Affiliation:
1. Division of Microbiological Studies, Food and Drug Administration, Washington, DC 20204,1 and
2. Food and Drug Administration Pacific Regional Laboratory, Northwest District, Bothell, Washington 980412
Abstract
ABSTRACT
Isolates of enterohemorrhagic
Escherichia coli
(EHEC) of serotype O104:H21 implicated in a 1994 outbreak of hemorrhagic colitis in Montana were analyzed for the presence of trait EHEC virulence markers. By using a multiplex PCR that specifically amplifies several genes, the O104:H21 strains were found to carry only the Shiga toxin 2 gene (
stx2
) and to express Stx2. They did not have the
eaeA
gene for γ-intimin, which is typically found in O157:H7, or the α- or β-intimin derivatives, which are common in other EHEC and enteropathogenic
E. coli
serotypes. Results of the multiplex PCR also indicated that the
ehxA
gene for enterohemolysin was absent from O104:H21. This, however, was not consistent with the results of a phenotypic assay that showed them to be hemolytic or a PCR analysis with another set of
ehxA
-specific primers, which indicated the presence of
ehxA
. To resolve this discrepancy, the
ehxA
region in O104:H21 and O157:H7 strains, to which the multiplex PCR primers anneal, was cloned and sequenced. Comparison of the sequences showed that the upstream primer binding site in the
ehxA
gene of O104:H21 was not identical to that of O157:H7. Specifically, there were several base mutations, including an A-to-G substitution at the 3′ end of the primer binding site. These base mutations are presumably not unique to O104:H21, since other enterohemolytic serotypes were also not detected with the
ehxA
primers used in the multiplex PCR. Comparison of the
ehxA
sequences of O104:H21 strains with those of other Stx-producing
E. coli
strains showed that they more closely resembled those of O8:H19 strains, which have cluster II
ehxA
genes, than those of O157:H7 strains, which have cluster I
ehxA
sequences. By modifying the upstream
ehxA
primer, the multiplex PCR was able to detect
ehxA
genes in both O157:H7 and O104:H21 strains.
Publisher
American Society for Microbiology
Cited by
49 articles.
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