Genetic analysis for virulence factors in Escherichia coli O104:H21 that was implicated in an outbreak of hemorrhagic colitis

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 gamma-intimin, which is typically found in O157:H7, or the alpha- or beta-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.

FIG. 1

Agarose gel electrophoresis of DNA fragments amplified by multiplex PCR specific for EHEC virulence genes and other trait markers of O157:H7 serotype. Lane 1, 100-bp DNA ladder (Life Technologies, Rockville, Md.); lane 2, 35150 (EHEC O157:H7), positive control; lane 3, E2348/69 (EPEC O127:H6), negative control; lane 4, 13C60 (EHEC O26:H11); lanes 5 (G5507), 6 (G5506), and 7 (G5508), strains of the O104:H21 serotype. The amplified products (sizes are in base pairs) from the O157:H7-positive control shown in lane 2 are, from top to bottom, stx2 (584 bp), eaeA (397 bp), stx1 (348 bp), uidA (252 bp), and ehxA (166 bp).

FIG. 2

Agarose gel electrophoresis of DNA fragments amplified from the ehxA gene by PCR with primers hlyA1 and hlyA4. Lane 1, 100-bp DNA ladder (Life Technologies); lane 2, 35150 (O157:H7); positive control; lane 3, DH5α, negative control; lanes 4 to 6, O104:H21 strains G5508, G5507, and G5506, respectively. The top two bands of the size ladder are 2,072 and 1,500 bp, respectively.

FIG. 3

Partial nucleotide sequence of ehxA gene showing the MFS-1F primer binding site. Sequences shown are hlyA (O157:H7), published by Schmidt et al. (25); O157:H7 (strain 35150); and O104:H21 (strain G5507). The mismatched bases are shaded. The region indicated by MFS-1Fb shows the new primer sequence, which, with MFS-1R, amplifies the ehxA genes from strains of both serotypes O157:H7 and O104:H21.

FIG. 4

Agarose gel electrophoresis of DNA fragments amplified by multiplex PCR with primer MFS-1Fb instead of primer MFS-1F for amplification of ehxA. Lane 1, 123-bp DNA ladder (Sigma, St. Louis, Mo.); lane 2, 35150 (O157:H7), positive control; lane 3, G5507 (O104:H21). The amplified products (sizes are in base pairs) from O157:H7 shown in lane 2 are, from top to bottom, stx2 (584 bp), eaeA (397 bp), stx1 (348 bp), uidA (252 bp), and ehxA (158 bp).