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. 1998 Jun;36(6):1729-32.
doi: 10.1128/JCM.36.6.1729-1732.1998.

Detection of Bacteroides fragilis enterotoxin gene by PCR

R Shetab  1 , S H Cohen, T Prindiville, Y J Tang, M Cantrell, D Rahmani, J Silva Jr
Affiliations

Detection of Bacteroides fragilis enterotoxin gene by PCR

R Shetab et al. J Clin Microbiol. 1998 Jun.

Abstract

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.

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Figures

FIG. 1
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.
FIG. 2
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.
FIG. 3
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).
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