Association of enterohemorrhagic Escherichia coli hemolysin with serotypes of shiga-like-toxin-producing Escherichia coli of human and bovine origins

Abstract

In this study we investigated whether the enterohemorrhagic Escherichia coli (EHEC) hemolysin gene ehxA could be used as an indicator of pathogenicity in Shiga-like-toxin-producing Escherichia coli (SLTEC) isolates. The isolates in a collection of 770 SLTEC strains of human and bovine origins were assigned to group 1 (230 human and 138 bovine SLTEC isolates belonging to serotypes frequently implicated in human disease), group 2 (85 human and 183 bovine isolates belonging to serotypes less frequently implicated in disease), and group 3 (134 bovine isolates belonging to serotypes not implicated in disease). PCR amplification was used to examine all of the SLTEC isolates for the presence of ehxA and the virulence-associated genes eae, slt-I, and slt-II. The percentages of human isolates in groups 1 and 2 that were positive for ehxA were 89 and 46%, respectively, and the percentages of bovine isolates in groups 1 to 3 that were positive for ehxA were 89, 51, and 52%, respectively. The percentages of human isolates in groups 1 and 2 that were positive for eae were 92 and 27%, respectively, and the percentages of bovine isolates in groups 1 to 3 that were positive for eae were 78, 15, and 19%, respectively. The frequencies of both ehxA and eae were significantly higher for group 1 isolates than for group 2 isolates. The presence of the ehxA gene was associated with serotype, as was the presence of the eae gene. Some serotypes, such as O117:H4, lacked both eae and ehxA and have been associated with severe disease, but only infrequently. The slt-I genes were more frequent in group 1 isolates than in group 2 isolates, and the slt-II genes were more frequent in group 2 isolates than in group 1 isolates. In a second experiment we determined the occurrence of the ehxA and slt genes in E. coli isolated from bovine feces. Fecal samples from 175 animals were streaked onto washed sheep erythrocyte agar plates. Eight E. coli-like colonies representing all of the morphological types were transferred to MacConkey agar. A total of 1, 080 E. coli isolates were examined, and the ehxA gene was detected in 12 independent strains, only 3 of which were positive for slt. We concluded that the ehxA gene was less correlated with virulence than the eae gene was and that EHEC hemolysin alone has limited value for screening bovine feces for pathogenic SLTEC because of presence of the ehxA gene in bovine isolates that are not SLTEC.

FIG. 1

Large plasmids (A) and Southern blot (B) of plasmid DNA hybridized with a probe for ehxA. Lanes M₁ and M₂ contained molecular weight markers. Lanes 1 through 3, lanes 4 and 5, and lanes 6 and 7 contained plasmid extracts of SLTEC isolates belonging to serotypes O121:H19, O113:H21, and O26:H11, respectively. Lane 8 contained plasmid DNA from a positive control O157:H7 SLTEC isolate known to contain a 90-kb EHEC plasmid. The isolates in lanes 1, 4, and 6 had an EHEC hemolysin-positive phenotype and were probe positive. The isolates in lanes 2, 3, 5, and 7 had an EHEC hemolysin-negative phenotype and were probe negative. Note that the EHEC plasmid in the eae-negative O113:H21 isolate was considerably larger than the approximately 90-kb plasmid in the SLTEC isolates which were eae positive.