Enterotoxigenic Bacteroides fragilis: a Rogue among Symbiotes · In early studies, oral inoculation...
Transcript of Enterotoxigenic Bacteroides fragilis: a Rogue among Symbiotes · In early studies, oral inoculation...
CLINICAL MICROBIOLOGY REVIEWS, Apr. 2009, p. 349–369 Vol. 22, No. 20893-8512/09/$08.00�0 doi:10.1128/CMR.00053-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Enterotoxigenic Bacteroides fragilis: a Rogue among SymbiotesCynthia L. Sears*
Divisions of Infectious Diseases and Gastroenterology, Department of Medicine, Johns Hopkins University School of Medicine,Baltimore, Maryland
INTRODUCTION .......................................................................................................................................................349ETBF INFECTIONS IN ANIMALS .........................................................................................................................349
Naturally Occurring ETBF Infections .................................................................................................................349Experimental ETBF Infections .............................................................................................................................349
ETBF ASSOCIATION WITH HUMAN CLINICAL DISEASE ............................................................................350Epidemiology and Clinical Presentation of ETBF-Associated Diarrheal Disease.........................................350Other ETBF Disease Associations........................................................................................................................351Diagnosis of ETBF Infection.................................................................................................................................354Therapy of ETBF-Associated Diarrheal Disease ................................................................................................356
BFT GENETICS AND PROTEIN STRUCTURE...................................................................................................356GENETICS OF ETBF ................................................................................................................................................359BFT MECHANISM OF ACTION.............................................................................................................................362
In Vitro Studies of Cell Lines, Polarized Epithelial Monolayers, and Human Colon..................................362Molecular Mechanism of Action of BFT .............................................................................................................363
PROPOSED MODEL FOR PATHOGENESIS OF ETBF DISEASE..................................................................364SUMMARY AND FUTURE CHALLENGES ..........................................................................................................366ACKNOWLEDGMENTS ...........................................................................................................................................366REFERENCES ............................................................................................................................................................366
INTRODUCTION
Bacteroides species comprise nearly half of the fecal floracommunity and are host symbionts critical to host nutrition(e.g., Bacteroides thetaiotaomicron) and mucosal and systemicimmunity (e.g., B. fragilis) (25, 35, 54, 55, 73, 98, 110, 127).Among Bacteroides species, B. fragilis strains are opportunisticpathogens, being the leading anaerobic isolates in clinical spec-imens, bloodstream infections, and abdominal abscesses de-spite comprising typically �1 to 2% of the cultured fecal flora(50, 62, 75, 87, 91). In 1984, while investigating the etiology oflamb diarrheal disease, Myers and colleagues provided the firstevidence that certain strains of B. fragilis were epidemiologi-cally associated with diarrheal disease (64). Studies by thesame investigators revealed that both the isolates and theirsterile culture supernatants stimulated intestinal secretion inlamb ligated intestinal loops (64, 69; see reference 105 for areview). The secretory responses in some ligated intestinalloops were so potent that the loops burst, a response reminis-cent of cholera toxin-stimulated secretory responses. The bio-logically active factor was proposed to be a heat-labile, �20-kDa protein toxin, now known to be one of a family of B.fragilis toxins (BFTs) (69, 106). B. fragilis strains eliciting intes-tinal secretion were named enterotoxigenic B. fragilis (ETBF)and their nonsecretory counterparts were termed nontoxigenicB. fragilis (NTBF). This review describes the progress over thesubsequent nearly 25 years in defining the role of ETBF in
human disease, the genetics and mechanism of action of BFT,and insights into the molecular evolution of ETBF strains.
ETBF INFECTIONS IN ANIMALS
Table 1 summarizes the data on ETBF infections in animals.
Naturally Occurring ETBF Infections
ETBF strains were initially identified during an investigationof newborn lamb diarrheic disease (64). Subsequent reportsindicated that calves, foals, and piglets were susceptible toETBF-associated diarrheal illnesses in the field (17, 65, 66, 69,105). In the small number of reports available, the diarrhealillnesses were largely self-limited, with little mortality, exceptpossibly in newborn lambs (64). The limitations of these re-ports include variable assessment for additional enteric patho-gens and a lack of epidemiology to assess ETBF carriage inasymptomatic livestock. Consistent with the native colonichabitat of B. fragilis, exfoliating colitis with an intense neutro-philic mucosal infiltrate was observed in a single piglet exam-ined by histopathology (17).
Experimental ETBF Infections
In early studies, oral inoculation of ETBF into newborn lambs,piglets, or importantly, gnotobiotic (germfree) piglets reproducedthe diarrheal illnesses observed in the field, providing furthersupport for the proposal that select B. fragilis strains stimulatedintestinal secretion and diarrheal disease (23, 64, 65). Histopa-thology from gnotobiotic piglets revealed that lesions were mostsevere in the colon, where crypt hyperplasia and neutrophilicinfiltrates were observed. By scanning electron microscopy, the
* Mailing address: Divisions of Infectious Diseases and Gastroen-terology, Department of Medicine, 1550 Orleans Street, CRB2 Build-ing, Suite 1M.05, Baltimore, MD 21231. Phone: (410) 614-0141. Fax:(410) 955-0740. E-mail: [email protected].
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colonic surface epithelium had a cobblestone appearance associ-ated with round, swollen epithelial cells and epithelial cell exfo-liation (23). Similar but more variable lesions were also observedprimarily in the distal half of the small intestine. No extraintesti-nal lesions were noted. Additional studies with infant and 2-week-old rabbits as well as adult rabbits with ligated ceca confirmed theenteropathogenicity of ETBF, but in these disease models bloodydiarrhea and mortality were frequently observed (17, 65–68).However, ETBF virulence was variable in rabbit models (66, 67),consistent with subsequent observations that sterile culture super-natants of ETBF exhibited variable biologic activity on HT29/C1cells (a human colonic epithelial tumor cell line) (13, 30, 82, 116,119). Histopathologic abnormalities in these non-gnotobiotic an-imal models also occurred only in the distal ileum and colon, withdisruption of the epithelial integrity and predominant neutro-philic or mixed neutrophilic and mononuclear cellular infiltratesin the lamina propria; animals colonized with NTBF strains ex-hibited normal colonic histopathology without inflammation bylight microscopy. ETBF adherence and/or invasion of colonocyteswas not observed by light or electron microscopy. Bacteremia hasnot been reported for these animal models (68).
Early studies indicated that mice (suckling and young adult)and hamsters do not exhibit secretory responses to ETBF (68,69). Recently, colonization of gnotobiotic mice with ETBF, butnot NTBF, has been shown to induce acute, sometimes lethal,colitis (71, 92). In contrast, conventional mice colonized withETBF develop rapid-onset, transient diarrhea lasting 3 to 4days. Subsequently, conventional mice colonized with ETBFexhibit persistent, asymptomatic colonization, with ongoinghistopathologic colitis present for as long as 16 months (90, 92)(Fig. 1). Additional studies show that purified BFT, albeit at apharmacologic dose (i.e., 10 �g), stimulates secretion and his-tologic enteritis in mouse ileal loops (42, 44).
In initial studies, ETBF or sterile culture supernatants wereinjected into ligated intestinal (predominantly jejunal) seg-ments of lambs, calves, or rabbits (64, 69). These studies con-firmed that ETBF stimulated intestinal secretion but, moreimportantly, provided the first evidence that ETBF secreted aheat-labile protein toxin responsible for stimulating intestinalsecretion (69). Subsequently, Obiso and colleagues demon-strated that purified BFT (1-�g to 50-�g doses) stimulatesdose-dependent secretion of sodium, chloride, and albumin inboth ileal and colonic ligated loops in rats, rabbits, and lambs(80). Colonic secretory responses exceeded ileal responses inall species. However, species-dependent BFT potency was ob-served, with the greatest fluid responses in the ilea of lambsand the colons of rabbits. The histology of ileal or colonicligated loops inoculated with 20 �g of purified BFT for eachspecies exhibited marked epithelial cell rounding and detach-ment, neutrophilic inflammation, and in some sections, necro-sis and hemorrhage. Time course analyses revealed that histo-logic changes (at 10 h) preceded detection of intestinalsecretion (examined at 18 h). Consistent with the subsequentdetermination that BFT is a zinc-dependent metalloproteasetoxin (see below), metal-chelating agents reduced (�90%) thesecretory and histologic responses to purified BFT in ligatedintestinal loops and zinc reconstituted them (�50%).
ETBF ASSOCIATION WITH HUMANCLINICAL DISEASE
Epidemiology and Clinical Presentation ofETBF-Associated Diarrheal Disease
ETBF strains were first isolated from humans with diarrhealillnesses in an uncontrolled study in 1987 (70) examining 10individuals with diarrhea of unknown origin (from Montana)and 34 infants (2 to 14 months of age; Navajo Area IndianHealth Service, Tuba City, AZ). Overall, ETBF isolates wereidentified in 8 of 44 patients (two adults and six children of �5years of age), with only one infant positive for a second entericorganism, enterotoxigenic Escherichia coli (ETEC). In this ini-
FIG. 1. ETBF induce murine colitis. Colonization of 4-week-oldC57BL/6 mice with ETBF for 2 weeks induces inflammation and hy-perplasia throughout the colon. Panels 1 to 3, mice inoculated withphosphate-buffered saline; panels 4 to 6, mice inoculated with ETBFstrain 86-5443-2-2. In panels 4 and 5, the rounded and detachingcolonic epithelial cells in ETBF-infected mice can be seen. (Repro-duced from reference 90 with permission of the publisher. CopyrightJohn Wiley and Sons Ltd.)
TABLE 1. Animal species susceptible to ETBF infection or BFTbiologic activity
Type of infection or expt and susceptible animal species
Naturally occurring ETBF infectionsa
Newborn lambsb
CalvesFoalsPiglets
Experimental ETBF infections or BFT inoculationsLambs (oral inoculationc and intestinal
loop inoculationd)Calves (intestinal loop inoculationd)Rabbits (oral inoculation, RITARD model, and intestinal
loop inoculationd,e)Rats (intestinal loop inoculationd)Gnotobiotic piglets (oral inoculationf)Mice (oral inoculation and ileal loop inoculationg)
a Presenting with diarrhea.b One diarrheic ewe reported (64).c All oral inoculations are with ETBF strains.d Jejunal, ileal, and/or colonic loops inoculated with ETBF, sterile ETBF
culture supernatants, or purified BFT (64, 69, 80).e Three-day-old (oral inoculation), 2-week-old (direct small bowel inocula-
tion), and adult (oral inoculation with ligated ceca) animals. RITARD, reversibleileal tie adult rabbit diarrhea (17, 65–68, 80).
f One to 2 days of age (23).g Conventional or gnotobiotic mice of more than 3 weeks of age (71, 90, 92).
Inoculation of ileal loops with purified BFT stimulated secretion (42, 44).
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tial study, most patients had self-limited diarrhea, but inter-mittent diarrhea of more than 3 years duration, diarrhea per-sisting 4 weeks, high fever, and frank/occult fecal blood werenoted in ETBF-positive patients (70). The first human studywith control subjects to investigate a putative association be-tween ETBF infection and human diarrheal disease was con-ducted in the pediatric outpatient clinics of the Apache Indianreservation at Whiteriver, AZ, in 1992 (95). The key results ofthis study were that children of �1 year of age did not developETBF-associated diarrhea, whereas ETBF isolates were asso-ciated with diarrheal illnesses in children between the ages of1 and 5 years. ETBF isolates from single families evaluated byrestriction enzyme analysis appeared to be genetically related.
Table 2 summarizes studies with control subjects evaluatingthe role of ETBF as an etiologic agent of diarrheal disease.Table 3 summarizes uncontrolled case series or reports ofhuman ETBF infection. The majority of controlled studiesconducted to date have evaluated ETBF as an agent of acutecommunity-acquired diarrheal disease in children (Table 2).One Swedish study assessed ETBF in adults hospitalized withacute diarrheal disease (128) compared to isolation of ETBFfrom healthy outpatient control adults. Overall, study designshave been variable, and many studies are hampered by smallnumbers of patients and controls, a lack of thorough studies forother enteric pathogens, and a wide range of recovery rates (6to 70%) for B. fragilis.
Of 17 studies with controls (Table 2), 5 did not show anassociation between ETBF and diarrheal disease in differingpatient populations (4, 8, 48, 77, 84). Of these studies, a fol-low-up study of the same patient population by the same au-thors subsequently did show an association of ETBF with di-arrheal disease (59a, 84), one study had a B. fragilis isolationrate of only 6% (77), and three studies were from Brazil (4, 8,48). Excluding the one study (128) focused on adults, all butone (10) of the remaining studies confirmed the original ob-servations of Sack et al. (95) suggesting that, similar to the casewith Clostridium difficile, there is no association between ETBFisolation and diarrheal illnesses in children of �1 year old,whereas diarrheal disease due to ETBF emerges after age 1.The mechanisms accounting for the apparent lack of pathoge-nicity of ETBF in very young children are unknown. However,there is marked variability in the frequencies at which ETBFstrains are associated with diarrhea in different geographiclocales (from 3.5% in Bangladesh to 28% in Italy). Only onelarge study to date has investigated ETBF in adults (defined asindividuals who are �15 years old), identifying ETBF in 27%of 728 patients with diarrhea and 12% of 194 healthy controls(P � 0.01) (128). In this study, 19% of adults of �30 years ofage, 10.6% of adults between the ages of 30 and 60, and 3.7%of adults older than 60 years were asymptomatically colonizedwith ETBF. Among adults between 15 and 30 years of age,ETBF fecal carriage rates were similar for those with andwithout diarrhea, whereas an association of ETBF with diar-rheal disease emerged in older patients. Other studies alsosuggest that asymptomatic colonization with ETBF is common(up to �30%) (7) (Table 2; also see Table 5). Similarly, 9.3%of 237 B. fragilis strains cultured from sewage influent in Boze-man, MT, were ETBF, suggesting moderately high endemiccarriage of ETBF (109). ETBF carriage for up to 16 monthswith fecal ETBF isolates identical by restriction enzyme anal-
ysis has been reported (95). Together, the data suggest thatETBF strains are globally distributed enteric pathogens caus-ing diarrhea in both children and adults. Similar to the case forother enteric pathogens, asymptomatic ETBF colonization iscommon. Geographic diversity in the recovery of entericpathogens is also well known (10a), although the reasons un-derlying the variable isolation rates have not been elucidated.With respect to detection of ETBF in stool, one importantvariable is likely the sensitivity of the diagnostic approachesused (see “Diagnosis of ETBF Infection”).
ETBF clinical illnesses are typically characterized as self-lim-ited with watery diarrhea; if noted, persistent diarrhea (�14 days)has been reported for a minority (0 to 22%) of patients. Table 4summarizes the range of clinical findings reported in studies ofETBF diarrheal illnesses. When assessed, ETBF diarrheal ill-nesses have been clinically indistinguishable from non-ETBF di-arrheal illnesses in the populations studied (10, 95, 128). A recentprospective analysis involving 73 ETBF-infected patients (43 chil-dren under age 15 and 30 adults) in Bangladesh (102) correlatedclinical findings with more detailed stool analyses to evaluate thepathogenesis of ETBF diarrheal illnesses. In this series, althoughillnesses were again self-limited (2 to 11 days), substantial abdom-inal pain (88%), tenesmus (66%), and nocturnal diarrhea (80%)were reported. In contrast, fever (�37.5°C) (7%) and fecal occultblood (8.5%) were infrequent. Leukocytosis was not detected.Stool analyses for leukocytes, fecal lactoferrin, and proinflamma-tory cytokines (tumor necrosis factor alpha and interleukin-8 [IL-8]) indicated that ETBF induced inflammatory diarrhea com-pared to stools from asymptomatic control individuals notinfected with ETBF. Consistent with the inflammatory mucosalresponse to ETBF infection, serum immunoglobulin A (IgA) andIgG antibodies as well as fecal IgA to BFT were detected. In onereport, two to four distinct, sequential episodes of ETBF-associ-ated diarrhea were reported for children in Bangladesh, suggest-ing that acquired immunity to ETBF is incomplete (85).
No studies have been designed to specifically determine ifETBF contributes to persistent or chronic diarrhea or to diar-rheal illnesses in travelers; limited observations are availableon the role of ETBF in diarrhea defined as nosocomial (oc-curring �3 days after hospital admission) (16, 57) or antibioticassociated (39, 86). In one study, ETBF strains were identifiedin stools of 9% of patients with non-C. difficile nosocomialdiarrhea, which was significantly greater than ETBF detectionin outpatient controls (2%; P � 0.04), but hospitalized controlswere not evaluated (16). C. difficile and ETBF were detectedsimultaneously in a small subset of fecal samples from patientswith antibiotic-associated diarrhea (57, 86). In one study ofchildren, there was no association of ETBF with antibiotic-associated diarrhea (39). Well-known extraintestinal or intes-tinal complications of enteric infections, such as reactive ar-thritis or diarrhea-predominant irritable bowel syndrome, havenot yet been reported for ETBF.
Other ETBF Disease Associations
Three recent but small reports raise the specter of adverse,long-term consequences of chronic ETBF colonization (Table5). In two reports examining patients with irritable bowel dis-ease (IBD) (7, 88), active (but not clinically quiescent) IBDwas associated with ETBF infection, although the differences
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TA
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ited
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ysis
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.,19
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ildre
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wer
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e(P
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;no
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ciat
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een
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BF
and
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ildre
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der
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der
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ithdi
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rols
wer
eE
TB
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sitiv
e(P
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001)
(diff
eren
cew
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whe
nch
ildre
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ithm
ultip
lein
fect
ions
wer
eex
clud
ed)
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ensi
vest
udie
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ente
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istr
atio
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ior
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sion
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pw
asa
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ound
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owis
olat
ion
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sof
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lis(2
0%)
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Joaq
uin
etal
.,19
95(1
01)
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anch
ildre
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ithan
dw
ithou
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–199
1
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578
age-
mat
ched
cont
rols
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lcul
ture
,HT
29/C
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llas
say
B.f
ragi
lisis
olat
edfr
om32
%an
d21
%of
child
ren
with
and
with
out
diar
rhea
,res
pect
ivel
y(P
�0.
001)
;4.
8%of
child
ren
of1
to10
year
sof
age
with
diar
rhea
and
1.5%
ofco
ntro
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ere
ET
BF
posi
tive
(P�
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);no
asso
ciat
ion
betw
een
ET
BF
and
diar
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inch
ildre
nun
der
1ye
arol
d
Lim
ited
anal
ysis
for
othe
ren
teri
cpa
thog
ens;
low
isol
atio
nra
tes
ofB
.fra
gilis
(�35
%)
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osti
etal
.,19
97(8
4)R
ando
mly
sele
cted
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lsto
ols
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itted
toa
mic
robi
olog
yla
bfr
omin
patie
nts
and
stoo
lsfr
omhe
alth
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ntro
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1994
–199
5
Ital
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child
ren
and
106
adul
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ych
ildre
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ts
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,HT
29/C
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llas
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ilar
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nra
tes
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thy
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ren
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tsan
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dad
ults
with
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rhea
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%)
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lstu
dy;m
ixed
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tpo
pula
tion
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acut
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mia
l,an
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chro
nic
diar
rhea
;B.f
ragi
lisis
olat
ion
rate
sof
�40
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%
Niy
ogie
tal
.,19
97(7
7)In
patie
ntch
ildre
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ithan
dw
ithou
tac
ute
diar
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,199
5–19
96
Kol
kata
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ia22
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tient
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ithdi
arrh
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172
cont
rols
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lcul
ture
,HT
29/C
1ce
llas
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Ove
rall,
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ofpa
tient
sw
ithdi
arrh
eaan
d1.
7%of
cont
rols
wer
eE
TB
Fpo
sitiv
e(d
iffer
ence
not
sign
ifica
nt);
noag
eas
soci
atio
nof
ET
BF
with
diar
rhea
was
iden
tified
Lim
ited
anal
ysis
for
othe
ren
teri
cpa
thog
ens;
B.f
ragi
lisw
asis
olat
edfr
omon
ly24
(6%
)ch
ildre
n,bu
t9
(38%
)of
the
isol
ates
wer
eE
TB
FM
enoz
ziet
al.,
1998
(59a
)In
patie
ntch
ildre
nw
ithdi
arrh
eaan
dag
e-m
atch
edco
ntro
lsw
ithou
tdi
arrh
ea(1
996–
1998
)
Parm
a,It
aly
227
patie
nts
with
diar
rhea
,23
7co
ntro
lsSt
oolc
ultu
re,H
T29
/C1
cell
assa
yB
.fra
gilis
was
isol
ated
from
27.7
%of
patie
nts
and
19%
ofco
ntro
ls(P
�0.
05);
ET
BF
was
asso
ciat
edw
ithdi
arrh
eafo
rch
ildre
nof
1to
5ye
ars
ofag
e(3
3.3%
vs16
.4%
�pat
ient
svs
cont
rols
�;P
�0.
01);
noco
rrel
atio
nof
ET
BF
with
antib
iotic
ther
apy
was
foun
d
Low
B.f
ragi
lisis
olat
ion
rate
s;lim
ited
clin
ical
deta
ils
Kat
oet
al.,
1999
(39)
Urb
anho
spita
l-bas
edst
udy
ofch
ildre
nne
gativ
efo
rco
mm
onba
cter
ial
ente
ropa
thog
ens,
1996
Nag
oya,
Japa
n41
6ch
ildre
n(1
37w
ithno
n-an
tibio
tic-a
ssoc
iate
ddi
arrh
ea,1
66w
ithan
tibio
tic-a
ssoc
iate
ddi
arrh
ea,1
13co
ntro
lsw
ithou
tdi
arrh
ea)
Stoo
lcul
ture
,PC
Rfo
rB
.fra
gilis
bft
onba
cter
iali
sola
tes,
HT
29/C
1ce
llas
say
Ove
rall,
ET
BF
was
asso
ciat
edw
ithno
n-an
tibio
tic-a
ssoc
iate
ddi
arrh
eain
child
ren
of1
to14
year
sof
age
(14.
9%vs
4.9%
inco
ntro
ls;P
�0.
05)
but
not
with
antib
iotic
-as
soci
ated
diar
rhea
(ass
esse
don
lyin
child
ren
upto
6ye
ars
ofag
e);n
oas
soci
atio
nbe
twee
nE
TB
Fan
ddi
arrh
eain
child
ren
unde
r1
year
old
Stoo
lcul
ture
spe
rfor
med
onst
ools
froz
enfo
r�
1m
o;B
.fr
agili
sis
olat
edfr
om23
to54
%of
child
ren
of�
1ye
arof
age
352 SEARS CLIN. MICROBIOL. REV.
on Decem
ber 3, 2020 by guesthttp://cm
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Dow
nloaded from
Alb
ert
etal
.,19
99(2
)H
ospi
tal-b
ased
stud
yof
child
ren
with
acut
edi
arrh
ea;c
omm
unity
child
ren
with
out
diar
rhea
wer
eus
edas
cont
rols
,199
3–19
94
Dha
ka,B
angl
ades
h81
4pa
tient
sw
ithdi
arrh
ea,
814
cont
rols
with
out
diar
rhea
Stoo
lcul
ture
,HT
29/C
1ce
llas
say
Ove
rall,
3.5%
ofpa
tient
sw
ithdi
arrh
eaan
d1.
5%of
cont
rols
wer
eE
TB
Fpo
sitiv
e(P
�0.
01);
ET
BF
was
not
asso
ciat
edw
ithdi
arrh
eain
child
ren
unde
r1
year
old
Ext
ensi
vest
udie
sof
ente
ric
path
ogen
s
Zha
nget
al.,
1999
(128
)A
dult
inpa
tient
s(�
15ye
ars
old)
with
diar
rhea
and
outp
atie
nthe
alth
yco
ntro
ls,1
996–
1997
Hud
ding
e,Sw
eden
(Sto
ckho
lmsu
burb
)72
8pa
tient
sw
ithdi
arrh
ea,
194
cont
rols
IMS-
PCR
,HT
29/C
1ce
llas
say
Ove
rall,
26.8
%of
patie
nts
with
diar
rhea
and
12.4
%of
cont
rols
wer
eE
TB
Fpo
sitiv
e(P
�0.
01);
ET
BF
was
asso
ciat
edw
ithdi
arrh
eain
adul
tsof
�30
year
sof
age
but
not
inth
ose
of15
to30
year
sof
age
Ext
ensi
vest
udie
sof
ente
ric
path
ogen
s;st
ools
wer
efr
ozen
for
anun
spec
ified
time
prio
rto
anal
ysis
for
ET
BF
;19%
ofpa
tient
sw
ithdi
arrh
eaan
d4%
ofco
ntro
lsto
okan
tibio
tics
prio
rto
eval
uatio
nC
acer
eset
al.,
2000
(10)
Out
patie
ntch
ildre
n(�
10ye
ars
old)
with
and
with
out
diar
rhea
,19
98–1
999
Leo
n,N
icar
agua
106
patie
nts
with
diar
rhea
,60
age-
mat
ched
cont
rols
IMS-
PCR
,HT
29/C
1ce
llas
say
Ove
rall,
8.4%
ofpa
tient
sw
ithdi
arrh
eaan
d0%
ofco
ntro
lsw
ere
ET
BF
posi
tive
(P�
0.03
);ch
ildre
nof
�1
year
ofag
ew
ithdi
arrh
eaha
dth
ehi
ghes
tE
TB
Fis
olat
ion
rate
s(1
1.1%
vs4.
6%fo
rch
ildre
nof
�1
year
ofag
e�d
iffer
ence
not
sign
ifica
nt�)
Onl
yen
teri
cpa
rasi
tes
wer
eev
alua
ted
inco
ntro
lchi
ldre
n;st
ools
wer
efr
ozen
for
anun
spec
ified
time
prio
rto
anal
ysis
for
ET
BF
;inc
ompl
ete
mat
chin
gof
patie
nts
and
cont
rols
;ant
ibio
ticad
min
istr
atio
npr
ior
tost
ool
sam
plin
gw
asa
pote
ntia
lco
nfou
nder
Bre
ssan
eet
al.,
2001
(8)
Hos
pita
lized
imm
unod
efici
ent
child
ren
with
orw
ithou
tdi
arrh
ea;h
ealth
yco
ntro
lchi
ldre
nfr
omda
yca
rece
nter
s(d
ates
not
spec
ified
)
Sao
Paul
o,B
razi
l56
imm
unod
efici
ent
child
ren,
74co
ntro
lsSt
oolc
ultu
re,P
CR
for
bft
gene
onba
cter
ial
isol
ates
,HT
29ce
llas
say
Onl
yon
ech
ildw
ithA
IDS
and
nodi
arrh
eaw
asE
TB
Fpo
sitiv
eO
ther
ente
ric
path
ogen
sno
tev
alua
ted;
B.f
ragi
lisis
olat
ion
rate
sof
18to
27%
;all
imm
unod
efici
ent
child
ren
wer
eon
antib
iotic
sw
hen
stud
ied
Ant
unes
etal
.,20
02(4
)U
rban
child
ren
with
and
with
out
diar
rhea
(1-y
ear
stud
y)
Rio
deJa
neir
o,B
razi
l66
child
ren
(0to
5ye
ars
old)
with
diar
rhea
,25
child
ren
with
out
diar
rhea
Stoo
lcul
ture
,PC
Rfo
rbf
tge
nein
bact
eria
lis
olat
es,H
T29
cell
assa
y
ET
BF
was
isol
ated
from
one
child
with
diar
rhea
Clin
ical
deta
ils,i
nclu
ding
age
dist
ribu
tion
ofch
ildre
n,no
tpr
esen
ted;
25.8
%an
d84
%of
diar
rhea
and
cont
rols
tool
s,re
spec
tivel
y,w
ere
cultu
repo
sitiv
efo
rB
.fra
gilis
Krz
yzan
owsk
yan
dA
rila
-Cam
pos,
2003
(48)
Hos
pita
lized
child
ren
with
acut
edi
arrh
ea,h
ealth
yco
ntro
lchi
ldre
nfr
omda
yca
rece
nter
s(d
ates
not
spec
ified
)
Sao
Paul
o,B
razi
l96
patie
nts
with
diar
rhea
,74
cont
rols
Stoo
lcul
ture
,HT
29/C
1ce
llas
say,
PCR
for
bft
gene
onba
cter
ial
isol
ates
2.1%
ofpa
tient
sw
ithdi
arrh
eaan
d0%
ofco
ntro
lsw
ere
ET
BF
posi
tive
Eva
luat
ion
ofot
her
ente
ric
path
ogen
sun
clea
r;B
.fra
gilis
isol
atio
nra
tes
of13
.5%
to24
%
Ngu
yen
etal
.,20
05(1
18)
Hos
pita
l-bas
edst
udy
ofch
ildre
nw
ithdi
arrh
eaan
dhe
alth
yco
ntro
lch
ildre
nfr
omda
yca
reor
heal
thce
nter
,20
01–2
002
Han
oi,V
ietn
am58
7pa
tient
sw
ithdi
arrh
ea,
249
age-
mat
ched
cont
rols
(all
�5
year
sol
d)
IMS-
PCR
(with
only
MA
bC
3,no
tM
Ab
4H8)
Ove
rall,
7.3%
ofpa
tient
sw
ithdi
arrh
eaan
d2.
4%of
cont
rols
wer
eE
TB
Fpo
sitiv
e(P
�0.
01);
noas
soci
atio
nof
ET
BF
with
diar
rhea
inch
ildre
nun
der
1ye
arol
d;fo
rch
ildre
nof
�1
year
ofag
e,8.
9%w
ithdi
arrh
eaan
d2.
9%of
cont
rols
wer
eE
TB
Fpo
sitiv
e(P
�0.
01)
Cam
pylo
bact
ersp
p.no
tso
ught
due
tola
ckof
faci
litie
s;st
ools
wer
efr
ozen
for
anun
spec
ified
time
prio
rto
anal
ysis
for
ET
BF
;B.f
ragi
lisis
olat
ion
rate
sno
tsp
ecifi
ed;i
ncom
plet
em
atch
ing
ofpa
tient
san
dco
ntro
lsPa
thel
aet
al.,
2005
(85)
Lon
gitu
dina
lstu
dyof
birt
hco
hort
,199
3–19
96R
ural
Ban
glad
esh
(Mir
zapu
r)25
2ch
ildre
n(�
2ye
ars
old)
Stoo
lcul
ture
,HT
29/C
1ce
llas
say
ET
BF
isol
ated
from
40/1
97(2
0.3%
)B
.fr
agili
s-po
sitiv
edi
arrh
eals
tool
san
d15
/185
(8.1
%)
B.f
ragi
lis-p
ositi
veno
ndia
rrhe
alst
ools
(P�
0.00
1);
ET
BF
asso
ciat
edw
ithac
ute
diar
rhea
ldis
ease
inch
ildre
nof
�1
year
ofag
e;16
.3%
ofal
lchi
ldre
nw
ere
infe
cted
with
ET
BF
inth
efir
st2
year
sof
life
Fir
stco
mm
unity
-bas
edsu
rvei
llanc
est
udy
ofE
TB
Fin
fect
ion;
exte
nsiv
eev
alua
tion
ofen
teri
cpa
thog
ens;
cultu
rem
etho
dolo
gych
ange
dov
erth
est
udy
peri
od;o
nly
1,08
3/7,
429
(14.
6%)
stoo
lspe
cim
ens
wer
eB
.fra
gilis
cultu
repo
sitiv
eov
ertim
e
Con
tinue
don
follo
win
gpa
ge
VOL. 22, 2009 ENTEROTOXIGENIC BACTEROIDES FRAGILIS 353
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nloaded from
did not reach statistical significance in one study (7). Of note,in the latter study, ETBF isolates were identified by PCR inmucosal washes of �30% of all individuals examined by en-doscopy (7). In an additional prospective, cross-sectional epi-demiologic report from Turkey (113), fecal ETBF was isolatedfrom 38% of 56 patients with colorectal cancer but only 12% of40 sex- and age-matched concurrent controls (P � 0.01).
Several studies, largely conducted in microbiology laboratories,have examined whether ETBF strains are isolated in excessamong bloodstream isolates (Table 6). In six of eight studies,ETBF isolation from blood exceeded isolation from other ex-traintestinal specimens, but the results reached statistical signifi-cance in only one study (38). Only one study (26) examinedwhether isolation of ETBF from blood was associated with evi-dence of excess clinical morbidity or mortality, and it did not findan association. However, when the studies are combined, 232 of1,325 extraintestinal B. fragilis isolates (17.5%) were identified asETBF, with 86 of 368 blood isolates (23.4%) and 146 of 957 otherextraintestinal isolates (15.3%) being ETBF (P � 0.0005; two-sided chi-square test), suggesting that ETBF may be isolatedmore frequently from bloodstream infections. Additional studieswill be necessary to determine if ETBF strains exhibit increasedvirulence in extraintestinal infections, consistent with their viru-lence in colonic disease.
Diagnosis of ETBF Infection
Table 7 summarizes the approaches to diagnosis of ETBFinfection. Diagnosis of ETBF infection from stool is difficult.Similar to C. difficile diagnosis, detection of the bft gene or itsbiologic activity is required to diagnose ETBF colonization ordisease. Most studies have used one of two approaches, eitherB. fragilis fecal isolation with testing for the bft gene (by PCR)or for BFT (in the HT29/C1 cell assay) or direct examinationfor the bft gene in DNA extracted from stool. The HT29/C1cell assay detects the biologic activity of BFT in culture super-natants of ETBF strains, demonstrating a sensitivity of 89%and a specificity of 100% compared to ETBF strain identifica-tion by the ability to stimulate secretion in the lamb intestinalloop assay (119) (Fig. 2). The HT29/C1 cell assay detects aslittle as 0.5 pM purified BFT; the half-maximal concentrationof BFT detected by the HT29/C1 cell assay is 12.5 pM BFT(97). Analysis of our collection of B. fragilis strains (n � 304strains) indicates that the presence of the bft gene correlateswell with the ability to detect BFT in the HT29/C1 cell assay(29). In rare instances (n � 2 strains), deletions/mutations inthe bft gene have been identified that result in a lack of syn-thesis and/or secretion of biologically active BFT by an ETBFstrain (29).
Available data further suggest that overnight anaerobic cul-tivation of stool in a nutrient broth promotes enhanced recov-ery of B. fragilis and ETBF (102). BFT biologic activity hasbeen detected directly in fecal supernatants from patients withdiarrhea by use of the HT29/C1 cell assay (84). Enzyme im-munoassays (EIAs) have been reported for detection of BFT,and in preliminary data, detection of BFT in stool samples byEIA has been reported (89, 116). A report of a combinedimmunomagnetic separation (to concentrate B. fragilis fromstool) and PCR approach (termed IMS-PCR) to detect ETBFwas encouraging (sensitivity, 50 CFU ETBF/g stool), but this
TA
BL
E2—
Con
tinue
d
Ref
eren
ce(a
utho
r,yr
�ref
eren
ceno
.�)St
udy
desi
gnL
ocat
ion
nE
TB
Fdi
agno
stic
met
hod
Mai
nre
sults
Stre
ngth
s/lim
itatio
nsof
stud
y
Dur
maz
etal
.,20
05(2
4)U
rban
outp
atie
ntch
ildre
nan
dad
ults
with
acut
edi
arrh
ea;h
ospi
taliz
edco
ntro
ls
Mal
atya
,Tur
key
117
child
ren
(0to
16ye
ars
old)
and
104
adul
tsw
ithdi
arrh
ea;1
02ch
ildre
nan
d95
adul
tsw
ithou
tdi
arrh
ea(t
ime
and
age
mat
ched
)
Nes
ted
PCR
for
bft
gene
onst
ool
sam
ples
ET
BF
asso
ciat
edw
ithdi
arrh
eain
child
ren
of1
to5
year
sof
age
(25%
vs9.
5%in
cont
rols
;P�
0.05
);no
asso
ciat
ion
ofE
TB
Fw
ithdi
arrh
eain
patie
nts
of�
1ye
arof
age
or�
5ye
ars
ofag
e
Dia
rrhe
a-as
soci
ated
E.c
oli
clas
ses
not
eval
uate
d;pa
tient
popu
latio
nw
asou
tpat
ient
s,bu
tco
ntro
lpop
ulat
ion
was
hosp
italiz
ed
Coh
enet
al.,
2006
(16)
Cas
e-co
ntro
lser
ies
(dat
esno
tsp
ecifi
ed)
Uni
vers
ityof
Cal
iforn
ia,D
avis
,Sa
cram
ento
,CA
152
patie
nts
with
noso
com
iald
iarr
hea
(with
out
C.d
iffici
le),
85ou
tpat
ient
cont
rols
unde
rgoi
ngsu
rvei
llanc
eco
lono
scop
y
Stoo
lcul
ture
,nes
ted
PCR
for
bft
gene
from
B.f
ragi
lisis
olat
esan
dfe
cal
DN
A
30/1
52(1
9.7%
)an
d28
/85
(32%
)di
arrh
eaan
dco
ntro
lsto
ols,
resp
ectiv
ely,
wer
ecu
lture
posi
tive
for
B.f
ragi
lis;4
patie
nts
wer
eE
TB
Fpo
sitiv
eby
cultu
re,b
ut14
/152
(9.2
%)
patie
nts
wer
epo
sitiv
eby
dire
ctst
oolP
CR
for
bft
sequ
ence
s;2.
3%(n
�2)
ofco
ntro
lsub
ject
sw
ere
posi
tive
for
ET
BF
(P�
0.04
)
Sugg
ests
that
ET
BF
may
cont
ribu
teto
noso
com
ial
diar
rhea
and
that
dire
ctst
ool
PCR
for
bft
gene
impr
oves
diag
nost
icse
nsiti
vity
aA
llst
udie
sin
clud
eda
cont
rolp
opul
atio
nan
dar
elis
ted
inch
rono
logi
calo
rder
.ET
BF
,ent
erot
oxig
enic
B.f
ragi
lis;I
MS-
PCR
,im
mun
omag
netic
sepa
ratio
n-PC
R;R
ITA
RD
,rev
ersi
ble
inte
stin
altie
adul
trab
bitd
iarr
hea;
MA
b,m
onoc
lona
lant
ibod
y;H
T29
cells
,par
enta
lHT
29ce
lllin
e;H
T29
/C1
cells
,clo
ned
HT
29ce
lllin
e.
354 SEARS CLIN. MICROBIOL. REV.
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nloaded from
method requires the use of two anti-B. fragilis antibodies forbest sensitivity, and one antibody has been lost (129; A. Wein-traub, Karolinska Institute, personal communication). Furthercommercial development of an EIA to detect fecal BFT or ofIMS-PCR has not occurred. Limited comparative data suggest,not unexpectedly, that direct stool-based PCR approaches aremore sensitive for the diagnosis of ETBF colonization or dis-ease than fecal cultivation of B. fragilis, followed by detectionof bft or BFT (16, 88, 107). Anaerobic stool culture of B. fragilisis affected by delays in processing of samples as well as (likely)
the general difficulty of anaerobic microbiology (95, 107). Us-ing stool culture, detection of ETBF is further affected by thefecal heterogeneity of B. fragilis strains, where both NTBF andETBF may coexist (16), although one report suggested dis-placement of fecal NTBF by ETBF in diarrheal disease (95).
Together, the data suggest that similar to detection ofETEC, testing of multiple fecal B. fragilis colonies for bft/BFTis necessary to enhance the diagnostic sensitivity if fecal cultureis the chosen diagnostic approach. Overall, it remains uncer-tain which method(s), and in particular which PCR approach,
TABLE 3. Miscellaneous clinical reportsa
Reference (author,yr �reference no.�) Study designb Location n ETBF diagnostic
method Main results Limitations
Myers et al., 1987(70)
Case series Montana and NavajoArea IndianHealth Service,AZ
10 patients withdiarrhea inMontana and34 infants (2to 14 monthsold) inArizona
Stool culture, LLIL,and rabbitinoculation
8 patients diagnosed with ETBF,with one infant having aconcurrent infection withETEC; persistent (4 weeks)and chronic (3 years) diarrheaas well as frank/occult fecalblood noted in some patients
No controls includedin the study
Meisel-Mikolajczyket al., 1994 (58)
Case series Warsaw, Poland 120 hospitalizedchildren (2weeks to 3.5years old), 56patients withdiarrhea
Stool culture,HT29/C1 cellassay
16.7% of stools were culturepositive for B. fragilis; twochildren were ETBF positive
Clinical details limited,but majority ofpatients hadpneumonia andantibiotic therapy;low B. fragilisisolation rate
Aucher et al., 1996(5)
Case report Poitiers, France 1 Cerebrospinal fluidculture, HT29/C1cell assay
Meningitis in a neonate withmedullary-colonic fistula, Hxbloody diarrhea; cross-reactionwith Haemophilus influenzaetype 6 latex agglutinationnoted
Single case of ETBFmeningitis; role ofBFT in diseasepathogenesisunknown
Leszczynski et al.,1997 (51)
Case series Warsaw, Poland 120 pregnantwomen
Vaginal culture,PCR for bft gene
6.6% of vaginal cultures werepositive for B. fragilis; oneETBF isolate was identified;no identified clinical sequelae
Single case of vaginalETBF colonization
Meisel-Mikolajczyket al., 1999 (57)
Case series Warsaw, Poland 50 cases ofnosocomialdiarrhea
Stool culture,HT29/C1 cellassay, PCR for bftgene in stool orbacterial isolates
17/50 (34%) stools were culturepositive for B. fragilis; 4/17(23%) patients had ETBF,among whom 3 patients werealso positive for toxigenic C.difficile; DNA from ETBFpositive stools was also PCRpositive for bft gene
No control populationstudied; role ofETBF in nosocomialdiarrhea not defined
Kato et al., 2000(40)
Collection of stoolsfrom individualswithout diarrhea(hospital visitors,employees,students, andnursing homeresidents)
Japan 361 stools(nondiarrheal)
Culture, PCR forbft gene
Overall, B. fragilis was isolatedfrom 38.8% of stools (range,21.1% for children of �1 yearold to 51.3% for 7- to 14-year-olds); 25/140 (17.8%) B.fragilis strains were ETBF(range, 0% for children of �1year old to 8.9% for 1- to 6-year-olds and 17- to 64-year-olds)
Clinical details (e.g.,antibiotic use) notpresented
Martirosian et al.,2001 (52)
Case series Poland 34 appendices Culture, HT29/C1cell assay, PCRfor bft gene
2/34 (5.9%) appendices (fromone adult and one child)carried ETBF; bothappendices were gangrenous
Role of ETBF indisease pathogenesisunknown
Pituch et al., 2003(86)
Case series, 2000–2001
Warsaw, Poland 332 patients withnosocomialdiarrhea (272adults, 60children)
Stool culture,HT29/C1 cellassay, PCR for bftgene on bacterialisolates
50/332 (15%) stools were culturepositive for B. fragilis; 9/50(18%) patients were positivefor ETBF, among whom 5patients were also positive fortoxigenic C. difficile
No control populationstudied; role ofETBF in nosocomialdiarrhea not defined
Antunes et al.,2004 (3)
Case series, 2000–2001
Rio de Janeiro,Brazil
334 hospitalizedchildren withdiarrhea
Stool culture, PCRfor bft gene inbacterial isolates
31/334 (9.3%) stools wereculture positive for B. fragilis;no ETBF identified
Low isolation rate forB. fragilis; no controlpopulation studied;clinical diarrhealdisease data notpresented
a Case reports and series lacking control populations. LLIL, lamb ligated intestinal loop assay.b Dates for studies not specified except in a few instances.
VOL. 22, 2009 ENTEROTOXIGENIC BACTEROIDES FRAGILIS 355
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will provide the best diagnostic sensitivity and specificity fordetection of ETBF disease or colonization. Table 8 summa-rizes the primers utilized to date for detection of the bft genein B. fragilis isolates or DNA extracted from stool. In someinstances, reported primer use has included multiple base pairmismatches, suggesting the possibility of decreased diagnosticsensitivity and specificity (86). Nested PCR is the most sensi-tive PCR method to detect the bft gene reported to date,detecting 102 to 103 CFU ETBF/g stool (108). The develop-ment of rapid, accurate, and sensitive diagnostic testing forETBF organisms will enhance assessments of the epidemiologyof these bacteria and their disease associations as well as beingan important prerequisite to consideration of therapeutic trialsfor ETBF disease.
Therapy of ETBF-Associated Diarrheal Disease
Because diagnosis of ETBF as an etiology of diarrheal dis-ease is presently limited to the research setting and the diag-nosis is often delayed even if sought, no controlled studies haveevaluated whether antibiotic therapy shortens ETBF-associ-ated diarrheal illnesses. Reports of antibiotic sensitivity testingof clinical ETBF strains are limited to 58 strains from Ban-gladesh and 9 strains from Nicaragua (10, 102). Whereas allstrains were resistant to ampicillin in Nicaragua, 97% of Ban-gladeshi ETBF strains were sensitive to ampicillin; conversely,all strains were sensitive to metronidazole in Nicaragua, but arelatively high proportion (7%) of ETBF strains were metro-nidazole resistant in Bangladesh. Approximately 10% of ETBFstrains were clindamycin resistant in both locales, and 26% ofETBF strains were resistant to tetracycline in Bangladesh (notevaluated in Nicaragua). Because available reports suggest thatETBF disease is self-limited, adequate oral rehydration ther-apy is central to clinical care, consistent with the therapeuticapproach to other diarrheal illnesses.
BFT GENETICS AND PROTEIN STRUCTURE
Three distinct alleles of bft have been identified, namely,bft-1, bft-2, and bft-3 (13, 31, 40, 46). The terminology bft-1,bft-2, and bft-3 was adopted to recognize the temporal se-quence in which the alleles were identified. ETBF strains maypossess two copies of a single bft genotype, but no ETBFstrains have yet been reported to contain mixed bft alleles. Thebft genes are chromosomal, with a G�C content of 39%, andare predicted to encode a 397-residue holotoxin with a calcu-lated molecular mass of ca. 44.5 kDa. The predicted BFTstructure is a preproprotein holotoxin (Fig. 3) (31, 46, 115,121). The initial 18 amino acids of the BFT holotoxin make upa signal peptide (preprotein domain) predicted as important indelivery of the holotoxin to the B. fragilis membrane, where the193-residue proprotein toxin domain is postulated (by analogyto other bacterial zinc-dependent metalloproteases [56]) to beinstrumental in proper protein folding and secretion of mature,biologically active BFT (31). The pre- and proprotein domainsof all BFTs exhibit high sequence homology (ca. 97 to 98%),consistent with conserved functions for these protein domains.Based on N-terminal sequencing of purified BFT, the propro-tein domain is cleaved at an Arg-Ala site (amino acids 211 and212) to release mature BFT from the bacterial cell (31, 115,
TA
BL
E4.
Clin
ical
obse
rvat
ions
ofE
TB
Fdi
arrh
eali
llnes
ses
Clin
ical
char
acte
rist
ic
Val
uefo
rgr
oup
from
indi
cate
dst
udy
refe
renc
ec
Apa
che
Indi
anch
ildre
nan
dad
ults
,AZ
(n�
33)a
(95)
Chi
ldre
nof
�5
yrof
age,
Dha
ka,
Ban
glad
esh
(n�
10)a
(94)
Chi
ldre
n(�
1yr
to�
10yr
),O
klah
oma
City
,O
K(n
�43
)a(9
9)
Adu
lts(�
15yr
),Sw
eden
(n�
91)a
(128
)
Chi
ldre
n(�
2yr
),N
icar
agua
(n�
9)a
(10)
Chi
ldre
n(�
5yr
),V
ietn
am(n
�19
)a(1
18)
Chi
ldre
n(�
2yr
),ru
ral
Ban
glad
esh
(n�
18E
TB
Fep
isod
esin
15ch
ildre
n)a
(85)
Chi
ldre
nan
dad
ults
,Dha
ka,
Ban
glad
esh
(n�
43ch
ildre
nof
�15
yran
d30
adul
ts)a
(102
)
Med
ian
dura
tion
ofdi
arrh
ea(d
ays)
33.
5—
203
—6
3e
Ran
ge(d
ays)
1–14
�1–
�15
——
2–20
—2–
392–
11M
edia
nno
.of
stoo
ls/d
ay6
8—
�5,
29%
;5–1
0,40
%;
�10
,30%
75
5—
Ran
ge3–
163–
15—
—4–
102–
93–
20—
Fev
er(%
)b58
—25
4444
536
7V
omiti
ng(%
)21
—33
4444
426
—A
bdom
inal
pain
(%)
——
—69
——
—88
Blo
ody
stoo
ls(%
)9
1042
14—
522
8f
Deh
ydra
tion
(%)
18—
—37
—68
619
Hos
pita
lizat
ion
(%)
1510
0A
llou
tpat
ient
s49
All
outp
atie
nts
—C
omm
unity
surv
eilla
nce
—O
ther
ente
ric
path
ogen
s(%
)4
547
5345
5655
8Pe
rsis
tent
diar
rhea
(%)d
—10
21—
22—
—0
aN
umbe
rof
patie
nts
with
ET
BF
-ass
ocia
ted
diar
rhea
onw
hich
data
pres
ente
dar
eba
sed.
The
brea
kdow
nof
child
ren
and
adul
tsin
refe
renc
e95
isun
clea
r.In
refe
renc
es94
,118
,and
128,
the
data
show
nar
efo
rpa
tient
sw
ithE
TB
Fal
one,
with
outo
ther
ente
ric
path
ogen
s.T
heto
taln
umbe
rsof
ET
BF
-ass
ocia
ted
diar
rhea
case
sre
port
edin
thes
ere
fere
nces
are
22,4
3,an
d19
5,re
spec
tivel
y.R
efer
ence
85re
port
sa
tota
lof4
0E
TB
Fdi
arrh
eal
epis
odes
in33
child
ren.
bW
hen
defin
ed,t
empe
ratu
reof
�38
°C.
c—
,dat
ano
tre
port
ed.
dD
efine
das
�14
days
ofdi
arrh
ea.
eSe
vent
y-ni
nepe
rcen
tof
the
popu
latio
nre
port
edno
ctur
nald
iarr
hea.
fE
ight
perc
ent
ofst
ools
wer
efe
calo
ccul
tbl
ood
posi
tive.
356 SEARS CLIN. MICROBIOL. REV.
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121). Consistent with these predictions, ca. 44-kDa and 20-kDaproteins are detected with anti-BFT antisera in lysates ofETBF strains (28, 29).
The predicted mature toxin domain of each bft allelecontains an extended zinc-binding metalloprotease motif,HEXXHXXGXXH, and a perfectly superimposable methio-nine residue close to the metalloprotease motif. These datasuggest that BFTs are members of the matrix metalloproteasesubfamily (matrixins) of the metzincin superfamily of zinc-dependent metalloprotease enzymes (61, 79). Limited homol-ogy between eukaryotic matrix metalloproteases and BFT ledto the hypothesis that BFT may be an ancestor of host matrixmetalloproteases (53). As predicted, the proteolytic activity ofBFT appears to be crucial to its biologic activity (61, 122), andBFT contains 1 g-atom of Zn2� per toxin molecule (61). Fur-thermore, zinc chelation reduces BFT biologic activity by ca.90% (47, 61, 80).
Point mutations to modify each of the conserved aminoacids of the extended metalloprotease motif as well as theconserved downstream methionine also reduce or eliminate
the biologic activities of BFT (28). Although BFT has beenreported to be autoproteolytic (61, 116), BFT metalloproteasepoint mutations do not alter the intracellular processing andsecretion of BFT by B. fragilis, suggesting that other intracel-lular B. fragilis proteases process the holotoxin to mature BFT(28). Additional mutational analysis of the C-terminal regionof BFT indicated that this region is intolerant to modest aminoacid deletions, suggesting that this region is also important forBFT activity (104). Truncation mutations removing only twoC-terminal amino acids reduced BFT biologic activity, andremoval of eight (or more) amino acids obliterated it. BFTmutants lacking eight or more C-terminal amino acids wereexpressed similar to wild-type toxin, but the mutant BFTs wereunstable (104).
The predicted amino acid sequences of the BFT holotoxinproteins reveal highly homologous proteins, with BFT-1 andBFT-2 being 95% similar and 92% identical (31). BFT-3 ismore closely related to BFT-2 (93% and 96% identical toBFT-1 and BFT-2, respectively) (13). The predicted proteindomains of the toxins, however, exhibit differing degrees of
TABLE 5. ETBF association with IBD or colorectal cancer
Reference (author,yr �reference no.�) Study design Location n
ETBFdiagnostic
methodMain results Limitations
Prindiville et al.,2000 (88)
Endoscopy-basedcase series,with controls
University ofCalifornia,Davis,Sacramento,CA
101 patients withIBD or diarrhea,69 controls
Stool culture,HT29 cellassay,nestedPCR forbft geneDNAextractedfrom stool
11/57 (19.3%) patients withactive IBD were positivefor ETBF by stool PCRvs 0/26 patients withinactive IBD (P � 0.01);5/18 (27.8%) patientswith diarrhea and 2/69(2.9%) controls wereETBF positive by stoolPCR (P � 0.0005); only4.8% with IBD and 11%with diarrhea wereETBF positive by cultureand HT29 cell assay
Limited study designdetails, includingsource of controls;stools were frozenfor 2 weeks priorto analysis byculture; B. fragilisisolation rates notreported
Bassett et al.,2004 (7)
Endoscopy-basedcase series,with controls
London,UnitedKingdom
35 patients with IBDand 37 controls;control populationincluded 19patients withculture-negativediarrhea and 18patients withoutdiarrhea, all ofwhom were beinginvestigated byendoscopy forabdominal pain orchanges in bowelhabits
Nested PCRfor bftgene onDNAextractedfromluminalwashingsor biopsies
Of 60 patients with luminalwashings available foranalysis, 10/28 (35.7%)“controls” (6/14 withdiarrhea and 4/14without diarrhea) and8/32 (25%) IBD patientswere ETBF positive; 7/25(28%) patients withactive IBD and 1/7(14.3%) patients withinactive IBD were ETBFpositive (difference notsignificant); 4/32 (12.5%)IBD patients and 6/33(18.2%) controls withcolonic biopsies wereETBF positive
Small study withlimited details oncontrolpopulation;populationdefined ascontrols includedpatients with andwithout diarrhea;colonic biopsiesand luminalwashings werefrozen for anunspecified timeprior to analysis
Ulger et al.,2006 (113)
Prospective,consecutivecase serieswithconcurrentcontrols
Istanbul,Turkey
73 patients withcolorectal cancer;59 healthycontrols (age andgender matched)
Stool culture,PCR forbft gene onfecal B.fragilisisolates
B. fragilis isolated from77% of patients and 68%of controls (differencenot significant); 21/56(38%) patients and 5/40(12%) controls wereETBF positive (P �0.009)
Small cross-sectionalstudy without fullcolon cancer riskfactor analysis
VOL. 22, 2009 ENTEROTOXIGENIC BACTEROIDES FRAGILIS 357
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TA
BL
E6.
Mic
robi
olog
yla
bora
tory
-bas
edst
udie
sa
Ref
eren
ce(a
utho
r,yr
�ref
eren
ceno
.�)St
udy
desi
gnL
ocat
ion
nE
TB
Fdi
agno
stic
met
hod
Mai
nre
sults
Lim
itatio
ns
Pant
osti
etal
.,19
94(8
2)M
icro
biol
ogy
labo
rato
ry-b
ased
stud
y(d
ates
not
spec
ified
)It
aly
146
B.f
ragi
lisis
olat
es(9
5ex
trai
ntes
tinal
,51
feca
l)H
T29
cell
assa
y11
.5%
ofex
trai
ntes
tinal
and
10%
offe
calB
.fra
gilis
isol
ates
wer
eE
TB
F
No
bloo
dis
olat
esex
amin
ed;
limite
dcl
inic
alde
tails
avai
labl
eK
arun
iaw
ati
etal
.,19
95C
olle
ctio
nof
B.f
ragi
lisis
olat
es,1
987–
1989
Japa
n14
1ex
trai
ntes
tinal
clin
ical
B.
frag
ilis
isol
ates
(25
bloo
dis
olat
es)
HT
29/C
1ce
llas
say
Ove
rall,
23.4
%of
B.f
ragi
lisis
olat
esw
ere
ET
BF
;9/2
5(3
6%)
bloo
dan
d24
/116
(20.
7%)
othe
rex
trai
ntes
tinal
isol
ates
wer
eE
TB
F(d
iffer
ence
not
sign
ifica
nt)
No
clin
ical
deta
ilsav
aila
ble
Mun
dyan
dSe
ars,
1996
(63)
Mic
robi
olog
yla
bora
tory
-bas
edst
udy,
1992
–199
4B
altim
ore,
MD
65ex
trai
ntes
tinal
clin
ical
B.
frag
ilis
isol
ates
(28
bloo
dis
olat
es)
Cul
ture
,HT
29C
1ce
llas
say,
BF
Ten
zym
e-lin
ked
imm
unos
orbe
ntas
say
Ove
rall,
6.2%
ofB
.fra
gilis
isol
ates
wer
eE
TB
F(i
nclu
ding
one
bloo
dis
olat
e)
�33
%of
B.f
ragi
lisis
olat
esfr
omth
est
udie
dtim
epe
riod
are
unav
aila
ble
Kat
oet
al.,
1996
(38)
Cen
tral
mic
robi
olog
yla
bora
tory
-bas
edst
udy,
1987
–198
9
Japa
n18
8un
sele
cted
B.f
ragi
lisis
olat
esfr
omex
trai
ntes
tinal
clin
ical
sam
ples
Cul
ture
,HT
29/C
1ce
llas
say,
PCR
for
bft
gene
Ove
rall,
18.6
%of
B.f
ragi
lisis
olat
esw
ere
ET
BF
;18/
64(2
8.1%
)bl
ood
and
17/1
24(1
3.7%
)ot
her
extr
aint
estin
alis
olat
esw
ere
ET
BF
(P�
0.05
)
No
clin
ical
deta
ilsav
aila
ble
Szok
eet
al.,
1997
(112
)M
icro
biol
ogy
labo
rato
ry-b
ased
stud
y,19
95–1
996
Szeg
ed,H
unga
ry13
4B
.fra
gilis
isol
ates
(40
from
outp
atie
nts
with
diar
rhea
with
out
anot
her
caus
e,20
from
heal
thy
cont
rols
tool
s,74
from
othe
rcl
inic
alsa
mpl
es)
Cul
ture
,HT
29ce
llas
say
40%
,15%
,and
20.3
%of
B.f
ragi
lisis
olat
esfr
omdi
arrh
eals
tool
s,co
ntro
lsto
ols,
and
extr
aint
estin
also
urce
s,re
spec
tivel
y,w
ere
ET
BF
;sto
olfil
trat
esw
ere
also
posi
tive
inH
T29
cell
assa
y
No
bloo
dis
olat
este
sted
;clin
ical
deta
ilsno
tav
aila
ble,
but
extr
aint
estin
alE
TB
Fte
nded
tobe
high
er-t
iter
BF
Tpr
oduc
ers
Chu
nget
al.,
1999
(13)
Mic
robi
olog
yla
bora
tory
-bas
edst
udy,
1995
–199
7Se
oul,
Kor
ea89
extr
aint
estin
alcl
inic
alB
.fr
agili
sis
olat
es(2
2bl
ood
isol
ates
)
HT
29/C
1ce
llas
say,
PCR
and
colo
nybl
othy
brid
izat
ion
for
bft
gene
Ove
rall,
38%
ofB
.fra
gilis
isol
ates
wer
eE
TB
F;1
2/22
(54.
5%)
bloo
dis
olat
esan
d22
/67
(33%
)ot
her
extr
aint
estin
alis
olat
es(P
�0.
07)
wer
eE
TB
F
No
clin
ical
deta
ilsav
aila
ble
Cla
ros
etal
.,20
00(1
4)M
icro
biol
ogy
labo
rato
ry-b
ased
stud
y(d
ates
not
spec
ified
)G
erm
any,
Sout
hern
Cal
iforn
ia93
extr
aint
estin
alcl
inic
alB
.fr
agili
sis
olat
es(1
3bl
ood
isol
ates
)
PCR
for
bft
gene
Ove
rall,
11%
ofB
.fra
gilis
isol
ates
wer
eE
TB
F;3
/13
(23%
)bl
ood
isol
ates
and
7/80
(9%
)ot
her
extr
aint
estin
alis
olat
es(d
iffer
ence
not
sign
ifica
nt)
wer
eE
TB
F
No
clin
ical
deta
ilsav
aila
ble
Fou
lon
etal
.,20
03(2
6)M
icro
biol
ogy
labo
rato
ry-b
ased
stud
yof
bloo
dis
olat
es(1
989–
2002
)an
dot
her
isol
ates
(200
0–20
03)
Bel
gium
166
extr
aint
estin
alcl
inic
alis
olat
es(1
03bl
ood
isol
ates
)PC
Rfo
rbf
tge
neO
vera
ll,24
.1%
ofB
.fra
gilis
isol
ates
wer
eE
TB
F;2
7/10
3(2
6.2%
)bl
ood
and
13/6
3(2
0.6%
)ot
her
extr
aint
estin
alis
olat
esw
ere
ET
BF
(diff
eren
ceno
tsi
gnifi
cant
)
Rev
iew
ofcl
inic
alda
ta,i
nclu
ding
shoc
kan
dm
orta
lity
rate
s,in
dica
ted
nodi
ffere
nces
betw
een
patie
nts
with
ET
BF
orN
TB
Fba
cter
emia
Obu
ch-W
oszc
zaty
nski
etal
.,20
04(8
1)M
icro
biol
ogy
labo
rato
ry-b
ased
stud
y(d
ates
not
spec
ified
)T
heN
ethe
rlan
ds,
Pola
nd21
0B
.fra
gilis
isol
ates
(78
extr
aint
estin
alis
olat
esfr
omT
heN
ethe
rlan
ds;1
32fe
cal
isol
ates
,mai
nly
from
antib
iotic
-ass
ocia
ted
diar
rhea
,fro
mPo
land
)
PCR
for
bft
gene
15%
ofB
.fra
gilis
isol
ates
from
The
Net
herl
ands
and
13%
ofis
olat
esfr
omPo
land
wer
eE
TB
F
No
clin
ical
deta
ilsav
aila
ble
Cla
ros
etal
.,20
06(1
5)M
icro
biol
ogy
labo
rato
ry-b
ased
stud
y(d
ates
not
spec
ified
)U
nite
dSt
ates
(CA
,IL
),G
erm
any
260
extr
aint
estin
alcl
inic
alB
.fr
agili
sis
olat
es(6
3bl
ood
isol
ates
,197
skin
and
soft
tissu
eis
olat
es)
PCR
for
bft
gene
Ove
rall,
12.3
%of
B.f
ragi
lisis
olat
esw
ere
ET
BF
;12/
63(1
9%)
bloo
dis
olat
esan
d20
/197
(10%
)sk
inan
dso
fttis
sue
isol
ates
wer
eE
TB
F(d
iffer
ence
not
sign
ifica
nt)
No
clin
ical
deta
ilsav
aila
ble
Avi
la-C
ampo
set
al.,
2007
(6)
Col
lect
ion
ofB
.fra
gilis
isol
ates
(dat
esno
tsp
ecifi
ed)
Uni
ted
Stat
es(C
A)
323
extr
aint
estin
alcl
inic
alis
olat
es(5
0bl
ood
isol
ates
,6fe
cali
sola
tes)
PCR
for
bft
gene
Ove
rall,
13.6
%of
B.f
ragi
lisis
olat
esw
ere
ET
BF
;4/5
0(8
%)
bloo
dis
olat
es(i
nclu
ding
one
cath
eter
tipis
olat
e)an
d40
/273
(14.
6%)
othe
rex
trai
ntes
tinal
isol
ates
wer
eE
TB
F;n
ofe
cali
sola
tes
wer
eE
TB
F
No
clin
ical
deta
ilsav
aila
ble
aSt
udie
sof
B.f
ragi
lisis
olat
eco
llect
ions
.Ref
eren
ces
for
stud
ies
incl
udin
gB
.fra
gilis
bloo
dis
olat
esar
esh
own
inbo
ld.
358 SEARS CLIN. MICROBIOL. REV.
on Decem
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identity, with only 2 to 5 amino acid changes between BFT-1,BFT-2, and BFT-3 in the preproprotein domains (13), whereasup to 25 amino acid changes occur in the mature toxin proteindomains (13, 31). Consistent with the predicted protein diver-sity in the mature BFT protein, BFT-1 and BFT-2 purify withdistinct biochemical profiles and differ in sodium dodecyl sul-fate-polyacrylamide gel electrophoresis mobility and two-di-mensional gel electrophoresis mobility, consistent with the pre-dicted uniqueness of the BFT-1 and BFT-2 proteins (121).BFT-2 also exhibits modest but consistently greater specificbiologic activity than BFT-1 in vitro, although the importanceof this observation to disease pathogenesis is unknown (121).BFT-1 and BFT-2 are trypsin resistant and stable over a widepH range (i.e., pHs 5 to 10) (97, 115, 121), potentially enablingthese toxins to resist degradation in animal and human guts.BFT-3 exhibits a purification profile and specific activity (bio-logic activity/mg protein) similar to those of BFT-2, consistentwith its greater homology to BFT-2 than BFT-1, but otherdetails on the properties of BFT-3 are not available (13).
Only limited investigations have characterized the epidemi-ology of the specific bft alleles of ETBF, but available dataindicate that all three bft alleles are globally distributed (Table9). Although the distribution of the bft-3 allele is not restrictedto Southeast Asia (6, 21), ETBF strains possessing bft-3 havebeen reported predominantly from Korea, Japan, or Vietnam,suggesting that regional evolution of ETBF may have occurred(13, 40, 76). Overall, the data suggest that the bft-1 allele ismost common among human ETBF strains evaluated to date.
GENETICS OF ETBF
Virulence genes of some organisms are clustered in uniquechromosomal loci, termed pathogenicity islands, that possessat least two virulence genes and have a G�C content differingfrom that of the host organism chromosome, with the lattersuggesting acquisition of the sequences from a foreign organ-ism (33). Study of our bank of NTBF (n � 191) and ETBF (n �113) strains, using probes derived from restriction enzymemapping of cosmid clones containing the bft gene, revealed fivedistinct patterns of hybridization, three of which predominated(Fig. 4). Strains with these genetic patterns were termed pat-tern I, II, and III B. fragilis strains (29). The chromosomes ofall ETBF strains (pattern I strains) possess a 6-kb DNA regionnot found in NTBF strains (29, 60). This 6-kb region containsthe bft gene and a second putative virulence gene, termed themetalloprotease II gene (mpII). Because this 6-kb DNA regioncontains two potential virulence genes and its G�C content is35%, differing significantly from the predicted ca. 43% G�Ccontent for the B. fragilis chromosome (11, 49), this 6-kb DNAregion, exclusively present in ETBF, was termed the B. fragilispathogenicity island or islet (due to its relatively small size)(BfPAI) (29, 60). Sequence analysis of the BfPAI revealed aca. 700-bp region upstream of bft with five putative B. fragilispromoter consensus sequences; this 700-bp promoter region is
TABLE 7. Approaches to diagnosis of ETBF infection
Diagnostic method Advantages Limitations
Stool culture B. fragilis isolation allowing directconfirmation of bft gene by PCR or ofBFT secretion by the HT29/C1 cell assay
Labor-intensive and expensive; delays diagnosisseveral days; dependent on anaerobicmicrobiology expertise
Stool PCR for bft gene Rapid compared to stool culture for B.fragilis
Requires fecal DNA extraction; sensitivitypotentially limited by fecal inhibitors of PCR;sensitivity enhanced by using overnightenrichment culture; absence of B. fragilisisolation for diagnosis confirmation
HT29/C1 cell assaya Detects BFT biologic activity directly in stoolor in culture supernatants of B. fragilisisolates; excellent correlation with lambintestinal loop assay
Expensive, labor-intensive, requires anaerobicmicrobiology expertise and subjectiveinterpretation of HT29/C1 cell assay unlessBFT-neutralizing antibody available
Enzyme-linked immunosorbentassay for fecal BFT
Potentially rapid diagnostic approach Only limited data support detection of BFT instool
IMS-PCR Potentially time-saving; B. fragilis isolationand PCR for bft gene can be performed inparallel on same sample
Requires noncommercial reagents; variableperformance to date
Intestinal loop assaysb Detects secretion stimulated by ETBF orBFT
Requires prior B. fragilis isolation; expensive andlabor-intensive; a research tool only
RITARD modelc Detects ETBF disease Requires prior B. fragilis isolation; expensive andlabor-intensive; a research tool only
a Parental HT29 cells sometimes substituted.b Usually lamb or rabbit.c RITARD, reversible ileal tie adult rabbit diarrhea.
FIG. 2. Effect of BFT on HT29/C1 cells in vitro. HT29/C1 cells (ahuman colonic carcinoma continuous cell line) exhibit morphologicalchanges, including cell rounding and dissolution of cell clusters, whentreated with BFT (5 nM). (Reprinted from reference 106 with permis-sion from Elsevier.)
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TABLE 8. Oligonucleotide primers used to identify ETBF strains and bft isoformsd
Primer use and name Primer sequence (5–3)b Primer 5 positionin bft (bp)
No. of mismatchesApproachc Reference(s)
bft-1 bft-2 bft-3
Identification of ETBFstrains
BF1 (forward) GACGGTGTATGTGATTTGTCTGAGAGA
654 3 3 3 PCR 83, 107
BF2 (reverse) ATCCCTAAGATTTTATTATCCCAAGTA
947 0 3 3
BF1 (forward) GACGGTGTATGTGATTTGTCTGAGAGA
654 3 3 3 IMS-PCR 129
BF2 (reverse) ATCCCTAAGATTTTATTATCCCAAGTA
947 0 3 3
GBF-201 GAACCTAAAACGGTATATGT 729a (real start codonat 646)
0 0 0 IMS-PCR 118
GBF-210 GTTGTAGACATCCCACTGGC 1096a (real start codonat 1013)
0 0 0
RS3 (forward outer) TGAAGTTAGTGCCCAGATGCAGG 705 0 1 1 Nested PCR 7, 16, 24, 108RS4 (reverse outer) GCTCAGCGCCCAGTATATGACC 1072 0 5 4RS1 (forward inner) TGCGGCGAACTCGGTTAATGC 729 1 1 1RS2 (reverse inner) AGCTGGGTTGTAGACATCCCA
CTGG1019 0 0 0
P1 (forward) CGCGGAATTCATGTTCTAATGAAGCTGAT
54 0 0 0 PCR 13
P5 (reverse) TGGTCTCGAGATCGCCATCTGCTATTTCC
1191 3 0 0
BFTF (forward) CGCGGCATTATTAGCTGCATGTTCTAATG
36 0 0 0 PCR 30
P4 (reverse) GATACATCAGCTGGGTTGTAGACATCCCA
1027 0 0 0
404, BF3 GAGCCGAAGACGGTGTATGTGATTTGT
646 5 5 5 PCR 86, 113
407, BF4 TGCTCAGCGCCCAGTATATGACCTAGT
1073 0 5 4
GBF-201 GAACCTAAAACGGTATATGT 729a (real start codonat 646)
0 0 0 PCR 39
GBF-210 GTTGTAGACATCCCACTGGC 1096a (real start codonat 1013)
0 0 0
BF5 (forward) GATGCTCCAGTTACAGCTTCCATTG
91 0 1 0 PCR 3
BF6 (reverse) CGCCCAGTATATGACCTAGTTCGTG
1066 0 3 2
Identification of bft isoformsP1 (forward) CGCGGAATTCATGTTCTAATGAA
GCTGAT54 0 0 0 RFLP 13
P5 (reverse) TGGTCTCGAGATCGCCATCTGCTATTTCC
1191 3 0 0
BF5 (forward) GATGCTCCAGTTACAGCTTCCATTG
91 0 1 0 RFLP andPCR
21
BF6 (reverse) CGCCCAGTATATGACCTAGTTCGTG
1066 0 3 2
BFT2R (reverse bft-2) TTGGATCATCCGCATGCCT 1087 5 0 2BFT3R (reverse bft-3) TTGGATCATCCGCATGGTT 1087 5 2 0GBF201 (forward
consensus)GAACCTAAAACGGTATATGT 646 0 0 0 PCR 40
GBF312 (reverse bft-1) CCTCTTTGGCGTCGC 835 0 5 5 118GBF322 (reverse bft-2) CGCTCGGGCAACTAT 820 4 0 3GBF334 (reverse bft-3) TGTCCCAAGTTCCCCAG 931 2 2 0GBF201 (forward
consensus)GAACCTAAAACGGTATATGT 646 0 0 0 PCR 6
BFT-TYPE1 (reverse bft-1) ATTGAACCAGGACATCCCT 960 0 6 5GBF322 (reverse bft-2) CGCTCGGGCAACTAT 820 4 0 3BFT-TYPE3 (reverse bft-3) CGTGTGCCATAACCCCA 931 1 1 0Oligoprobe bft-1 GGCGCTGAGCATACGGATAATT 1063 0 6 6 Hybridization 31Oligoprobe bft-2 GGTGCTAGGCATGCGGATGATC 1063 6 0 2
a The authors labeled the starting nucleotides for their primers as noted. However, the actual starting nucleotides for these primers are nucleotides 646 (for primerlabeled 729) and 1013 (for primer labeled 1096), based on the identified start codons leading to the correct reading frame for the signal peptide of BFT (31).
b The 5 positions in primers P1 and P5 correspond to the nucleotide shown in bold. The underlined sequences in primers P1 and P5 show the restriction sites forEcoRI and XhoI, respectively, created to clone the bft gene.
c RFLP, restriction fragment length polymorphism analysis.d Courtesy of A. Franco-Mora.
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required for maximal BFT production by ETBF strains, al-though the precise regulatory sequences have not yet beenmapped (30). mpII encodes a protein predicted to be similar insize to BFT and also predicted to be a zinc-dependent, cata-lytically active protein, but with only 56% similarity and 28%identity to the BFT proteins. To date, no in vitro biologicactivity for MPII has been identified, likely due in part to itspoor expression in vitro under growth conditions favoring ex-pression of bft (A. A. Franco, personal communication). Usingan mpII deletion mutant and other recombinant ETBF strains,
the in vitro HT29/C1 cell biologic activity of BFT is not de-pendent on or modified by MPII expression (A. A. Franco andC. L. Sears, unpublished observations). In vivo expression ofmpII or the role of MPII in ETBF disease pathogenesis has notyet been addressed.
The culture supernatants of ETBF strains grown in vitrovary significantly in HT29/C1 cell biologic activity (13, 30, 82,116, 119). Although the mechanism(s) accounting for this vari-ation is incompletely understood, at least two mechanismslikely contribute to the variable biologic activity, including bftcopy number (21) and transcriptional regulation of bft yieldingdifferent amounts of BFT secreted by ETBF strains (30). Incontrast, variation in the biologic activities of purified isoformsof BFT is only modest (13, 121). It is not known if similar
FIG. 3. Schematic of the structure of BFT holotoxin. Each of the BFT isotypes (BFT-1, BFT-2, and BFT-3) consists of three protein domains,i.e., the signal peptide, proprotein, and mature toxin. The holotoxin is cleaved by an as yet unidentified B. fragilis protease at amino acids (AAs)arginine (Arg)211-alanine (Ala)212 prior to release of the mature, �20-kDa BFT protein from the bacterial cells into the colon. H, histidine; G,glycine. (Reprinted from references 103 and 106 with permission from Elsevier.)
TABLE 9. Distribution of bft isotypes among ETBF strains
Reference (author, yr �reference no.�,location) and type of isolate
No. ofETBFstrains
% of strains withallele
bft-1 bft-2 bft-3
Chung et al., 1999 (13), KoreaIntestinal isolates 34 41 22 32
Kato et al., 2000 (40), JapanAsymptomatic control stools 36 72 14 14
Children with diarrheaNon-antibiotic-associated 19 63 26 10.5Antibiotic associated 11 36 54.5 9Cow 16 37.5 50 12.5Extraintestinal isolates 61 69 18 13
Total 143 63 24 13D’Abusco et al., 2000 (21), Italy
Extraintestinal isolates 28 61 36 4Fecal isolatesa 40 70 27.5 2.5
Nguyen et al., 2005 (76), Vietnam 49b 69 18 12Ulger et al., 2006 (114), Turkey 54c 91d 9 0Nakano et al., 2007 (72), Brazil 3e 100 0 0Avila-Campos et al. (6), United States 44f 68 25 7
a Primarily from children (n � 18) and adults (n � 10) with diarrhea; bft-2 wasidentified significantly more often in fecal ETBF isolates from children than inthose from adults.
b Primarily from children with diarrhea; no ETBF strains with the bft-3 genewere identified among the six control children positive for ETBF.
c Thirty-nine fecal ETBF strains (31 from colon cancer patients) and 15 ex-traintestinal ETBF strains.
d Eighty-seven percent, 88%, and 100% of ETBF isolates from colon cancerpatients, control patient feces, and extraintestinal sites, respectively, containedthe bft-1 gene.
e Fecal ETBF was isolated over 8 months from three children, one with AIDSand two immunocompetent children with acute diarrhea.
f Four blood, 25 intra-abdominal, 4 pulmonary, 5 skin and soft tissue, and 6miscellaneous ETBF isolates.
FIG. 4. Schematic of the molecular types of B. fragilis. Pattern I B.fragilis strains are ETBF strains possessing at least one 65-kb conjuga-tive transposon (86 CTn), within which is contained the 6-kb BfPAI.The BfPAI contains two genes, one encoding BFT (bft), demonstratedto be important to ETBF pathogenesis (92), and one encoding metal-loprotease II (mpII), a putative virulence protein. Pattern II B. fragilisstrains lack CTn86 and CTn9343 (or related sequences). Pattern III B.fragilis strains are NTBF strains that possess at least one 65-kb conju-gative transposon (9343 CTn). See the text and Fig. 5 for additionaldetails. (Reprinted from reference 103 with permission from Elsevier.)
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differences in BFT expression occur in vivo and/or impactclinical disease expression.
Both pattern II and pattern III B. fragilis strains are NTBF(29). Using the B. fragilis genome database, the DNA elementpresent in pattern III NTBF strains and a related elementpresent in all ETBF strains have been identified as members ofa new family of putative conjugative transposons, termedCTn9343 (from NTBF strain NCTC 9343) and CTn86 (fromETBF strain 86-5443-2-2) (27). Pattern II NTBF strains aredistinguished by the absence of either of these CTns. In ananalysis of 191 NTBF strains, 52% of NTBF strains were pat-tern II strains and 43% were pattern III strains. A small num-ber of NTBF strains (5%) displayed other genomic patterns(29). CTn9343 and CTn86 are approximately 64 kb, with openreading frames organized as modules with various G�C con-tents, suggesting regions where the genes may be of Bacteroidesorigin and regions acquired from other genetic sources. Theseputative CTns have very limited sequence similarity to otherdescribed Bacteroides species CTns and are distinct in severalproperties, including their predicted mechanism of transposi-tion, their lack of tetracycline regulation of CTn chromosomalexcision, and the absence of tetQ (27). Although circularizationof the CTns within ETBF and NTBF strains is readily identi-fied, indicating that the CTns can excise from the B. fragilischromosomes, transfer of CTn86 or CTn9343 to other organ-isms has not yet been observed (27; Franco, personal commu-nication). However, it is postulated that interorganism transferof these putative CTns with further acquisition of the BfPAImay be the mechanism by which ETBF strains evolved as adistinct class of B. fragilis strains (27). Additional data suggestthat additional CTns, related to but distinct from CTn86 andCTn9343, are present in both ETBF and NTBF (9) (Fig. 5). Itis unknown how or if these putative CTns modulate B. fragilisvirulence. Similarly, it is unknown what biologic advantage, ifany, CTn86 or the BfPAI confers upon ETBF strains. How-ever, mobilization of a plasmid containing bft plus its promoterregion into pattern III, but not pattern II, NTBF is efficient andyields high-level expression of BFT, indicating the differinggenetic potentials of these two populations of NTBF strainsand also suggesting that CTn9343 or another chromosomallocus unique to pattern III NTBF (and absent in pattern IINTBF) regulates BFT production (30).
By use of a variety of molecular techniques, B. fragilis strainshave also been classified into two phylogenetic divisions (32,34). Division I is characterized by the presence of the cepAgene (encoding a serine--lactamase of class A) and the ab-sence of the cfiA gene (encoding a metallo--lactamase of classB, conferring, for example, imipenem resistance), whereas di-vision II is characterized by the presence of cfiA and the ab-sence of the cepA gene. All ETBF strains, to date, are divisionI B. fragilis, as are ca. 80% of B. fragilis strains isolated inclinical studies (9, 32). Multilocus enzyme electrophoresis andcluster analysis indicate the ETBF strains are nonclonal, con-sistent with the higher recombination rates ascribed to divisionI B. fragilis (32).
Phylogenetic data indicate that B. fragilis strains are diverse,and functional studies and sequences of the genomes of twoNTBF strains identified DNA inversion regulatory mecha-nisms, suggesting that these organisms are highly adaptable,with rapid and dynamic variability in surface molecule expres-
sion patterns (11, 20, 49). B. fragilis can express up to at leasteight distinct capsular polysaccharides, a previously unprece-dented complexity for a single organism (18). It is unknown ifthe enteric pathogenicity of ETBF is modulated by expressionof specific surface characteristics (including capsular polysac-charides), for example, influencing adherence of ETBF to theintestinal mucosa and/or delivery of BFT in vivo.
BFT MECHANISM OF ACTION
In Vitro Studies of Cell Lines, Polarized EpithelialMonolayers, and Human Colon
To date, the in vitro biologic activity of BFT has been re-stricted to continuous epithelial cell lines capable of formingpolarized monolayers (119, 126). Predominantly intestinal celllines (HT29, HT29/C1, Caco-2, T84, SW480, and HCT116)
FIG. 5. Schematic representations of CTn elements found in ETBFand NTBF strains. Both ETBF and NTBF strains may possess a varietyof CTns related to those originally described for ETBF strain 86-5443-2-2 (CTn86) and NTBF strain 9343 (CTn9343) (27). Panels A and Bshow different patterns of CTns present in a collection of 123 ETBFand 73 NTBF strains, respectively. Gray boxes represent the left end ofCTn86, and black boxes represent the left end of CTn9343. (Reprintedfrom reference 9.)
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have been studied (78, 123, 126; S. Wu and C. L. Sears, un-published observations). More limited studies indicate thatrenal (MDCK) and pulmonary (Calu-3) cell lines that canpolarize in vitro also develop morphological changes and thusexhibit a biologic response to BFT (78; Wu and Sears, unpub-lished observations). Overall, subconfluent cloned HT29/C1cells have been studied in greatest detail due to their exquisiteconcentration-dependent sensitivity to BFT (63, 97). The half-maximal BFT concentration altering HT29/C1 cell morphologyis ca. 12.5 pM, with the onset of activity at 0.5 pM (97). Usingsubconfluent HT29/C1 cells, the hallmarks of the BFT biologicresponse are a rapid onset (within 10 to 15 min) and temper-ature-dependent (maximal activity detected at 37°C) morpho-logical changes in which cell rounding and swelling with loss ofcell-to-cell contact occur (Fig. 2) (61, 63, 97, 119, 122). Thesechanges are reminiscent of the morphological changes in thesurface epithelial cells of intestines infected with ETBF (Fig.1). Although the total F (filamentous)-actin content of cellstreated with BFT is unaltered (22, 47, 96, 97), marked redis-tribution of F-actin occurs, with decreased stress fibers andperipheral F-actin condensation observed in unpolarizedHT29 cells treated with BFT (22, 47). The mechanism(s) re-sponsible for the cell morphology changes stimulated by BFTis unknown, although a broad-spectrum tyrosine kinase inhib-itor was noted to delay the onset of BFT-induced cell mor-phology changes (124). Inhibitors of microtubules and endo-somal or Golgi trafficking do not alter induction of cellmorphology changes by BFT (22, 78, 97).
Although the bioactivity of BFT is not reversible by washingearly on, subconfluent HT29/C1 cells recover a normal appear-ance by light microscopy within 2 to 3 days after BFT treat-ment, indicating that the biologic activity of BFT on HT29/C1cells is reversible over time (97, 119, 122). The majority ofavailable evidence indicates that BFT is a nonlethal and non-cytotoxic protein, namely, BFT stimulates rather than dimin-ishes protein synthesis (12, 47), it does not stimulate cellularlactate dehydrogenase (LDH) or 51Cr release or the cellularuptake of vital dyes (trypan blue and propidium iodide) (12,47, 78, 120), and DNA synthesis continues normally (22). BFTtreatment of polarized colonic epithelial cell (HT29 and T84)monolayers in vitro resulted in delayed (�36 h after BFTtreatment) apoptosis of a minority of treated cells, althoughthe initial response to BFT was in fact shown to be inductionof an antiapoptotic protein (cellular inhibitor of apoptosis pro-tein 2 [cIAP2]) (43; see “Molecular Mechanism of Action ofBFT”). After BFT treatment of T84 cells or human colonbiopsies in vitro, a delayed loss of cell viability and apoptosis ofdetached (and thus dying) epithelial cells were noted (99, 100).Of potential importance to human ETBF disease, heterogene-ity was noted in the rate of onset (2 to 18 h) and severity ofepithelial morphological changes in human colon biopsiestreated with BFT, with no BFT response noted in 20% of theindividuals sampled (99).
BFT stimulates a concentration- and time-dependent in-crease in the permeability of epithelial monolayers (T84,MDCK, HT29, HT29/C1, and Caco-2 cells) (12, 78, 105). No-tably, BFT exhibits polar monolayer bioactivity, increasing per-meability rapidly and at lower toxin concentrations whenplaced on the basolateral membranes rather than the apicalmembranes of epithelial monolayers or human colons studied
in vitro (12, 78, 93, 105, 120; Wu and Sears, unpublishedobservations). In addition, basolateral but not apical BFT canrapidly but transiently increase the short-circuit current (indic-ative of chloride secretion) (12). BFT also stimulates predom-inantly basolateral release of proinflammatory CXC chemo-kines, consistent with a role for BFT in inducing mucosalinflammation (45, 124; see “Molecular Mechanism of Actionof BFT”).
The changes in T84 cell monolayer permeability are accom-panied by cellular morphological changes, with apical BFTcausing only relatively slow (with onset at 6 h), focal changeson the apical epithelial cell membranes. In contrast, basolat-eral BFT more rapidly (by 90 min) modifies the morphology ofevery cell in the monolayer, with cell swelling and unraveling ofthe apical membrane microvilli resulting in a striking domedcellular appearance (12, 47). The disappearance of the mi-crovilli is associated with a loss of F-actin from the apical poleof the cells, with a marked reassembly of the F-actin at thebasolateral pole of the cells (12). Concomitantly, as detailed bytransmission electron microscopy, BFT stimulates structuralchanges and even dissolution of the zonula occludens (tightjunction) and zonula adherens, electron-dense structures thatregulate the permeability of epithelial monolayers, whereasdesmosomes remain intact (12, 105). The redistribution ofF-actin and loss of the zonula occludens and zonula adherensreadily explain the measured increase in monolayer permeabil-ity, although full mechanistic details are not available. BFT-induced colon permeability may further expose the submucosato other bacterial luminal antigens and thus contribute to howETBF fosters colonic inflammation (7, 37, 88, 90, 113). Wellsand colleagues further reported that the basolateral mem-branes of BFT-treated HT29 cells permitted increased associ-ation and invasion of pathogenic enteric bacteria, except forListeria monocytogenes (120). BFT-treated HT29 cells arelikely resistant to L. monocytogenes invasion due to BFT-in-duced loss of cellular E-cadherin (see “Molecular Mechanismof Action of BFT”), one ligand for invasion of this bacterium(59). Lastly, BFT, presumably by modifying mucosal perme-ability, has been reported to act as a mucosal adjuvant, en-hancing the systemic antibody response to an intranasal anti-gen challenge in mice (117).
Molecular Mechanism of Action of BFT
To date, the only cellular protein demonstrated to be rapidlycleaved after treatment of colonic epithelial cells with BFT isthe zonula adherens protein E-cadherin (122, 125). In subcon-fluent HT29/C1 cells, the onset of E-cadherin cleavage is rapid,as it is detectable by 1 min and typically complete within 1 to2 h. BFT induces E-cadherin cleavage via two steps, the initialrelease of the E-cadherin ectodomain, which is dependent onbiologically active BFT, and subsequent processing of the in-tracellular E-cadherin fragment by host cell presenilin-1/�-secretase (a member of the intramembrane cleavage protease[iCLips] family) (125). Only E-cadherin presented on an intact,living cell is cleaved in response to BFT (i.e., in vitro cleavageof E-cadherin cannot be demonstrated), and this E-cadherincleavage requires cellular ATP, suggesting that protein confor-mation and/or other cellular properties contribute to the pro-teolytic event (122). Cellular recovery after BFT treatment
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correlates with resynthesis of E-cadherin. Cleavage of E-cad-herin also occurs in vivo in a murine model of ETBF diseaseand correlates with the onset of colonic inflammation anddisruption of the epithelial barrier by histopathology (92).
Besides its critical role in intercellular adhesion at thezonula adherens in intestinal tissue, E-cadherin is tethered atits intracellular domain to -catenin, a nuclear signaling pro-tein involved in both normal and dysregulated cellular growth(74). Proteolysis of E-cadherin in response to BFT induces-catenin nuclear localization, upregulation of c-myc (a -cate-nin-regulated oncogene) transcription and translation, and cel-lular proliferation of HT29/C1 cells (123). HT29/C1 cell pro-liferation is stimulated by as little as 0.5 nM BFT (123).Further studies suggest that -catenin signaling accounts foronly ca. 30 to 40% of BFT-induced cellular proliferation, in-dicating that other, as yet unidentified mechanisms contributeto BFT-initiated cell proliferation (123). BFT-treated HT29/C1cells are highly mobile, consistent with observations indicatingthat diminished E-cadherin on tumor cells enhances metastaticpotential (Wu and Sears, unpublished observations). In paral-lel, BFT also stimulates induction of the antiapoptotic proteincIAP2, mediated by p38 mitogen-activated protein kinase (p38MAPK) and cyclooxygenase-2 (COX-2) signaling with produc-tion of prostaglandin E2 (PGE2) (43). BFT-induced colonocyteproliferation and mobility, combined with resistance to apop-tosis, may contribute to the putative oncogenic potential ofBFT and support an initial report of an association betweenETBF colonization and colorectal carcinoma (113). An alter-native hypothesis is that antiapoptotic signaling by BFT in-creases the colonocyte life span, permitting further generationof proinflammatory signaling to the colonic submucosa (43).
Reported in vitro proteolytic substrates of BFT include G(monomeric)-actin, gelatin, azocoll, tropomyosin, collagen IV,human complement C3, and fibrinogen. However, no biologicsignificance of these substrates has yet been identified withregard to the cellular or intestinal mechanism of action of BFT(61, 79, 96). Furthermore, BFT induces shedding of a broadarray of HT29/C1 membrane proteins over time (125). How-ever, the identities of these shed proteins and whether they arecleaved by BFT or specifically due to BFT-induced cell signal-ing are unknown. Of other proteins specifically tested, BFTdoes not cleave the cell surface proteins occludin or claudin 1and 2 (both present in the zonula occludens) and 1-integrin (abasal intestinal epithelial cell protein) (122; Wu and Sears,unpublished observations) or the intracellular proteins -cate-nin, �-catenin, zonula occludens protein-1 (ZO-1), and actin(96, 122). Although these proteins are not BFT substrates,ZO-1, -catenin, and occludin, for example, redistribute incells by 1 hour after treatment with BFT (78, 122).
ETBF induces intestinal inflammation in animals and hu-mans (80, 102, 105). In addition to increased colonic perme-ability that may augment submucosal inflammation throughexposure to luminal bacterial antigens, ETBF likely inducesintestinal inflammation via BFT-stimulated intestinal epithelialcell release of proinflammatory chemokines, including IL-8(also called CXCL8 or [the murine equivalent] macrophageinflammatory protein 2 [MIP-2]), epithelial-neutrophil activat-ing protein 78 (ENA-78), CCL2, monocyte chemotactic pro-tein 1 (MCP-1), and growth-related oncogene alpha (GRO-�;also called CXCL1) (41, 42, 45, 100, 124). The time course of
chemokine release varies, with IL-8 appearing first (at 2 to 4 h)and GRO-� and ENA-78 appearing later (45, 124). Similarly,BFT also stimulates increased synthesis of these chemokinesfrom isolated human colonocytes (45). BFT also induces in-creased expression of the biologically inactive form of trans-forming growth factor beta (TGF-) by intestinal epithelialcells in vitro (100). It is postulated that inactive TGF- issubsequently processed by proteases in the intestinal mucosato active TGF- and contributes to mucosal repair in ETBFdisease. In contrast, no data have yet identified direct effects ofBFT on immune cells or myofibroblasts (92, 100).
NF- B appears to act as a central regulator of chemokineexpression in BFT-stimulated colonic epithelial cells in vitro(41, 45, 124), and NF- B activation in BFT-treated isolatedhuman colonocytes has been demonstrated (41). BFT inducesan unusual discrete supranuclear localization of NF- B, a find-ing also previously reported in response to IL-1 (36, 124). Theregulation of NF- B activation stimulated by BFT is complex,involving receptor and nonreceptor tyrosine kinases, MAPKs(p38, extracellular signal-related kinase, and c-Jun N-terminalkinase), Ras, and AP-1 (42, 124). NF- B is also reported tomediate BFT-induced colonic epithelial cell expression (in-cluding in human colonocytes) of COX-2 but not COX-1,resulting in increased levels of cellular PGE2 and cyclic AMP(cAMP).
Initial experiments implicate BFT-initiated NF- B activa-tion in colonic epithelial cells as the orchestrator of inflamma-tion and secretion in ETBF disease. First, inhibition of NF- Bactivation diminished BFT-induced chemokine release andpolymorphonuclear leukocyte transepithelial migration in co-lonic epithelial monolayers in vitro, suggesting that NF- Bsignaling was directly linked to polymorphonuclear leukocytemucosal influx in ETBF disease (41). Second, both p38 MAPKand COX-2 inhibition significantly decreased secretion inmouse ileal loops treated with BFT, implicating NF- B acti-vation by BFT as a key cellular coordinator of secretion inETBF disease. Inhibition of p38 MAPK activation also re-versed BFT-induced ileal inflammation in the mouse (42). Theeffect of COX-2 inhibition on murine ileal histology was notreported, although COX-2 inhibition did not diminish MIP-2production, suggesting at least some differential regulation ofsecretion and inflammation in response to ETBF infection(44).
PROPOSED MODEL FOR PATHOGENESIS OFETBF DISEASE
Figure 6 proposes a model for the pathogenesis of ETBFdisease. Available data suggest that among Bacteroides species,B. fragilis seeks a mucosal niche aided by its decoration withfucosylated molecules mimicking host proteins (19, 73). Al-though ETBF adherence in vivo or in vitro has not yet beenexamined, the pathogenesis of ETBF intestinal disease is ex-pected to be initiated by adherence of the organism to thecolonic mucosa, with local delivery of BFT. The histology ofETBF disease in animals has not identified adherent organ-isms, but available results are limited by formalin fixation,which can interfere with detection of mucosal adherence in thecolon (111). Given the in vitro potency of BFT in cell assays(97) and the limited quantities of BFT secreted into cultures in
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vitro (121), it seems probable that small amounts of BFTdelivered by adherent ETBF cells to the colonic epitheliummay be sufficient to modify colonic epithelial cell structure andfunction. Consistent with this postulate, recent data indicatethat bft expression is necessary and sufficient to induce colitis inmurine models (92). No quantitative data have yet correlatedETBF colonization or disease with levels of BFT expressed by
ETBF strains. It is predicted that BFT promotes disease bybinding to apical membrane receptors on colonic epithelialcells (CECs), initiating a burst of complex signal transductionresulting in rapid E-cadherin cleavage (122, 125, 126). E-cad-herin cleavage releases -catenin associated with the cytoplas-mic domain of E-cadherin. -Catenin nuclear translocationalong with activation of tyrosine kinases, MAPKs, and NF- B
FIG. 6. Model of ETBF colitis pathogenesis. ETBF colonizes the colon, where BFT is released and attaches to a specific colonic epithelial cell(CEC) receptor, triggering complex (and incompletely understood) CEC signal transduction involving -catenin, tyrosine kinases (TK), mitogen-activated protein kinases (MAPK), and NF- B. CEC signal transduction results in the cleavage of E-cadherin as well as in new CEC proteinsynthesis, with increased expression of c-Myc, cyclooxygenase-2 (COX-2), and chemokines/cytokines, including IL-8 and TGF-. E-cadherincleavage initiates decreased barrier function of the colonic mucosa, with the potential for increased exposure of the mucosal immune system toantigens of ETBF as well as the colonic flora fostering an inflammatory mucosal response. c-Myc expression stimulates CEC proliferation, at leastin part. Release of chemokines/cytokines by CECs into the submucosa enhances mucosal inflammation in response to ETBF colonization. Theprecise contributions of different mucosal immune cells to the inflammatory response to ETBF colon colonization are unknown, but data suggestthat both polymorphonuclear leukocytes and lymphocytes are important (41; S. Wu and C. L. Sears, submitted for publication). DC, dendritic cell;M�, macrophage; PMNs, polymorphonuclear leukocytes.
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results in nuclear signaling with new gene transcription (41, 42,100, 124). E-cadherin cleavage, along with cellular F-actin re-arrangement, further promotes colonic permeability (93) andaccess of innate mucosal immune cells to luminal bacterialantigens. This likely promotes mucosal inflammatory and se-cretory responses that are augmented by BFT-induced CECchemokine expression and PGE2 synthesis. Induction of c-Mycsynthesis further induces CEC proliferation (123). No datahave yet identified a role for BFT in pathogenesis beyond itsdirect CEC actions. The precise timing and order of the sig-naling cascades initiated by BFT remain to be deciphered, asdo the details of how or which specific signal transductionmechanisms contribute to the cell morphology changes, E-cadherin cleavage, new gene expression, and CEC prolifera-tion stimulated by BFT. Together, the data support the con-cept that ETBF strains, through secretion of BFT, areproinflammatory and oncogenic bacteria, at least in some hosts(99). Limited clinical observations are consistent with this con-cept (7, 88, 102, 113).
SUMMARY AND FUTURE CHALLENGES
ETBF organisms are common human colonic symbioteswhose potential to cause human disease is incompletely under-stood. In murine models, ETBF induces acute, self-limited,symptomatic colitis that transitions to long-term carriagewhere the murine host constrains but does not eliminate ETBFcolitis (92). Well-designed human investigations are needed toassess whether this paradigm occurs in humans and to deter-mine the impact of long-term carriage of ETBF on colonicstructure, function, and disease incidence. Based on availableclinical and experimental data, conditions where ETBF maycontribute to disease include IBD, colorectal cancer, and po-tentially other enigmatic conditions where colonic inflamma-tion may be pathogenic, such as necrotizing enterocolitis inneonates, postinfectious irritable bowel syndrome, antibiotic-associated diarrhea, and even perhaps malnutrition in childrenin the developing world. Possible mechanisms regulating anddefining the outcome of the host-ETBF interaction includedifferences in ETBF strain virulence, genetically determinedhost differences in adhesion, immune, or inflammatory re-sponses to ETBF, and/or modulation of ETBF virulence by theother host colonic flora. Studies to detect ETBF in patientpopulations should employ both sensitive molecular microbi-ologic fecal diagnosis and serology to detect anti-BFT antibod-ies that develop, at least for patients with acute ETBF diar-rheal disease (102). Whether anti-BFT antibodies are useful tofurther evaluate the epidemiology of ETBF human infection orcolonization is as yet unknown. There is surging interest in theimpact of symbiont bacteria, particularly the colonic flora, onnormal host physiology, immunology, and disease. AmongBacteroides species, the molecularly diverse B. fragilis strainsare distinguished by being critical symbionts and importanthuman pathogens. Future studies of the colonic flora, includ-ing ETBF, hold promise for illuminating the mechanisms gov-erning the essential yet sometimes pathogenic relationship be-tween flora and host.
ACKNOWLEDGMENTS
I thank R. Bradley Sack, Shaoguang Wu, and Augusto Franco-Morafor reviewing the manuscript and providing their insights. I also thankA. Franco-Mora for providing Table 8 in the manuscript and all thecontributors over time to studies from my laboratory.
I have no conflicts of interest to report.This work was supported by grants RO1 DK45496 and RO1
DK080817 and by a Senior Investigator Award from the Crohn’s andColitis Foundation.
REFERENCES
1. Reference deleted.2. Albert, M. J., A. S. Faruque, S. M. Faruque, R. B. Sack, and D. Ma-
halanabis. 1999. Case-control study of enteropathogens associated withchildhood diarrhea in Dhaka, Bangladesh. J. Clin. Microbiol. 37:3458–3464.
3. Antunes, E. N., E. O. Ferreira, L. S. Falcao, G. R. Paula, K. E. Avelar, D. E.Barroso, J. P. Leite, M. C. Ferreira, and R. M. Domingues. 2004. Non-toxigenic pattern II and III Bacteroides fragilis strains: coexistence in thesame host. Res. Microbiol. 155:522–524.
4. Antunes, E. N., E. Ferreira, D. Vallim, G. Paula, L. Seldin, A. Sabra, M.Gerreira, and R. Domingues. 2002. Pattern III non-toxigenic Bacteroidesfragilis (NTBF) strains in Brazil. Anaerobe 8:17–22.
5. Aucher, P., J. P. Saunier, G. Grollier, M. Sebald, and J. L. Fauchere. 1996.Meningitis due to enterotoxigenic Bacteroides fragilis. Eur. J. Clin. Micro-biol. Infect. Dis. 15:820–823.
6. Avila-Campos, M. J., C. Liu, Y. Song, M. C. Rowlinson, and S. M. Finegold.2007. Determination of bft gene subtypes in Bacteroides fragilis clinicalisolates. J. Clin. Microbiol. 45:1336–1338.
7. Basset, C., J. Holton, A. Bazeos, D. Vaira, and S. Bloom. 2004. Are Heli-cobacter species and enterotoxigenic Bacteroides fragilis involved in inflam-matory bowel disease? Dig. Dis. Sci. 49:1425–1432.
8. Bressane, M., L. Durigon, and M. Avila-Campos. 2001. Prevalence of theBacteroides fragilis group and enterotoxigenic Bacteroides fragilis in immu-nodeficient children. Anaerobe 7:277–281.
9. Buckwold, S. L., N. B. Shoemaker, C. L. Sears, and A. A. Franco. 2007.Identification and characterization of conjugative transposons CTn86 andCTn9343 in Bacteroides fragilis strains. Appl. Environ. Microbiol. 73:53–63.
10. Caceres, M., G. Zhang, A. Weintraub, and C.-E. Nord. 2000. Prevalenceand antimicrobial susceptibility of enterotoxigenic Bacteroides fragilis inchildren with diarrhoea in Nicaragua. Anaerobe 6:143–148.
10a.Centers for Disease Control and Prevention. 2008. Preliminary FoodNetdata on the incidence of infection with pathogens transmitted commonlythrough food—10 states, 2007. MMWR Morb. Mortal. Wkly. Rep. 57:366–370.
11. Cerdeno-Tarraga, A. M., S. Patrick, L. C. Crossman, G. Blakely, V. Abratt,N. Lennard, I. Poxton, B. Duerden, B. Harris, M. A. Quail, A. Barron, L.Clark, C. Corton, J. Doggett, M. T. Holden, N. Larke, A. Line, A. Lord, H.Norbertczak, D. Ormond, C. Price, E. Rabbinowitsch, J. Woodward, B.Barrell, and J. Parkhill. 2005. Extensive DNA inversions in the B. fragilisgenome control variable gene expression. Science 307:1463–1465.
12. Chambers, F. G., S. S. Koshy, R. F. Saidi, D. P. Clark, R. D. Moore, andC. L. Sears. 1997. Bacteroides fragilis toxin exhibits polar activity on mono-layers of human intestinal epithelial cells (T84 cells) in vitro. Infect. Immun.65:3561–3570.
13. Chung, G. T., A. A. Franco, S. Wu, G. E. Rhie, R. Cheng, H. B. Oh, and C. L.Sears. 1999. Identification of a third metalloprotease toxin gene in extraint-estinal isolates of Bacteroides fragilis. Infect. Immun. 67:4945–4949.
14. Claros, M. C., Z. C. Claros, Y. J. Tang, S. H. Cohen, J. Silva, Jr., E. J.Goldstein, and A. C. Rodloff. 2000. Occurrence of Bacteroides fragilis en-terotoxin gene-carrying strains in Germany and the United States. J. Clin.Microbiol. 38:1996–1997.
15. Claros, M. C., Z. Claros, D. Hecht, D. Citron, E. Goldstein, J. Silva, Jr., Y.Tang-Feldman, and A. Rodloff. 2006. Characterization of the Bacteroidesfragilis pathogenicity island in human blood culture isolates. Anaerobe12:17–22.
16. Cohen, S. H., R. Shetab, Y. J. Tang-Feldman, P. Sarma, J. Silva, Jr., andT. P. Prindiville. 2006. Prevalence of enterotoxigenic Bacteroides fragilis inhospital-acquired diarrhea. Diagn. Microbiol. Infect. Dis. 55:251–254.
17. Collins, J. H., M. E. Bergeland, L. L. Myers, and D. S. Shoop. 1989.Exfoliating colitis associated with enterotoxigenic Bacteroides fragilis in apiglet. J. Vet. Diagn. Investig. 1:349–351.
18. Comstock, L. E., and D. L. Kasper. 2006. Bacterial glycans: key mediatorsof diverse host immune responses. Cell 126:847–850.
19. Coyne, M. J., B. Reinap, M. M. Lee, and L. E. Comstock. 2005. Humansymbionts use a host-like pathway for surface fucosylation. Science 307:1778–1781.
20. Coyne, M. J., K. G. Weinacht, C. M. Krinos, and L. E. Comstock. 2003. Mpirecombinase globally modulates the surface architecture of a human com-mensal bacterium. Proc. Natl. Acad. Sci. USA 100:10446–10451.
21. d’Abusco, A. S., M. Del Grosso, S. Censini, A. Covacci, and A. Pantosti.
366 SEARS CLIN. MICROBIOL. REV.
on Decem
ber 3, 2020 by guesthttp://cm
r.asm.org/
Dow
nloaded from
2000. The alleles of the bft gene are distributed differently among entero-toxigenic Bacteroides fragilis strains from human sources and can be presentin double copies. J. Clin. Microbiol. 38:607–612.
22. Donelli, G., A. Fabbri, and C. Fiorentini. 1996. Bacteroides fragilis entero-toxin induces cytoskeletal changes and surface blebbing in HT-29 cells.Infect. Immun. 64:113–119.
23. Duimstra, J. R., L. L. Myers, J. E. Collins, D. A. Benfield, D. S. Shoop, andW. C. Bradbury. 1991. Enterovirulence of enterotoxigenic Bacteroides fra-gilis in gnotobiotic pigs. Vet. Pathol. 28:514–518.
24. Durmaz, B., M. Dalgalar, and R. Durmaz. 2005. Prevalence of enterotoxi-genic Bacteroides fragilis in patients with diarrhea: a controlled study.Anaerobe 11:318–321.
25. Eckburg, P. B., E. M. Bik, C. N. Bernstein, E. Purdom, L. Dethlefsen, M.Sargent, S. R. Gill, K. E. Nelson, and D. A. Relman. 2005. Diversity of thehuman intestinal microbial flora. Science 308:1635–1638.
26. Foulon, I., D. Pierard, G. Muyldermans, K. Vandoorslaer, O. Soetens, P.Rosseel, and S. Lauwers. 2003. Prevalence of fragilysin gene in Bacteroidesfragilis isolates from blood and other extraintestinal samples. J. Clin. Mi-crobiol. 41:4428–4430.
27. Franco, A. A. 2004. The Bacteroides fragilis pathogenicity island is containedin a putative novel conjugative transposon. J. Bacteriol. 186:6077–6092.
28. Franco, A. A., S. Buckwold, J. W. Shin, M. Ascon, and C. L. Sears. 2005.Mutation of the zinc-binding metalloprotease motif affects Bacteroides fra-gilis toxin activity without affecting propeptide processing. Infect. Immun.73:5273–5277.
29. Franco, A. A., R. K. Cheng, G. T. Chung, S. Wu, H. B. Oh, and C. L. Sears.1999. Molecular evolution of the pathogenicity island of enterotoxigenicBacteroides fragilis strains. J. Bacteriol. 181:6623–6633.
30. Franco, A. A., R. K. Cheng, A. Goodman, and C. L. Sears. 2002. Modulationof bft expression by the Bacteroides fragilis pathogenicity island and itsflanking region. Mol. Microbiol. 45:1067–1077.
31. Franco, A. A., L. M. Mundy, M. Trucksis, S. Wu, J. B. Kaper, and C. L.Sears. 1997. Cloning and characterization of the Bacteroides fragilis metal-loprotease toxin gene. Infect. Immun. 65:1007–1013.
32. Gutacker, M., C. Valsangiacomo, and J. C. Piffaretti. 2000. Identification oftwo genetic groups in Bacteroides fragilis by multilocus enzyme electro-phoresis: distribution of antibiotic resistance (cfiA, cepA) and enterotoxin(bft) encoding genes. Microbiology 146:1241–1254.
33. Hacker, J., G. Blum-Oehler, I. Muhldorfer, and H. Tschape. 1997. Patho-genicity islands of virulent bacteria: structure, function and impact onmicrobial evolution. Mol. Microbiol. 23:1089–1097.
34. Holton, J. 2008. Enterotoxigenic Bacteroides fragilis. Curr. Infect. Dis. Rep.10:99–104.
35. Hooper, L. V., and J. I. Gordon. 2001. Commensal host-bacterial relation-ships in the gut. Science 292:1115–1118.
36. Jobin, C., S. Haskill, L. Mayer, A. Panja, and R. B. Sartor. 1997. Evidencefor altered regulation of I kappa B alpha degradation in human colonicepithelial cells. J. Immunol. 158:226–234.
37. Karin, M., and F. R. Greten. 2005. NF-kappaB: linking inflammation andimmunity to cancer development and progression. Nat. Rev. Immunol.5:749–759.
38. Kato, N., H. Kato, K. Watanabe, and K. Ueno. 1996. Association of ente-rotoxigenic Bacteroides fragilis with bacteremia. Clin. Infect. Dis. 23(Suppl.1):S83–S86.
39. Kato, N., C. Liu, H. Kato, K. Watanabe, H. Nakamura, N. Iwai, and K.Ueno. 1999. Prevalence of enterotoxigenic Bacteroides fragilis in childrenwith diarrhea in Japan. J. Clin. Microbiol. 37:801–803.
40. Kato, N., C. X. Liu, H. Kato, K. Watanabe, Y. Tanaka, T. Yamamoto, K.Suzuki, and K. Ueno. 2000. A new subtype of the metalloprotease toxingene and the incidence of the three bft subtypes among Bacteroides fragilisisolates in Japan. FEMS Microbiol. Lett. 182:171–176.
41. Kim, J. M., S. J. Cho, Y. K. Oh, H. Y. Jung, Y. J. Kim, and N. Kim. 2002.Nuclear factor-kappa B activation pathway in intestinal epithelial cells is amajor regulator of chemokine gene expression and neutrophil migrationinduced by Bacteroides fragilis enterotoxin. Clin. Exp. Immunol. 130:59–66.
42. Kim, J. M., H. Y. Jung, J. Y. Lee, J. Youn, C. H. Lee, and K. H. Kim. 2005.Mitogen-activated protein kinase and activator protein-1 dependent signalsare essential for Bacteroides fragilis enterotoxin-induced enteritis. Eur.J. Immunol. 35:2648–2657.
43. Kim, J. M., J. Y. Lee, and Y. J. Kim. 2008. Inhibition of apoptosis inBacteroides fragilis enterotoxin-stimulated intestinal epithelial cells throughthe induction of c-IAP-2. Eur. J. Immunol. 38:2190–2199.
44. Kim, J. M., J. Y. Lee, Y. M. Yoon, Y. K. Oh, J. S. Kang, Y. J. Kim, and K. H.Kim. 2006. Bacteroides fragilis enterotoxin induces cyclooxygenase-2 andfluid secretion in intestinal epithelial cells through NF-kappaB activation.Eur. J. Immunol. 36:2446–2456.
45. Kim, J. M., Y. K. Oh, Y. J. Kim, H. B. Oh, and Y. J. Cho. 2001. Polarizedsecretion of CXC chemokines by human intestinal epithelial cells in re-sponse to Bacteroides fragilis enterotoxin: NF-kappa B plays a major role inthe regulation of IL-8 expression. Clin. Exp. Immunol. 123:421–427.
46. Kling, J. J., R. L. Wright, J. S. Moncrief, and T. D. Wilkens. 1997. Cloning
and characterization of the gene for the metalloprotease enterotoxin ofBacteroides fragilis. FEMS Microbiol. Lett. 146:279–284.
47. Koshy, S. S., M. H. Montrose, and C. L. Sears. 1996. Human intestinalepithelial cells swell and demonstrate actin rearrangement in response tothe metalloprotease toxin of Bacteroides fragilis. Infect. Immun. 64:5022–5028.
48. Krzyzanowsky, F., and M. Avila-Campos. 2003. Detection of non-entero-toxigenic and enterotoxigenic Bacteroides fragilis in stool samples fromchildren in Sao Paulo, Brazil. Rev. Inst. Med. Trop. Sao Paulo 45:225–227.
49. Kuwahara, T., A. Yamashita, H. Hirakawa, H. Nakayama, H. Toh, N.Okada, S. Kuhara, M. Hattori, T. Hayashi, and Y. Ohnishi. 2004. Genomicanalysis of Bacteroides fragilis reveals extensive DNA inversions regulatingcell surface adaptation. Proc. Natl. Acad. Sci. USA 101:14919–14924.
50. Lassmann, B., D. R. Gustafson, C. M. Wood, and J. E. Rosenblatt. 2007.Reemergence of anaerobic bacteremia. Clin. Infect. Dis. 44:895–900.
51. Leszczynski, P., A. van Belkum, H. Pituch, H. Verbrugh, and F. Meisel-Mikolajczyk. 1997. Vaginal carriage of enterotoxigenic Bacteroides fragilisin pregnant women. J. Clin. Microbiol. 35:2899–2903.
52. Martirosian, G., M. Bulanda, B. Wojcik-Stojek, P. Obuch-Woszczatynski,G. Rouyan, P. Heczko, and F. Meisel-Mikolajczyk. 2001. Acute appendici-tis: the role of enterotoxigenic strains of Bacteroides fragilis and Clostridiumdifficile. Med. Sci. Monit. 7:382–386.
53. Massova, I., L. P. Kotra, R. Fridman, and S. Mobashery. 1998. Matrixmetalloproteinases: structures, evolution, and diversification. FASEB J.12:1075–1095.
54. Mazmanian, S. K., C. H. Liu, A. O. Tzianabos, and D. L. Kasper. 2005. Animmunomodulatory molecule of symbiotic bacteria directs maturation ofthe host immune system. Cell 122:107–118.
55. Mazmanian, S. K., J. L. Round, and D. L. Kasper. 2008. A microbialsymbiosis factor prevents intestinal inflammatory disease. Nature 453:620–625.
56. McIver, K. S., E. Kessler, J. C. Olson, and D. E. Ohman. 1995. The elastasepropeptide functions as an intramolecular chaperone required for elastaseactivity and secretion in Pseudomonas aeruginosa. Mol. Microbiol. 18:877–889.
57. Meisel-Mikolajczyk, F., P. Leszczynski, A. van Belkum, H. Pituch, P.Obuch-Woszczatynski, and G. Rouyan. 1999. Enterotoxin-producing Bac-teroides fragilis (ETBF) strains in stool samples submitted for testing ofClostridium difficile and its toxins. Anaerobe 5:217–219.
58. Meisel-Mikolajczyk, F., M. Sebald, E. Torbicka, K. Rafalowska, and U.Zielinska. 1994. Isolation of enterotoxigenic Bacteroides fragilis strains inPoland. Acta Microbiol. Pol. 43:389–392.
59. Mengaud, J., H. Ohayon, P. Gounon, R. Mege, and P. Cossart. 1996.E-cadherin is the receptor for internalin, a surface protein required forentry of L. monocytogenes into epithelial cells. Cell 84:923–932.
59a.Menozzi, M. G., et al. October 1998. 2nd World Congress on AnaerobicBacteria and Infections, Nice, France, abstr. 5.007.
60. Moncrief, J. S., A. J. Duncan, R. L. Wright, L. A. Barroso, and T. D.Wilkins. 1998. Molecular characterization of the fragilysin pathogenicityislet of enterotoxigenic Bacteroides fragilis. Infect. Immun. 66:1735–1739.
61. Moncrief, J. S., R. Obiso, L. A. Barroso, J. J. Kling, R. L. Wright, R. L. VanTassell, D. M. Lyerly, and T. D. Wilkins. 1995. The enterotoxin of Bacte-roides fragilis is a metalloprotease. Infect. Immun. 63:175–181.
62. Moore, W. E. C., and L. V. Holdeman. 1974. Human fecal flora: the normalflora of 20 Japanese-Hawaiians. Appl. Microbiol. 27:961–979.
63. Mundy, L. M., and C. L. Sears. 1996. Detection of toxin production byBacteroides fragilis: assay development and screening of extraintestinal clin-ical isolates. Clin. Infect. Dis. 23:269–276.
64. Myers, L. L., B. D. Firehammer, D. S. Shoop, and M. M. Border. 1984.Bacteroides fragilis: a possible cause of acute diarrheal disease in newbornlambs. Infect. Immun. 44:241–244.
65. Myers, L. L., and D. S. Shoop. 1987. Association of enterotoxigenic Bacte-roides fragilis with diarrheal disease in young pigs. Am. J. Vet. Res. 48:774–775.
66. Myers, L. L., D. S. Shoop, and T. D. Byars. 1987. Diarrhea associated withenterotoxigenic Bacteroides fragilis in foals. Am. J. Vet. Res. 48:1565–1567.
67. Myers, L. L., D. S. Shoop, and J. E. Collins. 1990. Rabbit model to evaluateenterovirulence of Bacteroides fragilis. J. Clin. Microbiol. 28:1658–1660.
68. Myers, L. L., D. S. Shoop, J. E. Collins, and W. C. Bradbury. 1989. Diar-rheal disease caused by enterotoxigenic Bacteroides fragilis in infant rabbits.J. Clin. Microbiol. 27:2025–2030.
69. Myers, L. L., D. S. Shoop, B. D. Firehammer, and M. M. Border. 1985.Association of enterotoxigenic Bacteroides fragilis with diarrheal disease incalves. J. Infect. Dis. 152:1344–1347.
70. Myers, L. L., D. S. Shoop, L. L. Stackhouse, F. S. Newman, R. J. Flaherty,G. W. Letson, and R. B. Sack. 1987. Isolation of enterotoxigenic Bacteroidesfragilis from humans with diarrhea. J. Clin. Microbiol. 25:2330–2333.
71. Nakano, V., D. A. Gomes, R. M. Arantes, J. R. Nicoli, and M. J. Avila-Campos. 2006. Evaluation of the pathogenicity of the Bacteroides fragilistoxin gene subtypes in gnotobiotic mice. Curr. Microbiol. 53:113–117.
72. Nakano, V., T. A. Gomes, M. A. Vieira, R. C. Ferreira, and M. J. Avila-
VOL. 22, 2009 ENTEROTOXIGENIC BACTEROIDES FRAGILIS 367
on Decem
ber 3, 2020 by guesthttp://cm
r.asm.org/
Dow
nloaded from
Campos. 2007. bft gene subtyping in enterotoxigenic Bacteroides fragilisisolated from children with acute diarrhea. Anaerobe 13:1–5.
73. Namavar, F., E. B. Theunissen, A. M. Verweij-Van Vught, P. G. Peerbooms,M. Bal, H. F. Hoitsma, and D. M. MacLaren. 1989. Epidemiology of theBacteroides fragilis group in the colonic flora in 10 patients with coloniccancer. J. Med. Microbiol. 29:171–176.
74. Nelson, W. J., and R. Nusse. 2004. Convergence of Wnt, beta-catenin, andcadherin pathways. Science 303:1483–1487.
75. Nguyen, M. H., V. L. Yu, A. J. Morris, L. McDermott, M. W. Wagener, L.Harrell, and D. R. Snydman. 2000. Antimicrobial resistance and clinicaloutcome of Bacteroides bacteremia: findings of a multicenter prospectiveobservational trial. Clin. Infect. Dis. 30:870–876.
76. Nguyen, T. V., P. Le Van, C. Le Huy, K. N. Gia, and A. Weintraub. 2005.Detection and characterization of diarrheagenic Escherichia coli fromyoung children in Hanoi, Vietnam. J. Clin. Microbiol. 43:755–760.
77. Niyogi, S. K., P. Dutta, U. Mitra, and D. K. Pal. 1997. Association of entero-toxigenic Bacteroides fragilis with childhood diarrhoea. Indian J. Med. Res.105:167–169.
78. Obiso, R. J., Jr., A. O. Azghani, and T. D. Wilkins. 1997. The Bacteroidesfragilis toxin fragilysin disrupts the paracellular barrier of epithelial cells.Infect. Immun. 65:1431–1439.
79. Obiso, R. J., Jr., D. Bevan, and T. D. Wilkins. 1997. Molecular modelingand analysis of fragilysin, the Bacteroides fragilis toxin. Clin. Infect. Dis.25:S153–S155.
80. Obiso, R. J., Jr., D. M. Lyerly, R. L. Van Tassell, and T. D. Wilkins. 1995.Proteolytic activity of the Bacteroides fragilis enterotoxin causes fluid secre-tion and intestinal damage in vivo. Infect. Immun. 63:3820–3826.
81. Obuch-Woszczatynski, P., R. G. Wintermans, A. van Belkum, H. Endtz, H.Pituch, D. Kreft, F. Meisel-Mikolajczyk, and M. Luczak. 2004. Enterotoxi-genic Bacteroides fragilis (ETBF) strains isolated in The Netherlands andPoland are genetically diverse. Acta Microbiol. Pol. 53:35–39.
82. Pantosti, A., M. Cerquetti, R. Colangeli, and F. D’Ambrosio. 1994. Detec-tion of intestinal and extra-intestinal strains of enterotoxigenic Bacteroidesfragilis by the HT-29 cytotoxicity assay. J. Med. Microbiol. 41:191–196.
83. Pantosti, A., M. Malpeli, M. Wilks, M. G. Menozzi, and F. D’Ambrosio.1997. Detection of enterotoxigenic Bacteroides fragilis by PCR. J. Clin.Microbiol. 35:2482–2486.
84. Pantosti, A., M. G. Menozzi, A. Frate, L. Sanfilippo, F. D’Ambrosio, and M.Malpeli. 1997. Detection of enterotoxigenic Bacteroides fragilis and its toxin instool samples from adults and children in Italy. Clin. Infect. Dis. 24:12–16.
85. Pathela, P., K. Z. Hasan, E. Roy, K. Alam, F. Huq, A. K. Siddique, and R. B.Sack. 2005. Enterotoxigenic Bacteroides fragilis-associated diarrhea in chil-dren 0–2 years of age in rural Bangladesh. J. Infect. Dis. 191:1245–1252.
86. Pituch, H., P. Obuch-Woszczatynski, F. Luczak, and F. Meisel-Mikolajczyk.2003. Clostridium difficile and enterotoxigenic Bacteroides fragilis strains iso-lated from patients with antibiotic associated diarrhoea. Anaerobe 9:161–163.
87. Polk, F. B., and D. L. Kasper. 1996. Bacteroides fragilis subspecies in clinicalisolates. Ann. Intern. Med. 86:569–571.
88. Prindiville, T. P., R. A. Sheikh, S. H. Cohen, Y. J. Tang, M. C. Cantrell, andJ. Silva, Jr. 2000. Bacteroides fragilis enterotoxin gene sequences in patientswith inflammatory bowel disease. Emerg. Infect. Dis. 6:171–174.
89. Qadri, F., M. G. Mohi, A. Chowdhury, K. Alam, T. Azim, C. L. Sears, R. B.Sack, and M. J. Albert. 1996. Monoclonal antibodies to the enterotoxin ofBacteroides fragilis: production, characterization, and immunodiagnostic ap-plication. Clin. Diagn. Lab. Immunol. 3:608–610.
90. Rabizadeh, S., K. J. Rhee, S. Wu, D. Huso, C. M. Gan, J. E. Golub, X. Wu,M. Zhang, and C. L. Sears. 2007. Enterotoxigenic Bacteroides fragilis: apotential instigator of colitis. Inflamm. Bowel Dis. 13:1475–1483.
91. Redondo, M. C., M. D. Arbo, J. Grindlinger, and D. R. Snydman. 1995.Attributable mortality of bacteremia associated with the Bacteroides fragilisgroup. Clin. Infect. Dis. 20:1492–1496.
92. Rhee, K.-J., S. Wu, X. Wu, D. L. Huso, B. Karim, A. A. Franco, S. Rabi-zadeh, J. Golub, L. E. Mathews, J. Shin, R. B. Sartor, D. Golenbock, A.Hamad, C. M. Gan, F. Housseau, and C. L. Sears. 2009. Induction ofpersistent colitis by a human commensal, enterotoxigenic Bacteroides fragi-lis, in wild-type C57BL/6 mice. Infect. Immun. 77:1708–1718.
93. Riegler, M., M. Lotz, C. Sears, C. Pothoulakis, I. Castagliuolo, C. C. Wang,R. Sedivy, T. Sogukoglu, E. Cosentini, G. Bischof, W. Feil, B. Teleky, G.Hamilton, J. T. LaMont, and E. Wenzl. 1999. Bacteroides fragilis toxin 2damages human colonic mucosa in vitro. Gut 44:504–510.
94. Sack, R. B., M. J. Albert, K. Alam, P. K. B. Neogi, and M. S. Akbar. 1994.Isolation of enterotoxigenic Bacteroides fragilis from Bangladeshi childrenwith diarrhea: a controlled study. J. Clin. Microbiol. 32:960–963.
95. Sack, R. B., L. L. Myers, J. Almeido-Hill, D. S. Shoop, W. C. Bradbury, R.Reid, and M. Santosham. 1992. Enterotoxigenic Bacteroides fragilis: epide-miologic studies of its role as a human diarrhoeal pathogen. J. DiarrhoealDis. Res. 10:4–9.
96. Saidi, R. F., K. Jaeger, M. H. Montrose, S. Wu, and C. L. Sears. 1997.Bacteroides fragilis toxin alters the actin cytoskeleton of HT29/C1 cells invivo qualitatively but not quantitatively. Cell Motil. Cytoskelet. 37:159–165.
97. Saidi, R. F., and C. L. Sears. 1996. Bacteroides fragilis toxin rapidly intox-
icates human intestinal epithelial cells (HT29/C1) in vitro. Infect. Immun.64:5029–5034.
98. Salyers, A. A. 1984. Bacteroides of the human lower intestinal tract. Annu.Rev. Microbiol. 38:293–313.
99. Sanfilippo, L., T. J. Baldwin, M. G. Menozzi, S. P. Borriello, and Y. R.Mahida. 1998. Heterogeneity in responses by primary adult human colonicepithelial cells to purified enterotoxin of Bacteroides fragilis. Gut 43:651–655.
100. Sanfilippo, L., C. K. Li, R. Seth, T. J. Balwin, M. G. Menozzi, and Y. R.Mahida. 2000. Bacteroides fragilis enterotoxin induces the expression ofIL-8 and transforming growth factor-beta (TGF-beta) by human colonicepithelial cells. Clin. Exp. Immunol. 119:456–463.
101. San Joaquin, V. H., J. C. Griffis, C. Lee, and C. L. Sears. 1995. Associationof Bacteroides fragilis with childhood diarrhea. Scand. J. Infect. Dis. 27:211–215.
102. Sears, C. L., S. Islam, A. Saha, M. Arjumand, N. H. Alam, A. S. G. Faruque,M. A. Salam, J. Shin, D. Hecht, A. Weintraub, R. B. Sack, and F. Qadri.2008. Enterotoxigenic Bacteroides fragilis infection is associated with inflam-matory diarrhea. Clin. Infect. Dis. 47:797–803.
103. Sears, C. L. 2001. The toxins of Bacteroides fragilis. Toxicon 39:1737–1746.104. Sears, C. L., S. L. Buckwold, J. W. Shin, and A. A. Franco. 2006. The
C-terminal region of Bacteroides fragilis toxin is essential to its biologicalactivity. Infect. Immun. 74:5595–5601.
105. Sears, C. L., L. L. Myers, A. Lazenby, and R. L. Van Tassell. 1995. Ente-rotoxigenic Bacteroides fragilis. Clin. Infect. Dis. 20(Suppl. 2):S142–S148.
106. Sears, C., A. Franco, and S. Wu. 2005. Bacteroides fragilis toxins, p. 535–546.In J. Alouf and M. Popoff (ed.), The comprehensive sourcebook of bacterialprotein toxins. Academic Press, Oxford, England.
107. Sharma, N., and R. Chaudhry. 2006. Rapid detection of enterotoxigenicBacteroides fragilis in diarrhoeal faecal samples. Indian J. Med. Res. 124:575–582.
108. Shetab, R., S. H. Cohen, T. Prindiville, Y. J. Tang, M. Cantrell, D. Rah-mani, and J. Silva, Jr. 1998. Detection of Bacteroides fragilis enterotoxingene by PCR. J. Clin. Microbiol. 36:1729–1732.
109. Shoop, D. S., L. L. Myers, and J. B. LeFever. 1990. Enumeration of ente-rotoxigenic Bacteroides fragilis in municipal sewage. Appl. Environ. Micro-biol. 56:2243–2244.
110. Sonnenburg, J. L., J. Xu, D. D. Leip, C. H. Chen, B. P. Westover, J.Weatherford, J. D. Buhler, and J. I. Gordon. 2005. Glycan foraging in vivoby an intestine-adapted bacterial symbiont. Science 307:1955–1959.
111. Swidsinski, A., J. Weber, V. Loening-Baucke, L. P. Hale, and H. Lochs.2005. Spatial organization and composition of the mucosal flora in patientswith inflammatory bowel disease. J. Clin. Microbiol. 43:3380–3389.
112. Szoke, I., E. Dosa, and E. Nagy. 2006. Enterotoxigenic Bacteroides fragilis inHungary. Anaerobe 3:87–89.
113. Toprak, N. U., A. Yagci, B. M. Gulluoglu, M. L. Akin, P. Demirkalem, T.Celenk, and G. Soyletir. 2006. A possible role of Bacteroides fragilis entero-toxin in the aetiology of colorectal cancer. Clin. Microbiol. Infect. 12:782–786.
114. Ulger, T. N., D. Rajendram, A. Yagci, S. Gharbia, H. N. Shah, B. M.Gulluoglu, L. M. Akin, P. Demirkalem, T. Celenk, and G. Soyletir. 2006.The distribution of the bft alleles among enterotoxigenic Bacteroides fragilisstrains from stool specimens and extraintestinal sites. Anaerobe 12:71–74.
115. Van Tassell, R. L., D. M. Lyerly, and T. D. Wilkins. 1992. Purification andcharacterization of an enterotoxin from Bacteroides fragilis. Infect. Immun.60:1343–1350.
116. Van Tassell, R. L., D. M. Lyerly, and T. D. Wilkins. 1994. Characterizationof enterotoxigenic Bacteroides fragilis by a toxin-specific enzyme-linked im-munosorbent assay. Clin. Diagn. Lab. Immunol. 1:578–584.
117. Vines, R. R., S. S. Perdue, J. S. Moncrief, D. R. Sentz, L. A. Barroso, R. L.Wright, and T. D. Wilkins. 2000. Fragilysin, the enterotoxin from Bacte-roides fragilis, enhances the serum antibody response to antigen co-admin-istered by the intranasal route. Vaccine 19:655–660.
118. Vu, N. T., P. Le Van, C. Le Huy, and A. Weintraub. 2005. Diarrhea causedby enterotoxigenic Bacteroides fragilis in children less than 5 years of age inHanoi, Vietnam. Anaerobe 11:109–114.
119. Weikel, C. S., F. D. Grieco, J. Reuben, L. L. Myers, and R. B. Sack. 1992.Human colonic epithelial cells, HT29/C1, treated with crude Bacteroidesfragilis enterotoxin dramatically alter their morphology. Infect. Immun.60:321–327.
120. Wells, C. L., E. M. A. Van De Westerlo, R. P. Jechorek, B. A. Feltis, T. D.Wilkins, and S. L. Erlandsen. 1996. Bacteroides fragilis enterotoxin modu-lates epithelial permeability and bacterial internalization by HT-29 entero-cytes. Gastroenterology 110:1429–1437.
121. Wu, S., L. A. Dreyfus, A. O. Tzianabos, C. Hayashi, and C. L. Sears. 2002.Diversity of the metalloprotease toxin produced by enterotoxigenic Bacte-roides fragilis. Infect. Immun. 70:2463–2471.
122. Wu, S., K.-C. Lim, J. Huang, R. F. Saidi, and C. L. Sears. 1998. Bacteroidesfragilis enterotoxin cleaves the zonula adherens protein, E-cadherin. Proc.Natl. Acad. Sci. USA 95:14979–14984.
123. Wu, S., P. J. Morin, D. Maouyo, and C. L. Sears. 2003. Bacteroides fragilis
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enterotoxin induces c-Myc expression and cellular proliferation. Gastroen-terology 124:392–400.
124. Wu, S., J. Powell, N. Mathioudakis, S. Kane, E. Fernandez, and C. L. Sears.2004. Bacteroides fragilis enterotoxin induces intestinal epithelial cell secre-tion of interleukin-8 through mitogen-activated protein kinases and a ty-rosine kinase-regulated nuclear factor-kappaB pathway. Infect. Immun.72:5832–5839.
125. Wu, S., K. J. Rhee, M. Zhang, A. Franco, and C. L. Sears. 2007. Bacteroidesfragilis toxin stimulates intestinal epithelial cell shedding and �-secretase-dependent E-cadherin cleavage. J. Cell Sci. 120:1944–1952.
126. Wu, S., J. Shin, G. Zhang, M. Cohen, A. Franco, and C. L. Sears. 2006. The
Bacteroides fragilis toxin binds to a specific intestinal epithelial cell receptor.Infect. Immun. 74:5382–5390.
127. Xu, J., M. K. Bjursell, J. Himrod, S. Deng, L. K. Carmichael, H. C. Chiang,L. V. Hooper, and J. I. Gordon. 2003. A genomic view of the human-Bacteroides thetaiotaomicron symbiosis. Science 299:2074–2076.
128. Zhang, G., B. Svenungsson, A. Karnell, and A. Weintraub. 1999. Prevalenceof enterotoxigenic Bacteroides fragilis in adult patients with diarrhea andhealthy controls. Clin. Infect. Dis. 29:590–594.
129. Zhang, G., and A. Weintraub. 1998. Rapid and sensitive assay for de-tection of enterotoxigenic Bacteroides fragilis. J. Clin. Microbiol. 36:3545–3548.
Cynthia L. Sears (M.D.) received her med-ical degree from Thomas Jefferson MedicalCollege, followed by training in InternalMedicine at The New York Hospital in NewYork City. She trained in Infectious Dis-eases at the Memorial Sloan Kettering Can-cer Institute and the University of Virginia,where she was a member of the infectiousdiseases faculty until 1988. Subsequently,Dr. Sears joined the faculty at Johns Hop-kins University School of Medicine, whereshe is now a Professor of Medicine in the Divisions of InfectiousDiseases and Gastroenterology, Department of Medicine. As a resultof international experiences, she developed a clinical interest in food-borne and enteric infections. She has conducted laboratory as well asclinical research on enterotoxigenic Bacteroides fragilis (ETBF) overthe past 15 years. Her present work focuses on studies to define howETBF induces colitis and the link between chronic colonic inflamma-tion induced by bacteria and colonic tumorigenesis.
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