INTRODUCTION

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Bacteroides fragilis, an obligatory anaerobic bacterium found in high numbers among the normal intestinal flora of humans, is recognized as an important cause of intra-abdominal infections in humans (3). However, neither diarrheal disease nor extracellular toxin production due to B. fragilis was appreciated until 1984, when Myers and coworkers (4, 5) found that some strains of B. fragilis were associated with diarrheal diseases in young farm animals and humans by producing a factor with enterotoxigenic activity. Recent studies have shown that the rate of enterotoxigenic B. fragilis (ETBF) carriage is high both in adults and in children, regardless of whether diarrhea is present (7). The role of ETBF as a diarrheal agent has been described previously (2, 8, 9, 13, 14). It has also been suggested that ETBF may be endemic in communities (10). Therefore, more investigations on the identification, epidemiology, and pathogenic role of ETBF are necessary.

PCR has been used to identify ETBF in pure cultures and directly from feces (6, 11). The assays are based on amplification of the enterotoxin gene, which encodes a zinc-binding metalloprotease (2). Pantosti et al. (6) used primers deduced from the sequence of the gene in an assay in which stool samples were inoculated on selective media followed by a sweep of total growth. The sensitivity was 10⁴ to 10⁵ CFU/g of feces (6). Recently, Shetab et al. (11) published a description of a nested PCR assay for the detection of the B. fragilis enterotoxin gene with a detection limit of 100 to 1,000 CFU/g of stool. However, false-negative results may occur if PCR inhibitors, which are commonly present in stool samples, are not removed prior to amplification (16). The aim of the present study was to use well-characterized specific monoclonal antibodies (MAbs) coated on magnetic beads and subsequent specific capture of bacteria by immunomagnetic separation (IMS) in order to concentrate and separate the bacteria. Here, we describe an IMS-PCR assay, based on the use of B. fragilis-specific MAbs, followed by PCR amplification of the enterotoxin gene for the rapid, sensitive, and specific detection of ETBF directly from clinical samples.

	MATERIALS AND METHODS

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Bacterial strains. Clinical isolates of 21 ETBF strains were provided by J. M. Albert from the International Centre for Diarrhoeal Disease Research, Bangladesh, in Dhaka, Bangladesh. All the ETBF strains produced enterotoxin, as shown in an assay with the HT29/C1 cell line (see below). The nonenterotoxigenic Bacteroides strains investigated were B. fragilis NCTC 9343 and B. thetaiotaomicron NCTC 10582 (National Collection of Type Cultures, London, United Kingdom); B. fragilis VPI 5631, B. fragilis VPI 4225, B. fragilis VPI 4117, and B. fragilis VPI 2552 (Virginia Polytechnic Institute and State University, Blacksburg); and B. fragilis ATCC 23745, B. ovatus ATCC 8483, and B. vulgatus ATCC 8482 (American Type Culture Collection, Rockville, Md.). In addition, Salmonella typhi IS 501, Shigella flexneri 4bR, Escherichia coli (serotypes O:35, O:104, O:136, and O:138), and Vibrio cholerae O139 strains were from the strain collection at the Division of Clinical and Oral Bacteriology, Huddinge University Hospital.

Production and purification of MAbs. The MAbs used in this study were basically prepared as described earlier (1). Two different MAbs were used: MAb 4H8 (immunoglobulin G3 [IgG3]), which binds to an immunodominant epitope in the lipopolysaccharide (LPS) of a majority of B. fragilis isolates (17), and MAb C3 (IgG2b), which most likely binds to a common epitope present in the inner core region of B. fragilis LPS (18). Both MAbs were purified from serum-free medium with a protein A column (Sigma Chemical Company, St. Louis, Mo.).

Coagglutination test. Staphylococcus aureus Cowan I bacteria (Sigma) were sensitized individually with 0.1 ml of MAb C3 (1 mg/ml) and 0.1 ml of MAb 4H8 (1 mg/ml).

Preparation of spiked fecal samples and clinical fecal samples for IMS. ETBF-negative stool samples were collected. In order to estimate the sensitivity of the IMS-PCR assay under experimental conditions, serial 10-fold dilutions of the ETBF D-94 strain were prepared in phosphate-buffered saline (PBS), and the mixture was added to a preweighed stool sample in order to obtain concentrations ranging from 10⁶ to 10 CFU/ml. Briefly, 0.1 ml of diluted strain D-94 was added to 0.9 ml of diluted feces (1 g/5 ml in PBS). Similar procedures were also followed with ETBF-negative stool samples spiked with other strains tested in the study (see Table 2). The spiked fecal samples (ca. 1 ml) were inoculated into prereduced peptone yeast glucose (PYG) broth medium (4.5 ml) containing 0.05% (wt/vol) kanamycin (Sigma), and the mixture was incubated at 37°C for 24 to 48 h. After incubation, the broth medium was centrifuged at 600 × g for 10 min. The supernatant was collected and centrifuged at 3,000 × g for 10 min, and the pellet was suspended in 80 µl of PBS for incubation with magnetic beads.

IMS. Rat anti-mouse IgG2b-coated Dynabeads M450 and sheep anti-mouse IgG-coated Dynabeads M280 (Dynal, Oslo, Norway) were used. The amounts of the MAbs required for coating of the immunomagnetic particles were determined according to the manufacturer's recommendations, with minor modifications. Briefly, 1 µl of MAb C3 (1 mg/ml) was mixed with 25 µl of rat anti-mouse IgG2b-coated Dynabeads M450 (4 × 10⁸ beads/ml), and 1.5 µl of MAb 4H8 (1 mg/ml) was mixed with 25 µl of sheep anti-mouse IgG-coated Dynabeads M280 (6 × 10⁸ to 7 × 10⁸ beads/ml). The volumes were adjusted to 100 µl with PBS, and the beads were incubated overnight with bidirectional rotation at 4°C. The coated beads were washed three times with PBS containing 0.05% Tween 20 (Merck, Schuchardt, Munich, Germany) and were resuspended in PBS with 0.5% bovine serum albumin (Sigma) at 10⁷ beads/ml. The beads were stored at 4°C until they were used.

PCR assay. The primers used in the study were those described by Pantosti et al. (6). The forward primer (BF1; 5'-dGACGGTGTATGTGATTTGTCTGAGAGA-3') and the reverse primer (BF2; 5'-dATCCCTAAGATTTTATTATTATCCCAAGTA-3') were synthesized by Interactiva Biotechnologie GmbH (Ulm, Germany). The specific amplified 294-bp band of DNA is expected.

Enterotoxin assay with HT29/C1 cells. To assess whether enterotoxin was produced, after the separation from each fecal sample 20 µl of the magnetic beads was inoculated into brain heart infusion broth (BHI) supplemented with hemin (5 µg/ml), and the mixture was incubated at 37°C for 48 h in an anaerobic cabinet. The broth cultures were centrifuged at 3,000 × g for 10 min, and the supernatants were filtered through a 0.20-µm-pore-size filter (Sartorius AG, Göttingen, Germany). The filtered supernatants were frozen immediately and were kept at 20°C until use.

	RESULTS

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Coagglutination tests of ETBF strains with two MAbs. Both coagglutination test reagents were used for all 21 ETBF strains (Table 1). All strains except ETBF D-93 and ETBF D-115 were positive by tests with MAb C3. However, these two strains were positive when MAb 4H8 was used. By tests with both MAbs, all 21 strains were correctly identified as B. fragilis. Fourteen strains reacted with both coagglutination test reagents (Table 1).

Sensitivity of IMS-PCR for detection of ETBF. The sensitivity of the IMS-PCR assay was investigated with fecal samples spiked with 10-fold dilutions of ETBF D-94. The DNA from bacterial cells isolated by IMS was subjected to PCR. As determined by electrophoresis, the lowest original dilution of ETBF bacteria that could be detected by IMS-PCR was 10² CFU/ml (Fig. 1). This corresponds to ~50 CFU per g of fecal sample. No amplification of DNA from nonspiked fecal samples could be seen.

View larger version (31K): [in this window] [in a new window]   FIG. 1.   IMS-PCR products from spiked fecal samples prepared with serial dilutions of ETBF D-94. Lanes 1 to 6, dilutions from 106 to 10 CFU/ml, respectively; lane 7, positive control; lane 8, negative control; lanes M, DNA molecular mass markers (Marker V; Boehringer Mannheim). The 294-bp product correlates with the amplified portion of the enterotoxin gene.

Detection of ETBF in spiked fecal samples by IMS-PCR. Fecal samples spiked with different strains were tested. PCR followed by electrophoresis showed that only in tests with the ETBF strains listed in Table 2 was the amplification product detected (294 bp) (Fig. 2). None of the fecal samples spiked with other bacterial strains was positive. Nonspiked fecal samples were also negative.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   IMS-PCR products from spiked fecal samples and clinical samples. Lanes: 1, ETBF D-100 (104 CFU/ml); 2, ETBF D-104 (104 CFU/ml); 3, nonenterotoxigenic B. fragilis NCTC 9343; 4, E. coli O:104, 5, B. vulgatus; 6, fecal sample a; 7, fecal sample b; 8, control sample; 9, positive control; 10, negative control; M, DNA molecular mass markers. The 294-bp product correlates with the amplified portion of the enterotoxin gene.

Detection of ETBF in fecal samples by IMS-PCR and HT29/C1 cell assay. Five fecal samples from patients with diarrhea and five fecal samples from healthy controls were tested by the IMS-PCR assay. Amplification products were found in tests with three of five samples from patients with diarrhea (Fig. 2). The samples from healthy controls were negative.

	DISCUSSION

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ETBF has been considered to be an important cause of diarrheal disease in humans as a result of the production of an enterotoxin (5, 8). The epidemiology of ETBF or its pathogenic role is not fully understood. No final proof has yet been provided to define the pathogenic importance of this microorganism in diarrheal diseases. To evaluate the association between diarrhea and the presence of ETBF, it is necessary to detect the microorganism from among the complex mixture of bacterial flora in the feces and to investigate its ability to produce enterotoxin. A direct PCR assay has been used to detect ETBF from fecal samples (6, 11). However, the PCR inhibitors present in feces seem to affect the sensitivity of this method and can cause false-negative results (16). Therefore, we developed a combined IMS and PCR assay for the specific, sensitive, and rapid detection of ETBF in feces.

The MAbs used in this study (MAb 4H8 and MAb C3) were described earlier: MAb 4H8, which specifically binds to an immunodominant epitope of LPSs from B. fragilis (17), and MAb C3, which most likely recognizes an epitope in the inner core region of LPSs from B. fragilis (18). Mixtures of magnetic beads individually coated with one of the two MAbs were used in order to increase the capacity to capture B. fragilis in the IMS procedure. Before applying the fecal samples to IMS, the samples were inoculated into PYG broth containing kanamycin. An antibiotic agent was used to inhibit aerobic bacteria and to select for B. fragilis. With the aid of magnetic beads, the captured B. fragilis strains are concentrated, and the substances which may be inhibitory to PCR analyses are effectively removed. Combined with PCR, the assay provided a very sensitive method for the detection of ETBF in fecal samples, with a detection limit of ~50 CFU/g of feces.

Although the time required for analysis is extended, we believe that incubation of the captured B. fragilis in PYG medium is necessary. The minimum time from receipt of the sample to the final PCR result was 36 h if the incubation in PYG medium was carried out for 24 h. This can be compared to the time required to perform the conventional method, i.e., the cell culture cytotoxicity assay, which takes between 48 and 60 h.

The fecal samples spiked with ETBF were positive by IMS-PCR, and the amplified 294-bp product was detected. None of the samples spiked with other (nonenterotoxigenic) B. fragilis isolates was positive. In addition, three of five clinical fecal samples from patients with diarrhea yielded the amplification products, and these samples were also tested by the HT29/C1 cell assay, which confirmed the enterotoxin production, thus showing that the IMS-PCR assay is specific for the detection of ETBF in clinical samples.

In order to fully evaluate the sensitivity and specificity of the method described here, a study with larger numbers of samples must be conducted. We are currently evaluating this method by analyzing a large collection of fecal samples from patients with diarrhea and control subjects.