title: Standardization of enterococci density estimates by EPA qPCR methods and comparison of beach action value exceedances in river waters with culture methods.
abstract: The U.S. EPA has published recommendations for calibrator cell equivalent (CCE) densities of enterococci in recreational waters determined by a qPCR method in its 2012 recreational water quality criteria (RWQC). The CCE quantification unit stems from the calibration model used to estimate enterococci densities in recreational beach waters in the EPA National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study and directly informed the derivation of the RWQC recommendations. Recent studies have demonstrated that CCE estimates from the method can vary when using different cultured Enterococcus cell preparations in calibrator samples. These differences have been attributed to differences in the quantities of targeted gene copies (target sequences) that are recovered per nominal calibrator cell by DNA extraction. Standardization of results from the calibration model will require the estimation of target sequence recoveries from the calibrator and water samples. In addition, comparisons of water sample results with the RWQC values will require a knowledge of target sequence recoveries from the NEEAR study calibrator samples. In this study recoveries of target sequences and the mean target sequence/cell ratio for the NEEAR study calibrator samples were retrospectively estimated with a corroborated standard curve. A modification of the calibration model was then used to estimate enterococci target sequence quantities in water samples from eight midwestern U.S. rivers. CCE estimates were obtained by dividing these target sequence quantities by the mean NEEAR study target sequence/cell ratio. This target sequence-based quantification approach resulted in a high degree of agreement in beach action decisions (determinations of whether bacterial fecal indicator densities are above or below RWQC-recommended values) from CCE results of the qPCR method and from culture dependent enumeration of both enterococci and Eschericia coli in the corresponding water samples.
authors.affiliation: U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, Cincinnati, OH, United States. Electronic address: haugland.rich@epa.gov.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Haugland
authors.affiliation: U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, Cincinnati, OH, United States.
authors.forename: Shawn D
authors.initials: SD
authors.lastname: Siefring
authors.affiliation: U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, Cincinnati, OH, United States.
authors.forename: Manju
authors.initials: M
authors.lastname: Varma
authors.affiliation: U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, Cincinnati, OH, United States.
authors.forename: Alfred P
authors.initials: AP
authors.lastname: Dufour
authors.affiliation: U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, Cincinnati, OH, United States.
authors.forename: Kristen P
authors.initials: KP
authors.lastname: Brenner
authors.affiliation: U.S. Environmental Protection Agency, Office of Research and Development, National Health Effects and Environmental Effects Research Laboratory, Chapel Hill, NC, United States.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Wade
authors.affiliation: U.S. Environmental Protection Agency, Office of Research and Development, National Health Effects and Environmental Effects Research Laboratory, Chapel Hill, NC, United States.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Sams
authors.affiliation: Ohio River Valley Water Sanitary Commission, Cincinnati, OH, United States.
authors.forename: Stacey
authors.initials: S
authors.lastname: Cochran
authors.affiliation: Ohio River Valley Water Sanitary Commission, Cincinnati, OH, United States.
authors.forename: Steve
authors.initials: S
authors.lastname: Braun
authors.affiliation: U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, Cincinnati, OH, United States.
authors.forename: Mano
authors.initials: M
authors.lastname: Sivaganensan
mesh_terms: D058491:Bacterial Load / Q000379:methods / Q000592:standards*; D001493:Bathing Beaches; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D016983:Enterococcus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D045483:Rivers / Q000382:microbiology*; D014481:United States
chemical_list: D004269:DNA, Bacterial
keywords: EPA methods; Enterococcus; Recreational water quality criteria; qPCR