Skip to main content
Log in

Comparison of Fecal Indicator Bacteria Densities in Marine Recreational Waters by QPCR

  • Published:
Water Quality, Exposure and Health Aims and scope Submit manuscript

Abstract

The US EPA is currently investigating the use of quantitative PCR (qPCR) analysis techniques to estimate densities of fecal indicator bacteria (FIB) in recreational waters. Present water quality guidelines, based on culturable FIB, prevent same day water quality determination, whereas results from qPCR-based approaches are available within several hours. Epidemiological studies at Publicly-Owned Treatment Works (POTW)-impacted freshwater beaches have also indicated correlations between qPCR determined Enterococcus densities and swimming-related illness rates. Similar qPCR assays are now available for several other accepted or emerging FIB groups. This study provides an initial assessment of qPCR estimated Enterococcus, Bacteroidales, E. coli and Clostridium spp. densities in marine water and sand samples collected over one summer from two POTW-impacted recreational beaches. Relative target sequence densities of these organisms in the samples did not correspond with their relative estimated cell densities. These observations were attributable to differences in target sequences recovered from the calibrator cells of the different types of organisms. Comparative cycle threshold (CT) qPCR analyses of whole cell calibrator samples provide a simple and standardizable approach for estimating both total cell and target sequence densities of different types of FIB in water. Cell density estimates obtained by this approach are subject to uncertainty due to potential variability in absolute numbers of target sequences in the target organisms under different physiological or environmental conditions, but still may allow for informative comparisons with the target sequence estimates.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Anderson KL, Whitlock JE, Harwood VJ (2005) Persistence and differential survival of fecal indicator bacteria in subtropical waters and sediments. Appl Environ Microbiol 71(6):3041–3048

    Article  CAS  Google Scholar 

  • Applied Biosystems (1997) ABI Prism 7700 Sequence Detection System. User Bulletin #2. Applied Biosystems Corporation, Foster City, CA, USA

  • Bej AK, DiCesare JL, Haff L, Atlas RM (1991) Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid. Appl Environ Microbiol 57(4):1013–1017

    CAS  Google Scholar 

  • Bernhard AE, Field KG (2000) A PCR assay to discriminate human and ruminant feces on the basis of host differences in bacteroides-prevotella gene encoding 16S rRNA. Appl Environ Microbiol 66(10):4571–4574

    Article  CAS  Google Scholar 

  • Bisson JW, Cabelli VJ (1979) Membrane filter enumeration method for Clostridium perfringens. Appl Environ Microbiol 37(1):55–66

    CAS  Google Scholar 

  • Boehm AB (2007) Enterococci concentrations in diverse coastal environments exhibit extreme variability. Environ Sci Technol 41(24):8227–8232

    Article  CAS  Google Scholar 

  • Brinkman NE, Haugland RA, Wymer L, Byappanahalli M, Whitman RL, Vesper SJ (2003) Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water. Appl Environ Microbiol 69(3):1775–1782

    Article  CAS  Google Scholar 

  • Cole JR, Chai B, Farris RJ, Wang Q, Kulam-Syed-Mohideen AS, McGarrell DM, Bandela AM, Cardenas E, Garrity GM, Tiedje JM (2007) The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data. Nucleic Acids Res 35:169–172

    Article  Google Scholar 

  • Desmarais TR, Solo-Gabriele HM, Palmer CJ (2002) Influence of soil on fecal indicator organisms in a tidally influenced subtropical environment. Appl Environ Microbiol 68(3):1165–1172

    Article  CAS  Google Scholar 

  • Dick LK, Field KG (2004) Rapid estimation of numbers of fecal Bacteroidetes by use of a quantitative PCR assay for 16S rRNA genes. Appl Environ Microbiol 70(9):5695–5697

    Article  CAS  Google Scholar 

  • Donovan E, Unice K, Roberts JD, Harris M, Finley B (2008) Risk of gastrointestinal disease associated with exposure to pathogens in the water of the Lower Passaic River. Appl Environ Microbiol 74(4):994–1003

    Article  CAS  Google Scholar 

  • Dufour AP (1984) Health effects criteria for fresh recreational waters. US Environmental Protection Agency, Washington, DC

  • Fiksdal L, Maki JS, LaCroix SJ, Staley JT (1985) Survival and detection of Bacteroides spp., prospective indicator bacteria. Appl Environ Microbiol 49(1):148–150

    CAS  Google Scholar 

  • Fujioka RS, Shizumura LK (1985) Clostridium perfringens, a reliable indicator of stream water quality. J Water Pollut Control Fed 57(10):986–992

    Google Scholar 

  • Guy RA, Payment P, Krull U, Horgen PA (2003) Real-time PCR for quantification of giardia and cryptosporidium in environmental water samples and sewage. Appl Environ Microbiol 69(9):5178–5185

    Article  CAS  Google Scholar 

  • Gyles C, Johnson R, Gao A, Ziebell K, Pierard D, Aleksic S, Boerlin P (1998) Association of enterohemorrhagic Escherichia coli hemolysin with serotypes of shiga-like-toxin-producing Escherichia coli of human and bovine origins. Appl Environ Microbiol 64(11):4134–4141

    CAS  Google Scholar 

  • Haugland R, Siefring S, Varma M, Atikovic E, Brenner K, Dufour A (2006) Evaluation of matrix effects from nationwide inland surface waters on quantitation polymerase chain reaction analysis for fecal indicator bacteria. National Beaches Conference, Niagara Falls, NY, 11–13 October

  • Haugland RA, Siefring S, Wymer L, Brenner K, Dufour A (2005) Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis. Water Res 39:559–568

    Article  CAS  Google Scholar 

  • Heid C, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994

    Article  CAS  Google Scholar 

  • Jackson JH, Schmidt TM, Herring PA (2008) A systems approach to model natural variation in reactive properties of bacterial ribosomes. BMC Syst Biol 2:62

    Article  CAS  Google Scholar 

  • Khan IU, Gannon V, Kent R, Koning W, Lapen DR, Miller J, Neumann N, Phillips R, Robertson W, Topp E, van Bochove E, Edge TA (2007) Development of a rapid quantitative PCR assay for direct detection and quantification of culturable and non-culturable Escherichia coli from agriculture watersheds. J Microbiol Methods 69(3):480–488

    Article  CAS  Google Scholar 

  • Kildare BJ, Leutenegger CM, McSwain BS, Bambic DG, Rajal VB, Wuertz S (2007) 16S rRNA-based assays for quantitative detection of universal, human-, cow-, and dog-specific fecal Bacteroidales: a Bayesian approach. Water Res 41(16):3701–3715

    Article  CAS  Google Scholar 

  • Kim JH, Grant SB (2004) Public mis-notification of coastal water quality: a probabilistic evaluation of posting errors at Huntington Beach, California. Environ Sci Technol 38(9):2497–2504

    Article  CAS  Google Scholar 

  • Klappenbach JA, Saxman PR, Cole JR, Schmidt TM (2001) rrndb: the ribosomal RNA operon copy number database. Nucleic Acids Res 29(1):181–184

    Article  CAS  Google Scholar 

  • Kreader CA (1995) Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution. Appl Environ Microbiol 61:1171–1179

    CAS  Google Scholar 

  • Kurmayer R, Kutzenberger T (2003) Application of real-time PCR for quantification of microcystin genotypes in a population of the toxic cyanobacterium microcystis sp. Appl Environ Microbiol 69(11):6723–6730

    Article  CAS  Google Scholar 

  • Layton A, McKay L, Williams D, Garrett V, Gentry R, Sayler G (2006) Development of Bacteroides 16S rRNA gene taqman-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water. Appl Environ Microbiol 72(6):4214–4224

    Article  CAS  Google Scholar 

  • Ludwig W, Schleifer KH (2000) How quantitative is quantitative PCR with respect to cell counts? Syst Appl Microbiol 23(4):556–562

    CAS  Google Scholar 

  • Matsuki T, Watanabe K, Fujimoto J, Takada T, Tanaka R (2004) Use of 16S rRNA gene-targeted group-specific primers for real-time PCR analysis of predominant bacteria in human feces. Appl Environ Microbiol 70(12):7220–7228

    Article  CAS  Google Scholar 

  • Mazel D, Houmard J, Castets AM, Tandeau de Marsac, N. (1990) Highly repetitive DNA sequences in cyanobacterial genomes. J Bacteriol 172(5):2755–2761

    CAS  Google Scholar 

  • Medema GJ, Bahar M, Schets FM (1997) Survival of Cryptosporidium parvum, Escherichia coli, Faecal enterococci and Clostridium perfringens in river water: influence of temperature and autochthonous microorganisms. Water Sci Tech 35(11–12):249–252

    CAS  Google Scholar 

  • Noble RT, Griffith J, Blackwood D, Fuhrman J, Gregory J, Hernandez X, Liang X, Bera A, Schiff K (2006) Multitiered approach using quantitative PCR to track sources of fecal pollution affecting Santa Monica Bay, California. Appl Environ Microbiol 72(2):1604–1612

    Article  CAS  Google Scholar 

  • Noble RT, Moore DF, Leecaster MK, McGee CD, Weisberg SB (2003) Comparison of total coliform, fecal coliform and Enterococcus bacterial indicator response for ocean recreational water quality testing. Water Res 37:1637–1643

    Article  CAS  Google Scholar 

  • Okabe S, Okayama N, Savichtcheva O, Ito T (2007) Quantification of host-specific Bacteroides-Prevotella 16S rRNA genetic markers for assessment of fecal pollution in freshwater. Appl Microbiol Biotechnol 74(4):890–901

    Article  CAS  Google Scholar 

  • Read SC, Clarke RC, Martin A, De Grandis SA, Hii J, McEwen S, Gyles CL (1992) Polymerase chain reaction for detection of verocytotoxigenis Escherichia coli isolated from animal and food sources. Mol Cell Probes 6(2):153–161

    Article  CAS  Google Scholar 

  • Rinttilä T, Kassinen A, Malinen E, Krogius L, Plava A (2004) Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR. J Appl Microbiol 97:1166–1177

    Article  CAS  Google Scholar 

  • Savichtcheva O, Okabe S (2006) Alternative indicators of fecal pollution: relations with pathogens and conventional indicators, current methodologies for direct pathogen monitoring and future application perspectives. Water Res 40(13):2463–2476

    Article  CAS  Google Scholar 

  • Shanks OC, Atikovic E, Blackwood AD, Lu J, Noble RT, Domingo JS, Siefring S, Sivaganesan M, Haugland RA (2008) Quantitative PCR for detection and enumeration of genetic markers of bovine fecal pollution. Appl Environ Microbiol 74(3):745–752

    Article  CAS  Google Scholar 

  • Shannon KE, Lee DY, Trevors JT, Beaudette LA (2007) Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment. Sci Total Environ 382(1):121–129

    Article  CAS  Google Scholar 

  • Shibata T, Solo-Gabriele HM, Fleming LE, Elmir S (2004) Monitoring marine recreational water quality using multiple microbial indicators in an urban tropical environment. Water Res 38(13):3119–3131

    Article  CAS  Google Scholar 

  • Siefring S, Varma M, Atikovic E, Wymer L, Haugland R (2007) 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(2):225–237

    Article  CAS  Google Scholar 

  • Smith C, Hill V (2009) Dead-end hollow-fiber ultrafiltration for recovery of diverse microbes from water. Appl Environ Microbiol 75(16):5284–5289

    Article  CAS  Google Scholar 

  • Sivaganesan M, Haugland R, Chern E, Shanks O (2009) Alternative Bayesian real-time PCR strategies for generation of calibration equations for absolute quantification of nucleic acids. BMC Bioinformatics. Submitted for publication

  • US Environmental Protection Agency (1986) Ambient water quality criteria for bacteria—1986. EPA 440/5-84/002. Criteria and Standards Division, US Environmental Protection Agency, Washington, DC

  • Wade TJ, Calderon RL, Brenner KP, Sams E, Beach M, Haugland R, Wymer L, Dufour A (2008) High sensitivity of children to swimming-associated gastrointestinal illness. Epidemiology 19(3):375–383

    Article  Google Scholar 

  • 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(1):24–8

    Article  Google Scholar 

  • Wade TJ, Pai N, Eisenberg JN, Colford JM Jr (2003) Do US Environmental Protection Agency water quality guidelines for recreational waters prevent gastrointestinal illness? A systematic review and meta-analysis. Environ Health Perspect 111(8):1102–1109

    Google Scholar 

  • Walters S, Field K (2009) Survival and persistence of human and ruminant-specific faecal Bacteroidales in freshwater microcosms. Environ Microbiol 11(6):1410–1421

    Article  Google Scholar 

  • Wang RF, Cao WW, Cerniglia CE (1996) PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples. Appl Environ Microbiol 62(4):1242–1247

    CAS  Google Scholar 

  • Wymer LJ, Dufour AP, Brenner KP, Martinson JW, Stutts WR, Schaub SA (2004) The EMPACT beaches project. Results and recommendations from a study on the microbiological monitoring of recreational waters. EPA 600/R-04/023, 2004. US Environmental Protection Agency, Office of Research and Development, Washington, DC

  • Yamahara KM, Layton BA, Santoro AE, Boehm AB (2007) Beach sands along the California coast are diffuse sources of fecal bacteria to coastal waters. Environ Sci Technol 41(13):4515–4521

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Richard A. Haugland.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chern, E.C., Brenner, K.P., Wymer, L. et al. Comparison of Fecal Indicator Bacteria Densities in Marine Recreational Waters by QPCR. Water Expo. Health 1, 203–214 (2009). https://doi.org/10.1007/s12403-009-0019-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12403-009-0019-2

Navigation