An interlaboratory study on the detection methods for enterotoxigenic Escherichia coli in vegetables using enterotoxin gene screening and selective agars for ETEC-specific isolation

Enterotoxigenic Escherichia coli (ETEC) causes acute diarrhea and is transmitted through contaminated food and water; however, systematic procedures for its specific detection in foods have not been established. To establish an efficient detection method for ETEC in food, an interlaboratory study using ETEC O148 and O159 as representative serogroups was first conducted with 13 participating laboratories. A series of tests including enrichment, real-time PCR assays, plating on selective agars, and concentration by immunomagnetic separation followed by plating onto selective agar (IMS-plating methods) were employed. This study particularly focused on the detection efficiencies of real-time PCR assays for enterotoxin genes (sth, stp, and lt), IMS-plating methods, and direct plating onto sorbitol MacConkey agar and CHROMagar STEC medium, supplemented with tobramycin, which is a novel modification in the preparation of a selective agar. Cucumber and leek samples inoculated with ETEC O148 and O159, either at 4–7 CFU/25 g (low levels) or at 21–37 CFU/25 g (high levels) were used as samples with uninoculated samples used as controls. At high inoculation levels, the sensitivities of sth, stp, and lt detection, direct-plating, and IMS-plating methods in cucumber inoculated with O148 and in both foods inoculated with O159 were 100%. In leek inoculated with high levels of O148, the sensitivities of sth, stp, and lt detection, direct-plating, and the IMS-plating method were 76.9%, 64.1%, and 74.4%, respectively. At low inoculation levels, the sensitivities of sth, stp, and lt detection, direct plating, and IMS-plating method in cucumber inoculated with O148 and in both foods inoculated with O159 were in the range of 87.2–97.4%. In leek inoculated with low levels of O148, the sensitivities of sth, stp, and lt detection, direct plating, and the IMS-plating method were 59.0%, 33.3%, and 38.5%, respectively. Thus, ETEC in food contaminated with more than 21 CFU/25 g were detected at high rate (over 74%) using real-time PCR assays and IMS-plating onto selective agar. Therefore, screening sth, stp, and lt genes followed by isolation of STEC using the IMS-plating method may be an efficient method for ETEC detection.