Emerging Infectious Diseases
[Volume 6 No.2 / March - April 2000]

Dispatches

Bacteroides fragilis Enterotoxin Gene Sequences in Patients with
Inflammatory Bowel Disease

Thomas P. Prindiville, Rafik A. Sheikh, Stuart H. Cohen,
Yajarayma J. Tang, Mary C. Cantrell, and Joseph Silva, Jr.
University of California, Davis Medical Center, Sacramento,
California, USA

We identified enterotoxigenic Bacteroides fragilis in stool
specimens of patients with inflammatory bowel disease and other
gastrointestinal disorders. The organism was detected in 11
(13.2%) of 83 patients with inflammatory bowel disease. Of 57
patients with active disease, 19.3% were toxin positive; none of
those with inactive disease had specimens positive for
enterotoxigenic Bacteroides fragilis gene sequences.

Bacteroides fragilis, a gram-negative rod, constitutes 1% to 2% of
the normal colonic bacterial microflora in humans (1,2). It is
frequently associated with extraintestinal infections such as
abscesses and soft tissue infections, as well as diarrheal diseases in
animals and humans (3-6). Enterotoxigenic B. fragilis (ETBF) is an
emerging enteric pathogen associated with diarrheal diseases in
children, adults, and animals (4,7-9). The pathogen is unusual in
children during the first year of life, but diarrhea associated with it
is common in children 1 to 5 years of age, which suggests early
acquisition of the organism and maternal protection. The
pathogenicity of B. fragilis is related to its production of a potent
enterotoxin, a zinc-containing metalloprotease with a molecular
weight of 20,000 (10). The enterotoxin is tight-junction specific;
causes rounding, swelling, and pyknosis of cultured enterocytes;
and induces a fluid response in ligated intestinal loops and a
cytotoxic response in the HT-29 colon cell line (7). Ulcerative
colitis and Crohn disease are inflammatory diseases of the
gastrointestinal tract characterized by spontaneous remissions and
relapses. Many microbial pathogens, particularly Mycobacterium
paratuberculosis, paramyxoviruses, and Listeria monocytogenes
have been implicated in the etiology of inflammatory bowel
disease (IBD) (11). In addition, enteric pathogens such as
Campylobacter jejuni, Salmonella, Shigella, Yersinia, and
Escherichia coli have also been associated with relapses of IBD
(12). We investigated the prevalence of ETBF in 83 patients with
idiopathic IBD, in 18 patients with routine culture-negative
diarrhea, and in a control population of 69 outpatients (Table 1).

Table 1. Demographic characteristics of patients with
inflammatory bowel disease (IBD)

The Study

The study protocol was approved by the Human Subjects Research
Review Committee of the University of California, Davis. All
samples were collected after informed consent was obtained. Of
the 83 patients in the IBD group, 60 had Crohn disease, and 23 had
ulcerative colitis (Table 2). Active disease was present in 68.6% of
these patients on endoscopy (20 ulcerative colitis and 37 Crohn
disease patients) in the form of mucosal erythema and ulceration.
In the miscellaneous diarrhea group, 10 patients had irritable bowel
syndrome; however, no mucosal erythema, edema, or ulceration
was observed. None of the control patients had a history of
diarrhea, and no erythema or ulceration was observed. Fecal
specimens from the IBD group were collected endoscopically from
patients undergoing colonoscopy or flexible sigmoidoscopy for
evaluation of symptoms of diarrhea or abdominal pain. The fecal
specimens from the 18 diarrhea patients with negative routine stool
cultures and the 69 controls were collected by endoscopy. Fecal
specimens were cultured for B. fragilis in the selective medium
Bacteroides Bile Esculin agar. Positive cultures were identified by
using the RapID ANA II Panel (REMEL, Inc, Lenexa, KS). Plates
were incubated anaerobically at 37C for 48 hours. The presence of
B. fragilis enterotoxin in the isolates was detected in the HT-29
colon cell line (13,14). HT-29 cells were grown and maintained in
RPMI medium with glutamine (Gibco, Life Technologies, Inc.,
Grand Island, NY) supplemented with penicillin (100 IU/ml),
streptomycin (100 g/ml) (Sigma, Saint Louis, MO), and heat-
inactivated fetal bovine serum (Hyclone Laboratories, Inc., Logan,
UT) at 12% in 25-ml flasks at 37C in 5% CO 2 . For the
cytotoxicity assay, HT-29 cells were suspended in 20 ml of
medium for each plate, and 180 l/well were placed into 96-well
tissue culture plates (Corning Glass Works, Corning, NY). The
cells were allowed to attach and grow for 2 to 3 days. Supernatants
from B. fragilis isolates grown on Brain Heart Infusion Broth were
filtered through a 0.45-m Acrodisk syringe filter (Gelman
Sciences, Ann Arbor, MI), and 20-l serial dilutions were placed
into the wells in duplicate. The plates were incubated at 37C for 3
to 4 hours under 5% CO 2 and examined for typical cytopathic
changes. Cultures were considered positive for B. fragilis
enterotoxin if a visible cytopathic effect was neutralized by specific
antiserum. The highest dilution of the culture supernatant
producing cytopathic changes in at least 50% of the cells after 3 to
4 hours of incubation was considered the cytotoxic titer. For the
neutralization assay, dilutions of 1:25 anti-enterotoxin rabbit
antiserum in phosphate-buffered saline were mixed with culture
supernatants positive for enteroxin. After incubation for 30 minutes
at 37C, 20 l of each mixture was inoculated into HT-29 cells as
in the cytotoxicity assay. Neutralization was indicated by the lack
of cytotoxic effect.

Table 2. Bacteroides fragilis enterotoxin gene amplification
products in patients with inflammatory bowel disease (IBD)

DNA was extracted from the fecal specimens and amplified by
using specific primers (14) to detect B. fragilis enterotoxin gene
sequences.1 Chi-square was used to determine statistical
significance

ETBF was cultured from four patients with IBD and two patients
with diarrhea. However, B. fragilis enterotoxin gene sequences
were detected in the stools of 11 (13.25%) of 83 patients with
idiopathic IBD (five with Crohn disease and six with ulcerative
colitis). All 11 patients positive for ETBF had active disease by
endoscopic and histologic tests (Table 2). No ETBF was found in
patients with inactive disease. The Crohn disease patients who
were positive for enterotoxin gene sequences had superficial
mucosal disease. In the control group, 2 (2.9%) of 69 patients were
positive for B. fragilis enterotoxin gene sequences. Enterotoxin
amplification products were also detected in the specimens of 5
(27.7%) of 18 patients with diarrhea due to miscellaneous causes
(Table 2). All the specimens were positive when amplified with
primers specific for the 16S rRNA gene of enteric bacteria.

Conclusions

The normal colonic microflora of humans is a complex ecosystem
of approximately 500 species of aerobic and anaerobic
microorganisms. Although the gut of the newborn infant is sterile,
Bacteroides speciesthe predominant anaerobic constituent of the
colonic floraappear at approximately 10 days, are established by
2 weeks, and usually remain constant lifelong (1,2). In breast-fed
infants, Bifidobacterium are the predominant population, and
Bacteroides group organisms remain undetectable. However, after
weaning, the Bacteroides group organisms increase and
Bifidobacterium organisms decrease substantially (16).

The exact etiology of idiopathic IBD is still unknown, although a
potential role for infectious agents or toxins that may stimulate an
inflammatory response has been suggested (16,17). For example,
Peptostreptococcus, Coprococcus, and Bacteroides sp. have been
reported in patients with Crohn disease (17). A role for these
microorganisms in the disease process is also suggested by the
clinical responses of some patients to antibiotics (18). Observations
of B. thetaiotaomicron in patients with ulcerative colitis (16) and
B. vulgatus in guinea pigs with experimentally induced ulcerative
colitis (19) suggest that microorganisms may influence the
development or maintenance of intestinal inflammation in IBD.

In this study, B. fragilis enterotoxin gene sequences were detected
by nested polymerase chain reaction (PCR) in the stools of 13.2%
of patients with inflammatory bowel disease and 2.9% controls.
The low recovery of ETBF in culture may be due to the length of
time from specimen collection to processing in the laboratory
(most specimens were kept frozen for at least 2 weeks before
culturing). Similar results have been reported by Sack et al. (9),
who found a marked reduction in the recovery of B. fragilis with
time. Amplification of enterotoxin gene directly in the stools of
these patients appears to be a more sensitive detection method. In a
previous study (14), we found 100% correlation between PCR and
enterotoxin production in isolates; we therefore did not routinely
perform the HT-29 cell assay in specimens of these patients.

In the IBD group, all the patients positive for ETBF had active
disease, which suggests an association with disease activation or
flare-up. ETBF was also found in patients with ulcerative proctitis,
collagenous colitis, and microscopic colitis. In patients with Crohn
disease, ETBF was usually seen in the colonic superficial
inflammatory disease type. The presence of ETBF in IBD patients
may represent alterations of endogenous bacterial flora, which may
be related to either the etiology or flare-up of the disease or both.
Colonization with ETBF may be acquired early in life or may be a
de novo infection related to flare-ups of the disease.

Acknowledgment

The authors thank David M. Lyerly for providing the anti-
enterotoxin rabbit antiserum.

Dr. Prindiville is an associate professor of medicine in the Division
of Gastroenterology at the University of California, Davis,
California, USA.

Address for correspondence: Stuart H. Cohen, Department of
Internal Medicine, Division of Infectious Diseases, 4150 V Street,
Patient Services and Support Building, Suite 500, Sacramento, CA
95817, USA; fax: 916-734-7766; e-mail:
stcohen@ucdavis.edu.

1 A 100-mg sample of stool was suspended in 400 l of TES buffer
(50 mM Tris [pH 8], 5 mM EDTA, 50 mM NaCl) and centrifuged
at 1,000 x g for 3 min to remove large particles. The supernatant
was then centrifuged at 5,000 x g for 7 min. The pellet was washed
once in 200 l of TES, and centrifuged at 5,000 x g for 3 min, and
the supernatant was discarded. The pellet was suspended in 100 l
of sterile H 2 O and boiled for 10 min, then centrifuged at 1,000 x
g for 2 min and the supernatant containing the DNA extracted
twice with phenol: chloroform: isoamyl alcohol (25:24:1) and
precipitated with ethanol. The sequences of the primers and probes
used and PCR conditions were as described (14). Amplification
with the outer primers RS-3 (5'
TGAAGTTAGTGCCCAGATGCAGG 3') and RS-4 (5'
GCTCAGCGCCCAGTATA TGACC 3') yielded a 367-bp product.
Amplification of this product with the inner primers RS-1 (5'
TGCGGCGAACTCGGTTAATGC 3') and RS-2
(5'AGCTGGGTTGTAGACATCCCACTGG' 3') amplified a 290-
bp product. The reaction mixtures were prepared in 1X PCR buffer
(50 mM KCl, 20 mM Tris HCl, 2.5 mM MgCl 2 , 100 g bovine
serum albumin per ml [pH 8.4]) and contained per reaction 20
pmol of the respective primers, 0.1 mM concentrations each of 2'-
deoxynucleoside 5'-triphosphate, 2 U of recombinant DNA
polymerase (rTaq) (Perkin Elmer, Norwalk, CT), and 10 l purified
fecal DNA. The final reaction volume was adjusted to 100 l with
sterile deonized water. The PCR profile included a denaturing step
at 95C for 30 sec, followed by a 60C annealing step for 30 sec,
with extension at 72C for 30 sec. The outer PCR was performed
for 35 cycles in a thermal cycler (MJ Research). Amplification
with the inner primers was done for 30 cycles. Negative controls
included a blank containing all PCR reagents with no DNA. As
control for amplifiable DNA in the stool specimens, primers
targeting the 16S rRNA gene of enteric bacteria were used as
described by Kato et al. (15).
  
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Emerging Infectious Diseases
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