INTRODUCTION

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Bacteroides fragilis is a gram-negative, anaerobic bacillus which constitutes about 1 to 2% of the normal bacterial flora in humans (5). B. fragilis has been associated with soft-tissue infections, abscesses, and bacteremia in humans (16). Some strains of B. fragilis produce an enterotoxin which causes acute diarrhea in young lambs, calves, pigs, and foals (7-9). Enterotoxigenic B. fragilis (ETBF) has also been found to cause diarrhea in children (12, 14). Sack et al. reported the isolation of ETBF in 12% of children with diarrhea in Bangladesh compared with 6% of controls (12). In a similar study, San Joaquin et al. found a strong association between diarrheal diseases in children and ETBF (14). In both studies, ETBF was associated with diarrheal cases in children >1 year of age. The enterotoxin from B. fragilis induces a fluid response in ligated intestinal loops and a cytotoxic response in HT-29 cells (7, 17, 19). Detection and identification of ETBF is by culture of stool specimens on selective medium and analysis of B. fragilis supernatants for enterotoxin activity (12). Recovery of B. fragilis from stool samples decreases with time of storage of the specimen prior to culturing (13). In addition, many isolates lose viability during subculturing; thus, an enterotoxin assay cannot be performed.

Recently, the enterotoxin gene of B. fragilis has been cloned, sequenced, and identified as producing a zinc metalloprotease of 44.4 kDa (4). In this study, we report the development of a PCR assay to detect gene sequences of B. fragilis enterotoxin directly from stool specimens. This rapid, sensitive, and specific assay can be clinically useful in studying the role of this organism in diarrheal diseases of children and adults.

	MATERIALS AND METHODS

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Bacterial strains and culture media. B. fragilis ATCC 43858, 43859, and 43860 were obtained from the American Type Culture Collection. B. fragilis W1 and W2 were kindly provided by F. Meisel-Mikolajczyk from the Medical Academy of Warsaw, Poland. These five isolates are enterotoxigenic in humans. Fifteen other isolates obtained from patients with diarrhea were also analyzed in this study. Twenty-five B. fragilis isolates were obtained from the University of California, Davis, School of Veterinary Medicine, and were recovered from horses with diarrhea. B. distasonis, B. ovatus, B. thetaiotaomicron, B. uniformis, and Escherichia coli were obtained from the Clinical Laboratories at the University of California, Davis Medical Center, Sacramento. Clostridium difficile P-224 is a toxigenic isolate from our culture collection. All B. fragilis group isolates were grown on the selective medium Bacteroides bile esculin agar (Anaerobe Systems, San Jose, Calif.) for 36 to 48 h at 37°C under anaerobic conditions. Isolates were confirmed to be B. fragilis if they were catalase positive and indole negative. E. coli was grown on Brucella anaerobic plates. C. difficile was grown in the selective medium cycloserine-cefoxitin-fructose agar as described elsewhere (15).

DNA extraction from pure cultures and stool specimens. DNA was extracted from isolated colonies of B. fragilis as described previously (15). DNA was extracted from spiked stools by a modification of the methods of Balatbat et al. and Kato et al. (1, 3). Briefly, 1 g of stool which tested negative for B. fragilis was spiked with ETBF at concentrations ranging from 10 to 10⁶ cells/g of stool. For DNA extraction, 100 mg of stool was suspended in 400 µl of TES buffer (50 mM Tris [pH 8], 5 mM EDTA, 50 mM NaCl) and centrifuged at 2,000 × g for 5 min to remove large particles. The supernatant was then centrifuged at 7,000 × g for 5 min to pellet bacterial cells. The pellet was resuspended in TES plus 25% sucrose and incubated with 5 µl of lysozyme (100 mg/ml) for 1 h at 37°C. The suspension was centrifuged at 7,000 × g, and the pellet was resuspended in TES containing 0.8% Sarkosyl and 100 µg of proteinase K/ml and incubated at 60°C for 2 h. After digestion with proteinase K, the supernatant was extracted twice with phenol-chloroform-isoamyl alcohol (24:24:1), and the DNA was precipitated with 2 volumes of absolute ethanol.

DNA amplification by PCR. The sequences of the oligonucleotide primers and probe used in this study are listed in Table 1. These oligonucleotides were designed according to the B. fragilis enterotoxin published sequence (4). A nested PCR approach was used to amplify a 290-bp fragment of the ETBF gene in stool specimens. The PCR method of Mullis and Faloona was used for amplification with the thermostable DNA polymerase (rTaq; Perkin-Elmer Cetus) (6). For the first amplification reaction, the outer primers RS-3 and RS-4 were used. The reaction mixtures were prepared in 1× PCR buffer (50 mM KCl, 20 mM Tris-HCl, 2.5 mM MgCl₂, 100 µg of bovine serum albumin per ml [pH 8.4]) and contained per reaction 20 pmol of the respective primers, 0.1 mM concentrations of each 2'-deoxynucleoside 5'-triphosphate, 2 U of recombinant DNA polymerase (rTaq), and 10 µl of purified DNA from stools. For the second round of amplifications, the reaction mixture was prepared as described above, except that the inner primers RS-1 and RS-2 were used and 5 µl of the amplified product from the first PCR was used as the source of DNA. For amplification of DNA from pure cultures, 5 µl of the DNA preparation was amplified with the external (RS-3 and RS-4) or internal (RS-1 and RS-2) primers as described above. The reaction mixtures were covered with 150 µl of mineral oil to prevent evaporation. The PCR profile included a denaturing step at 95°C for 30 s, followed by annealing of the primers at 60°C for 30 s, with extension at 72°C for 30 s. For the outer PCR, amplification was done for 35 cycles in a thermal cycler (MJ Research). Amplification with the inner primers was done for 30 cycles. Negative controls consisted of a blank containing all PCR reagents but no DNA. As control for amplifiable DNA in the stool specimen, primers targeting the 16S rRNA of enteric bacteria were used as described elsewhere (3).

Detection of amplified products. Amplification products were visualized by running 9 µl of the reaction mixture in 2% agarose gels in Tris-borate-EDTA buffer. Size markers in all gels were from the 123-bp DNA ladder (Bethesda Research Laboratories, Grand Island, N.Y.). Gels were run at a constant 110 V for 90 min, stained in an ethidium bromide solution (0.5 µg/ml) for 30 min, destained for 30 min, and photographed under UV light with a Land camera (Polaroid, Cambridge, Mass.).

Southern blot analysis. PCR products were confirmed as B. fragilis enterotoxin gene sequences by internal probe hybridization. A nonradioactive method with horseradish peroxidase, a biotin-labeled oligonucleotide (RS-7), and an enzyme chemiluminescence detection system (Amersham, Arlington Heights, Ill.) were used as previously described (2).

Enterotoxin assay. The cytotoxicity assay for detecting B. fragilis enterotoxin was performed as described previously (10, 17, 18). HT-29 cells were grown and maintained in RPMI medium with glutamine (Gibco, Life Technologies, Inc., Grand Island, N.Y.) supplemented with penicillin (100 IU/ml), streptomycin (Sigma, Saint Louis, Mo.) (100 µg/ml) and heat-inactivated fetal bovine serum (Hyclone Laboratories, Inc., Logan Utah) at 12% in 25-ml flasks at 37°C under 5% CO₂. For the cytotoxicity assay, HT-29 cells from one to two flasks were used for each plate. Cells were resuspended in 20 ml of medium for each plate to be inoculated and aliquoted (180 µl/well) into 96-well tissue culture plates (Corning Glass Works, Corning, N.Y.). The cells were then allowed to attach and grow for 2 to 3 days. Supernatants from bacterial cultures grown on brain heart infusion broth were filtered through 0.45-µm Acrodisk syringe filters (Gelman Sciences, Ann Arbor, Mich.), and 20 µl of serial twofold dilutions was inoculated into the wells in duplicate. The plates were incubated at 37°C under 5% CO₂ for 3 to 4 h and then examined for the presence of typical toxin-induced cytopathic changes. Culture supernatants were considered positive for B. fragilis enterotoxin if a cytopathic effect was visible that was neutralized by specific antiserum. The cytotoxic titer was the highest dilution of the culture supernatant that affected at least 50% of the cells after 3 to 4 h of incubation. For the neutralization assay, anti-enterotoxin rabbit antiserum was diluted 1:25 in phosphate-buffered saline. Doubling dilutions of this product were mixed with culture supernates found to be positive in the cytotoxicity assay. After incubation at 37°C for 30 min, 20 µl of each mixture was inoculated into HT-29 cells as in the cytoxicity assay. Neutralization was indicated by the lack of any cytotoxic effect.

	RESULTS

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Amplification of enterotoxigenic strains of B. fragilis. A PCR assay was developed to detect ETBF. We designed and tested two sets of primers for later use in a nested assay. With a pair of outer primers (RS-3 and RS-4), we amplified the expected 367-bp DNA fragment in eight known enterotoxigenic strains of B. fragilis isolated from humans and nine strains isolated from horses with diarrhea. Amplification with the inner primers (RS-1 and RS-2) yielded the expected 290-bp DNA fragment (Fig. 1A). Nonspecific amplification products were observed with DNA from 28 nonenterotoxigenic isolates (these isolates did not show any cytotoxic activity in HT-29 cells). Confirmation of specificity of the product was by Southern blot analysis with a biotin-labeled internal probe (RS-7). As seen in Fig. 1B, only DNA amplification products from toxigenic strains hybridized with the probe. None of the amplicons observed with the nonenterotoxigenic isolates probed positive.

View larger version (59K): [in this window] [in a new window]   FIG. 1.   Ethidium-bromide-stained 2% agarose gel (A) and Southern blot (B) analysis of PCR products of DNA from selected enterotoxigenic and nonenterotoxigenic B. fragilis isolates amplified with the inner primers RS-1 and RS-2 and the outer primers RS-3 and RS-4. Lanes 1 and 17, 123-bp DNA ladder; lanes 2 to 5 and 9 to 12, DNA from enterotoxigenic isolates of B. fragilis amplified with the outer (lanes 2 to 5) and inner (lanes 9 to 12) primers, respectively; lanes 6 to 8 and 13 to 15, DNA from nonenterotoxigenic isolates of B. fragilis amplified with the outer (lanes 6 to 8) and inner (lanes 13 to 15) primers, respectively.

Sensitivity of PCR in detecting ETBF. For the sensitivity experiment in stool specimens, a nested PCR approach was used as described in Materials and Methods. The sensitivity of our assay in detecting ETBF was determined in two sets of experiments. In the first experiment, ETBF at concentrations ranging from 10 to 10⁶ cells was inoculated into 1 g of stool from a healthy person. Using a nested PCR approach, we detected 100 to 1,000 cells of ETBF per g of stool (Table 2 and Fig. 2). No amplification product was detected in DNA extracted from the unspiked stool by the nested PCR method for B. fragilis. The 16S rRNA gene of enteric bacteria was amplifiable in this specimen, indicating the lack of inhibitors of rTaq polymerase in the sample.

View larger version (82K): [in this window] [in a new window]   FIG. 2.   Sensitivity of the nested PCR technique in detecting ETBF DNA contained in 102 to 106 cells/g of stool. Lanes 1 and 12, 123-bp DNA marker ladder; lane 3, 108 cells of ETBF in 1 g of stool; lanes 4 through 8, 106, 105, 104, 103, and 102 cells of ETBF/g of stool, respectively; lane 9, 100 ng of ETBF DNA; lane 10, negative control.

Specificity of the PCR. To determine the specificity of the PCR, amplification reactions were carried out with DNA from other species in the B. fragilis group, E. coli, and toxigenic C. difficile. As shown in Fig. 3A, no amplification products of the expected size were obtained with DNA from B. distasonis, B. ovatus, B. uniformis, and B. thetaiotaomicron, E. coli, or toxigenic C. difficile after amplification with the primers RS-3 and RS-4. DNA extracted from all these species remained negative when probed with the internal probe (Fig. 3B).

View larger version (45K): [in this window] [in a new window]   FIG. 3.   Specificity of PCR in differentiation of B. fragilis from other B. fragilis group isolates and from E. coli and C. difficile. (A) Ethidium-bromide-stained 2% agarose gel of PCR products and (B) Southern blot analysis of products in panel A after amplification with the primers RS-3 and RS-4. Lanes 1 and 12, 123-bp DNA ladder. DNA from B. distasonis (lane 2), B. ovatus (lane 3), B. thetaiotaomicron (lane 4), B. uniformis (lane 5), ETBF (lanes 6 to 8), E. coli (lane 9), and toxigenic C. difficile (lane 10).

Correlation between PCR and enterotoxin assay. We obtained a 100% correlation between PCR and the enterotoxin assay in HT-29 cells. All the isolates positive by PCR produced enterotoxin (data not shown).

	DISCUSSION

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B. fragilis is emerging as an etiologic agent of diarrhea in animals and in humans (7-9, 12, 14). Although B. fragilis constitutes about 1 to 2% of the normal intestinal flora, it has been associated with extraintestinal infections such as abscesses, soft-tissue infections, and bacteremias (5). In a recent communication, Pantosi et al. reported the isolation of ETBF from the stools of healthy children and adults as well as from those of children and adults with diarrhea (11). These investigators, however, were able to detect B. fragilis enterotoxin directly in only a portion of the stool specimens positive for ETBF by culture. Detection of B. fragilis enterotoxin in stools is dependent on amount of toxin produced, sensitivity of the assay, and stability of the toxin, which is susceptible to degradation by proteases (18).

In this study, we described a rapid, sensitive, and specific method for detection of ETBF directly in stool specimens. Sensitivity of the nested PCR allowed us to detect as little as 1 pg of enterotoxigenic DNA or 100 to 1,000 cells of ETBF per g of stool. Currently, identification of ETBF isolates is done by culturing in selective medium (Bacteroides bile esculin) and testing the isolates for the presence of enterotoxin by either a cytotoxicity assay in HT-29 cells or the lamb ileal loop test (17). Culturing on selective medium requires the presence of 10⁴ CFU, although recovery of isolates from specimens is dependent on time of culturing from obtainment of the fecal sample (13). The cytotoxicity assay in HT-29 cells has been reported to be 89% sensitive compared to the lamb ileal loop test (19). The latter biological assay is obviously more expensive and labor-intensive. Specific DNA amplification of ETBF from stool specimens eliminates the need for culturing the organism and for further biochemical tests for identification. We obtained a 100% correlation between PCR and the enterotoxin assay in HT-29 cells. Thus, a single test can be used to detect and identify enterotoxigenic isolates of B. fragilis. Considering the time required to culture, purify the isolate, and test for enterotoxin production in vitro with a tissue culture assay (5 to 6 days), the PCR assay is a rapid test. The time required to carry out this assay, approximately 48 to 72 h, can be shortened as the assay is refined. The clinical significance of ETBF as a cause of diarrhea in humans is not clear. In two separate studies, ETBF has been significantly associated with diarrhea in children >1 year of age in Bangladesh and in an Apache population in the United States (12, 14). In a recent study, a high rate of carriage of ETBF was found in healthy subjects and in subjects with diarrhea in Italy, both children and adults (11). We believe our PCR assay is of clinical importance and significance, as it will detect ETBF in patient samples in which the organism may be undetected by the conventional tests. At present, we continue to optimize and refine this assay, including screening of new primers. This assay may contribute to the elucidation of the role and epidemiology of ETBF in diarrheal and intestinal infectious diseases in humans. Studies are in progress in our laboratory to determine the incidence rates of B. fragilis in several forms of diarrheal diseases.