Applied Microbiology and Biotechnology

, Volume 102, Issue 22, pp 9793–9802 | Cite as

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-DiotEmail author
Methods and protocols


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.


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



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.


  1. Bankier C, Cheong Y, Mahalingam S, Edirisinghe M, Ren G, Cloutman-Green E, Ciric L (2018) A comparison of methods to assess the antimicrobial activity of nanoparticle combinations on bacterial cells. PLoS One 13:e0192093. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Baoguang L, Chen JQ (2013) Development of a sensitive and specific qPCR assay in conjunction with propidium monoazide for enhanced detection of live Salmonella spp. in food. BMC Microbiol 13:273. CrossRefGoogle Scholar
  3. Berney M, Hammes F, Bosshard F, Weilenmann HU, Egli T (2007) Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight kit in combination with flow cytometry. Appl Environ Microbiol 73:3283–3290. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Cangelosi GA, Meschke JS (2014) Dead or alive: molecular assessment of microbial viability. Appl Environ Microbiol 80:5884–5891. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Ding T, Suo Y, Xiang Q, Zhao X, Chen S, Ye X, Liu D (2017) Significance of viable but nonculturable Escherichia coli: induction, detection and control. J Microbiol Biotechnol 27:417–428. CrossRefPubMedGoogle Scholar
  6. Emerson JB, Adams RJ, Roman CMB, Brooks B, Coil DA, Dahlhausen K, Ganz HH, Hartmann EM, Hsu T, Justice NB, Paulino-Lima IG, Luongo JC, Lymperopoulou DS, Gomez-Silvan C, Rothschild-Mancinelli B, Balk M, Huttenhower C, Nocker A, Vaishampayan P, Rothschild LJ (2017) Schrödinger's microbes: tools for distinguishing the living from the dead in microbial ecosystems. Microbiome 5:86. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Evans DJ, Evans DG, DuPont HL, Orskov I (1977) Patterns of loss of enterotoxigenicity by Escherichia coli isolated from adults with diarrhea: suggestive evidence for an interrelationship with serotype. Infect Immun 17:105–111PubMedPubMedCentralGoogle Scholar
  8. Gensberger ET, Polt M, Konrad-Koszler M, Kinner P, Sessitsch A, Kostic T (2014) Evaluation of quantitative PCR combined with PMA treatment for molecular assessment of microbial water quality. Water Res 67:367–376. CrossRefPubMedGoogle Scholar
  9. Gill A (2017) The importance of bacterial culture to food microbiology in the age of genomics. Front Microbiol 8:777. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Guerra A, Etienne-Mesmin L, Livrelli V, Denis S, Blanquet-Diot S, Alric M (2012) Relevance and challenges in modeling human gastric and small intestinal digestion. Trends Biotechnol 30:591–600. CrossRefPubMedGoogle Scholar
  11. Huijsdens XW, Linskens RK, Mak M, Meuwissen SG, Vandenbroucke-Grauls CM, Savelkoul PH (2002) Quantification of bacteria adherent to gastrointestinal mucosa by real-time PCR. J Clin Microbiol 40:4423–4427CrossRefGoogle Scholar
  12. Kennedy D, Cronin UP, Wilkinson MG (2011) Responses of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus to simulated food processing treatments, determined using fluorescence-activated cell sorting and plate counting. Appl Environ Microbiol 77(13):4657–4668. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Li L, Mendis N, Trigui H, Oliver JD, Faucher SP (2014) The importance of the viable but non-culturable state in human bacterial pathogens. Front Microbiol 5:258. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Lothigius A, Sjoling A, Svennerholm AM, Bolin I (2010) Survival and gene expression of enterotoxigenic Escherichia coli during long-term incubation in sea water and freshwater. J Appl Microbiol 108:1441–1449. CrossRefPubMedGoogle Scholar
  15. Reyneke B, Ndlovu T, Khan S, Khan W (2017) Comparison of EMA-, PMA- and DNase qPCR for the determination of microbial cell viability. Appl Microbiol Biotechnol 101:7371–7383. CrossRefPubMedGoogle Scholar
  16. Roussel C, Cordonnier C, Galia W, Le Goff O, Thévenot J, Chalancon S, Alric M, Thevenot-Sergentet D, Leriche F, Van de Wiele T, Livrelli V, Blanquet-Diot S (2016) Increased EHEC survival and virulence gene expression indicate an enhanced pathogenicity upon simulated pediatric gastrointestinal conditions. Pediatr Res 80:734–743. CrossRefPubMedGoogle Scholar
  17. Roussel C, Sivignon S, Van de Wiele T, Blanquet-Diot S (2017) Foodborne enterotoxigenic Escherichia coli: from gut pathogenesis to new preventive strategies involving probiotics. Future Microbiol 12:73–93. CrossRefPubMedGoogle Scholar
  18. Shi H, Xu W, Luo Y, Chen L, Liang Z, Zhou X, Huang K (2011) The effect of various environmental factors on the etidium monazite and quantitative PCR method to detect viable bacteria. J Appl Microbiol 111:1194–1204. CrossRefPubMedGoogle Scholar
  19. Su CP, Jane WN, Wong HC (2013) Changes of ultrastructure and stress tolerance of Vibrio parahaemolyticus upon entering viable but nonculturable state. Int J Food Microbiol 160:360–366. CrossRefPubMedGoogle Scholar
  20. Tamburini S, Ballarini A, Ferrentino G, Moro A, Foladori P, Spilimbergo S, Jousson O (2013) Comparison of quantitative PCR and flow cytometry as cellular viability methods to study bacterial membrane permeabilization following supercritical CO2 treatment. Microbiology 159:1056–1066. CrossRefPubMedGoogle Scholar
  21. Trudeau K, Vu KD, Shareck F, Lacroix M (2012) Capillary electrophoresis separation of protein composition of γ-irradiated food pathogens Listeria monocytogenes and Staphylococcus aureus. PLoS One 7:e32488. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Villareal MLM, Padilha M, Vieira ADS, de Melo Franco BDG, Martinez RCR, Saad SMI (2013) Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by propidium monoazide – qPCR. PLoS One 8:e82102. CrossRefGoogle Scholar
  23. Zeng B, Zhao G, Cao X, Yang Z, Wang C, Hou L (2012) Formation and resuscitation of viable but nonculturable Salmonella typhi. Biomed Res Int 2013:907170–907177. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Zhao X, Zhong J, Wei C, Lin CW, Ding T (2017) Current perspectives on viable but non-culturable state in foodborne pathogens. Front Microbiol 8:580. CrossRefPubMedPubMedCentralGoogle Scholar

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
    Email author
  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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