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Comparison of conventional plating, PMA-qPCR, and flow cytometry for the determination of viable enterotoxigenic Escherichia coli along a gastrointestinal in vitro model

  • C. Roussel
  • W. Galia
  • F. Leriche
  • S. Chalancon
  • S. Denis
  • T. Van de Wiele
  • S. Blanquet-Diot
Methods and protocols
  • 53 Downloads

Abstract

Recent technological advances for bacterial viability assessment using molecular methods or flow cytometry can provide meaningful interest for the demarcation between live and dead microorganisms. Nonetheless, these methods have been scarcely applied to foodborne pathogens and never for directly assessing their viability within the human digestive environment. The purpose of this study was to compare two methods based on membrane integrity (propidium monoazide (PMA) q-PCR and Live/Dead flow cytometry) and the classical plate-count method to determine the viability of a common foodborne pathogen, enterotoxigenic Escherichia coli (ETEC), during its transit trough simulated human gastrointestinal environment. Viable ETEC counts in the gastric and small intestinal compartments of the gastrointestinal TIM model indicated a consensus between the three tested methods (PMA-qPCR, flow cytometry, and plate counts). In a further step, flow cytometry analysis appeared as the preferred method to elucidate ETEC physiological states in the in vitro digestive environment by discriminating four subpopulations, while PMA-qPCR can only distinguish two. The defined viable/altered ETEC population was found during all in vitro digestions, but mainly in the gastric compartment. Being able to discriminate the particular physiological states of pathogenic microorganisms in the digestive environment is of high interest, because if some cells are not observable on culture media, they might keep their ability to express virulence functions.

Keywords

Enterotoxigenic Escherichia coli Digestive environment Viability Plating PMA-qPCR Flow cytometry 

Notes

Acknowledgements

This work was supported by a fellowship from Ministère de la Recherche (France) to Charlène Roussel.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • C. Roussel
    • 1
    • 2
  • W. Galia
    • 3
  • F. Leriche
    • 4
  • S. Chalancon
    • 1
  • S. Denis
    • 1
  • T. Van de Wiele
    • 2
  • S. Blanquet-Diot
    • 1
  1. 1.UMR UCA-INRA 454 MEDIS, Microbiology Digestive Environment and HealthUniversity of Clermont-AuvergneClermont-FerrandFrance
  2. 2.CMET, Center for Microbial Ecology and TechnologyGhent UniversityGhentBelgium
  3. 3.UMR 5557 Microbial Ecology, Research group on bacterial opportunistic pathogens and environment, CNRSLyonFrance
  4. 4.Unité de recherche FromagèreVetAgro SupLempdesFrance

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