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Pathogens and
Toxins in Foods
CHALLENGES AND INTERVENTIONS
This page intentionally left blank
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: [email protected]
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
664.001
579—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 • vii 10. Pathogenic Vibrios in Seafood • 146

Preface • xi Anita C. Wright and Keith R. Schneider

11. Yersinia enterocolitica and Yersinia

1. Bacillus cereus and Other pseudotuberculosis • 164
Bacillus spp. • 1 Maria Fredriksson-Ahomaa, Miia Lindström,
Mansel W. Griffiths and Hannu Korkeala

2. Campylobacter jejuni and Other 12. Other Bacterial Pathogens: Aeromonas,

Campylobacters • 20 Arcobacter, Helicobacter, Mycobacterium,
Nelson A. Cox, L. Jason Richardson, Plesiomonas, and Streptococcus • 181
and Michael T. Musgrove Elaine M. D’Sa and Mark A. Harrison

3. Clostridium botulinum • 31 13. Food-Borne Parasites • 195

Michael W. Peck Dolores E. Hill and J. P. Dubey

4. Clostridium perfringens • 53 14. Human Pathogenic Viruses in

Vijay K. Juneja, John S. Novak, and Food • 218
Ronald J. Labbe Lee-Ann Jaykus and Blanca Escudero-Abarca

5. Diarrheagenic Escherichia coli • 71 15. Seafood Toxins • 233

Catherine S. Beauchamp and John N. Sofos Sherwood Hall

6. Listeria monocytogenes • 95 16. Biogenic Amines in Foods • 248

Anna C. S. Porto-Fett, Jeffrey E. Call, K. Koutsoumanis, C. Tassou, and G.-J. E. Nychas
Peter M. Muriana, Timothy A. Freier,
and John B. Luchansky 17. Fungal and Mushroom Toxins • 275
Charlene Wolf-Hall
7. Salmonella • 108
Stan Bailey, L. Jason Richardson, Nelson A. Cox, 18. Critical Evaluation of Uncertainties of
and Douglas E. Cosby Gluten Testing: Issues and Solutions for Food
Allergen Detection • 286
8. Staphylococcal Food Poisoning • 119 Carmen Diaz-Amigo and Jupiter M. Yeung
Keun Seok Seo and Gregory A. Bohach
19. Naturally Occurring Toxins in
9. Shigella • 131 Plants • 301
Keith A. Lampel Andrea R. Ottesen and Bernadene A. Magnuson

v
vi CONTENTS

20. Chemical Residues: Incidence in the 26. Interventions for Hazard Control
United States • 314 in Retail-Handled Ready-To-Eat
Stanley E. Katz and Paula Marie L. Ward Foods • 411
Alexandra Lianou and John N. Sofos
21. A European Food Safety Perspective
on Residues of Veterinary Drugs and 27. Interventions for Hazard Control
Growth-Promoting Agents • 326 at Food Service • 436
Martin Danaher and Deirdre M. Prendergast O. Peter Snyder, Jr.

22. Prions and Prion Diseases • 343 28. Recent Developments in Rapid
Dragan Momcilovic Detection Methods • 450
Lawrence D. Goodridge and Mansel W.
23. Interventions for Hazard Control in Foods Griffiths
Preharvest • 357
Jarret D. Stopforth, Balasubrahmanyam Kottapalli, 29. Molecular Subtyping and Tracking of
and John N. Sofos Food-Borne Bacterial Pathogens • 460
Brandon A. Carlson and Kendra K.
24. Interventions for Hazard Control in Foods Nightingale
during Harvesting • 379
Mayra Márquez-González, Kerri B. Harris, 30. Food Safety Management
and Alejandro Castillo Systems • 478
Virginia N. Scott and Yuhuan Chen
25. Interventions for Hazard Control during
Food Processing • 396
Ifigenia Geornaras and John N. Sofos Index • 493
 CONTRIBUTORS

Stan Bailey Martin Danaher

Biomerieux, Inc., Hazelwood, MI 63042 Food Safety Department, Teagasc, Ashtown Food
Research Centre, Ashtown, Dublin 15, Ireland
Catherine S. Beauchamp
Colorado Premium Foods, 2035 2nd Ave., Carmen Diaz-Amigo
Greeley, CO 80631 Center for Food Safety and Applied Nutrition, U.S.
Food and Drug Administration, Laurel, MD 20708
Gregory A. Bohach
Department of Microbiology, Molecular Biology, Elaine M. D’Sa
and Biochemistry, University of Idaho, Moscow, Department of Foods and Nutrition, The University
ID 83844 of Georgia, Athens, GA 30602

Jeffrey E. Call J. P. Dubey

Microbial Food Safety Research Unit, U.S. U.S. Department of Agriculture, Agricultural Research
Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute,
Service, Eastern Regional Research Center, Animal Parasitic Diseases Laboratory, Building 1001,
Wyndmoor, PA 19038 BARC-East, Beltsville, MD 20705-2350

Brandon A. Carlson Blanca Escudero-Abarca

Department of Animal Sciences, Colorado State Department of Food, Bioprocessing and Nutrition
University, Fort Collins, CO 80523 Sciences, North Carolina State University, Raleigh,
NC 27695-7624
Alejandro Castillo
Department of Animal Science, Texas A&M Maria Fredriksson-Ahomaa
University, College Station, TX 77843-2471 Institute of Hygiene and Technologie of Food of
Animal Origin, Ludwig-Maximilians University
Yuhuan Chen Munich, Schoenleutnerstrasse 8, D-85764
Grocery Manufacturers Association, 1350 I St. Oberschleissheim, Germany
N.W., Suite 300, Washington, DC 20005
Timothy A. Freier
Douglas E. Cosby Corporate Food Safety and Regulatory Affairs,
USDA-ARS-Bacterial Epidemiology and Cargill, Inc., Minneapolis, MN 55440-9300
Antimicrobial Resistant Research Unit, Russell
Research Center, Athens, GA 30605 Ifigenia Geornaras
Center for Meat Safety & Quality, Food Safety
Nelson A. Cox Cluster of Infectious Diseases Supercluster,
USDA-ARS-Poultry Microbiological Safety Research Department of Animal Sciences, Colorado State
Unit, Russell Research Center, Athens, GA 30605 University, Fort Collins, CO 80523-1171

vii
viii CONTRIBUTORS

Lawrence D. Goodridge K. Koutsoumanis

Department of Animal Sciences, Colorado State Faculty of Agriculture, Department of Food
University, Fort Collins, CO 80523-1171 Science and Technology, Laboratory of Food
Microbiology and Hygiene, Aristotle University of
Mansel W. Griffiths Thessaloniki, P.O. Box 265, 54124, Thessaloniki,
Canadian Research Institute for Food Safety and Greece
Department of Food Science, University of Guelph,
Guelph, Ontario, Canada N1G 2W1 Ronald J. Labbe
Department of Food Science, University of
Sherwood Hall Massachusetts, Amherst, MA 01003-1021
Chemical Contaminants Branch HFS-716,
Division of Bioanalytical Chemistry, Office of Keith A. Lampel
Regulatory Science, Center for Food Safety and Food and Drug Administration, HFS-710,
Applied Nutrition, U.S. FDA, College Park, 5100 Paint Branch Parkway, College Park,
MD 20740 MD 20740

Kerri B. Harris Alexandra Lianou

Department of Animal Science, Texas A&M Laboratory of Food Microbiology and Hygiene,
University, College Station, TX 77843-2471 Department of Food Science and Technology,
Faculty of Agriculture, Aristotle University of
Mark A. Harrison Thessaloniki, Thessaloniki 54124, Greece
Department of Food Science and Technology,
The University of Georgia, Athens, GA 30602 Miia Lindström
Department of Food and Environmental Hygiene,
Dolores E. Hill University of Helsinki, P.O. Box 66, 00014 Helsinki
U.S. Department of Agriculture, Agricultural Research University, Finland
Service, Animal and Natural Resources Institute,
Animal Parasitic Diseases Laboratory, Building 1044, John B. Luchansky
BARC-East, Beltsville, MD 20705-2350 Microbial Food Safety Research Unit, U.S.
Department of Agriculture, Agricultural Research
Lee-Ann Jaykus Service, Eastern Regional Research Center,
Department of Food, Bioprocessing and Nutrition Wyndmoor, PA 19038
Sciences, North Carolina State University, Raleigh,
NC 27695-7624 Bernadene A. Magnuson
Cantox Health Sciences International, 2233
Vijay K. Juneja Argentia Road, Suite 308, Mississauga, Ontario,
Microbial Food Safety Research Unit, Eastern Canada L5N 2X7
Regional Research Center, Agricultural Research
Service, U.S. Department of Agriculture, 600 Mayra Márquez-González
E. Mermaid Lane, Wyndmoor, PA 19038 Department of Animal Science, Texas A&M
University, College Station, TX 77843-2471
Stanley E. Katz
Department of Biochemistry and Microbiology, Dragan Momcilovic
Rutgers, The State University of New Jersey, School Center for Veterinary Medicine, Food and Drug
of Environmental & Biological Sciences, 76 Lipman Administration, Rockville, MD 20855
Drive, New Brunswick, NJ 08901-8525
Peter M. Muriana
Hannu Korkeala Department of Animal Science, Food and
Department of Food and Environmental Hygiene, Agricultural Products Research and Technology
University of Helsinki, P.O. Box 66, 00014 Helsinki Center, Oklahoma State University, Stillwater,
University, Finland OK 74078

Balasubrahmanyam Kottapalli Michael T. Musgrove

Kraft Foods, 200 Deforest Ave., East Hanover, Egg Safety and Quality Research Unit, Russell
NJ 07936 Research Center, Athens, GA 30605
 CONTRIBUTORS ix

Kendra K. Nightingale Keun Seok Seo

Department of Animal Sciences, Colorado State Department of Microbiology, Molecular Biology,
University, Fort Collins, CO 80523 and Biochemistry, University of Idaho, Moscow,
ID 83844
John S. Novak
American Air Liquide, Delaware Research & O. Peter Snyder, Jr.
Technology Center, 200 GBC Drive, Newark, Hospitality Institute of Technology and
DE 19702-2462 Management, 670 Transfer Road, Suite 21A, St.
Paul, MN 55114
G.-J. E. Nychas
Agricultural University of Athens, Department John N. Sofos
of Food Science and Technology, Laboratory of
Center for Meat Safety & Quality, Food Safety
Microbiology & Biotechnology of Foods, Iera
Cluster of Infectious Diseases Supercluster,
Odos 75, Athens 11855, Hellas
Department of Animal Sciences, Colorado State
University, 1171 Campus Delivery, Fort Collins,
Andrea R. Ottesen
CO 80523-1171
Plant Sciences and Landscape Architecture,
University of Maryland, College Park,
Jarret D. Stopforth
MD 20742
PURAC, Arkelsedijk 46, 4206 AC, Gorinchem,
The Netherlands
Michael W. Peck
Institute of Food Research, Norwich Research Park,
C. Tassou
Colney, Norwich NR4 7UA, United Kingdom
National Agricultural Research Foundation,
Anna C. S. Porto-Fett Institute of Technology of Agricultural Products,
Microbial Food Safety Research Unit, U.S. S. Venizelou 1, Lycovrisi 14123, Athens, Hellas
Department of Agriculture, Agricultural Research
Service, Eastern Regional Research Center, Paula Marie L. Ward
Wyndmoor, PA 19038 Department of Biochemistry and Microbiology,
Rutgers, The State University of New Jersey, School
Deirdre M. Prendergast of Environmental & Biological Sciences, 76 Lipman
Food Safety Department, Teagasc, Ashtown Food Drive, New Brunswick, NJ 08901-8525
Research Centre, Ashtown, Dublin 15, Ireland
Charlene Wolf-Hall
L. Jason Richardson Great Plains Institute of Food Safety, 1523
USDA-ARS-Poultry Microbiological Safety Research Centennial Blvd., 114A Van Es Hall, North Dakota
Unit, Russell Research Center, Athens, GA 30605 State University, Fargo, ND 58105

Keith R. Schneider Anita C. Wright

University of Florida, Food Science and Human University of Florida, Food Science and Human
Nutrition Department, 359 FSHN Bldg., Nutrition Department, 359 FSHN Bldg., Newell
Newell Drive, Gainesville, FL 32611 Drive, Gainesville, FL 32611

Virginia N. Scott Jupiter M. Yeung

Grocery Manufacturers Association, 1350 I St. Grocery Manufacturers Association, Washington,
N.W., Suite 300, Washington, DC 20005 DC 20005
This page intentionally left blank
 PREFACE

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

xi
xii PREFACE

Accordingly, this book brings together these latest The credit for making this book a reality goes to
advances and should be of special benefit to those the authors and coauthors of various chapters. We
looking for a resource along with or in place of addi- commend the authors for their endeavor in compiling
tional classroom training. This book should be a the necessary information in their chapters. We hope
valuable tool for those who are directly or indirectly that the excellent work of the contributors will serve as
involved in the production, handling, processing, dis- a basis for new and innovative approaches to control-
tribution, and serving of food; control of hazards and ling food-borne hazards and significantly contributing
spoilage of food products; inspection of food process- to technologies that decrease the incidence of food-
ing facilities; or research studies on microbial control borne illnesses. Furthermore, we look forward to the
or inactivation. Those in academic, industrial, and breakthrough discoveries that will emerge in the future
government institutions, including federal, state, pri- as a result of the information presented in this book.
vate, and local agencies, as well as food consultants
and lobbyists, should find the book helpful in their Vijay K. Juneja
work. John N. Sofos
Pathogens and Toxins in Foods: Challenges and Interventions
Edited by V. K. Juneja and J. N. Sofos
© 2010 ASM Press, Washington, DC

Chapter 1

Bacillus cereus and Other Bacillus spp.

Mansel W. Griffiths

BACILLUS SPP. study may result in the identification of additional

genospecies.
Bacillus spp. are gram-positive, endospore-form- A second group of species of major interest is the
ing facultatively anaerobic bacteria. The resistance of B. cereus group, which is comprised of B. cereus,
their spores to adverse conditions has resulted in B. thuringiensis, B. mycoides, and B. anthracis. A psy-
widespread distribution of the organism. They have chrotrophic species, B. weihenstephanensis, has also
been isolated from air, soil, and water, as well as ani- been isolated (Lechner et al., 1998). While the mem-
mal and plant material. The ubiquitous nature of bers of this group are easily distinguished from other
these organisms makes contamination of food mate- spore-formers by their inability to produce acid from
rials a common occurrence. mannitol and their production of lecithinase, they are
In 1980, with the publication of the Approved difficult to distinguish from each other. Cells of these
Lists of Bacterial Names, taxonomic experts cooper- organisms are wider than 1 m, sporangia are not
ated in establishing a comprehensive and agreed-upon swollen, and their spores are ellipsoidal. B. cereus
list of accepted names of bacterial species. At this and B. thuringiensis are usually motile, whereas
time, 38 species of aerobic, endospore-forming bacte- B. cereus, B. thuringiensis, and B. mycoides are
ria were listed, of which 31 were allocated to the hemolytic and penicillin resistant. B. anthracis is exclu-
genus Bacillus (Skerman et al., 1980). Since 1980, the sively lysed by the gamma phage. Many of the so-
taxonomy of these organisms has been restructured called typical reactions, such as the rhizoid growth of
based on the application of better enrichment and B. mycoides, the formation of crystalline parasporal
isolation procedures that take into account their inclusion bodies by B. thuringiensis, and the pathoge-
physiology and nutritional and cultural requirements nicity of B. anthracis are culture dependent and/or
and on the development of molecular methods, espe- plasmid encoded. There is still significant controversy
cially 16S rRNA/DNA sequence analysis. The latter surrounding the taxonomy of the B. cereus group.
has led to the separation of groups of species from the Helgason et al. (2000) have proposed that B. anthra-
core genus Bacillus to form new genera (Fritze, cis, B. cereus, and B. thuringiensis should be regarded
2004). as one species with subspecies, while Jackson et al.
The genus Bacillus can be split into two groups: (1999) found an extensive genotypic diversity among
the B. subtilis group and the B. cereus group. Spe- the species, suggesting the presence of even more spe-
cies of the B. subtilis group are closely related and cies (or subspecies) within this group.
include B. subtilis, B. licheniformis, and B. pumilus. Multiple locus sequence typing of strains of
These species are generally mesophilic, with cells B. cereus has revealed three lineages, with the major-
less than 1 m wide, sporangia not swollen, and ity of food-related isolates belonging to lineage I
ellipsoidal spores. All members of the group are (Cardazzo et al., 2008). This work also demon-
placed in 16S rRNA/DNA group 1. Only some of the strated that there is widespread horizontal gene
species in this group can be differentiated based on transfer among the strains, and this has contributed
classical phenotypic tests. However, all species can significantly to the evolution of toxin-encoding
be differentiated by molecular techniques, and further genes.

Mansel W. Griffiths • Canadian Research Institute for Food Safety and Department of Food Science, University of Guelph, Guelph,
Ontario, Canada N1G 2W1.

1
2 GRIFFITHS

Characteristics of Bacillus cereus action of, or response to, one nutrient receptor on the
Arguably the most important characteristic of spore; or those that prevent the action of, or response
Bacillus spp. is their ability to form refractile to, several or all of the spore’s nutrient receptors
endospores. These spores are more resistant than veg- (Cortezzo et al., 2004). Organic acids can be used to
etative cells to heat, drying, food preservatives, and control germination and outgrowth of B. cereus
other environmental challenges. The bacteria of the spores in foods, but only when the storage tempera-
genus Bacillus are usually free living, that is, not host ture is 8C (Del Torre et al., 2001).
adapted, and their spores are widely distributed Their hydrophobic nature, coupled with the pres-
throughout nature. The spores of B. cereus are ellip- ence of appendages on their surfaces, enables spores to
soidal and central to subterminal and do not distend adhere to several types of surfaces, including epithelial
the sporangia (Griffiths and Phillips, 1990b). cells (Faille et al., 2002; Klavenes et al., 2002; Peng
et al., 2001; Stalheim and Granum, 2001; Tauveron
The spore et al., 2006). However, damage to the exosporium,
Spores from strains commonly associated with such as that which would occur during sporulation
food poisoning have a decimal reduction time at 95C under unfavorable conditions or by shear stress that
(D95) of approximately 24 minutes, with other strains may occur during processes such as microfluidization
showing a wider range of heat resistance (D95 of 1.5 to (Feijoo et al., 1997b), results in a decrease in the ability
36 minutes) (Meer et al., 1991). The z value varies of the spore to adhere to surfaces (Faille et al., 2007).
between about 6C and 9C (Byrne et al., 2006). How-
ever, there appears to be substantial heterogeneity Vegetative growth
among spore populations in their response to heat Early in the growth cycle vegetative cells are
treatment (Cronin and Wilkinson, 2008). Models gram positive, but cells may become gram variable
describing the effects of temperature and pH on ther- when in late log or stationary phase. Colonies on
mal inactivation (Fernandez et al., 1999, 2002) and agar media have a dull or frosted appearance.
germination and outgrowth (Gaillard et al., 2005) of The emergence of psychrotrophic strains of the
B. cereus spores have been generated. There was little organism, which have been classified as Bacillus
difference in the heat resistances of spores in the pres- weihenstephanensis, now means that the temperature
ence and absence of nisin, which is known to prevent range for growth is between approximately 5C and
spore outgrowth, but spores were 10 times more sen- 50C (Lechner et al., 1998). Generation times at 7C
sitive to high-pressure treatments (517 MPa) when lie between 9.4 and 75 hours, but in boiled rice at
nisin was present (Cruz and Montville, 2008). The 30C the generation times can range between 26 and
spore is also more resistant to irradiation, and the 57 minutes (Dufrenne et al., 1995; Meer et al., 1991).
dose for a 90% reduction in count lies between 1.25 The organism can produce toxin at low temperatures,
and 4 kGy. The corresponding dose for vegetative cells but this phenomenon appears to be strain dependent
is 0.17 to 0.65 kGy (De Lara et al., 2002). (Fermanian et al., 1997). Several strains can grow
Spore germination can occur over the tempera- slowly at sodium chloride concentrations of 10%,
ture range of 5C to 50C in cooked rice and between with a minimum water activity (aw) for growth
1C and 59C in laboratory media (Griffiths and between 0.91 and 0.93. B. cereus has an absolute
Schraft, 2002). Bacillus cereus spores germinate in requirement for amino acids as growth factors, but
response to particular nutrients, such as glycine or a vitamins are not required. The organism grows over a
neutral l-amino acid and purine ribosides, which they pH range of approximately 4.4 to 9.3. These limits for
sense by receptors encoded by the gerA family of growth are not absolute and are dependent on several
operons (Hornstra et al., 2005), or in response to factors, including strain (Baker and Griffiths, 1993).
physical treatments, such as temperature (Griffiths A surface structure of B. cereus cells, the S-layer, is
and Phillips, 1990b) and high pressures of 500 MPa involved in the adhesion of the organism to host cells
(Black et al., 2007). It has been demonstrated that and increased radiation resistance (Kotiranta et al.,
simulation of passage through the gastrointestinal 2000). It has also been shown that the ability of
tract at 37C resulted in better germination of spores B. cereus cells to tolerate bile depends on strain and the
of mesophilic rather than psychrotrophic strains of food in which the cells are present (Clavel et al., 2007).
B. cereus (Wijnands et al., 2006b). The ability of
spores to survive transit through the intestinal tract CHARACTERISTICS OF BACILLUS CEREUS
also depends on the food matrix and stomach acidity FOOD POISONING
(Clavel et al., 2004). Germination can be inhibited by
a variety of compounds, and these can be assigned to Two distinct types of illness have been attributed
either of two broad groups: those that inhibit the to the consumption of foods contaminated with
 CHAPTER 1 • BACILLUS CEREUS AND OTHER BACILLUS SPP. 3

B. cereus: (i) the diarrheal syndrome, which has an spp. This accounted for 3.4% of outbreaks and 1.6%
incubation time of 4 to 16 h and is manifested as of cases of bacterial food-borne illness reported dur-
abdominal pain and diarrhea that usually subsides ing this period (Anonymous, 2008).
5
within 12 to 24 h (the infective dose is reported as 10
7
to 10 cells ingested) and (ii) the emetic syndrome, which Pathogenesis of the diarrheal syndrome
has an incubation time of 1 to 5 h, causing nausea and Goepfert et al. (1972) were the first to relate the
vomiting that last for 6 to 24 h. To cause emetic B. cereus mechanism of pathogenicity of B. cereus to a possible
food poisoning, the food involved will typically contain enterotoxin responsible for fluid accumulation in the
5 8
10 to 10 cells/g. In some B. cereus outbreaks there ligated rabbit ileal loop (LRIL) test measured after
appears to be a clear overlap of the diarrheal and emetic injection of B. cereus cultures or culture supernatants
syndromes (Kramer and Gilbert, 1989). (Spira and Goepfert, 1972). Initial attempts to purify
As well as causing enteric illness, B. cereus has the enterotoxin of B. cereus revealed a protein com-
been responsible for postoperative infections, espe- plex, hemolysin BL (HBL), consisting of three pro-
cially in immunocompromised patients. Although a teins, termed B, L1, and L2, that exhibited positive
rare event, such an infection can result in bacteremia, results in the LRIL and vascular permeability assays
septicemia, endocarditis, meningitis, and pneumonia, (Beecher and Wong, 1994, 1997; Ryan et al., 1997).
among other symptoms (Drobniewski, 1993; Schoeni All three components were apparently required to
and Wong, 2005). The organism is also commonly produce maximal fluid accumulation in the LRIL
associated with eye infections, such as posttraumatic assay. The HBL enterotoxin is very heterogeneous,
endophthalmitis and metastatic endophthalmitis, and and some B. cereus strains may produce more than
can very quickly result in irreversible tissue damage one set of the three HBL components (Beecher and
(Schoeni and Wong, 2005). There have been some Wong, 2000; Schoeni and Wong, 1999). The nucle-
reports of neonatal infections due to B. cereus, and it otide and deduced amino acid sequences have been
has been proposed that the systemic complications reported for all three components of HBL (Heinrichs
observed with these cases are associated with entero- et al., 1993; Okstad et al., 1999; Ryan et al., 1997).
toxins (Girisch et al., 2003). The proteins exhibit 20 to 24% homology and have
Bacillus cereus Diarrheal Syndrome a structure that is almost entirely -helix, indicating
that they have evolved from the duplication of a sin-
The disease gle gene (Schoeni and Wong, 2005).
The diarrheal illness caused by B. cereus is self- The HBL enterotoxin complex produces a
limiting, and no treatment is necessary. In severe unique discontinuous pattern of hemolysis on blood
cases, fluid replacement therapy may be required. agar (Beecher and Macmillan, 1990; Beecher and
This type of B. cereus food poisoning was first Wong, 1994, 1997), which involves the B and L1
described in detail by Hauge (1955), who found that components.
an outbreak of gastroenteritis in a hospital was asso- As well as causing fluid accumulation in the
ciated with vanilla pudding contaminated with high LRIL assay, HBL is dermonecrotic (Beecher and
8
numbers (up to 10 CFU/ml) of B. cereus. Since that Wong, 1994) and cytotoxic (Beecher et al., 1995).
first publication, reports on the incidence of B. cereus The toxin acts by binding to the membrane of eukary-
diarrheal disease have increased worldwide. otes, where it oligomerizes to form pores (Beecher
The incidence of food-borne illness caused by and Wong, 1997). A possible pathway of pore forma-
B. cereus varies from country to country. Although tion has been suggested based on the X-ray crystal
information on the extent of the problem is scant, it structure of the B component (Madegowda et al.,
has been reported that outbreaks due to B. cereus can 2008).
be the most common cause of food-borne illness in In contrast, Granum’s research group in Norway
some countries, such as The Netherlands (Simone has purified and characterized a nonhemolytic entero-
et al., 1997) and Taiwan (Chang and Chen, 2003). In toxin (Nhe) (Granum et al., 1996; Lund and Granum,
France it has been estimated that there are 219 to 701 1996). This toxin is also composed of three protein
cases of B. cereus-associated food-borne illness annu- components (39, 45, and 105 kDa), which demon-
ally, resulting in 26 to 84 hospitalizations (Vaillant strate homology with each other and the components
et al., 2005). Data for the United States suggest that of HBL (Schoeni and Wong, 2005). The 105-kDa
more than 27,000 cases of B. cereus gastrointestinal protein is a protease that is not part of the Nhe com-
infections may occur annually (Mead et al., 1999). plex (Lund and Granum, 1999). The genes encoding
For the period between 1998 and 2006, the Centers the Nhe complex are situated in an operon contain-
for Disease Control and Prevention reported 90 out- ing three open reading frames (ORFs), nheA, nheB,
breaks and 1,623 cases of infection caused by Bacillus and nheC (Granum et al., 1999). NheB is the only
4 GRIFFITHS

protein of the complex that binds to Vero cells, but and colleagues (Shinagawa, 1990; Shinagawa et al.,
all three proteins are needed for activity. The function 1991), has been cloned and sequenced by Asano et al.
of NheC is not yet understood, but it probably func- (1997). It has been suggested that EntFM has similar
tions as a “catalyst,” either by bringing NheA and properties to a phosphatase-associated protein with
NheB together following binding of NheB to the tar- cell wall hydrolase activity from B. subtilis (Margot
get cells or by enhancing conformational changes et al., 1998; Schoeni and Wong, 2005).
(Lindback et al., 2004). Whereas the exact mode of A cytotoxic protein (CytK) implicated in necrotic
action is unclear, plasma membrane disruption was enteritis has been isolated from a B. cereus strain
observed with epithelia exposed to Nhe from culture involved in a severe outbreak of B. cereus diarrheal
supernatants of B. cereus, but not with those exposed disease, which resulted in three deaths. The protein
to supernatants from a mutant strain lacking NheB was very similar to ß-barrel channel-forming toxins
and NheC (Fagerlund et al., 2008). The purified Nhe found in Staphylococcus aureus and Clostridium per-
components also combined to form large pores in fringens (Lund et al., 2000). Again this protein was
lipid bilayers, which led to the hypothesis that, capable of forming pores in lipid bilayers (Hardy
because of their common structural and functional et al., 2001). The cytK gene was detected in 73% of
properties, the HBL/Nhe and ClyA families of toxins B. cereus isolates involved in diarrheal disease, but
belong to a superfamily of pore-forming cytotoxins. only in 37% of isolates from food not implicated in
Sequencing and gene expression assays show that the disease (Guinebretiere et al., 2002, 2006). Subse-
genes responsible for both HBL and Nhe are tran- quently, two variants of CytK, CytK1 and CytK2,
scribed from one operon, with maximum enterotoxin have been identified. The isolated CytK2 proteins
activity produced during late exponential or early were hemolytic, cytotoxic, and capable of forming
stationary growth (Granum et al., 1999). Early work membrane pores, but their toxicity was about 20%
suggested that enterotoxigenic B. cereus isolates pro- of that of CytK1 (Fagerlund et al., 2004; Guinebre-
duce either both Nhe and HBL toxins or only one tiere et al., 2006). Brillard and Lereclus (2004)
(Lund and Granum, 1997). A more extensive study, reported that cytK was transcribed more strongly in
in which PCR primer pairs targeting hblC, hblD, an isolate of B. cereus linked to human illness than in
hblA, nheA, nheB, nheC, cytK, and entFM were used a reference strain and that cytK was controlled by the
to analyze 411 B. cereus strains and 205 B. thuringi- global regulator PlcR.
ensis strains, confirmed this finding and identified It was originally thought that the B. cereus diar-
four groups, based on the presence of virulence genes. rheal syndrome was the result of intoxication, due to
In group I, all eight genes were present; groups II and the ingestion of toxin produced during growth of
III contained isolates devoid of all hbl genes and cytK B. cereus in the food. However, it has been postulated
genes, respectively; and group IV strains lacked both that the disease results from ingestion of B. cereus
the hbl and cytK genes. Both nhe and entFM genes cells that grow and produce enterotoxin within the
were present in all isolates tested (Ngamwongsatit intestinal tract of the patient (toxico-infection) (Gra-
et al., 2008). num and Lund, 1997; Griffiths and Schraft, 2002;
Agata et al. (1995) described yet another pro- Schraft and Griffiths, 2006). This claim is based on
tein, B. cereus enterotoxin T (BcET), which showed three observations. (i) Most strains produce entero-
positive reactions in vascular permeability reaction toxin in significant amounts only after reaching cell
7
VPR and LRIL tests. The protein and the encoding concentrations of 10 /ml, while the infectious dose in
DNA sequence are not related to HBL or Nhe. How- many food-borne outbreaks has been determined to
3 4
ever, additional research indicated that this protein is be 10 to 10 /ml. (ii) The toxin is rapidly degraded at
not likely to cause diarrheal disease (Choma and Gra- pH 3 (a pH similar to that found in the stomach) and
num, 2002) and that the bceT gene was, in fact, not a hydrolyzed by the proteolytic enzymes of the diges-
single gene but four independent, ligated DNA frag- tive tract (Granum et al., 1993). However, these stud-
ments (Hansen et al., 2003). One of these fragments ies were conducted using enterotoxin produced in
had 93% homology to an ORF (ORF 101) located defined or semidefined media, and it should be noted
within the pathogenic island of the Bacillus anthracis that enterotoxin produced and ingested in a food
pXO1 virulence plasmid. The authors concluded that matrix may be protected from damage through
the enterotoxic activity of the original cloned bceT low pH and enzymes (Baker and Griffiths, 1995).
construct could be due to either this fusion gene or (iii) Incubation times of more than 24 h have been
the fragment with homology to ORF 101. observed for many outbreaks, and this is too long for
A fourth possible B. cereus enterotoxin (EntFM), simple enterotoxin action and may reflect the time
which was thought to be similar to the gene encoding required for spore germination. However, several
the 45-kDa cytotoxic protein described by Shinagawa B. cereus isolates are able to grow well under anaerobic
 CHAPTER 1 • BACILLUS CEREUS AND OTHER BACILLUS SPP. 5

conditions at 37C and produce significant levels of pore formation in the plasma membrane, has been
enterotoxin within 6 h (Glatz and Goepfert, 1976; proposed by Fagerlund et al. (2008). B. cereus entero-
 
Granum et al., 1993). Other studies have shown that toxins reverse absorption of fluid, Na , and Cl by
enterotoxin was not produced in defined medium epithelial cells and cause malabsorption of glucose
during anaerobic growth (Beattie and Williams, and amino acids, as well as necrosis and mucosal
2002). Both HBL and Nhe production are influenced damage. It has been suggested that the effects on fluid
by carbon source and growth rate of the organism absorption are due to stimulation of adenylate cyclase
during anaerobic growth (Ouhib et al., 2006). At low (Kramer and Gilbert, 1989).
oxidoreduction potentials, there is a strong stimula-
tion of HBL production and, to a lesser extent, Nhe
Bacillus cereus Emetic Syndrome
production. Nhe was produced first, early in the
exponential growth phase, and HBL was synthesized The disease
later, during the early-stationary-growth phase (Zigha A second type of B. cereus gastroenteritis, the
et al., 2006). This response is controlled by a two- emetic syndrome, was first identified in the 1970s and
component regulatory system, ResDE, and by a pro- was associated with the consumption of fried rice
tein similar to the Fur redox regulator of B. subtilis (Kramer and Gilbert, 1989). The emetic illness has an
(Duport et al., 2006; Zigha et al., 2007). incubation time of 1 to 5 h and is manifested by nau-
It may well be possible that both modes of patho- sea and vomiting that last for 6 to 24 h. Diarrhea is
genesis may be possible, especially since several diar- observed infrequently. The symptoms are usually self-
rhegenic enterotoxins have been described. For limiting, and treatment is seldom necessary. To cause
example, a toxico-infection might be the mode of 5
emesis, the food involved will typically contain 10 to
action for a more severe form of B. cereus diarrhege- 8
10 cells/g. On rare occasions, emetic B. cereus may
nic syndrome described by Granum (1997). In one also cause more severe disease. An outbreak was
outbreak, 17 out of 24 people were affected after eat- described in which a 17-year-old boy exhibited
ing stews, and 3 of the patients were hospitalized, B. cereus emetic syndrome and died of liver failure
1 for 3 weeks. The infective dose observed in this within 2 days (Mahler et al., 1997). His father suf-
4 5
outbreak was 10 to 10 cells, which is lower than that fered hyperbilirubinemia and rhabdomyolysis, but he
usually associated with B. cereus. The time to onset of recovered. High levels of B. cereus emetic toxin were
symptoms for these patients was more than 24 hours, found in the pan used to reheat the food and in the
also longer than commonly observed for B. cereus. In boy’s liver and bile. B. cereus was also cultured from
another outbreak, competitors at a skiing event in both intestinal contents and pan residues. It was con-
Norway became ill after drinking milk. The illness cluded that liver failure was due to the emetic toxin
was restricted to young skiers between the ages of 16 causing inhibition of hepatic mitochondrial fatty-acid
and 19, while the older coaches and officials did not oxidation. A subsequent outbreak linked to emetic
exhibit symptoms. Again, the incubation period was B. cereus and involving five children from a family in
greater than 24 hours in some cases and symptoms Belgium who had eaten a contaminated pasta salad
persisted for between 2 and several days. Andersson led to the death of the youngest child due to liver
et al. (1998a) postulate that this more severe form of failure (Dierick et al., 2005).
B. cereus gastroenteritis results from adhesion of spores
to the epithelial cells, followed by germination and
enterotoxin production within the intestinal tract. Pathogenesis of the emetic syndrome
The longer incubation time seen in these cases may be Early experiments relying on monkey feeding trials
due to the time required for germination of the spores. identified the cause of the emetic syndrome as a toxin
It has been demonstrated that spores of B. cereus because cell-free supernatants produced the same
strains with high hydrophobicity are more adherent symptoms as cell cultures (Kramer and Gilbert, 1989).
to CaCo-2 cells than spores with low hydrophobicity For many years the structure of the emetic toxin was
and that two of these highly hydrophobic strains were elusive, partly because there was no convenient assay.
isolated during the outbreak involving meat stew It was thought to be a lipid and was known to be
(Anderson et al., 1998a). After adhesion, the spores nonantigenic and extremely resistant to heat, alkaline
were able to germinate and produce enterotoxins. and acid pH, and proteolysis. The emetic toxin has
Only limited studies have been carried out on now been identified as a ring-structured peptide com-
the mode of action of Bacillus enterotoxin. The posed of three repeat sequences of four amino and/or
enterotoxins identified for B. cereus are capable of oxy-acids. The dodecadepsipeptide, termed cereulide,
forming pores in lipid bilayers, and a mechanism of has a molecular mass of 1.2 kDa and the following
cytotoxicity for Nhe, comprising osmotic lysis following composition: [d-O-leu-d-ala-l-O-val-l-val]3.
6 GRIFFITHS

It closely resembles the potassium ionophore chromatography and mass spectroscopy (Haggblom
valinomycin (Agata et al., 1994). The toxin was sta- et al., 2002), are now available. Kawamura-Sato et al.
ble at pH 2 to 11, withstood heating at 121C for (2005) have described a quantitative assay for cereu-
90 min, and was not inactivated by treatment with lide based on uncoupling of the respiratory activity of
trypsin and pepsin (Shinagawa et al., 1995, 1996). rat liver mitochondria.
Oxygen is required for the production of the toxin Unlike the diarrheal strains of B. cereus that are
(Jaaskelainen et al., 2004). Cereulide was formed extremely heterogeneous, emetic strains have very
when B. cereus emetic strains were present at cell low diversity and consist of a single, distinct cluster
6
numbers of 10 CFU/g in products with aw values of of isolates that are unable to degrade starch and do
0.953 and pH values of 5.6 (Jaaskelainen et al., not ferment salicin (Ehling-Schulz et al., 2005a).
2003). High levels of toxin (0.3 to 5.5 g/g) were
produced during growth of B. cereus in rice-containing
pastries at ambient temperatures (21 to 23C). How- Incidence and Transmission through Food
ever, cereulide was not formed in products stored at The mild and transient character of the B. cereus
4 to 8C (Jaaskelainen et al., 2003). food-borne diseases likely results in the disease being
Cereulide stimulates the vagus afferent by binding vastly underreported. None of the reports on food-
to 5-HT3 receptors and induces swelling of mitochon- borne disease outbreaks caused by B. cereus distin-
dria, with toxic effects due to potassium-ionophoretic guishes between emetic and diarrheal syndromes, but
properties (Agata et al., 1994; Mikkola et al., 1999; it is likely that the outbreaks reflect the diarrheal syn-
Sakurai et al., 1994). Cytotoxicity is accompanied by drome. The incidence of B. cereus food-borne dis-
inhibition of RNA synthesis and cell proliferation at eases seems to be higher in countries of northern
cereulide concentrations of 2 nM (Andersson et al., Europe (20 to 33%) and in Canada (14%). England
2007). At high doses, cereulide can cause a massive and Wales, Japan, and the United States (1.2 to 4.4%)
degeneration of hepatocytes (Yokoyama et al., 1999). have a clearly lower incidence. The reasons for these
The toxin also inhibits natural killer cells in vitro, differences are not known. Foods most often impli-
which suggests that the toxin may possess immuno- cated as vehicles for transmission of the diarrheal
modulating properties (Paananen et al., 2002). syndrome include meat and meat dishes, vegetables,
Cereulide is produced by a nonribosomal pep- cream, spices, poultry, and eggs. The emetic syndrome
tide synthetase (Ehling-Schulz et al., 2005b), which is is most frequently associated with starchy foods,
encoded by a cereulide synthetase (ces) gene cluster especially rice dishes (Kramer and Gilbert, 1989).
(Ehling-Schulz et al., 2006). These genes are found The amount of emetic toxin in 13 of 14 food
only in emetic strains of B. cereus and are located on samples implicated in emetic-type illness ranged
a 208-kb megaplasmid. The ces gene cluster (24 kb) from 0.01 to 1.28 g/g (Agata et al., 2002). When
comprises seven coding sequences (CDSs). These challenge studies were performed using an emetic
include the typical nonribosomal peptide synthetase toxin-producing strain of B. cereus, the organism
genes, encoding a phosphopantetheinyl transferase was capable of rapid growth in boiled rice and pro-
and two enzymes for the activation and incorpora- duced emetic toxin at both 30 and 35C. In farina-
tion of monomers in the growing peptide chain, a ceous foods, the production of emetic toxin was as
CDS encoding a putative hydrolase (upstream region), high as that in the foods implicated in illness. Low
and an ABC transporter (downstream). Regions levels of emetic toxin were detectable in eggs and
flanking the ces gene locus have high homology to meat and their products, and a small quantity of
virulence plasmids of B. cereus, B. thuringiensis, and toxin was detectable in milk and soy milk, when not
B. anthracis. This has led to the conclusion that, aerated. Bacterial growth and toxin production were
besides the insecticidal and anthrax toxins of B. thu- inhibited in foods cooked with vinegar, mayonnaise,
ringiensis and B. anthracis, respectively, a third type and catsup, presumably due to the low pH gener-
of B. cereus group toxins exists, and these toxins are ated by acetic acid (Agata et al., 2002). When potato
encoded on megaplasmids. puree and penne pasta were inoculated with
The emetic toxin can be assayed by determining cereulide-producing strains of B. cereus and stored
vacuolation induced in HEp-2 cells (Hughes et al., without shaking, higher cereulide concentrations
1988), and this has been aided by the development were achieved (4 g/g in puree and 3.2 g/g in
of colorimetric modifications (Finlay et al., 1997; penne) than in boiled rice (2 g/g) (Rajkovic et al.,
8
Mikami et al., 1994; Szabo et al., 1991). A rapid bio- 2006), despite counts in excess of 10 CFU/g being
assay, which tests toxicity of cereulide to boar sper- attained in all three products. With aeration, cereu-
matozoa (Andersson et al., 1998b, 2004), and a chemical lide production in these products was more than
assay, which is based on high-performance liquid 10-fold lower. Cereulide was not observed following
 CHAPTER 1 • BACILLUS CEREUS AND OTHER BACILLUS SPP. 7

growth of emetic strains of B. cereus in aerated milk, Of seven strains isolated from these products, one pro-
but levels reached 1.1 g/ml in statically incubated duced diarrheal toxin and four caused lethality in
milk. Thus, a number of factors, including food mice. Because of the ubiquitous distribution of the
type, temperature, pH, aeration, and strain, deter- organism, it is virtually impossible to obtain raw prod-
mine the amount of cereulide that will be produced. ucts that are free from B. cereus spores. Vegetable
Cereulide is also remarkably resistant to heat and purees have been implicated in outbreaks of B. cereus
alkaline pH (Rajkovic et al., 2008). illness (Lund et al., 2000), and the source of the con-
It has also been shown that emetic strains of tamination is the raw vegetable and the texturing
B. cereus were not able to grow at temperatures agents, such as milk protein and starch, used in the
below 10C but were able to grow at 48C. In gen- formulation (Guinebretiere et al., 2003). No spores
eral, spores of emetic strains also were more resistant were detected on the processing equipment.
to heat (90C) (Carlin et al., 2006). Carlin et al. sug- It was also postulated that B. cereus could be
gested that these properties make emetic strains a present in paper pulp and the products made from it;
special risk in heat-processed foods or preheated however, it has been determined that the level of cere-
foods that are kept warm, but they should not pose a ulide retained in paper is too low to present a risk to
risk in refrigerated foods. human health (Hoornstra et al., 2006).
An infant food containing cereal and dried infant The dairy industry faces problems due to B.
milk formula was associated with illness in two cereus because it is virtually impossible to exclude the
infants, which presented as rapid projectile vomiting organism from milk; their spores readily attach to
(Duc et al., 2005). Both B. subtilis and B. cereus were processing equipment, and pasteurization does not
isolated from the product, but cereulide was not eliminate them (Andersson et al., 1995). Indeed, in
detected. This led the investigators to suggest that the many cases, pasteurization may result in activation
cause of the illness was due either to a new cereulide- and germination of spores (Griffiths and Phillips,
type toxin or to the high levels of B. subtilis present. 1990b; Hanson et al., 2005), which has resulted in
It has been reported that infant food containing both shelf-life issues with milks pasteurized at tempera-
cereal and milk powder are able to support greater tures in excess of the recommended minimum for
levels of cereulide production than other infant for- high-temperature, short holding time processing.
mulas and that mishandling and temperature abuse Fortunately, it seems that the majority of B. cereus
of reconstituted formula were high-risk activities strains isolated from dairy products are not highly
(Shaheen et al., 2006). cytotoxic (Arnesen et al., 2007).
Altayar and Sutherland (2006) found that 45.8% The appearance of psychrotrophic strains in the
of 271 samples of soils, animal feces, and raw and dairy industry has added a new dimension to B. cereus
processed vegetables were positive for B. cereus. Of surveillance in food. Studies indicate that both raw
the 325 isolates obtained, only 3 were positive for and pasteurized milk will harbor psychrotrophic B.
emetic toxin production. All the positive isolates cereus, with a prevalence of 9 to 37% in raw milk and
came from washed or unwashed potato skins, and 2 to 35% in pasteurized milk (te Giffel et al., 1995),
one strain was psychrotrophic. and that the source of the contamination of raw milk
was primarily silage (te Giffel et al., 2002). Subsequent
work has suggested that the presence of B. cereus in
PRESENCE, GROWTH, AND SURVIVAL pasteurized milk may be the result of postpasteuriza-
OF B. CEREUS IN FOODS tion contamination at the filler (Eneroth et al., 2001).
These psychrotrophic strains have an average genera-
Presence of B. cereus in Foods
tion time of 17 h at 6C and produce enterotoxin dur-
Members of the B. cereus group are ubiquitously ing extended storage at slightly abusive temperatures
distributed in the environment, mainly because of their (Christiansson et al., 1989; Griffiths, 1990; Griffiths
spore-forming capabilities. Thus, B. cereus can easily and Phillips, 1990a). In coffee creamers stored at
contaminate various types of foods, especially prod- ambient temperatures, B. cereus can grow to levels of
ucts of plant origin. The organism is also frequently public health concern within 11 h (Feijoo et al.,
isolated from milk and dairy products, meat and meat 1997a). For example, Odumeru et al. (1997) found
products, pasteurized liquid egg, rice, ready-to-eat that 38% of pasteurized milks stored at 10C until
vegetables, and spices (te Giffel et al., 1996). The last their expiry date contained B. cereus enterotoxin. Sim-
can be a source of contamination of prepared meals ilarly, te Giffel et al. (1997) reported that 40% of pas-
(Banerjee and Sarkar, 2004). B. cereus was also iso- teurized milks sampled from household refrigerators
lated from vacuum-packaged and film-packaged soya- in The Netherlands were contaminated with B. cereus
and cereal-based vegetarian foods (Fang et al., 1999). and that about 75% of these were enterotoxigenic.
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