Table Of ContentClostridia
in
Gastrointestinal
Disease
Editor
S. Peter Borriello, Ph.D.
Research Fellow
Division of Communicable Diseases
Clinical Research Center
Harrow, Middlesex
England
Boca Raton London New York
CRC Press is an imprint of the
Taylor & Francis Group, an informa business
First published 1985 by CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742
Reissued 1918 by CRC Press
© 1985 by CRC Press is an imprint of Taylor & Francis Group, an Informa business
No claim to original U.S. Government works
This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish
reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the
consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this
publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has
not been acknowledged please write and let us know so we may rectify in any future reprint.
Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form
by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording,
or in any information storage or retrieval system, without written permission from the publishers.
For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.
com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a
not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a
photocopy license by the CCC, a separate system of payment has been arranged.
Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and
explanation without intent to infringe.
Library of Congress Cataloging in Publication Data
Main entry under title:
Clostridia in gastrointestinal disease.
Bibliography: p.
Includes index.
1. Intestines—Diseases—Etiology. 2. Clostridium
diseases. 3. Intestines—Microbiology. 4. Intestines—
Diseases—Animal models. I. Borriello, S. Peter.
[DNLM: 1. Clostridium—pathogenicity. 2. Clostridium
Infections—complications. 3. Gastrointestinal
Diseases—etiology. WC 368 C645]
RC862.C47C56 1985 616.3’3 84-11425
ISBN 0-8493-5656-3
A Library of Congress record exists under LC control number: 84011425
Publisher’s Note
The publisher has gone to great lengths to ensure the quality of this reprint but points out that some imperfections in the original copies
may be apparent.
Disclaimer
The publisher has made every effort to trace copyright holders and welcomes correspondence from those they have been unable to
contact.
ISBN 13: 978-1-315-89161-3 (hbk)
ISBN 13: 978-1-351-07071-3 (ebk)
Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the
CRC Press Web site at http://www.crcpress.com
FOREWORD
Although numerically the clostridia form only a very small component of the total gut
flora the genus produces one of the most potent collection of toxins known and represents
one of the most lethal collection of potential opportunist pathogens harboured in the gut.
They are ideally situated to take advantage of any compromise in the hosts normal defense
mechanisms to cause gastrointestinal disease. The recent discovery of Clostridiwn difficile
as a cause of antibiotic-associated diarrhea and colitis has served to refocus peoples attention
of the pathogenic potential of the clostridia. It also demonstrates our ignorance of the
importance of clostridia in gut disease as stated clearly in Chapter 1, "Although antimicro-
bials have been producing diarrhea for more than 40 years, the role of C. difficile . . .
has been revealed only within the past 4 years. This single fact unequivocally demonstrates
the ignorance of medical science as regards the importance of intestinal clostridia . . . "
The aim of this book is to bring together the information available on established clostridial
diseases of the gastrointestinal tract, including the more recent observations with respect to
mechanisms of action and to critically review the data available which implicate clostridia
in gastrointestinal diseases of unknown etiology such as infantile necrotizing enterocolitis
and large bowel cancer. Information on the wide range of gut diseases in animals, both
natural and laboratory induced, in which clostridia have been shown to be involved or are
implicated, has been included, as in many instances these observations serve to help delineate
the etiologies of human disease.
S. P. Borriello
THE EDITOR
S. Peter Bordello, Ph.D., MiBioL, is a lecturer in the Division of Communicable
Diseases at the Clinical Research Centre at Harrow in Middlesex, England. He graduated
with an honors degree in Microbiology at University College, University of London in 1975
and received his Ph.D. degree from the University of London registered through St. Thomas'
Hospital Medical School in 1981. Formerly a member of the scientific staff of the Public
Health Laboratory Service, Bacterial Metabolism Research Laboratory, London, he took up
his present post in 1979. His major area of interest is the bacterial flora of the gastrointestinal
tract in health and disease, including in particular antibiotic-associated diarrhea and colitis
and colon cancer. His recent research has included the discovery of the cause of a colitis
in rabbits and a new cause of antibiotic-associated diarrhea in man. Current interests also
include microbial metabolism of dietary components, microbial interactions, and the etiology
of sexually transmitted diseases of unknown cause.
Dr. Borriello is a member of the Institute of Biology, American Society of Microbiology,
the Society for Intestinal Microbial Ecology and Disease, the Anaerobe Discussion Group
and the Pathological Society of Great Britain and Ireland. He has been a member of the
Anaerobe Discussion Group steering committee since 1980 and is Chairman of the Publi-
cations Committee for the Society for Intestinal Microbial Ecology and Disease. Dr. Borriello
also serves as an outside reviewer for a number of journals and has edited or co-edited two
other books as well as having published over 50 scientific articles.
CONTRIBUTORS
Stephen S. Arnon, M.D. Michael J. Hill, Ph.D.
Senior Investigator Public Health Laboratory Service
Infant Botulism Research Project PHLS Centre for Applied Microbiology
California Department of Health Services and Research
Berkeley, California Bacterial Metabolism Research
Laboratory
Edward Balish, Ph.D. Salisbury, Wiltshire, England
Professor
Departments of Surgery and Medical Robert M. Kliegman, M.D.
Microbiology Assistant Professor
University of Wisconsin Medical School Department of Pediatrics
Madison, Wisconsin Case Western Reserve University
Rainbow Babies Children's Hospital
Cleveland, Ohio
S. Peter Bordello, Ph.D.
Lecturer
H. Elliott Larson, M.D., F.R.C.P.
Division of Communicable Diseases
Member, Scientific Staff
Clinical Research Centre
Division of Communicable Diseases
Harrow, Middlesex, England
Clinical Research Centre
Harrow, Middlesex, England
R. J. Carman, Ph.D.
Research Microbiologist
Ashley B. Price, M.A., B.M., B.Ch.,
Department of Animal Unit
F.R.C. Pathol.
Charing Cross Hospital Medical School
Consultant Pathologist
London, England
Department of Pathology
Northwick Park Hospital
Sydney M. Finegold, M.D. Harrow, Middlesex, England
Chief, Infectious Disease Section
VA Wadsworth Medical Center M. F. Stringer, Ph.D.
Professor of Medicine Senior Microbiologist
Professor of Microbiology and Food Hygiene Laboratory
Immunology Public Health Laboratory Service
UCLA School of Medicine London, England
Los Angeles, California
P. D. Walker, Ph.D.
W. Lance George, M.D. Head, Bacteriology R&D Department
Assistant Chief Wellcome Research Laboratories
Infectious Disease Section Beckenham, Kent, England
Director, Clinical Microbiology
Laboratory Charles E. Yale, M.D.
VA Wadsworth Medical Center Professor and Vice Chairman
Associate Professor of Medicine Department of Surgery
UCLA School of Medicine University of Wisconsin Medical School
Los Angeles, California Madison, Wisconsin
TABLE OF CONTENTS
Chapter 1
Clostridia in the Human Gastrointestinal Flora 1
W. Lance George and Sidney M. Finegold
Chapter 2
Infant Botulism 39
Stephen S. Arnon
Chapter 3
Evidence for Clostridial Involvement in Pneumatosis Cystoides Intestinalis 59
Charles E. Yale and Edward Balish
Chapter 4
Role of Clostridia in the Pathogenesis of Neonatal Necrotizing Enterocolitis 67
Robert M. Kliegman
Chapter 5
Pig- Bel 93
Peter D. Walker
Chapter 6
Clostridium perfringens Type A Food Poisoning 117
Michael F. Stringer
Chapter 7
Pseudomembranous and Antibiotic-Associated Colitis 145
S. Peter Borriello and H. Elliott Larson
Chapter 8
Clostridia and Human Colorectal Cancer 165
Michael J. Hill
Chapter 9
Histopathology of Clostridial Gut Diseases in Man 177
Ashley B. Price
Chapter 10
Clostridial Diseases of the Gastrointestinal Tract in Animals 195
S. Peter Borriello and Robert J. Carman
Chapter 11
Newly Described Clostridial Diseases of the Gastrointestinal Tract: Clostridium
Perfringens Enterotoxin-Associated Diarrhea and Neutropenic Enterocolitis
Due to Clostridium Septicum 223
S. P. Borriello
Index 231
1
Chapter 1
CLOSTRIDIA IN THE HUMAN GASTROINTESTINAL FLORA
W. Lance George and Sydney M. Finegold
TABLE OF CONTENTS
I. Introduction 2
II. Variations in Different Studies of Bowel Flora 2
III. Total Counts of Clostridia and Species of Clostridium at Various Sites in the
Gastrointestinal Tract 3
A. Healthy Infants and Children 3
1. Feces — Total Counts of Colstridium 3
2. Feces — Species of Clostridium 3
B. Adults 13
1. Stomach — Total Counts of Clostridium 13
2. Duodenum — Total Counts of Clostridium 13
3. Jejunum — Total Counts of Clostridium 13
4. Ileum — Total Counts of Clostridium 14
5. Proximal and Mid-colon — Total Counts of Clostridium 14
6. Feces — Total Counts of Clostridium 14
7. Vermiform Appendix — Species of Clostridium 14
8. Feces — Species of Clostridium 18
IV. Factors that Affect the Presence of Clostridia in the Human Bowel Flora 26
A. Age 27
B. Diet 27
1. Infants 27
2, Adults 27
C. Abnormalities of Gut Anatomy and Motility 29
D. Effect of Cathartics and Diarrhea upon Bowel Flora 30
E. Effect of Antimicrobials on Clostridia in Feces 30
1. Penicillins 30
2. Cephalosporins 30
3. Lincosamides 31
4. Tetracyclines 31
5. Erythromycin 31
6. Metronidazole 31
7. Other Agents 32
V. Summary 32
References 33
2 Clostridia in Gastrointestinal Disease
I. INTRODUCTION
Clostridia are frequently present in the normal flora of the human gastrointestinal (GI)
tract. Certain species of Clostridium (present either as normal flora or as exogenous "in-
vaders'*) in the GI tract may be of great consequence to the host — both as potential sources
of extra-GI infection and gut-related illnesses; the latter group of diseases will be reviewed
in subsequent chapters. The purpose of this chapter is to review clostridia as components
of the "normal" human GI flora and to review factors which may affect the presence of
clostridia in the gut.
II. VARIATIONS IN DIFFERENT STUDIES OF BOWEL FLORA
Moore and Holdeman1 have reported that the "total number of different kinds of bacteria
in the intestinal tract at any one time probably exceeds 400 to 500 species, but most are
represented by less than 108 cells per gram of feces (less than 1/1000 of the bacterial
population)". An investigator attempting to study the fecal flora is confronted with the need
to isolate species of bacteria that may be present in appreciable numbers (104 to 1010 per
gram), but that may be outnumbered as much as 10 million to 1 by other components of
the flora. Selective media for clostridia (and other bacteria) are quite important in this regard.2
It is not known, however, whether most of the species of Clostridium that may be present
in the gut will grow on the various selective media available. Heat shock is commonly used
as a selective means to recover clostridia from feces because spores are generally resistant
to such treatment; however, the resistance of spores to heat is somewhat variable, and
vegetative cells of clostridia usually do not survive heating. The effect of heating may
therefore reduce the viable counts of a given species of Clostridium or even eradicate it
altogether.
Limitations in methodology in some of the studies that we reviewed include the following:
1. Failure to use selective media.
2. Inoculation of media only with high dilutions of the specimen (if the lowest dilution
used is 107, then the lowest count of any Clostridium that could be detected would
be 107 or 108).
3. Use of less than optimal anaerobic transport of specimens.
4. Delays in processing of the specimen.
5. Freezing of the specimen prior to study (freezing is likely to kill vegetative ceils,
although spores would most likely survive).
6. Failure to homogenize the specimen (the distribution of organisms in feces is irregular).
7. Failure to base counts on the "dry weight" of the sample (to correct for variable
moisture content).2
These variables are quite significant and undoubtedly have a substantial impact on the data
reported.
Variations between the different populations studied may also have a significant impact
on the results of bowel flora studies; these variations may either be within a given study or
between studies to be compared. Variations include age of the subjects, type of diet, general
state of health, presence of GI disease (e.g., ulcerative colitis and sprue), and country of
residence. Such information, whenever available, was included in our review.
Improved methods for identification of clostridia (and other anaerobes) have been devel-
oped over the past 10 to 15 years; it is difficult to determine the reliability of identification
techniques that were used prior to 1970. For example, C, tetani was isolated rather frequently
from feces in several studies performed in the early part of this century, yet was not recovered
3
from apparently similar populations studied in the past 10 years (vide infra). This rather
striking difference may represent either a change in the composition of fecal flora, or may
be a reflection of incorrect identification in earlier studies. We have mentioned a number
of limitations in various studies; the purpose of such comments is not to denigrate the fine
work performed by many investigators, but rather to point out factors which we feel may
be important to the reader.
Despite the above-mentioned limitations, a great deal of information exists regarding the
presence of clostridia in the human GI tract. Many investigators use the term "normal bowel
flora", which is a rather elusive concept. An organism that is persistently present in the gut
of a given individual could be said to be normal flora. On the other hand, an organism that
is present only occasionally could be considered to be transient flora. Clostridial spores are
rather resistant to the harsh conditions in the upper GI tract (i.e., gastric acid and enzymes
in gut secretions); therefore, it is possible that when clostridia are recovered from bowel
contents or feces, they may represent transient flora more frequently than is the case with
nonspore-forming bacteria.
III. TOTAL COUNTS OF CLOSTRIDIA AND SPECIES OF CLOSTRIDIUM
AT VARIOUS SITES IN THE GASTROINTESTINAL TRACT
A. Healthy Infants and Children
Very few studies of the gut flora of infants and children, other than those of feces, have
been done. Challacombe et al.3 were unable to recover clostridia from either the gastric or
duodenal juices of 13 infants studied. Brook et al.4 recovered C. perfringens on three
occasions, and C. bifermentans and C. limosum on one occasion each from the gastric
contents of 35 newborn infants studied within 10 min of delivery.
1. Feces — Total Counts of Clostridium
Although the human GI tract is sterile at birth, it rapidly becomes colonized by bacteria.
The incidence and total counts of clostridia in feces of infants at various times during the
first 2 months of life are shown in Table 1. From these data, it is evident that the gut
frequently becomes colonized within the first 1 to 2 days of life. Such colonization often
persists for extended periods.
Most studies of older infants indicate that clostridia can be recovered from the feces of
80 to 100% of infants between 2 and 12 months of age; counts are usually 105 or higher
per gram of feces (Table 2).
2. Feces — Species of Clostridium
The species of Clostridium isolated from the feces of healthy infants are shown in Table
3. A total of 21 different species have been reported. A limitation of these data is the
relatively small number of patients studied. The most detailed studies of infant flora appear
to be those of Snyder25 who in 1940 recovered clostridia (6 different species) from all of
22 infants studied, and of Stark and Lee,14 who recovered clostridia (12 different species)
from all of 10 infants.
Clostridium species recovered from either feces or infection derived from the gut flora of
infants who were ill are shown in Table 4. Most of these infants had necrotizing enterocolitis
(NEC) and probably received antimicrobial therapy prior to having cultures performed. The
role of clostridia in neonatal NEC (to be discussed in a subsequent chapter) is unclear; the
data from infants with NEC are included in Table 4 because they may simply reflect the
normal presence of clostridia in the gut. Additional studies of normal infants and infants
with NEC are clearly needed.
