DETECTION OF HEMOLYSIN VARIANTS OF SHIGA TOXIN-PRODUCING ESCHERICHIA-COLI BY PCR AND CULTURE ON VANCOMYCIN-CEFIXIME-CEFSULODIN BLOOD AGAR

The presence of a hemolysin-encoding gene, elyA or hlyA, from Shiga to xin-producing Escherichia coli (STEC) was detected by PCR in each of 9 5 strains tested. PCR products of elyA from human STEC isolates of ser ovars frequently detected in Germany, such as O157:H-, O103:H2, O103:H -, O26:Hll, and O26:H-, showed nucleotide sequences identical to previ ously reported ones for O157:H7 and O111:H- strains. Compared to them, four elyA amplicons derived from human isolates of rare STEC serovars showed identity of about 98% but lacked an AluI restriction site. How ever, the nucleotide sequence of an amplicons derived from a porcine O 138:K81:H- STEC strain was identical to the corresponding region of hl yA, encoding alpha-hemolysin, from E. coli, This hlyA amplicon showed 68% identity with the nucleotide sequence of the corresponding elyA fr agment. It differed from the elyA PCR product in restriction fragments generated by AluI, EcoRI, and MluI, Of the 95 representative STEC str ains, 88 produced hemolysin on blood agar supplemented with vancomycin (30 mg/liter), cefixime (20 mu g/liter), and cefsulodin (3 mg/liter) (BVCC), The lowest added numbers of two to six STEC CFU per g of stool or per mi of raw milk were detectable on BVCC plates after seeding of the preenrichment broth, modified tryptic soy broth (mTSB) supplement ed with novobiocin (10 mg/liter), with 16 STEC strains. These strains represented the seven prevailing serovars diagnosed from German patien ts. However, with ground-beef samples, PCR was essential to identify t he lowest added numbers of two to six STEC CFU among colonies of hemol yzing Enterobacteriaceae, such as Serratia spp, acid alpha-hemolysin-p roducing E. coli. We conclude that preenrichment of stool and food sam ples in mTSB for 6 h followed by overnight culturing on BVCC is a simp le method for the isolation and presumptive identification of STEC.