Selective media and real-time PCR assays for the effective detection of enterotoxigenic Escherichia coli in vegetables

Enterotoxigenic Escherichia coli (ETEC) is a major foodborne pathogen. Along with water, vegetables are one of the major food sources related to infections. Effective detection methods for ETEC in food, however, have not yet been established. This study aimed to evaluate ETEC detection methods focusing on the major serogroups (O6, O25, O27, O148, O153, O159, and O169) with steps of enrichment, isolation, and real-time PCR targeting genes encoding the heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). ETEC strains (n = 20) were grown to 7.0–8.9 log CFU/mL in modified E. coli broth (mEC) at 42 °C for 18 h. The strains formed colonies typically representing E. coli on sorbitol MacConkey agar and Shiga toxin-producing E. coli on CHROMagar STEC base agar. The minimum detection levels for real-time PCR assays targeting LT and ST genes were 1.9–3.1 log CFU/mL of vegetable culture. Vegetables inoculated with 2.0 log CFU/g ETEC were cultured in mEC, and then ST and LT genes were detected in the culture by real-time PCR assays at low threshold cycle (Ct) values; further, ETEC in the culture was isolated by plating on agars. This study thus demonstrated effective detection methods for ETEC in vegetables.