Fragilysin Research Articles

Adverse effects of fenpropathrin on the intestine of common carp (Cyprinus carpio L.) and the mechanism involved

Fenpropathrin (FEN), a pyrethroid pesticide, is frequently detected in natural water bodies, unavoidable pose adverse effects to aquatic organisms. However, the harmful effects and potential mechanisms of FEN on aquatic species are poorly understood. In this study, common carp were treatment with FEN at 0.45 and 1.35 μg/L for 14 d, and the toxic effects and underlying mechanisms of FEN on the intestine of carp were revealed. RNA-seq results showed that FEN exposure cause a wide range of transcriptional alterations in the intestine and the differentially expressed genes were mainly enrichment in the pathways related to immune and metabolism. Specifically, FEN exposure induced pathological damage and altered submicroscopic structure of the intestine, elevated the levels of Bacteroides fragilis enterotoxin, altered the contents of claudin-1, occludin, and zonula occluden-1 (ZO-1), and causing injury to the intestinal barrier. In addition, inflammation-related index TNF-α in the serum and IL-6 in the intestinal tissues were generally increased after FEN exposure. Moreover, FEN exposure promoted an increase in reactive oxygen species (ROS), altered the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), upregulated the contents of malondialdehyde (MDA) in the intestines. The apoptosis-related parameter cytochrome c, caspase-9, and caspase-3 were significantly altered, indicating that inflammation reaction, oxidative stress, and apoptosis may be involved in the toxic mechanism of FEN on carp. Moreover, FEN treatment also altered the intestinal flora community significantly, which may affect the intestinal normal physiological function and thus affect the growth of fish. Overall, the present study help to clarify the intestinal reaction mechanisms after FEN treatment, and provide a basis for the risk assessment of FEN.

Bacteroides fragilis Enterotoxin Induces Autophagy through an AMPK and FoxO3-Pathway, Leading to the Inhibition of Apoptosis in Intestinal Epithelial Cells.

Macroautophagy/autophagy is essential for preserving cellular homeostasis by recycling nutrients and removing spoiled or aged proteins and organelles. It also has an essential role in defense mechanisms against microbial infections. However, the role of autophagy in enterotoxigenic Bacteroides fragilis infection remains largely unknown. In this study, we explored the role of B. fragilis enterotoxin (BFT) in the autophagic process of intestinal epithelial cells (IECs). The LC3-I of human HCT-116 IECs was converted to LC3-II by BFT stimulation. In addition, BFT-exposed cells showed the decreased expression of p62 in a time-dependent manner and increased levels of ATG5 and ATG12 gradually. Evidence of an enhanced autophagic process was supported by autophagosomes co-localized with LC3-lysosome-associated protein 2 in BFT-stimulated cells. The AMP-activated protein kinase (AMPK) and Forkhead box O3 (FoxO3a) axis were required for BFT-induced autophagy activation. In contrast with the activation of autophagy at 3-6 h after BFT exposure, IECs induced apoptosis-related signals at 12-48 h. HCT-116 IECs suppressing the formation of autophagosomes significantly activated apoptosis signals instead of autophagy early after BFT exposure. These data suggest that BFT can activate autophagy through the AMPK-FoxO3a pathway and the autophagy may suppress apoptosis during early exposure of IECs to BFT.

Bacteroides fragilis Enterotoxin Upregulates Matrix Metalloproteinase-7 Expression through MAPK and AP-1 Activation in Intestinal Epithelial Cells, Leading to Syndecan-2 Release.

Bacteroides fragilis enterotoxin (BFT) produced by enterotoxigenic B. fragilis (ETBF) causes colonic inflammation. BFT initially contacts intestinal epithelial cells (IECs) and affects the intestinal barrier. Although molecular components of the gut epithelial barrier such as metalloproteinase-7 (MMP-7) and syndecan-2 are known to be associated with inflammation, little has been reported about MMP-7 expression and syndecan-2 shedding in response to ETBF infection. This study explores the role of BFT in MMP-7 induction and syndecan-2 release in IECs. Stimulating IECs with BFT led to the induction of MMP-7 and the activation of transcription factors such as NF-κB and AP-1. MMP-7 upregulation was not affected by NF-κB, but it was related to AP-1 activation. In BFT-exposed IECs, syndecan-2 release was observed in a time- and concentration-dependent manner. MMP-7 suppression was associated with a reduction in syndecan-2 release. In addition, suppression of ERK, one of the mitogen-activated protein kinases (MAPKs), inhibited AP-1 activity and MMP-7 expression. Furthermore, the suppression of AP-1 and ERK activity was related to the attenuation of syndecan-2 release. These results suggest that a signaling cascade comprising ERK and AP-1 activation in IECs is involved in MMP-7 upregulation and syndecan-2 release during exposure to BFT.

Intestinal Epithelial Cells Exposed to Bacteroides fragilis Enterotoxin Regulate NF-κB Activation and Inflammatory Responses through β-Catenin Expression.

The Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), interacts with intestinal epithelial cells and can provoke signals that induce mucosal inflammation. Although β-catenin signaling is reported to be associated with inflammatory responses and BFT is known to cleave E-cadherin linked with β-catenin, little is known about the β-catenin-mediated regulation of inflammation in ETBF infection. This study was conducted to investigate the role of β-catenin as a cellular signaling intermediate in the induction of proinflammatory responses to stimulation of intestinal epithelial cells with BFT. Expression of β-catenin in intestinal epithelial cells was reduced relatively early after stimulation with BFT and then recovered to normal levels relatively late after stimulation. In contrast, phosphorylation of β-catenin in BFT-exposed cells occurred at high levels early in stimulation and decreased as time passed. Concurrently, late after stimulation the nuclear levels of β-catenin were relatively higher than those early after stimulation. Suppression of β-catenin resulted in increased NF-κB activity and interleukin-8 (IL-8) expression in BFT-stimulated cells. However, suppression or enhancement of β-catenin expression neither altered the phosphorylated IκB kinase α/β complex nor activated activator protein 1 signals. Furthermore, inhibition of glycogen synthase kinase 3β was associated with increased β-catenin expression and attenuated NF-κB activity and IL-8 expression in BFT-exposed cells. These findings suggest the negative regulation of NF-κB-mediated inflammatory responses by β-catenin in intestinal epithelial cells stimulated with BFT, resulting in attenuation of acute inflammation in ETBF infection.

Intake of Bifidobacterium longum and Fructo-oligosaccharides prevents Colorectal Carcinogenesis.

IntroductionWe aimed to investigate the effects of intake of yogurt containing Bifidobacterium longum (BB536-y) and fructo-oligosaccharides (FOS) in preventing colorectal carcinogenesis in healthy subjects, and the preventive effects of short-chain fatty acids (SCFA), whose production was enhanced by the intake of BB536-y and FOS, in human colon cancer cell lines.Materials and methodsThe subjects were 27 healthy persons who were divided into a group taking yogurt containing BB536 (BB536-y group; n = 14) and a group taking yogurt containing BB536 and FOS (BB536-y with FOS group; n = 13) once a day for 5 weeks. The feces were sampled before and after the intake to analyze the amount of SCFA in the feces and the profile of intestinal flora, such as putrefactive bacteria and Bacteroides fragilis enterotoxin (ETBF). Subsequently, human colon cancer cell lines (DLD-1 cells, WirDr cells) were cultured in the presence of SCFA (butyric acid, isobutyric acid, acetic acid) in order to evaluate the cell growth-inhibitory activity of SCFA (WST-8 assay) by calculating the IC50 value from the dose-response curve.ResultsIntake of BB536-y increased the total amount of SCFA in the feces and significantly suppressed the detection rate of ETBF and growth of putrefactive bacteria. Intake of BB536-y with FOS was associated with a higher Bifidobacterium detection rate than that of BB536-y alone. The contents of butyric acid, isobutyric acid, and acetic acid, namely, of SCFA, were also decreased. Analysis of the results of culture of DLD-1 cells and WirDr cells in the presence of butyric acid, isobutyric acid, and acetic acid revealed that each of the substances showed significant cell growth-inhibitory activity, with the activity being the highest for butyric acid, followed by that for isobutyric acid and acetic acid.ConclusionThese findings suggest that intake of both BB536-y and BB536-y with FOS prevents colorectal carcinogenesis.How to cite this article: Ohara T, Suzutani T. Intake of Bifidobacterium longum and Fructo-oligosaccharides prevents Colorectal Carcinogenesis. Euroasian J Hepato-Gastroenterol 2018;8(1):11-17.

Bacteroides fragilis Enterotoxin Induces Formation of Autophagosomes in Endothelial Cells but Interferes with Fusion with Lysosomes for Complete Autophagic Flux through a Mitogen-Activated Protein Kinase-, AP-1-, and C/EBP Homologous Protein-Dependent Pathway

Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), plays an essential role in mucosal inflammation. Although autophagy contributes to the pathogenesis of diverse infectious diseases, little is known about autophagy in ETBF infection. This study was conducted to investigate the role of BFT in the autophagic process in endothelial cells (ECs). Stimulation of human umbilical vein ECs (HUVECs) with BFT increased light chain 3 protein II (LC3-II) conversion from LC3-I and protein expression of p62, Atg5, and Atg12. In addition, BFT-exposed ECs showed increased indices of autophagosomal fusion with lysosomes such as LC3-lysosome-associated protein 2 (LAMP2) colocalization and the percentage of red vesicles monitored by the expression of dual-tagged LC3B. BFT also upregulated expression of C/EBP homologous protein (CHOP), and inhibition of CHOP significantly increased indices of autophagosomal fusion with lysosomes. BFT activated an AP-1 transcription factor, in which suppression of AP-1 activity significantly downregulated CHOP and augmented autophagosomal fusion with lysosomes. Furthermore, suppression of Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase (MAPK) significantly inhibited the AP-1 and CHOP signals, leading to an increase in autophagosomal fusion with lysosomes in BFT-stimulated ECs. These results suggest that BFT induced accumulation of autophagosomes in ECs, but activation of a signaling pathway involving JNK, AP-1, and CHOP may interfere with complete autophagy.

Bacteroides fragilis Enterotoxin Upregulates Heme Oxygenase-1 in Intestinal Epithelial Cells via a Mitogen-Activated Protein Kinase- and NF-κB-Dependent Pathway, Leading to Modulation of Apoptosis.

The Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), interacts with intestinal epithelial cells and can provoke signals that induce mucosal inflammation. Although expression of heme oxygenase-1 (HO-1) is associated with regulation of inflammatory responses, little is known about HO-1 induction in ETBF infection. This study was conducted to investigate the effect of BFT on HO-1 expression in intestinal epithelial cells. Stimulation of intestinal epithelial cells with BFT resulted in upregulated expression of HO-1. BFT activated transcription factors such as NF-κB, AP-1, and Nrf2 in intestinal epithelial cells. Upregulation of HO-1 in intestinal epithelial cells was dependent on activated IκB kinase (IKK)-NF-κB signals. However, suppression of Nrf2 or AP-1 signals in intestinal epithelial cells did not result in significant attenuation of BFT-induced HO-1 expression. HO-1 induction via IKK-NF-κB in intestinal epithelial cells was regulated by p38 mitogen-activated protein kinases (MAPKs). Furthermore, suppression of HO-1 activity led to increased apoptosis in BFT-stimulated epithelial cells. These results suggest that a signaling pathway involving p38 MAPK-IKK-NF-κB in intestinal epithelial cells is required for HO-1 induction during exposure to BFT. Following this induction, increased HO-1 expression may regulate the apoptotic process in responses to BFT stimulation.

The myeloid immune signature of enterotoxigenic Bacteroides fragilis-induced murine colon tumorigenesis.

Enterotoxigenic Bacteroides fragilis (ETBF), a human commensal and candidate pathogen in colorectal cancer (CRC), is a potent initiator of IL-17-dependent colon tumorigenesis in MinApc+/-mice. We examined the role of IL-17 and ETBF on the differentiation of myeloid cells into myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), which are known to promote tumorigenesis. The myeloid compartment associated with ETBF-induced colon tumorigenesis in Min mice was defined using flow cytometry and gene expression profiling. Cell sorted immature myeloid cells were functionally assayed for inhibition of T cell proliferation and iNOS expression in order to delineate MDSC populations. A comparison of ETBF infection to that of other oncogenic bacteria (Fusobacterium nucleatum or pks+ E. coli) revealed a specific, ETBF-associated colonic immune infiltrate. ETBF-triggered colon tumorigenesis is associated with an IL-17-driven myeloid signature characterized by subversion of steady-state myelopoiesis in favor of the generation of protumoral monocytic (MO)-MDSCs. Combined action of the Bacteroides fragilis enterotoxin BFT and IL-17 on colonic epithelial cells promoted the differentiation of MO-MDSCs, which selectively upregulated Arg1 and Nos2, produced NO and suppressed T cell proliferation. Evidence of a pathogenic inflammatory signature in humans colonized with ETBF may allow for the identification of populations at risk for developing colon cancer.

Substrate Cleavage Profiling Suggests a Distinct Function of Bacteroides fragilis Metalloproteinases (Fragilysin and Metalloproteinase II) at the Microbiome-Inflammation-Cancer Interface

Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)↓) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.

Long-term storage and safe retrieval of DNA from microorganisms for molecular analysis using FTA matrix cards

We assessed the potential use of Whatman FTA® paper as a device for archiving and long-term storage of bacterial cell suspensions of over 400 bacterial strains representing 61 genera, the molecular applications of immobilised DNA on FTA paper, and tested its microbial inactivation properties. The FTA paper extracted bacterial DNA is of sufficiently high quality to successfully carryout the molecular detection of several key genes including 16S rRNA, esp ( Enterococcus surface protein), Bft ( Bacteroides fragilis enterotoxin) and por (porin protein) by PCR and for DNA fingerprinting by random amplified polymorphic DNA-PCR (RAPD-PCR). To test the long-term stability of the FTA immobilised DNA, 100 of the 400 archived bacterial samples were randomly selected following 3 years of storage at ambient temperature and PCR amplification was used to monitor its success. All of the 100 samples were successfully amplified using the 16S rDNA gene as a target and confirmed by DNA sequencing. Furthermore, the DNA was eluted into solution from the FTA cards using a new alkaline elution procedure for evaluation by real-time PCR-based assays. The viability of cells retained on the FTA cards varied among broad groups of bacteria. For the more fragile gram-negative species, no viable cells were retained even at high cell densities of between 10 7 and 10 8 colony forming units (cfu) ml − 1 , and for the most robust species such as spore-formers and acid-fast bacteria, complete inactivation was achieved at cell densities ranging between 10 1 and 10 4 cfu ml − 1 . The inactivation of bacterial cells on FTA cards suggest that this is a safe medium for the storage and transport of bacterial nucleic acids.

A possible role of Bacteroides fragilis enterotoxin in the aetiology of colorectal cancer

The prevalence of enterotoxigenic Bacteroides fragilis (ETBF) was investigated in stool specimens from 73 patients with colorectal cancer and from 59 control patients. Stool specimens were cultured on Bacteroides Bile Esculin agar and B. fragilis was identified by conventional methods. After DNA extraction, the enterotoxin gene (bft) was detected by PCR in 38% of the isolates from colorectal cancer patients, compared with 12% of the isolates from the control group (p 0.009). This is the first study demonstrating an increased prevalence of ETBF in colorectal cancer patients.

Cloning and characterization of the gene for the metalloprotease enterotoxin of Bacteroides fragilis

Cloning and characterization of the gene for the metalloprotease enterotoxin of Bacteroides fragilis

Engineering of the gut commensal bacterium Bacteroides ovatus to produce and secrete biologically active murine interleukin-2 in response to xylan

The aim of this work was to engineer a gut commensal bacterium, Bacteroidesovatus, to produce and secrete a biologically active cytokine in a regulated manner as a basis for novel immunotherapies for chronic gut disorders. Bacteroides ovatus was engineered to produce murine interleukin-2 (MuIL2) intracellularly in response to xylan in culture media by inserting the MuIL2 gene into the xylanase operon of the organism. A second strain was engineered to secrete MuIL2 by adding Bacteroides fragilis enterotoxin secretion signal sequence to the protein. The recombinant strains produced MuIL2 only in the presence of xylan as determined by ELISA of cell lysates and culture supernatants. The IL2-dependent cell line CTLL-2 was used to demonstrate that MuIL2 produced by both B. ovatus strains was biologically active. This activity could be blocked by an anti-IL2 neutralizing antibody. The xylan-inducible nature of this system was demonstrated by RT-PCR. Bacteroides ovatus was successfully engineered to produce and secrete biologically active MuIL2 in a xylan-inducible manner. The production and secretion of a biologically active mammalian protein by a member of the gut microflora could lead to the development of new long-term immunotherapies for inflammatory gut diseases.

Bacteroides fragilis enterotoxin induces intestinal epithelial cell secretion of interleukin-8 through mitogen-activated protein kinases and a tyrosine kinase-regulated nuclear factor-kappaB pathway.

Enterotoxigenic Bacteroides fragilis (ETBF) secretes a 20-kDa metalloprotease toxin termed B. fragilis toxin (BFT). ETBF disease in animals is associated with an acute inflammatory response in the intestinal mucosa, and lethal hemorrhagic colitis may occur in rabbits. In this study, we confirmed recent reports (J. M. Kim, Y. K. Oh, Y. J. Kim, H. B. Oh, and Y. J. Cho, Clin. Exp. Immunol. 123:421-427, 2001; L. Sanfilippo, C. K. Li, R. Seth, T. J. Balwin, M. J. Menozzi, and Y. R. Mahida, Clin. Exp. Immunol. 119:456-463, 2000) that purified BFT stimulates interleukin-8 (IL-8) secretion by human intestinal epithelial cells (HT29/C1 cells) and demonstrate that stimulation of IL-8 production is dependent on biologically active BFT and independent of serum. Induction of IL-8 mRNA expression occurs rapidly and ceases by 6 h after BFT treatment, whereas IL-8 secretion continues to increase for at least 18 h. Our data suggest that BFT-stimulated IL-8 secretion involves tyrosine kinase-dependent activation of nuclear factor-kappaB (NF-kappaB) as well as activation of the mitogen-activated protein kinases (MAPKs), p38 and extracellular signal-related kinase. Simultaneous activation of NF-kappaB and MAPKs appears necessary for secretion of IL-8 by HT29/C1 cells treated with BFT.

Bacteroides fragilis enterotoxin (BFT) induces intestinal epithelial cell secretion of interleukin-8 (IL-8) through a nuclear factor-kB, mitrogen activated protein kinase (MAPK) co-activation pathway

BACKGROUND: Recent studies have demonstrated that BFT induces IL-8 expression in human intestinal epithelial cells in vitro and implied a significant association between detection of the bft gene in stool specimens of patients with inflammatory bowel disease (IBD) and disease activity. These data suggest that colonization with enterotoxigenic Bacteroides fragilis may stimulate intestinal inflammation in humans. AIMS: To evaluate the kinetics of IL-8 induction stimulated by BFT in intestinal epithelial cells, and to invesugate the intracellular signaling events leading to increased IL-8 level. METHODS: Human colon epithelial cell lines, T84 and HT29/C1, were stimulated by BFT under polarized or subconfluent growth conditions in the presence or absence of (1) p38 and/or ERK MAP kinase inhibitors (SB203580, U126), or (2) the tyrosine kinase inhibitor (Geinstein). ELISA and semi-quantitative RT-PCR were used to evaluate the production of IL-8. Activation of the p38 MAP kinase pathway was confirmed by detection of the active form of p38 kinase with Western blot. NF-kB pathway activation was examined by EMSA and RelA nuclear translocation using confocal microscopy. RESULTS: IL-8 mRNA and protein are induced by biologically active BFT in a concentration-dependent and serum-independent manner. IL-8 is mainly secreted from the basolateral membranes of T84 monolayers. Basolaterally applied BFT is 25 times more potent than apical BFT in stimulating IL-8 secretion. In HT29/ C1 cells, SB203580 (10 p,M) and U126 (5 IzM) abolish 90% and 70% of 1k-8 production, respectively. Genlstein (100 IzM) completely blocks BFT-stimulated 1L-8 production. BFT stimulated nuclear translocation of NF-kB is not affected by blocking of p38 kinase and ERK pathways. CONCLUSION: BFT induces 1L-8 production in epithelial cells through coactivation of NF-kB and MAP kinase signaling pathways.

Occurrence of Bacteroides fragilis enterotoxin gene-carrying strains in Germany and the United States.

Ninety-three Bacteroides fragilis isolates from different geographic locations were analyzed for the presence of an enterotoxin-encoding gene. It was shown that blood culture isolates were more likely to carry this gene than strains from other sources. All enterotoxin-positive strains belonged to the PCR fingerprint group I.

Bacteroides fragilis enterotoxin gene sequences in patients with inflammatory bowel disease.

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

Bacterial toxins modifying the actin cytoskeleton.

Numerous bacterial toxins recognize the actin cytoskeleton as a target. The clostridial binary toxins (Iota and C2 families) ADP-ribosylate the actin monomers causing the dissociation of the actin filaments. The large clostridial toxins from Clostridium difficile, Clostridium sordellii and Clostridium novyi inactivate, by glucosylation, proteins from the Rho family that regulate actin polymerization. In contrast, the cytotoxic necrotic factor from Escherichia coli activates Rho by deamidation and increases the formation of actin filaments. The enterotoxin of Bacteroides fragilis is a protease specific for E-cadherin and it promotes the reorganization of the actin cytoskeleton. The bacterial toxins that modify the actin cytoskeleton induce various cell disfunctions including changes in cell barrier permeability and disruption of intercellular junctions.

Heterogeneity in responses by primary adult human colonic epithelial cells to purified enterotoxin of Bacteroides fragilis

Background—Enterotoxigenic strains ofBacteroides fragilis (ETBF) have been implicated in diarrhoeal illness in livestock and children, but their role in adult human colonic disease is unknown.Aims—To investigate responses by primary adult...

Detection of Bacteroides fragilis enterotoxin gene by PCR.

Bacteroides fragilis constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of B. fragilis have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of B. fragilis in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the B. fragilis enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach, B. fragilis enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic B. fragilis isolates or B. distasonis, B. thetaiotaomicron, B. uniformis, B. ovatus, Escherichia coli, or Clostridium difficile. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic B. fragilis/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic B. fragilis and may be used clinically to determine the role of B. fragilis in diarrheal diseases.