The main purpose of this work was to evaluate culture enrichment conditions, with particular regard to those reported in ISO/TS 13136:2012, for STEC detection in food. The culture media evaluated included mTSB with novobiocin 0-16mg/l (mTSB+N0-16) or acriflavin 12mg/l (mTSB+A12); BPW; mBPWp with acriflavin 10mg/l, cefsulodin 10mg/l, vancomycin 8mg/l (mBPWp+ACV); and mBPWp with cefsulodin 10mg/l, vancomycin 8mg/l (mBPWp+CV). They were used for the growth of STEC O157, O26, O103, O111, O145 and O104 in pure cultures or in artificially contaminated food matrices (ground beef, mung bean sprouts). STEC detection was accomplished using commercially available multiplex real-time PCR assays targeting stx1-stx2 and eae, and serogroup-associated genes. More rapid multiplication of STEC in pure cultures occurred in mBPWp+CV, while an inhibitory effect of novobiocin and acriflavin was observed for some STEC serogroups in media with these selective agents. mBPWp+CV allowed the detection of all serogroups in bean sprouts when inoculated at levels as low as 1CFU/25g. A reduced novobiocin concentration of 2mg/l in mTSB was required for STEC detection in ground beef samples. A temperature of 42°C for the entire duration of the enrichment or 44°C after an initial phase of 6h at 37°C was important to limit the multiplication of non-target bacteria. Results of this study suggest that media and protocols should be adapted to the food being analyzed, since protocols provided in official reference methods may produce insufficient sensitivity.
Keywords: Enrichment medium; Ground beef; ISO/TS 13136:2012; Real-time PCR; STEC; Sprouts.
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