IDENTIFICATION OF NEW VEROCYTOTOXIN TYPE-2 VARIANT B-SUBUNIT GENES INHUMAN AND ANIMAL ESCHERICHIA-COLI ISOLATES

The sequence of a verocytotoxin 2 VT2) variant gene that was untypeabl e by the B subunit PCR and restriction fragment length polymorphism an alysis (PCR-RFLP) method described by Tyler et al, (S, D, Tyler, W, M, Johnson, H, Lior, G, Wang, and K, R, Rozee, J, Clin, Microbiol, 29:13 39-1343, 1991) was determined and compared with published sequences. I t was highly homologous to two recently reported VT2 variant sequences . The PCR-RFLP method described by Tyler et al, was extended to includ e these new sequences. New VT2 variants were identified in 65 of 359 V T-producing Escherichia coli (VTEC) with newly designed primers (VT2-c m and VT2-f) and were characterized as well by restriction analysis of the amplification products obtained with another VT2-specific primer pair (VT2-e and VT2-f), The VT genes harbored by 64 of these isolates proved to be untypeable by Tyler's PCR-RFLP method because no amplific ation was obtained with the primers used with this method (VT2-c and V T2-d), The last isolate harbored the new variant gene in addition to V T2vh-a. None of the isolates harboring these new toxin genes belonged to serogroups O157, O26, O103, O111, and O145. All 65 isolates were ne gative for the eaeA gene and were significantly less frequently entero hemolytic or positive for the enterohemorrhagic E, coli (EHEC) virulen ce plasmid than non-O157 VTEC isolates harboring other VT2 genes. They were also less frequently isolated from patients with EHEC-associated symptoms. The extended PCR-RFLP typing method is a useful tool to ide ntify less-virulent VTEC isolates and for VT genotyping in epidemiolog ical studies with non-O157 strains.