Highly Specific Sewage-Derived Quantitative PCR Assays Target Sewage-Polluted Waters.

The identification of sewage contamination in water has primarily relied on the detection of human-associated using markers within the V2 region of the 16S rRNA gene. Despite the establishment of multiple assays that target the HF183 cluster (i.e., ) and other organisms (e.g., ), the potential for more human-associated markers in this genus has not been explored in depth. We examined the population structure in sewage and animal hosts across the V4V5 and V6 hypervariable regions. Using near-full-length cloned sequences, we identified the sequences in the V4V5 and V6 hypervariable regions that are linked to the HF183 marker in the V2 region and found these sequences were present in multiple animals. In addition, the V4V5 and V6 regions contained human fecal marker sequences for organisms that were independent of the HF183 cluster. The most abundant in untreated sewage was not human associated but pipe derived. Two TaqMan quantitative PCR (qPCR) assays targeting the V4V5 and V6 regions of this organism were developed. Validation studies using fecal samples from seven animal hosts ( = 76) and uncontaminated water samples ( = 30) demonstrated the high specificity of the assays for sewage. Freshwater were also identified in uncontaminated water samples, demonstrating that measures of total do not reflect fecal pollution. A comparison of two previously described human assays (HB and HF183/BacR287) in municipal wastewater influent and sewage-contaminated urban water samples revealed identical results, illustrating the assays target the same organism. The detection of sewage-derived provided an independent measure of sewage-impacted waters. are major members of the gut microbiota, and host-specific organisms within this genus have been used extensively to gain information on pollution sources. This study provides a broad view of the population structure of within sewage to contextualize the well-studied HF183 marker for a human-associated The study also delineates host-specific sequence patterns across multiple hypervariable regions of the 16S rRNA gene to improve our ability to use sequence data to assess water quality. Here, we demonstrate that regions downstream of the HF183 marker are nonspecific but other potential human-associated markers are present. Furthermore, we show the most abundant in sewage is free living, rather than host associated, and specifically found in sewage. Quantitative PCR assays that target organisms specific to sewer pipes offer measures that are independent of the human microbiome for identifying sewage pollution in water.