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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Microbiol. | doi: 10.3389/fmicb.2018.01321

Identification of novel biomarkers for priority serotypes of Shiga toxin-producing Escherichia coli and the development of multiplex PCR for their detection

 Matthias Kiel1, Pierre Sagory-Zalkind2, Céline Miganeh2,  Christoph Stork1,  Andreas Leimbach1,  Camilla Sekse3, Alexander Mellmann1, Francois Rechenmann2 and  Ulrich Dobrindt1*
  • 1Universität Münster, Germany
  • 2Genostar Bioinformatics Solutions, France
  • 3Norwegian Veterinary Institute, Norway

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.

Keywords: STEC, O157, non-O157, multiplex PCR, Comparative genomics, Strain typing, Escherichia coli

Received: 09 Mar 2018; Accepted: 30 May 2018.

Edited by:

Jennifer Ronholm, McGill University, Canada

Reviewed by:

Angelica Reyes-Jara, Universidad de Chile, Chile
Alexander Gill, Health Canada, Canada  

Copyright: © 2018 Kiel, Sagory-Zalkind, Miganeh, Stork, Leimbach, Sekse, Mellmann, Rechenmann and Dobrindt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Dr. Ulrich Dobrindt, Universität Münster, Münster, Germany, dobrindt@uni-muenster.de