Host specificity and sensitivity of the established and novel sewage-associated marker genes in human and non-human fecal samples.

Microbial source tracking (MST) methods measure fecal contamination levels and identify possible sources using quantitative polymerase chain reaction (qPCR) that targets host-associated fecal microorganisms. To date, most established MST assays for human sources, especially bacterial markers, have shown some non-human 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 USA. 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 non-human fecal samples across 14 hosts collected from a subtropical region of Australia were tested for marker host specificity, host sensitivity and concentration. All sewage samples were positive for all six marker genes tested in this study. Bacterial markers were more prevalent in human feces than viral markers. 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 3-5 orders of magnitude lower than in sewage. When considering host specificity, sensitivity, and concentrations in source samples, the HF183, Lachno3 and crAssphage CPQ_056 were the most suitable assays in this study for sewage contamination tracking in subtropical waters of Australia. [ABSTRACT FROM AUTHOR]