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) Ͼ HPyV (0.99) Ͼ crAssphage CPQ_056 (0.98) Ͼ HF183 (0.96) Ͼ Lachno3 (0.95) Ͼ 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 trackingCitation Ahmed W, Gyawali P, Feng S, McLellan SL. 2019. Host specificity and sensitivity of established and novel sewageassoc...

Superior performance of human wastewater-associated viral markers compared to bacterial markers in tropical environments

“…The HPyV marker, targeting both BK and JC viruses, has shown 100% specificity for human sewage, with no cross-detection in any of the nonhuman fecal samples from Thailand (this study) or in other geographical regions (Ahmed et al, 2019a; McQuaig et al, 2009; Staley et al, 2012). Exceptions have been found only in one of 30 bird fecal samples and one of ten pig wastewater samples in Australia (Ahmed et al, 2019a, 2010).…”

“…The HPyV marker, targeting both BK and JC viruses, has shown 100% specificity for human sewage, with no cross-detection in any of the nonhuman fecal samples from Thailand (this study) or in other geographical regions (Ahmed et al, 2019a; McQuaig et al, 2009; Staley et al, 2012). Exceptions have been found only in one of 30 bird fecal samples and one of ten pig wastewater samples in Australia (Ahmed et al, 2019a, 2010). CrAssphage showed excellent specificity in this study; however, previous reports have indicated cross-detection in one composite swine sample from earlier work in Thailand (Kongprajug et al, 2019b) and in dog, cat, gull, poultry, and cattle samples from other geographical regions (Ahmed et al, 2018b, 2018a; Stachler et al, 2017).…”

“…Therefore, both were not expected to be present in human feces. In Australia, Lachno3 and crAssphage were also found in 68.9% and 37.9% of individual human fecal samples ( n = 29), respectively, but they were both present in 100% of WWTP-inf samples (n = 3), confirming that these markers are more suitable for tracking sewage pollution rather than individual human feces (Ahmed et al, 2019a).…”

“…Of these, quantitative polymerase chain reaction (qPCR) is the most widely used method (Harwood et al, 2014). Among human-associated viral MST markers, human polyomaviruses (HPyVs) have been used as indicators of sewage pollution in many regions (Ahmed et al, 2019a; McQuaig et al, 2009; Rachmadi et al, 2016; Staley et al, 2012). HPyVs are nonenveloped, icosahedral, double-stranded DNA viruses, belonging to the Polyomaviridae family (Moens et al, 2017).…”

“…In the USA and Australia, human-associated bacteria in the family Lachnospiraceae (Lachno3) and the genus Bacteroides (BacV6-21) have also been proposed as MST markers for human fecal pollution, and qPCR assays have recently been developed to detect them (Ahmed et al, 2019a; Feng et al, 2018; Feng and McLellan, 2019). Lachnospiraceae are anaerobic bacteria belonging to the order Clostridiales , which are prevalent in the human gut microbiota.…”