December 2022 • Journal of Environmental Health 11 control wells in each plate with those containing samples or standards. Data Analysis and Interpretation Each run was assessed visually for performance using ABI 7500 Fast software. Runs were screened for amplification in negative controls, high standard deviation among replicates, successful amplification in positive controls, and a standard curve constructed from plasmids. For each plate that was considered a successful run, results were exported into Excel using ABI 7500 Fast software. Analysis of the results and graphics were produced using RStudio (2015 version). Results Quality Assurance and Controls Quality assurance and controls were implemented at various stages of the project to ensure the reliability of the data. Field blanks revealed no evidence of contamination at any stage of the sample handling process. The salmon DNA used as a control spike revealed environmental inhibition in all undiluted samples, which was addressed by a dilution factor of 5, after which no samples showed interference. Similarly, diluted samples showed no evidence of inhibition, as internal amplification controls were appropriately detected. Two assays failed to pass the screening for successful runs and indicated nonspecific amplification. Steps taken to optimize both the GFC and GFD assays (Table 1) failed to improve performance, resulting in nonspecific amplification or no amplification. Due to these failings, we did not include these assays in further analyses. General Indicators of Fecal Contamination Traditional monitoring for E. coli at the three watershed sites revealed the occurrence of elevated bacterial counts as defined by the Connecticut bathing beach standard of 104 CFU/100 ml (Table 2). The Lower Farm River had lower E. coli levels relative to the other locations, but still had elevated levels in 50% of the samples. E. coli levels at the Goodwives River exceeded regulatory limits in 75% of samples, while samples collected in Sasco Brook suggested impairment 67% of the time. E. coli levels were higher at both the Goodwives River and Sasco Brook in summer months, with samples in July and August exceeding 10,000 CFU/100 ml at one or both sites. The GenBac3 marker is found in members of the phylumBacteroidetes but is not associated with a specific host. Organisms from the phylumBacteroidetes such as E. coli are found in the gut of many animals, although the bacteria are also known to occur in the environment without contributions of fecal matter (Fiksdal et al., 1985). Like E. coli, the GenBac3 marker is an indicator of fecal contamination from multiple sources, and the two are often correlated (Bower et al., 2005; Savichtcheva et al., 2007). We found only a weak relationship, however, between E. coli and GenBac3 (Figure 2). We found a slightly higher correlation between the levels of E. coli and the general marker GenBac3 (R2 = .44) at Goodwives River. This correlation, however, is largely influenced by the elevated GenBac3 counts andE. coli levels in July and August, whereas there is little to no correlation when considering other samples from the same sites (R2 = .19) when high counts were removed from the analysis. Host-Specific Markers In addition to identifying general indicators of fecal bacteria, we also examined the presence of host-specific markers that provide information on the source of contamination Raw Counts Generated for the Two General Markers Quantified in the Three Targeted Watersheds Date Lower Farm River, Branford Goodwives River, Darien Sasco Brook, Westport E. coli Levels (CFU/100 ml) GenBac3 Counts (Markers/100 ml) E. coli Levels (CFU/100 ml) GenBac3 Counts (Markers/100 ml) E. coli Levels (CFU/100 ml) GenBac3 Counts (Markers/100 ml) 1/19/2016 12 346 14 320 54 3,024 2/16/2016 14 330 650 337 70 903 3/29/2016 190 1,568 38 153 52 558 4/26/2016 84 138 900 1,460 470 1,490 5/10/2016 80 94 82 47 74 455 6/23/2016 92 321 308 439 520 102 7/26/2016 168 369 22,000 1,248 1,100 401 8/22/2016 760 566 13,600 1,704 19,600 1,346 9/21/2016 140 208 1,200 301 300 194 10/18/2016 80 237 116 241 132 154 11/21/2016 350 490 138 133 350 1,940 12/19/2016 138 2,630 138 350 350 1,242 Note. The two general markers quantified in this study were not host associated. TABLE 2
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