December 2022 • Journal of Environmental Health 9 host-specific genetic markers), it is possible to identify species that contribute to fecal contamination in a body of water (Bernhard & Field, 2000). This approach could provide water quality managers with possible contamination sources, which is a valuable starting point to begin source tracking and eventual mitigation in a targeted way. In our study, three local health departments in Connecticut collaborated to use host-specific genetic markers to provide information on possible contamination sources in three watersheds in coastal Connecticut. All three selected watersheds outlet into the Long Island Sound (LIS), an Atlantic Ocean tidal estuary that the U.S. Congress declared to be of national significance. Water quality in LIS is threatened by localized urbanization and the >9 million people who live within the watershed area (Save the Sound, 2022). The LIS estuary has been the focus of many remediation eorts (Schimmel et al., 1999; State of Connecticut, 2020), yet still experiences frequent elevated fecal bacteria counts, especially after rainfall events. By analyzing water samples from each watershed, we attempted to identify the sources of bacteria, evaluate the actual risk the bacteria pose to public health, better understand fluctuations in bacterial counts aecting the water quality of LIS, and establish mitigation programs based on these results. Methods Watershed Selection The Sasco Brook (Westport), Lower Farm River (Branford), and Goodwives River (Darien) watersheds (Figure 1) all have experienced unexplained elevated bacterial counts that were especially pronounced after precipitation events. Both Sasco Brook and Goodwives River have been identified by the Connecticut Department of Energy and Environmental Protection (CT DEEP; State of Connecticut, 2019) as impaired water bodies for not meeting state water quality standards for fecal coliform bacteria (i.e., a class of FIB that includes E. coli). The Lower Farm River site has also experienced elevated FIB levels and is of special interest because of recreational and commercial shellfishing. As all three watersheds feed into LIS, addressing impairments in water quality at these sites may also help to alleviate pressures on this estuary of national significance. Standardized Sample Collection, Processing, and Monitoring of Traditional Fecal Indicator Bacteria Sampling locations were selected near the mouth of each watershed. Water samples were collected once a month between January and December 2016 at low or ebbing tides to avoid tidal influence. For each sample, approximately 500 ml of water was collected in a sterile container from between 6 and 12 in. below the surface of the water. Samples were placed in an insulated cooler on ice for transport to the Harbor Watch Laboratory in Westport, Connecticut. E. coli enumeration was conducted at the Harbor Watch Laboratory using m-FC media following standard method 9222D (National Environmental Methods Index, n.d.). Individual CFUs were counted to estimate bacterial abundance in the water samples. For the genetic analysis, two independent 100-ml water subsamples were vacuum filtered on a 0.2-μm pore size polycarbonate filter (GE Osmotic 04CP04700) to concentrate bacterial cells. Following filtration, the filter was removed aseptically and placed into cryo-safe tubes with glass beads or in a sterile polypropylene tube with a screw cap. These filters were stored at -80 °C in the Connecticut Agricultural Experiment Station (CAES). DNA extractions were conducted on the first set of filters at CAES as previously described (Shanks et al., 2016). Analysis With qPCR Analysis for host-specific genetic markers was conducted at the Center for Genetic Analyses of Biodiversity in the Yale Institute for Biospheric Studies. Quantitative PCR (qPCR) was used (ABI 7500 Fast Real-Time PCR) to amplify all markers using either SYBR Green or TaqMan chemistry (Table 1). TaqMan reactions were a total volume of 20 μl consisting of 10 μl of TaqMan Fast Universal Master Mix (ThermoFisher 4352042), 500 nmol/l of each primer, and 250 nmol/l of probe. SYBR Green assays were conducted similarly, with 20 μl reactions consisting of Map of Connecticut Showing the Location of the Three Targeted Watersheds FIGURE 1 Goodwives River, Darien Sasco Brook, Westport Lower Farm River, Branford
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