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The presence of a hemolysin-encoding gene,elyA orhlyA , from Shiga toxin-producingEscherichia coli (STEC) was detected by PCR in each of 95 strains tested. PCR products ofelyA from human STEC isolates of serovars frequently detected in Germany, such as O157:H−, O103:H2, O103:H−, O26:H11, and O26:H−, showed nucleotide sequences identical to previously reported ones for O157:H7 and O111:H− strains. Compared to them, fourelyA amplicons derived from human isolates of rare STEC serovars showed identity of about 98% but lacked anAlu I restriction site. However, the nucleotide sequence of an amplicon derived from a porcine O138:K81:H− STEC strain was identical to the corresponding region ofhlyA , encoding alpha-hemolysin, fromE. coli . ThishlyA amplicon showed 68% identity with the nucleotide sequence of the correspondingelyA fragment. It differed from theelyA PCR product in restriction fragments generated byAlu I,Eco RI, andMlu I. Of the 95 representative STEC strains, 88 produced hemolysin on blood agar supplemented with vancomycin (30 mg/liter), cefixime (20 μg/liter), and cefsulodin (3 mg/liter) (BVCC). The lowest added numbers of two to six STEC CFU per g of stool or per ml of raw milk were detectable on BVCC plates after seeding of the preenrichment broth, modified tryptic soy broth (mTSB) supplemented with novobiocin (10 mg/liter), with 16 STEC strains. These strains represented the seven prevailing serovars diagnosed from German patients. However, with ground-beef samples, PCR was essential to identify the lowest added numbers of two to six STEC CFU among colonies of hemolyzingEnterobacteriaceae , such asSerratia spp. and alpha-hemolysin-producingE. coli . We conclude that preenrichment of stool and food samples in mTSB for 6 h followed by overnight culturing on BVCC is a simple method for the isolation and presumptive identification of STEC.

Detection of Hemolysin Variants of Shiga Toxin-Producing Escherichia coli by PCR and Culture on VancomycinCefixime-Cefsulodin Blood Agar