Identification of Genetic Markers for Differentiation of Shiga Toxin-Producing, Enteropathogenic, and Avirulent Strains ofEscherichia coliO26

Abstract

Shiga toxin-producing Escherichia coli (STEC) O26 is one of the top five enterohemorrhagic E. coli (EHEC) O groups most often associated with hemorrhagic colitis and hemolytic uremic syndrome (HUS) worldwide. STEC O26 is considered to have evolved from enteropathogenic (EPEC) O26 strains through the acquisition of Shiga toxin (Stx)-encoding genes. Our PCR data identified several STEC-like strains expressing all features of STEC except Stx production and carrying remnants of Stx phages that were probably derivatives of EHEC O26. EHEC and EPEC O26 strains phenotypically resemble O26 EHEC-like and apathogenic E. coli O26 strains and are therefore undistinguishable by cultural methods. A clear discrimination between the different O26 groups is required for diagnostics in patients and for control of food safety. To develop an assay for specific detection of EHEC and EHEC-like O26 strains, we used a high-throughput PCR approach for selection of discriminative genetic markers among 33 tested genes mostly encoding type III secretion system effector proteins. The genes ECs1822, nleH1-2, nleA, nleC, nleH1-1, nleG, nleG2, nleG6-1, nleG6-2, espJ, espM2, nleG8-2, espG, ent (or espL2), nleB, nleE, efa1, and espB were detected at different frequencies in O26 EHEC, EHEC-like, and EPEC strains, indicating the possible role of these genes in virulence of human pathogenic O26 strains. The espK and espN genes were detected only in EHEC and EHEC-like O26 strains. espK was present in 99.14% of EHEC and 91.14% of EHEC-like O26 strains and was hence the best candidate as a genetic marker for characterizing these pathogroups. These data were corroborated by a genotyping real-time PCR test based on allelic discrimination of the arcA (aerobic respiratory control protein A) gene. The results indicate that a combination of molecular detection tools for O26 wzx (wzx(O26)), eae-beta, stx, espK, and arcA genotyping is highly discriminative for clear identification of EHEC and EHEC-like E. coli O26 strains. This simple diagnostic test might be applicable in hospital service laboratories or public health laboratories to test strains isolated from stools of patients suffering from diarrhea.

Use of Clustered Regularly Interspaced Short Palindromic Repeat Sequence Polymorphisms for Specific Detection of Enterohemorrhagic Escherichia coli Strains of Serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 by Real-Time PCR

“…It is likely that these strains are remnants of EHEC strains which have lost their stx genes on subculture. It was previously described that such strains are frequent among E. coli O26:H11 isolates and still carry sequences of truncated stx phages in their genomes (3).…”

Identification of Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli in diarrhoeic calves and comparative genomics of O5 bovine and human STEC

“…Scores of AE-STEC O:H serotypes have been associated with HC and/or HUS in humans, but a few ones are also responsible for diarrhoea in young calves, namely O26:H11 and O111:H-and also O5:H-and O118:H16 that are considered as emerging serotypes in humans. In addition, some EPEC belonging mainly to the O26:H11 serotype also isolated from ruminants, including diarrhoeic calves could be AE-STEC having lost their stx genes (Bugarel et al, 2011;Moxley and Smith, 2010;Mainil and Fairbrother, 2014;Tozzoli and Scheutz, 2014;Beutin and Fach, 2015;Ferdous et al, 2015). Nevertheless, since most of those data are from the years 1980 and 1990, the purpose of this study was therefore to update the information on AE-STEC and EPEC from diarrhoeic calves.…”

“…Another interesting finding is the high number of Ehly-positive EPEC that were identified. Since Ehly is coded by pEHEC plasmid-located genes, future studies should identify the plasmid profiles of those EPEC and the ehxA gene localization (Tozzoli and Scheutz, 2014) and complete their virulotyping to confirm that they are not AE-STEC having lost the stx gene(s) (Bugarel et al, 2011;Ferdous et al, 2015).…”

Characteristics of Emerging Human-Pathogenic Escherichia coli O26:H11 Strains Isolated in France between 2010 and 2013 and Carrying the stx 2d Gene Only

“…Chromosomally encoded virulence factors such as the locus of enterocyte effacement (LEE) effectors and some type III secretion system effectors were also found to be conserved in the phylogenetic group ST21 (21). In the context of a molecular risk assessment strategy, we previously described a combination of molecular markers for specific identification of EHEC and EHEC-like O26:H11 strains.…”

“…In the context of a molecular risk assessment strategy, we previously described a combination of molecular markers for specific identification of EHEC and EHEC-like O26:H11 strains. Assays for these markers included wzx O26 , fliC H11 , eae-␤, stx, espK, and arcA single-nucleotide polymorphism (SNP) genotyping (21), as well as a set of PCR tests (SP_O26_C and SP_O26_D) targeting the clus-tered regularly interspaced short palindromic repeat (CRISPR) locus of EHEC O26:H11 (22).…”