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QPCR Determined Fecal Indicator Bacterial Densities in Marine Waters from Two Recreational Beaches
Citation:
CHERN, E. C., K. P. Brenner, L. J. WYMER, AND R. A. HAUGLAND. QPCR Determined Fecal Indicator Bacterial Densities in Marine Waters from Two Recreational Beaches. Presented at EPA National Beaches Conference 2009, Huntington Beach, CA, April 20 - 22, 2009.
Impact/Purpose:
The BEACH Act of 2000 (proposed for renewal in 2008) directs the U.S. EPA to establish more expeditious methods for the detection of pathogen indicators in coastal waters, as well as new water quality criteria based on these methods. Molecular based technologies such as real-time quantitative PCR (QPCR) have the ability to provide measurements of fecal indicator bacteria nucleic acids within a few hours and thus could lead to more accurate and health protective advisories to the public due to their greater timeliness. The National Epidemiological and Environmental Assessment of Recreational (NEEAR) Waters Studies, performed by U.S. EPA and CDC in 2003-2007 have demonstrated a correlation between swimming-related gastrointestinal illness rates and QPCR measurements of Enterococcus and Bacteroidales fecal bacteria at POTW-impacted fresh water and marine beaches. These results suggest that the QPCR technology can provide rapid determinations of fecal pollution levels that are predictive of swimming-related health risks at human waste impacted beaches. Limited available evidence suggests that genetic marker levels from bacterial groups that are generally found in all warm blooded animals, such as Enterococcus and Bacteroidales, may not correlate with health risks at beaches that are not impacted by human waste in the same manner as has been demonstrated thus far in the NEEAR studies. This potential distinction between human and non-human waste impacted beaches has raised questions about the validity of a national water quality criterion based solely on the relationships established in the NEEAR studies. It has been proposed that a similar test method for markers from human-specific fecal indicators is needed to identify beaches that would be subject water quality criteria established from the NEEAR studies. Beaches that are not impacted by human waste could be subject to different, yet to be specified criteria. The first objective of this study is to develop and evaluate qPCR assays and test methods for the detection and quantification of genetic markers from indicator bacteria that are associated with human fecal waste and to determine the occurrence and densities of these markers in waste waters and fecal material from different animal sources. The second objective is to similarly develop and/or evaluate qPCR assays and test methods for genetic markers from two other groups of general fecal indicator bacteria that historically have been widely used or are favored in specific regions. These bacterial groups include E. coli which has been accepted and widely used for freshwater monitoring and C. perfringens which has been favored in tropical and subtropical regions. The final objective is to determine the densities of these human and general fecal indicator markers, as well as previously identified markers for enterococci and total Bacterioidales,in a wide range of surface and recreational waters including archived samples from the previous NEEAR studies. Results from these studies will be used to determine correlations between indicator marker densities obtained by the best available new qPCR assays and test methods and swimming associated health risks by the National Health and Environmental Effects Laboratory and for the development of new national water quality criteria by the Office of Water.
Description:
The use of real-time qPCR to determine fecal indicator bacteria (FIB) densities is currently being investigated by the U.S. EPA. The present recreational water quality guidelines, based on culturable FIB, prevent same day determinations of water quality whereas results from the qPCR method can be available within several hours. Epidemiological studies at POTW-impacted freshwater beaches have shown a strong correlation between qPCR determined Enterococcus densities and swimming-related illness rates. This study provides an initial assessment of qPCR estimated Enterococcus, Bacteroidales, E. coli and Clostridium densities in marine water from two recreational beaches sampled over one summer. The estimated geometric mean cell densities per 100 ml of marine water from both beaches across sampling visits were 3.28 x 10 1, 1.71 x 103, 7.37 x 102, and 9.26 x 102 for Enterococcus, Bacteroidales, E. coli and Clostridium, respectively. These cell equivalent density estimates, determined using whole cell calibrator samples by a comparative cycle threshold (CT) approach, did not correspond with the relative target sequence density estimates of the different FIB in the samples which gave geometric means of 1.28 x 103, 2.35 x 104, 3.04 x 102, and 1.03 x 104 for Enterococcus, Bacteroidales, E. coli and Clostridium, respectively. This discrepancy was determined to be attributable to differences in recovery of target sequences from cells of the different organisms. QPCR analyses using whole cell calibrator samples provides a simple approach for comparing both total cell and target sequence density estimates of different FIB groups in water samples.