have proven to be more reliable means of identification and distinction for Arcobacter (and between Campylobacter and Arcobacter) species. cryaerophilus 1B at 60% (Harmon and Wesley. (1996) postulated that the genus has a significant reservoir in poultry products. butzleri at 8%. A total of 71. 1996). They include growth temperature.S. followed by A. to possibly go undetected. nonsporing rods and have been known to cause gastritis (rarely bacteremia or appendicitis) in humans and abortions. 1992).and species-specific DNA probes complementary to the highly conserved 16S rRNA molecule or the glyA gene.. butzleri was found to be the most prevalent species in 79.3%) and postchilled carcasses (9. Lammerding et al.8% of Arcobacter isolates showed multiple antimicrobial resistance. 1996. aerotolerant. Their role in the causation of disease is underlined by higher isolation rates from aborted fetuses and cases of porcine infertility than from healthy animals (Ellis et al. (2000) studied the shedding of Campylobacter and Arcobacter spp. cryaerophilus has been associated with cases of infectious abortion in cattle herds in Germany (Wesley. since in contrast to Campylobacter strains. together with the fact that classical laboratory testing is geared toward the detection of thermophilic Campylobacter spp. for epidemiological typing (Harrasset al.. containment of intestinal contents and fecal material will limit the spread of this emerging pathogen in farm animal operations.) has also been used. Its presence in red meat animals has also been documented. The presence of Arcobacter spp.1% of the samples. make up the rRNA superfamily VI of the epsilon class of Proteobacteria. and gastritis in livestock (Wesley. and aborted animal fetuses (Wesley. in disease under suitable conditions..8%) compared to prechilled (61. A skirrowii was not isolated in this study.52% of dairy cattle fecal samples (Wesley.9%) of A. Other tests suggested include arylsulfatase and pyrazinamidase activities and susceptibility to polymyxin B. 1994). Consequently. 1994). A. mesophilic. Antigenically identical strains have been recovered from diseased and healthy animals.3% to be positive for Arcobacter. The disease can be acquired from travel in developing areas/countries. underlining the possibly opportunistic role of Arcobacter spp. humans and animals with enteritis. Musmanno et al. A. crosscontamination. 1993). allowing Arcobacter spp. Son et al. in the feces of dairy cattle in various U. cryaerophilus 1A (2. Tertiary treatment of sewage effluent with 2 ppm chlorine dioxide removes 99. at the prescalding. sulfidicus (Wirsen et al.. the inability to hydrolyze hippurate. With the exception of A. states and found 14. 2002). 1993). Arcobacter spp. nitrofigilis. Surveys of cattle have detected Arcobacter spp. Arcobacter recovery rates were highest in prescalded carcasses (96. chicken flocks. and pork). butzleri isolates showed resistance to clindamycin.

0 and for A. cryaerophilus.5 to 8. pimento leaf essential oil (6%) reduced A.0 to 7. and clove aquaresin also inhibited growth at 6.37 and 2. butzleri in hospitalized patients who responded to antibiotics and in which A. It has been postulated that some of these campylobacters may be misidentified Arcobacter spp. skirrowii. In 1983. Organic acid concentration and type significantly affected the effectiveness of antimicrobial activity. Nisin (500 IU/ml) was inhibitory. it has been postulated that the genus is sensitive to the effects of ambient drying (Nachamkin and Blaser. with citric acid being more effective (Phillips.. respectively. Calculated z values ranged from 5. was suggested. Lactic and citric acids at concentrations of 0.1 was reported for exponential phase A. Low doses of irradiation (1. Due to the lower rate of recovery of Arcobacter from red meats that have a reduced surface moisture level. citric acid monohydrate.87 log10 using 4% lactic acid. Hancock (2000) studied the effects of spices on A. 2005). butzleri levels by 1.11 logs over 2 and 5 days. and 2% were found to be inhibitory to the growth in culture of A. Spread. Acetic acid. followed by thymol. Heat resistance studies have shown that recommended temperatures for safe cooking of foods should be sufficient to kill Arcobacter spp. cryaerophilus strains was 7. 1999). respectively. at less than 0.5 kGy) were found to be sufficient to eliminate the presence of A. Chemical. On pork loins. butzleri in ground pork. was inhibitory in vitro against three strains of A.28°C. The species studied were found to be cold sensitive. (D’Sa and Harrison. butzleri populations by 1. butzleri strains studied in vitro. butzleri and A. butzleri (D’Sa and Harrison. it was determined that the pH growth range for these isolates was 5. as it was found to possess a slightly higher radiation resistance than that of Campylobacter jejuni. both in vitro and sprayed onto fresh pork loins inoculated with A. Lior serogroup 1 predominates in samples of food and from patients with illness. In the marination study. A few other cases of A.5%. 1.02%) of A. caffeic acid. Cinnamon aquaresin (1. butzleri. tannic acid. are also significant contributors to the global incidence of diarrheal illness. or Characteristics The same food processing and handling practices that are recommended to reduce Campylobacter problems should control problems that could be associated with Arcobacter species. butzleri in vitro and in spice marinades on fresh pork loins. butzleri was the only pathogen isolated from an outbreak of diarrheal illness in Italian schoolchildren (Vandamme et al.13%) were the most inhibitory to the two strains of A. butzleri and two human isolates of A. The effect of NaCl on growth and survival of the genus was variable.5. while sodium citrate was more effective than sodium lactate. influencing microbial colonization. Recent Advances in Biological. Pimento leaf essential oil (3. in combination with other means of reducing these bacterial species on food.56%) and cinnamon oleoresin (3. Inclusion of both spices and organic acids as “hurdle” components. Given the high incidence of Campylobacter spp. butzleri. and one strain of A.CHAPTER 12 • OTHER BACTERIAL PATHOGENS 185 Arcobacter.20 to 6. carvacrol.0% for A. two strains of A.5.5 and 3. The optimum pH range for A.25%. it is possible that Arcobacter spp. 2001). and Physical Interventions To Guard against the Pathogens Cervenka et al. A. 2001). butzleri strains was 6. elucidated by the fact that a mild heat treatment followed by cold shock acted synergistically to produce a large reduction in cell numbers. Cinnamon aquaresin (3%) reduced A. In a study carried out by D’Sa (2002) using four human isolates of A. A. . have been identified as the leading cause of bacterial food-borne diarrheal illness in humans (CDC. butzleri studied. 2002). butzleri populations were reduced by 0. Survival at NaCl concentrations up to 5% was noted at 25°C for some Arcobacter strains.5 to 4. butzleri cells (Hilton et al. A z value of 8.13%). 2000). Thus. feed and dietary supplements may alter gut homeostasis. (2008) found that cinnamaldehyde. barbeque spice aquaresin. butzleri was isolated as the sole pathogen. There have been a few studies focused on the inhibition or elimination of Arcobacter in foods. 1993).016%) and survival (0. in raw poultry and in the environment and the ability of arcobacters to grow at mesophilic temperatures (as low as 15°C). Low levels of sodium tripolyphosphate can prevent growth (0.03 and 1.34 logs over the same time span. The organism is also cold sensitive and may be sublethally injured at lower temperatures. butzleri. Intrinsic and Extrinsic Factors That Affect Survival and Growth in Food Products and Contribute to Outbreaks Campylobacter spp. and eugenol.0. USDA/FSIS. and lactic acid were inhibitory to A. 2005. cryaerophilus. with upper growth limits of 3. cryaerophilus. point to the pathogenic and invasive capacity of the organism (D’Sa. 2007). Food Processing Operations That Influence the Numbers..0 to 7. Hancock (2000) also studied the efficacy of organic acids.

1996. Considering the similarities of many of the characteristics of the genera Arcobacter and Campylobacter. Metronidazole-clarithromycin antibiotic combinations have been used commonly to treat infections. spiral. H. pylori infection in developed countries (0. HELICOBACTER Characteristics of Organism and Type of Illness Helicobacter pylori. 1997) that grow optimally at 35 to 37°C but not at 25°C. 2003). is unknown. their frequency has been sporadic to date. including mucosaassociated lymphoid tissue lymphoma (Hardin and Wright. Sources and Incidence in the Environment and Foods A definitive source of the organisms is unknown (Hill. and stomach cancer. and combined PCR-enzyme-linked immunosorbent assays targeting both the 16s and 23s rRNA regions have been developed and tested as a means of identification and distinction of Arcobacter spp. H... Noncultivable cells have been detected in the oral cavity and in feces and may be significant in the assessment of the routes of transmission of the organism. the species has been known to produce viable but noncultivable forms that retain infective capacity (Bode et al. Antolin et al. and a P-type adenosinetriphosphatase..5 to 1% (Jiang and Doyle. The organism attaches to gastric mucosa and using various factors. first isolated in 1983.05% ferrous sulfate and 0. microaerophilic. 1995. including its own LPS. 2002). It colonizes stomach walls. 2004a).. 1993). The organisms are gram-negative. Addition of 0. an inflammatory reaction is induced that initiates a cascade of reactions. Following gastric colonization.186 D’SA AND HARRISON Discriminative Detection Methods for Confirmation and Trace-Back of Contaminated Products Multiplex PCR assays. 2002). Winters and Slavik. nonsporing. PCR-restriction fragment length polymorphism assays. chronic gastritis. Marshall et al. CardarelliLeite et al.. Food-borne transmission has been postulated by several authors but not definitively proven. 2002). 1999. pylori infections require an array of bacterial and host factors. 1997. and is one of the most common bacterial infections in humans (Suerbaum and Michetti. especially peptic ulcers. they also are controlled by the same food handling and processing practices. fastidious. is recognized as the major cause of peptic ulcer disease globally. It has been the target of intensive study because of its link to human illness. and iatrogenic routes. Under adverse conditions. 2001). Asymptomatic H. pylori disease (Petersen and Krogfelt. it is reasonable to consider species of Arcobacter potential food-borne hazards. While the two genera are frequently found on similar foods and share many growth and survival traits. is the type species and most well-known species of this genus. resulting in the recognizable symptoms of infection. Studies have demonstrated the ability of H. 2001).5%) was found to be on the decline. urease (which alters the gastric microenvironment).3 to 0. significant among these are flaA and flaB (genes coding for flagellin). The optimal NaCl concentration for growth is 0.. environmental. oral-oral. 2000). Whether this is related to the clinical and laboratory methods used. motile (with polar flagella