Identification of Novel Biomarkers for Priority Serotypes of Shiga Toxin-Producing Escherichia coli and the Development of Multiplex PCR for Their Detection
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 ph...
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doaj-9a0779edb1884f658d97764091a0a4832020-11-25T02:25:19ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2018-06-01910.3389/fmicb.2018.01321372698Identification of Novel Biomarkers for Priority Serotypes of Shiga Toxin-Producing Escherichia coli and the Development of Multiplex PCR for Their DetectionMatthias Kiel0Pierre Sagory-Zalkind1Céline Miganeh2Christoph Stork3Andreas Leimbach4Camilla Sekse5Alexander Mellmann6François Rechenmann7Ulrich Dobrindt8Institute of Hygiene, University of Münster, Münster, GermanyGenostar Bioinformatics, Montbonnot-Saint-Martin, FranceGenostar Bioinformatics, Montbonnot-Saint-Martin, FranceInstitute of Hygiene, University of Münster, Münster, GermanyInstitute of Hygiene, University of Münster, Münster, GermanyNorwegian Veterinary Institute, Oslo, NorwayInstitute of Hygiene, University of Münster, Münster, GermanyGenostar Bioinformatics, Montbonnot-Saint-Martin, FranceInstitute of Hygiene, University of Münster, Münster, GermanyIt 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.https://www.frontiersin.org/article/10.3389/fmicb.2018.01321/fullSTECO157non-O157multiplex PCRcomparative genomics |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Matthias Kiel Pierre Sagory-Zalkind Céline Miganeh Christoph Stork Andreas Leimbach Camilla Sekse Alexander Mellmann François Rechenmann Ulrich Dobrindt |
spellingShingle |
Matthias Kiel Pierre Sagory-Zalkind Céline Miganeh Christoph Stork Andreas Leimbach Camilla Sekse Alexander Mellmann François Rechenmann Ulrich Dobrindt Identification of Novel Biomarkers for Priority Serotypes of Shiga Toxin-Producing Escherichia coli and the Development of Multiplex PCR for Their Detection Frontiers in Microbiology STEC O157 non-O157 multiplex PCR comparative genomics |
author_facet |
Matthias Kiel Pierre Sagory-Zalkind Céline Miganeh Christoph Stork Andreas Leimbach Camilla Sekse Alexander Mellmann François Rechenmann Ulrich Dobrindt |
author_sort |
Matthias Kiel |
title |
Identification of Novel Biomarkers for Priority Serotypes of Shiga Toxin-Producing Escherichia coli and the Development of Multiplex PCR for Their Detection |
title_short |
Identification of Novel Biomarkers for Priority Serotypes of Shiga Toxin-Producing Escherichia coli and the Development of Multiplex PCR for Their Detection |
title_full |
Identification of Novel Biomarkers for Priority Serotypes of Shiga Toxin-Producing Escherichia coli and the Development of Multiplex PCR for Their Detection |
title_fullStr |
Identification of Novel Biomarkers for Priority Serotypes of Shiga Toxin-Producing Escherichia coli and the Development of Multiplex PCR for Their Detection |
title_full_unstemmed |
Identification of Novel Biomarkers for Priority Serotypes of Shiga Toxin-Producing Escherichia coli and the Development of Multiplex PCR for Their Detection |
title_sort |
identification of novel biomarkers for priority serotypes of shiga toxin-producing escherichia coli and the development of multiplex pcr for their detection |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Microbiology |
issn |
1664-302X |
publishDate |
2018-06-01 |
description |
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. |
topic |
STEC O157 non-O157 multiplex PCR comparative genomics |
url |
https://www.frontiersin.org/article/10.3389/fmicb.2018.01321/full |
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