Rapid and sensitive assay for detection of enterotoxigenic Bacteroides fragilis

Abstract

Bacteroides fragilis is an obligatory anaerobic, gram-negative bacterium found among the normal intestinal flora of humans. Enterotoxigenic strains of B. fragilis (ETBF) have been associated with diarrheal diseases in humans and animals. The enterotoxin of ETBF induces fluid changes in ligated intestinal segments and cytotoxic response in HT29/C1 cells. By using a pair of monoclonal antibodies (MAbs; MAb C3 and MAb 4H8) specific for the lipopolysaccharide of B. fragilis, an assay based on immunomagnetic separation (IMS) in combination with PCR (IMS-PCR) was developed. After DNA extraction, a 294-bp fragment was amplified. The specificity of the IMS-PCR assay was evaluated by adding previously isolated and confirmed ETBF strains to normal fecal samples. All fecal samples to which ETBF strains were added were positive, showing a 100% specificity. The spiked fecal samples were also used for evaluation of the sensitivity of the assay. The detection limit was found to be approximately 50 CFU/g of feces. By this method 10 clinical fecal samples (5 from patients with diarrhea and 5 from healthy controls) were examined. The results of PCR were in accordance with the results of the HT29/C1 cell assay for all samples. The minimum time to retrieval of the final result by the IMS-PCR method is 36 h. The proposed IMS-PCR assay is rapid and sensitive for the direct detection of ETBF in stool samples.

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.

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.