Characterization of cytolethal distending toxin genes and expression in shiga toxin-producing Escherichia coli strains of non-O157 serogroups

Abstract

We identified cytolethal distending toxin and its gene (cdt) in 17 of 340 non-O157 Shiga toxin-producing Escherichia coli (STEC) strains (serotypes O73:H18, O91:H21, O113:H21, and O153:H18), all of which were eae negative. cdt is either chromosomal and homologous to cdt-V (serotypes O73:H18, O91:H21, and O113:H21) or plasmidborne and identical to cdt-III (serotype O153:H18). Among eae-negative STEC, cdt was associated with disease (P = 0.003).

FIG. 1.

Amino acid sequence differences between CDT-III from STEC O153:H18 (strain 9063/02) (accession number AY365044) and CDT-V from STEC O91:H21 (strain 9282/01) (accession number AY365042), O113:H21 (strain 5249/01) (accession number AY365043), and O73:H18 (strain 2996/96) (accession number AY365045). The exchanges in the amino acids are marked by connecting lines between the three CDT ORFs. The amino acid sequence homologies between CDT-III from STEC O153:H18 and CDT-V from STEC O91:H21 and O113:H21 (the latter two share 100% identical CDT-V sequences) are given above the three depicted ORFs. The amino acid sequence homologies between CDT-III from STEC O153:H18 and CDT-V from STEC O73:H18 are given below the three ORFs.

FIG.2.

Agarose gel electrophoresis (A) and hybridization with the cdtB probe (B) of EcoRV-digested genomic DNA from cdt-harboring STEC strains and controls. Lanes S, molecular mass markers (digoxigenin-labeled DNA Molecular Weight Marker II; Roche Biochemicals). In lanes 1 to 8, the following STEC strains (serotypes and cdt alleles are shown in parentheses) are displayed: lane 1, strain 9282/01 (serotype O91:H21, allele cdt-V); lane 2, 2596/02 (O91:H21, cdt-V); lane 3, 5249/01 (O113:H21, cdt-V); lane 4, 2896/01 (O113:H21, cdt-V); lane 5, 2996/96 (O73:H18, cdt-V); lane 6, 9063/02 (O153:H18, cdt-III); lane 7, 7272/02 (O153:H18, cdt-III); lane 8, 5107/00 (O153:H18, cdt-III). Lanes 9 to 11 contain control strains as follows: lane 9, strain 493/89 (SF STEC serotype O157:H⁻, allele cdt-V); lane 10, 1404 (NTEC O78:H?, cdt-III); lane 11, E. coli C600 (negative control). Arrows in panel A indicate the EcoRV fragments that hybridized with the cdtB probe.

FIG. 3.

Plasmid profiles (A) and plasmid hybridization with the cdtB probe (B) of cdt-harboring STEC strains and controls. Lane S, molecular mass markers (plasmids R27 [169 kb] and R100 [90 kb] [Roche Molecular Biochemicals] and a 54-kb plasmid from strain V517 [17]). In lanes 1 to 8, the following STEC strains (serotypes and cdt alleles are shown in parentheses) are displayed: lane 1, strain 9282/01 (serotype O91:H21, allele cdt-V); lane 2, 2596/02 (O91:H21, cdt-V); lane 3, 5249/01 (O113:H21, cdt-V); lane 4, 2896/01 (O113:H21, cdt-V); lane 5, 2996/96 (O73:H18, cdt-V); lane 6, 9063/02 (O153:H18, cdt-III); lane 7, 7272/02 (O153:H18, cdt-III); lane 8, 5107/00 (O153:H18, cdt-III). Lanes 9 to 11 contain control strains as follows: lane 9, strain 493/89 (SF STEC serotype O157:H⁻, allele cdt-V); lane 10, 1404 (NTEC O78:H?, cdt-III); lane 11, E. coli C600 (negative control). The arrows in panel A indicate the plasmids that hybridized with the cdtB probe. The sizes of the hybridizing plasmids are given in panel B.