Applied Microbiology and Biotechnology

, Volume 98, Issue 20, pp 8707–8718 | Cite as

A propidium monoazide–quantitative PCR method for the detection and quantification of viable Enterococcus faecalis in large-volume samples of marine waters

  • Khaled W. Salam
  • Mutasem El-Fadel
  • Elie K. Barbour
  • Pascal E. SaikalyEmail author
Methods and protocols


The development of rapid detection assays of cell viability is essential for monitoring the microbiological quality of water systems. Coupling propidium monoazide with quantitative PCR (PMA-qPCR) has been successfully applied in different studies for the detection and quantification of viable cells in small-volume samples (0.25–1.00 mL), but it has not been evaluated sufficiently in marine environments or in large-volume samples. In this study, we successfully integrated blue light-emitting diodes for photoactivating PMA and membrane filtration into the PMA-qPCR assay for the rapid detection and quantification of viable Enterococcus faecalis cells in 10-mL samples of marine waters. The assay was optimized in phosphate-buffered saline and seawater, reducing the qPCR signal of heat-killed E. faecalis cells by 4 log10 and 3 log10 units, respectively. Results suggest that high total dissolved solid concentration (32 g/L) in seawater can reduce PMA activity. Optimal PMA-qPCR standard curves with a 6-log dynamic range and detection limit of 102 cells/mL were generated for quantifying viable E. faecalis cells in marine waters. The developed assay was compared with the standard membrane filter (MF) method by quantifying viable E. faecalis cells in seawater samples exposed to solar radiation. The results of the developed PMA-qPCR assay did not match that of the standard MF method. This difference in the results reflects the different physiological states of E. faecalis cells in seawater. In conclusion, the developed assay is a rapid (∼5 h) method for the quantification of viable E. faecalis cells in marine recreational waters, which should be further improved and tested in different seawater settings.


Enterococcus faecalis Marine recreational waters Propidium monoazide Quantitative PCR 



This work was supported by the National Council for Scientific Research, Lebanon and the University Research Board at the American University of Beirut. The authors thank Lucy Semerjian, Nesta Sagherian, and Houssam Shaib for their advice throughout the study. The authors also thank Mahmoud Hakim and Salam Abyad for their help in the design of the light apparatus.

Supplementary material

253_2014_6023_MOESM1_ESM.pdf (175 kb)
ESM 1 (PDF 175 kb)


  1. Agudelo RM, Codony F, Adrados B, Fittipaldi M, Peñuela G, Morató J (2010) Monitoring bacterial faecal contamination in waters using multiplex real-time PCR assay for Bacteroides spp. and faecal enterococci. Water SA 36:127–132Google Scholar
  2. Ahmed W, Stewart J, Gardner T, Powell D (2008) A real-time polymerase chain reaction assay for quantitative detection of the human-specific enterococci surface protein marker in sewage and environmental waters. Environ Microbiol 10:3255–3264PubMedCrossRefGoogle Scholar
  3. Bae S, Wuertz S (2009a) Discrimination of viable and dead fecal Bacteroidales bacteria by quantitative PCR with propidium monoazide. Appl Environ Microbiol 75:2940–2944PubMedCrossRefPubMedCentralGoogle Scholar
  4. Bae S, Wuertz S (2009b) Rapid decay of host-specific fecal Bacteroidales cells in seawater as measured by quantitative PCR with propidium monoazide. Water Res 43:4850–4859PubMedCrossRefGoogle Scholar
  5. Berney M, Vital M, Hülshoff I, Weilenmann H-U, Egli T, Hammes F (2008) Rapid, cultivation-independent assessment of microbial viability in drinking water. Water Res 42:4010–4018PubMedCrossRefGoogle Scholar
  6. Bogosian G, Aardema ND, Bourneuf EV, Morris PJ, O’Neil JP (2000) Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state. J Bacteriol 182:5070–5075PubMedCrossRefPubMedCentralGoogle Scholar
  7. Byappanahalli MN, Whitman RL, Shively DA, Nevers MB (2010) Linking non-culturable (qPCR) and culturable enterococci densities with hydrometeorological conditions. Sci Total Environ 408:3096–3101PubMedCrossRefGoogle Scholar
  8. Cawthorn D-M, Witthuhn RC (2008) Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide. J Appl Microbiol 105:1178–1185PubMedCrossRefGoogle Scholar
  9. Cenciarini-Borde C, Courtois S, La Scola B (2009) Nucleic acids as viability markers for bacteria detection using molecular tools. Future Microbiol 4:45–64PubMedCrossRefGoogle Scholar
  10. Delgado-Viscogliosi P, Solignac L, Delattre J-M (2009) Viability PCR, a culture-independent method for rapid and selective quantification of viable Legionella pneumophila cells in environmental water samples. Appl Environ Microbiol 75:3502–3512PubMedCrossRefPubMedCentralGoogle Scholar
  11. Ferretti JA, Tran HV, Cosgrove E, Protonentis J, Loftin V, Conklin CS, Grant RN (2011) Comparison of Enterococcus density estimates in marine beach and bay samples by real-time polymerase chain reaction, membrane filtration and defined substrate testing. Mar Pollut Bull 62:1066–1072PubMedCrossRefGoogle Scholar
  12. Fittipaldi M, Codony F, Adrados B, Camper AK, Morató J (2011) Viable real-time PCR in environmental samples: can all data be interpreted directly? Microb Ecol 61:7–12PubMedCrossRefGoogle Scholar
  13. Frahm E, Obst U (2003) Application of the fluorogenic probe technique (TaqMan PCR) to the detection of Enterococcus spp. and Escherichia coli in water samples. J Appl Microbiol 52:123–131CrossRefGoogle Scholar
  14. Gedalanga PB, Olson BH (2009) Development of a quantitative PCR method to differentiate between viable and nonviable bacteria in environmental samples. Appl Microbiol Biotechnol 82:587–596PubMedCrossRefGoogle Scholar
  15. Griffin DW, Lipp EK, McLaughlin MR, Rose JB (2001) Marine recreation and public health microbiology: quest for the ideal indicator. Biosci 51:817–825CrossRefGoogle Scholar
  16. Hammes F, Egli T (2010) Cytometric methods for measuring bacteria in water: advantages, pitfalls and applications. Anal Bioanal Chem 397:1083–1095PubMedCrossRefGoogle Scholar
  17. Hammes F, Berney M, Egli T (2011) Cultivation-independent assessment of bacterial viability. Adv Biochem Eng Biotechnol 124:123–150PubMedGoogle Scholar
  18. Haugland RA, Siefring SC, Wymer LJ, Brenner KP, Dufour AP (2005) Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis. Water Res 39:559–568PubMedCrossRefGoogle Scholar
  19. He J-W, Jiang S (2005) Quantification of enterococci and human adenoviruses in environmental samples by real-time PCR. Appl Environ Microbiol 71:2250–2255PubMedCrossRefPubMedCentralGoogle Scholar
  20. Keer JT, Birch L (2003) Molecular methods for the assessment of bacterial viability. J Microbiol Methods 53:175–183PubMedCrossRefGoogle Scholar
  21. Kell DB, Kaprelyants AS, Weichart DH, Harwood CR, Barer MR (1998) Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie Van Leeuwenhoek 73:169–187PubMedCrossRefGoogle Scholar
  22. Kim S-W, Raynor PC, Kuehn TH, Goyal SM, Ramakrishnan MA, Anantharaman S, Farnsworth JE (2008) Optimizing the recovery of surrogates for bacterial bioterrorism agents from ventilation filters. Clean 36:601–608Google Scholar
  23. Kobayashi H, Oethinger M, Tuohy MJ, Hall GS, Bauer TW (2009) Improving clinical significance of PCR: use of propidium monoazide to distinguish viable from dead Staphylococcus aureus and Staphylococcus epidermidis. J Orthop Res 27:1243–1247PubMedCrossRefGoogle Scholar
  24. Krüger N-J, Buhler C, Iwobi AN, Huber I, Ellerbroek L, Appel B, Stingl K (2014) “Limits of control”—crucial parameters for a reliable quantification of viable Campylobacter by real-time PCR. PLoS ONE 9:e88108PubMedCrossRefPubMedCentralGoogle Scholar
  25. Lleò MM, Tafi MC, Canepari P (1998) Nonculturable Enterococcus faecalis cells are metabolically active and capable of resuming active growth. Syst Appl Microbiol 21:333–339PubMedCrossRefGoogle Scholar
  26. Lleò MM, Bonato B, Signoretto C, Canepari P (2003) Vancomycin resistance is maintained in enterococci in the viable but nonculturable state and after division is resumed. Antimicrob Agents Chemother 47:1154–1156PubMedCrossRefPubMedCentralGoogle Scholar
  27. Lleò MM, Benedetti D, Tafi MC, Signoretto C, Canepari P (2007) Inhibition of the resuscitation from viable but non-culturable state in Enterococcus faecalis. Environ Microbiol 9:2313–2320PubMedCrossRefGoogle Scholar
  28. Luo J-F, Lin W-T, Guo Y (2010) Method to detect only viable cells in microbial ecology. Appl Microbiol Biotechnol 81:377–384CrossRefGoogle Scholar
  29. McFeters GA, Yu FP, Pyle BH, Stewart PS (1995) Physiological assessment of bacteria using fluorochromes. J Appl Microbiol 21:1–13CrossRefGoogle Scholar
  30. Nkuipou-Kenfack E, Engel H, Fakih S, Nocker A (2013) Improving efficiency of viability-PCR for selective detection of live cells. J Microbiol Methods 93:20–24PubMedCrossRefGoogle Scholar
  31. Nocker A, Camper AK (2009) Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques. FEMS Microbiol Lett 291:137–142PubMedCrossRefGoogle Scholar
  32. Nocker A, Cheung C-Y, Camper AK (2006) Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J Microbiol Methods 67:310–320PubMedCrossRefGoogle Scholar
  33. Nocker A, Sossa KE, Camper AK (2007a) Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR. J Microbiol Methods 70:252–260PubMedCrossRefGoogle Scholar
  34. Nocker A, Sossa-Fernandez P, Burr MD, Camper AK (2007b) Use of propidium monoazide for live/dead distinction in microbial ecology. Appl Environ Microbiol 73:5111–5117PubMedCrossRefPubMedCentralGoogle Scholar
  35. Nocker A, Mazza A, Masson L, Camper AK, Brousseau R (2009) Selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology. J Microbiol Methods 76:253–261PubMedCrossRefGoogle Scholar
  36. Pan Y, Breidt F (2007) Enumeration of viable Listeria monocytogenes cells by real-time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells. Appl Environ Microbiol 73:8028–8031PubMedCrossRefPubMedCentralGoogle Scholar
  37. Pruzzo C, Tarsi R, Lleò MM, Signoretto C, Zampini M, Colwell RR, Canepari P (2002) In vitro adhesion to human cells by viable but nonculturable Enterococcus faecalis. Curr Microbiol 45:105–110PubMedCrossRefGoogle Scholar
  38. Sassoubre LM, Nelson KL, Boehm AB (2012) Mechanisms for photoinactivation of Enterococcus faecalis in seawater. Appl Environ Microbiol 78:7776–7785PubMedCrossRefPubMedCentralGoogle Scholar
  39. Shapiro SS, Wilk MB (1965) An analysis of variance test for normality. Biometrika 52:591–611CrossRefGoogle Scholar
  40. Siefring S, Varma M, Atikovic E, Wymer L, Haugland RA (2008) Improved real-time PCR assays for the detection of fecal indicator bacteria in surface waters with different instrument and reagent systems. J Water Health 6:225–237PubMedCrossRefGoogle Scholar
  41. Taskin B, Gul Gozen A, Duran M (2011) Selective quantification of viable Escherichia coli bacteria in biosolids by quantitative PCR with propidium monoazide modification. Appl Environ Microbiol 77:4329–4335PubMedCrossRefPubMedCentralGoogle Scholar
  42. U.S. Environmental Protection Agency (2009) Method 1600: Enterococci in water by membrane filtration using membrane-Enterococcus indoxyl-β-D-glucoside agar (mEI). U.S. Environmental Protection Agency, Washington, DCGoogle Scholar
  43. Varma M, Field R, Stinson M, Rukovets B, Wymer L, Haugland R (2009) Quantitative real-time PCR analysis of total and propidium monoazide-resistant fecal indicator bacteria in wastewater. Water Res 43:4790–4801PubMedCrossRefGoogle Scholar
  44. Vesper S, McKinstry C, Hartmann C, Neace M, Yoder S, Vesper A (2008) Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA). J Microbiol Methods 72:180–184PubMedCrossRefGoogle Scholar
  45. Wade TJ, Calderon RL, Sams E, Beach M, Brenner KP, Williams AH, Dufour AP (2006) Rapidly measured indicators of recreational water quality are predictive of swimming-associated gastrointestinal illness. Environ Health Perspect 114:24–28PubMedCrossRefPubMedCentralGoogle Scholar
  46. Wagner AO, Malin C, Knapp BA, Illmer P (2008) Removal of free extracellular DNA from environmental samples by ethidium monoazide and propidium monoazide. Appl Environ Microbiol 74:2537–2539PubMedCrossRefPubMedCentralGoogle Scholar
  47. Yamahara KM, Walters SP, Boehm AB (2009) Growth of Enterococci in unaltered, unseeded beach sands subjected to tidal wetting. Appl Environ Microbiol 75:1517–1524PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Khaled W. Salam
    • 1
  • Mutasem El-Fadel
    • 1
  • Elie K. Barbour
    • 2
  • Pascal E. Saikaly
    • 3
    Email author
  1. 1.Department of Civil and Environmental EngineeringAmerican University of BeirutBeirutLebanon
  2. 2.Department of Animal and Veterinary SciencesAmerican University of BeirutBeirutLebanon
  3. 3.Water Desalination and Reuse Center, Division of Biological and Environmental Sciences and EngineeringKing Abdullah University of Science and TechnologyThuwalKingdom of Saudi Arabia

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