Abstract

Microbial source tracking (MST) methods measure fecal contamination levels and identify possible sources using quantitative PCR (qPCR) that targets host-associated fecal microorganisms. To date, most established MST assays for human sources, especially bacterial markers, have shown some nonhuman host cross-reactions. Recently developed assays, such as the crAssphage CPQ_056, Lachnospiraceae Lachno3, and Bacteroides BacV6-21, have more limited information on host sensitivity and host specificity for human or sewage sources, particularly in countries other than the United States. In this study, we rigorously evaluated six sewage-associated MST assays (i.e., Bacteroides HF183, human adenovirus [HAdV], human polyomavirus [HPyV], crAssphage CPQ_056, Lachno3, and BacV6-21) to show advantages and disadvantages of their applications for MST. A total of 29 human and 3 sewage samples and 360 nonhuman fecal samples across 14 hosts collected from a subtropical region of Australia were tested for marker host specificity, host sensitivity, and concentrations. All sewage samples were positive for all six marker genes tested in this study. Bacterial markers were more prevalent than viral markers in human feces. Testing against animal hosts showed human feces (or sewage)-associated marker gene specificity was HAdV (1.00) > HPyV (0.99) > crAssphage CPQ_056 (0.98) > HF183 (0.96) > Lachno3 (0.95) > BacV6-21 (0.90), with marker concentrations in some animal fecal samples being 3 to 5 orders of magnitude lower than those in sewage. When considering host specificity, sensitivity, and concentrations in source samples, the HF183, Lachno3, and crAssphage CPQ_056 tests were the most suitable assays in this study for sewage contamination tracking in subtropical waters of Australia.

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