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Non-O157 enterohemorrhagic Escherichia coliin priority serogroups O26, O45, O103, O111, O121, and O145 are increasingly recognized as important human pathogens. In the present study, a panel of monoclonal antibodies (MAbs) to the lipopolysaccharide O antigens of E. coliin serogroups O26, O45, O103, O111, O121, and O145 was produced. The specificity was evaluated by examining the reactivity of the MAbs with 50 E. colistrains and 42 non-E. colibacteria, and several MAbs highly specific for E. colistrains in each of the six non-O157 priority serogroups were identified. The use of these highly specific MAbs may be of considerable value for determining whether an E. coliisolate belongs to one of the six priority non-O157 serogroups, for developing specific detection assays for these organisms, and for characterizing the lipopolysaccharide O antigens of isolates in these serogroups.

A simple immunoenzymatic enterohemorrhagic Escherichia coli(EHEC) colony check (ECC) assay was developed for the presumptive identification of priority EHEC colonies isolated on plating media from enrichment broth cultures of foods. With this approach, lipopolysaccharide extracted from a colony is spotted on the grid of a polymyxin-coated polyester cloth strip, and bound E. coliserogroup O26, O45, O103, O111, O121, O145, and O157 antigens are subsequently detected by sequential reactions with a pool of commercially available peroxidase-conjugated goat antibodies and tetramethylbenzidine substrate solution. Each strip can accommodate up to 15 colonies, and test results are available within 30 min. Assay performance was verified using colonies from a total of 73 target EHEC isolates covering the range of designated priority serogroups (all of which were reactive), 41 nontarget E. coliisolates including several nontarget Shiga toxin-producing E. coliserogroups (all unreactive), and 33 non-E. colistrains (all unreactive except two bacterial strains possessing O-antigenic structures in common with those of the priority EHEC). The ECC assay was reactive with target colonies grown on several types of selective and nonselective plating media designed for their cultivation. These results support the use of the ECC assay for high-throughput screening of colonies isolated on plating media for detecting priority EHEC strains in foods.

A method has been developed for the detection in beef trim of priority Shiga toxin–producing E. coli(STEC) strains, defined as E. colipossessing the virulence factors stx1and/or stx2and intimin (eae), with O serogroups O26, O45, O103, O111, O121, O145, or O157. The method is based on recovery of the target bacteria by overnight enrichment in a broth optimized for recovery of O157 and non-O157 STEC, followed by screening using multiplex PCR techniques targeting (i) stx1, stx2, and eae(STE PCR) and (ii) gene sequences associated with the seven priority O serogroups (Poly O PCR), and then direct plating of broth samples positive in both STE and Poly O PCR onto Rainbow agar. Colonies on agar media were screened batchwise for STEC by the STE PCR, and presumptive isolates were characterized using a multiplex PCR and cloth-based hybridization array system targeting key virulence and O serogroup-specific markers. Using one representative strain of each priority O serogroup individually inoculated in beef trim samples, the method exhibited a limit of detection approaching 1 to 2 viable STEC cells per 65 g. None of the uninoculated trim samples produced positive results with either of the screening PCR procedures or on analysis of colonies recovered on plating media. STEC-negative samples were readily identified by screening PCR within 24 h, with a turnaround time of fewer than 4 days for confirmation of positives. The inclusivity and exclusivity characteristics of the screening PCR techniques were verified using a total of 65 different priority STEC strains: 24 nonpriority STEC, 15 non-STEC bacteria, and only those strains bearing the targeted characteristics produced screening PCR-positive results.

A cloth-based hybridization array system (CHAS) was developed for the identification of foodborne colony isolates of seven priority enterohemorrhagic Escherichia coli(EHEC-7) serogroups targeted by U.S. food inspection programs. Gene sequences associated with intimin; Shiga-like toxins 1 and 2; and the antigenic markers O26, O45, O103, O111, O121, O145, and O157 were amplified in a multiplex PCR incorporating a digoxigenin label, and detected by hybridization of the PCR products with an array of specific oligonucleotide probes immobilized on a polyester cloth support, with subsequent immunoenzymatic assay of the captured amplicons. The EHEC-7 CHAS exhibited 100% inclusivity and 100% exclusivity characteristics with respect to detection of the various markers among 89 different E. colistrains, with various marker gene profiles and 15 different strains of non–E. colibacteria.