Host Specificity and Sensitivity of Established and Novel Sewage-Associated Marker Genes in Human and Nonhuman Fecal Samples

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.IMPORTANCE Large financial investments are required to remediate fecal contamination sources in waterways, and accurate results from field studies are crucial to build confidence in MST approaches. Host specificity and sensitivity are two main performance characteristics for consideration when choosing MST assays. Ongoing efforts for marker assay validation will improve interpretation of results and could shed light on patterns of occurrence in nontarget hosts that might explain the underlying drivers of cross-reaction of certain markers. For field applications, caution should be taken to choose appropriate MST marker genes and assays based on available host specificity and sensitivity data and background knowledge of the contaminating sources in the study area. Since many waterborne pathogens are viruses, employing both viral and bacterial markers in investigations could provide insight into contamination dynamics and ecological behavior in the environment. Therefore, combined usage of marker assays is recommended for more accurate and informative sewage contamination detection and fecal source resolution.

Keywords: microbial source tracking.

FIG 1

Concentrations of sewage-associated marker genes in nonhuman fecal samples. (A) Gene copies (GC) of HF183, Lachno3, BacV6-21, and crAssphage CPQ_056 markers in animal hosts. Results are calculated in GC/ng of DNA and GC/g of feces. Each animal host is represented in unique color and shape. Error bars represent marker GC means ± standard deviations. (B) Overall nonhuman sample test results for six markers where the bar height represents sample numbers. Quantifiable sample numbers are shown in maroon bars, negative sample numbers are shown in light blue bars, and detectable but not quantifiable (positive) numbers are shown in gray bars.

FIG 2

Concentrations of sewage-associated markers in sewage and human feces. The y axis represents log₁₀ gene copy (GC) numbers, and the x axis shows groups of human feces and sewage. Each marker is shown as results of GC/ng DNA and GC/g of feces or per liter of sewage to better visualize between unequal group sizes. Horizontal line represents mean value, and band represents 95% highest density intervals.