Purpose: The objectives of this study were to develop antibodies against different STEC serogroups and produce immunomagnetic separation (IMS) and latex reagents. Furthermore, methodologies were developed to detect and isolate STEC O104 and enteroaggregative STEC (EAEC-STEC) O104:H4 in sprouts, utilizing multiplex PCR assays and the IMS and latex reagents.
Methods: Antibodies against the top six non-O157 STEC serogroups (O26, O45, O103, O111, O21, and O145), as well as STEC O104 and O157 were generated in rabbits and used to produce IMS and latex beads for the different pathogens. Specificity testing was performed using the target pathogens and non-target E. coli. A method was developed for detection of STEC O104 and EAEC-STEC O104:H4 in sprouts, consisting of a selective enrichment, two real-time multiplex PCR assays (stx2, aggR, and wzx104 genes for detection of EAEC-STEC O104:H4 and stx1-2, ehxA, and wzx104 for STEC O104), followed by IMS, isolation from selective agars, and confirmation by latex agglutination and PCR.
Results: The IMS and latex reagents against all of the STEC serogroups showed good specificity and were useful for isolating the target pathogens, as shown by plating onto selective and non-selective agars. Cold stressed STEC and EAEC-STEC O104 were detected and isolated from sprout samples inoculated at a level of less than 1 CFU/g. All presumptive colonies were confirmed by agglutination using the O104 latex particles and the multiplex PCR assays.
Significance: The IMS and latex reagents and the methods developed in this study improve the ability of the food industry and regulatory agencies worldwide to detect and isolate important STEC from food.