Abstract
It would be desirable to have an unambiguous scheme for the typing of Shiga toxin-producing Escherichia coli (STEC) isolates to subpopulations. Such a scheme should take the high genomic plasticity of E. coli into account and utilize the stratification of STEC into subgroups, based on serotype or phylogeny. Therefore, our goal was to identify specific marker combinations for improved classification of STEC subtypes. We developed and evaluated two bioinformatic pipelines for genomic marker identification from larger sets of bacterial genome sequences. Pipeline A performed all-against-all BLASTp analyses of gene products predicted in STEC genome test sets against a set of control genomes. Pipeline B identified STEC marker genes by comparing the STEC core proteome and the “pan proteome” of a non-STEC control group. Both pipelines defined an overlapping, but not identical set of discriminative markers for different STEC subgroups. Differential marker prediction resulted from differences in genome assembly, ORF finding and inclusion cut-offs in both workflows. Based on the output of the pipelines, we defined new specific markers for STEC serogroups and phylogenetic groups frequently associated with outbreaks and cases of foodborne illnesses. These included STEC serogroups O157, O26, O45, O103, O111, O121, and O145, Shiga toxin-positive enteroaggregative E. coli O104:H4, and HUS-associated sequence type (ST)306. We evaluated these STEC marker genes for their presence in whole genome sequence data sets. Based on the identified discriminative markers, we developed a multiplex PCR (mPCR) approach for detection and typing of the targeted STEC. The specificity of the mPCR primer pairs was verified using well-defined clinical STEC isolates as well as isolates from the ECOR, DEC, and HUSEC collections. The application of the STEC mPCR for food analysis was tested with inoculated milk. In summary, we evaluated two different strategies to screen large genome sequence data sets for discriminative markers and implemented novel marker genes found in this genome-wide approach into a DNA-based typing tool for STEC that can be used for the characterization of STEC from clinical and food samples.
Highlights
Shiga toxin-producing Escherichia coli (STEC) have a serious global health impact
Our genome-wide search for discriminative STEC markers identified new targets for detection and typing of different STEC subgroups. The combination of these novel chromosomal regions specific for the serogroup and for corresponding clonal groups with the STEC standard markers stx and eae resulted in a robust, specific and reliable typing of the clinically most relevant STEC variants and can improve risk analysis of STEC isolates by in silico typing based on NGS data or by multiplex PCR (mPCR)
Correct and timely identification of STEC isolates is crucial for food microbiology for market access testing as well as for surveillance of STEC-mediated disease
Summary
Shiga toxin-producing Escherichia coli (STEC) have a serious global health impact. An infection with STEC can lead to diarrhea, hemorrhagic colitis and in some cases hemolytic uremic syndrome (HUS) (Croxen et al, 2013). STEC have the potential to cause large outbreaks with hundreds of hospitalizations and deaths (Terajima et al, 2014; Fruth et al, 2015; Heiman et al, 2015; Yeni et al, 2016). Most of these outbreaks and severe cases of disease worldwide are caused by a limited number of strains including serogroup O157 and the so-called “Big Six” serogroups O26, O45, O103, O111, O121, and O145 (Brooks et al, 2005; Karch et al, 2005). Many other STEC variants are pathogenic (Blanco et al, 2004; Johnson et al, 2006; Mellmann et al, 2008)
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