Genetic analysis of enteropathogenic and enterohemorrhagic Escherichia coli serogroup O103 strains by molecular typing of virulence and housekeeping genes and pulsed-field gel electrophoresis

Abstract

We investigated the genetic relationships of 54 Escherichia coli O103 strains from humans, animals, and meat by molecular typing of housekeeping and virulence genes and by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) of seven housekeeping genes revealed seven profiles, I through VII. MLST profiles I plus III cover 45 Shiga toxin-producing E. coli (STEC) O103:H2 strains from Australia, Canada, France, Germany, and Northern Ireland that are characterized by the intimin (eae) epsilon gene and carry enterohemorrhagic E. coli (EHEC) virulence plasmids. MLST profile II groups five human and animal enteropathogenic E. coli (EPEC) O103:H2 strains that were positive for intimin (eae) beta. Although strains belonging to MLST groups II and I plus III are closely related to each other (92.6% identity), major differences were found in the housekeeping icdA gene and in the virulence-associated genes eae and escD. E. coli O103 strains with MLST patterns IV to VII are genetically distant from MLST I, II, and III strains, as are the non-O103 E. coli strains EDL933 (O157), MG1655 (K-12), and CFT073 (O6). Comparison of MLST results with those of PFGE and virulence typing demonstrated that E. coli O103 STEC and EPEC have recently acquired different virulence genes and DNA rearrangements, causing alterations in their PFGE patterns. PFGE typing was very useful for identification of genetically closely related subgroups among MLST I strains, such as Stx2-producing STEC O103 strains from patients with hemolytic uremic syndrome. Analysis of virulence genes contributed to grouping of E. coli O103 strains into EPEC and STEC. Novel virulence markers, such as efa (EHEC factor for adherence), paa (porcine adherence factor), and cif (cell cycle-inhibiting factor), were found widely associated with E. coli O103 EPEC and STEC strains.

FIG. 1.

fliC PCR: agarose gel electrophoresis of undigested and HhaI-digested fliC PCR products of E. coli H2, H7, and H11 reference strains and corresponding O103 strains. Lanes M, molecular weight marker (Hyperladder IV; Bioline, Luckenwalde, Germany); lanes 1 and 2, undigested fliC PCR products of strain Bi7455-41 (O43:H2) and 1557/00 (O103:H2); lanes 3 and 4, HhaI-digested products from lanes 1 and 2; lanes 5 and 6, undigested fliC PCR products of strain U5-41 (O1:K1:H7) and CA92-7164 (O103:[H7]); lanes 7 and 8, HhaI-digested products from lanes 5 and 6; lanes 9 and 10, undigested fliC PCR products of strain Su4321-41 (O13:K11:H11) and 92-1695 (O103:[H11]); lanes 11 and 12, HhaI-digested products from lanes 9 and 10.

FIG. 2.

Comparison of PFGE (uppercase letters) and MLST (Roman numerals) patterns. The MLST dendrogram is based on the analysis of seven housekeeping genes and was calculated with the S.T.A.R.T software. For this purpose, allele numbers of all sequences were determined, and a distance matrix was calculated. MLST types are indicated at the branches of the tree. The labeled rectangles indicate PFGE clusters as described in the text.

FIG. 3.

Split decomposition analysis using a distance matrix calculated with the alleles of housekeeping genes of 57 E. coli strains. The Roman numerals correspond to the MLST profiles in Table 4.

FIG. 4.

Split decomposition analysis with single genes using the DNA sequences of 57 strains. The splits depict conflicts in the phylogenetic descent of strains. O103 represents all other E. coli O103 strains in this study.

FIG. 5.

Dendrogram of PFGE patterns calculated with the Dice coefficient. The grey rectangles at the left indicate PFGE clusters and the percentages of similarity. Strains and MLST profiles are indicated on the right side.

FIG. 6.

Agarose gel electrophoresis of PCR products with primers specific for cif and cif-flanking regions. Lanes 1 and 14, molecular size markers (1-kb ladder). A, primers cif-int-s and cif-int-as; B, primers cif-rbs-s and cif-as; C, primers phage-s and ybhb-s. Lanes 2, 6, and 10, strain E22; lanes 3, 7, and 11, PMK5; lanes 4, 8, and 12, GSO740-1; lanes 5, 9, and 13, CB8086.