Stx2 subtyping of Shiga toxin-producing Escherichia coli isolated from cattle in France: detection of a new Stx2 subtype and correlation with additional virulence factors

Abstract

At least 11 Stx2 variants produced by Shiga toxin-producing Escherichia coli (STEC) isolated from patients and animals have been described. The Stx2 subtyping of STEC isolated from healthy cows positive for stx(2) (n = 104) or stx(2) and stx(1) (n = 63) was investigated. Stx2vh-b, Stx2 (renamed Stx2-EDL933), and Stx2vh-a were the subtypes mostly detected among the bovine isolates (39.5, 39, and 25.5%, respectively). Stx2e was not present, and subtypes included in the Stx2d group (Stx2d-OX3a, Stx2d-O111, and Stx2d-Ount) were found infrequently among the isolates examined (8.5%). A combination of two distinct Stx2 subtypes was observed among 23.5% of the strains. For the first time, a combination of three subtypes (Stx2-EDL933/Stx2vh-b/Stx2d and Stx2vh-a/Stx2vh-b/Stx2d) was detected (3.5% of the isolates). In addition, bovine STEC harboring stx(1) and one or two stx(2) genes appeared highly cytotoxic toward Vero cells. A new Stx2 subtype (Stx2-NV206), present among 14.5% of the isolates, showed high cytotoxicity for Vero cells. Two amino acid residues (Ser-291 and Glu-297) important for the activation of Stx2 by human intestinal mucus were conserved on the Stx2-NV206 A subunit. The gene encoding Ehx enterohemolysin was prominent among STEC harboring stx(2)-EDL933 alone (78%) or a combination of stx(2)-EDL933 and stx(2)vh-b (85%). In addition, Stx2-EDL933 and/or Stx2vh-b subtypes were highly associated with other putative virulence factors such as Stx1 and EspP extracellular serine protease, but not with EAST1 enterotoxin.

FIG. 1

Subtyping of Stx2 subtypes by RFLP-PCR. (A) Schematic representation of the restriction patterns obtained with the RFLP-PCR method developed by Tyler et al. (25). (B) Predicted sizes of the amplified products obtained with the VT2c and VT2d oligonucleotide primers and restricted with HaeIII, RsaI, and NciI endonuclease. The method developed to screen the Stx2vh-a subtype should also detect Stx2c and Stx2-OX3b because the nucleotide sequences of primers and sites of the restriction endonucleases used in the RFLP-PCR method were conserved among Stx2vh-a, Stx2c, and Stx2-OX3b genes. Similarly, the protocol used to discriminate Stx2-EDL933 should also detect Stx2-O48. The nucleotide sequence of Stx2-O113 is not available in the databases.