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

Handle URI:
http://hdl.handle.net/10754/563710
Title:
A propidium monoazide–quantitative PCR method for the detection and quantification of viable Enterococcus faecalis in large-volume samples of marine waters
Authors:
Salam, Khaled W.; El-Fadel, Mutasem E.; Barbour, Elie K.; Saikaly, Pascal ( 0000-0001-7678-3986 )
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. © 2014 Springer-Verlag Berlin Heidelberg.
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division; Water Desalination and Reuse Research Center (WDRC); Environmental Science and Engineering Program; Environmental Biotechnology Research Group
Publisher:
Springer Nature
Journal:
Applied Microbiology and Biotechnology
Issue Date:
23-Aug-2014
DOI:
10.1007/s00253-014-6023-x
Type:
Article
ISSN:
01757598
Sponsors:
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.
Appears in Collections:
Articles; Environmental Science and Engineering Program; Water Desalination and Reuse Research Center (WDRC); Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorSalam, Khaled W.en
dc.contributor.authorEl-Fadel, Mutasem E.en
dc.contributor.authorBarbour, Elie K.en
dc.contributor.authorSaikaly, Pascalen
dc.date.accessioned2015-08-03T12:07:20Zen
dc.date.available2015-08-03T12:07:20Zen
dc.date.issued2014-08-23en
dc.identifier.issn01757598en
dc.identifier.doi10.1007/s00253-014-6023-xen
dc.identifier.urihttp://hdl.handle.net/10754/563710en
dc.description.abstractThe 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. © 2014 Springer-Verlag Berlin Heidelberg.en
dc.description.sponsorshipThis 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.en
dc.publisherSpringer Natureen
dc.subjectEnterococcus faecalisen
dc.subjectMarine recreational watersen
dc.subjectPropidium monoazideen
dc.subjectQuantitative PCRen
dc.titleA propidium monoazide–quantitative PCR method for the detection and quantification of viable Enterococcus faecalis in large-volume samples of marine watersen
dc.typeArticleen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.contributor.departmentWater Desalination and Reuse Research Center (WDRC)en
dc.contributor.departmentEnvironmental Science and Engineering Programen
dc.contributor.departmentEnvironmental Biotechnology Research Groupen
dc.identifier.journalApplied Microbiology and Biotechnologyen
dc.contributor.institutionDepartment of Civil and Environmental Engineering, American University of BeirutBeirut, Lebanonen
dc.contributor.institutionDepartment of Animal and Veterinary Sciences, American University of BeirutBeirut, Lebanonen
kaust.authorSaikaly, Pascalen
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