Polymerase Chain Reaction을 이용한 국내 Bacteroides fragilis 분리균에서의 장독소 유전자 검출

Abstract

Bacteroides fragilis is a Gram negative nonsporulating anaerobic rod bacterium that makes up about 1 to 2% of the normal human colonic microflora. In 1984, Myer et al. reported that some strains of B. fragilis produce enterotoxin and cause diarrheal disease in cattle and human. Since then it has been termed enterotoxigenic B. fragilis (ETBF). In this study, we tried to detect enterotoxin gene from 37 B. fragilis strains, isolated in Korean patients, to confirm the existence of ETBF and usefulness of PCR as a rapid diagnosis method. By this method, we identified 9 ETBF strains and confirmed their pathogenesis by cytotoxicity test. No significant cross-reactivity with other anaerobes or aerobes was observed. Thus, the PCR method may be considered useful for the sensitive and rapid detection of anaerobic infections. And the entire amplified PCR mixture was ligated into a pT7Blue T-vector and transformed into E. coli. When the nucleotide sequences of cloned PCR products were compared with reported enterotoxin gene, pBF529 inserted DNA sequence was nearly in good agreement with it but pBF570 inserted DNA sequence showed some difference at nucleotide 270-300. A search for nucleotide sequence homologies revealed that pBF529 exhibited 99%, but pBF570 indicated only 90% identity with reported enterotoxin gene. According to these results, it was suggested that ETBF toxin can be differentiated into at least 2 subtypes.