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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

Abstract

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.

Keywords

Enterococcus faecalis Marine recreational waters Propidium monoazide Quantitative PCR 

Notes

Acknowledgments

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)

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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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