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.

An interlaboratory study on efficient detection of Shiga toxin-producing Escherichia coli O26, O103, O111, O121, O145, and O157 in food using real-time PCR assay and chromogenic agar.

To establish an efficient detection method for Shiga toxin (Stx)-producing Escherichia coli (STEC) O26, O103, O111, O121, O145, and O157 in food, an interlaboratory study using all the serogroups of detection targets was firstly conducted. We employed a series of tests including enrichment, real-time PCR assays, and concentration by immunomagnetic separation, followed by plating onto selective agar media (IMS-plating methods). This study was particularly focused on the efficiencies of real-time PCR assays in detecting stx and O-antigen genes of the six serogroups and of IMS-plating methods onto selective agar media including chromogenic agar. Ground beef and radish sprouts samples were inoculated with the six STEC serogroups either at 4-6CFU/25g (low levels) or at 22-29CFU/25g (high levels). The sensitivity of stx detection in ground beef at both levels of inoculation with all six STEC serogroups was 100%. The sensitivity of stx detection was also 100% in radish sprouts at high levels of inoculation with all six STEC serogroups, and 66.7%-91.7% at low levels of inoculation. The sensitivity of detection of O-antigen genes was 100% in both ground beef and radish sprouts at high inoculation levels, while at low inoculation levels, it was 95.8%-100% in ground beef and 66.7%-91.7% in radish sprouts. The sensitivity of detection with IMS-plating was either the same or lower than those of the real-time PCR assays targeting stx and O-antigen genes. The relationship between the results of IMS-plating methods and Ct values of real-time PCR assays were firstly analyzed in detail. Ct values in most samples that tested negative in the IMS-plating method were higher than the maximum Ct values in samples that tested positive in the IMS-plating method. This study indicates that all six STEC serogroups in food contaminated with more than 29CFU/25g were detected by real-time PCR assays targeting stx and O-antigen genes and IMS-plating onto selective agar media. Therefore, screening of stx and O-antigen genes followed by isolation of STECs by IMS-plating methods may be an efficient method to detect the six STEC serogroups.