Development of a High Resolution Virulence Allelic Profiling (HReVAP) Approach Based on the Accessory Genome of Escherichia coli to Characterize Shiga-Toxin Producing E. coli (STEC)

Abstract

Shiga-toxin producing Escherichia coli (STEC) strains possess a large accessory genome composed of virulence genes existing in multiple allelic variants, which sometimes segregate with specific STEC subpopulations. We analyzed the allelic variability of 91 virulence genes of STEC by Real Time PCR followed by melting curves analysis in 713 E. coli strains including 358 STEC. The 91 genes investigated were located on the locus of enterocyte effacement (LEE), OI-57, and OI-122 pathogenicity islands and displayed a total of 476 alleles in the study population. The combinations of the 91 alleles of each strain were termed allelic signatures and used to perform cluster analyses. We termed such an approach High Resolution Virulence Allelic Profiling (HReVAP) and used it to investigate the phylogeny of STEC of multiple serogroups. The dendrograms obtained identified groups of STEC segregating approximately with the serogroups and allowed the identification of subpopulations within the single groups. The study of the allelic signatures provided further evidence of the coevolution of the LEE and OI-122, reflecting the occurrence of their acquisition through a single event. The HReVAP analysis represents a sensitive tool for studying the evolution of LEE-positive STEC.

Keywords: STEC subtyping; accessory genome; allelic variants; bioinformatics; phylogenesis.

Figure 1

Allelic variability of the PAIs assayed. Number of alleles identified for each of the ORFs assayed and trend lines. (A) Alleles identified in the ORFs harbored by the LEE locus. (B) Alleles identified in the ORFs harbored by the OI-122. (C) Alleles identified in the ORFs harbored by the OI-57.

Figure 2

Amplification of HReVAP targets in STEC and EPEC strains. The results are reported as percentage of positive strains for different groups of samples, according to the color scale reported in the figure legend. (A) Results of the ORFs harbored by the LEE locus. (B) Results of the ORFs harbored by the OI-122. (C) Results of the ORFs harbored by the OI-57.

Figure 3

Clustering of the allelic signatures obtained with the HReVAP from STEC strains belonging to SPTs A and B. The different serogroups are labeled according to the following color legend: dark blue for O157, red for O26, green for O111, pale blue for O103, purple for O145, and black for O121. (A) Dendrogram of the allelic signatures from all the 91 ORFs. (B) Dendrogram of the allelic signatures obtained with the ORFs of the LEE locus. (C) Dendrogram of the allelic signatures obtained with the ORFs of the OI-122. (D) Dendrogram of the allelic signatures obtained with the ORFs of the OI-57.

Figure 4

Clustering of the allelic signatures obtained with the HReVAP from EPEC strains. The most represented serogroups are labeled according to the following color legend: red for O26, violet for O55, and blue-green for O127. (A) Dendrogram of the allelic signatures from all the 91 ORFs. (B) Dendrogram of the allelic signatures obtained with the ORFs of the LEE locus. (C) Dendrogram of the allelic signatures obtained with the ORFs of the OI-122. (D) Dendrogram of the allelic signatures obtained with the ORFs of the OI-57.

Figure 5

Clustering of the allelic signatures obtained with the HReVAP from the eae-negative STEC strains. Dendrogram of the allelic signatures obtained with the ORFs of the OI-57. The most represented serogroups are labeled according to the following color legend: crimson for O113, green for O111, pale blue for O103, orange for O91, and brown for O117.

Figure 6

Comparison of the dendrograms obtained by HReVAP, MLST, and WG-SNP typing. The different serogroups are labeled according to the following color legend: dark blue for O157, red for O26, green for O111, pale blue for O103, purple for O145, and black for O121. (A) Dendrogram of the allelic signatures obtained with the HReVAP typing. (B) Dendrogram obtained from the MLST typing. (C) Dendrogram obtained from the WG-SNP typing.