ΦB124-14 Bacteroides Bacteriophage-Like Quantitative Real-Time PCR Assays for Human Sewage Pollution Measurement

The ability to measure human sewage in environmental and wastewater samples is important to protect public health and natural water resources. A recent study reports the complete genome sequence of ΦB124-14, a bacteriophage capable of infecting a narrow subset of Bacteroides spp. closely associated with the human gut. To investigate the use of ΦB124-14 for fecal indicator and source identification applications, the entire genome was interrogated for potential human-associated genetic regions using a combination of bioinformatic and laboratory testing approaches. To assess bioinformatic predictions, 53 primer sets were subject to systematic testing using 100 fecal samples from ten animal sources, primary influent sewage from 36 geographical locations across the continental United States, and environmental surface waters with known human sewage impact. Based on candidate primer set end-point PCR analyses and next generation amplicon sequencing, two novel hydrolysis probe-based quantitative real-time PCR assays (qPCR), PS28 and PS30, were designed and evaluated. Both qPCR assays exhibited a sensitivity of 86.1%, a specificity of 100%, and successfully detected ΦB124-14-like genetic markers in sewage impacted environmental water samples. PS28 and PS30 performance was then compared to top performing DNA-based viral (CPQ_056 and CPQ_064) and bacterial (HF183/BacR287 and HumM2) human-associated qPCR assays. Findings indicate ΦB124-14 bacteriophage-like qPCR assays exhibit superior specificity, but markers consistently occur at lower concentrations in United States primary influent sewage samples. Furthermore, paired measurements of ΦB124-14 and crAssphage bacteriophage-like sequences in high volume (10 L) primary influent sewage samples ( n = 38) indicate significant correlations ranging from r = 0.593 (p < 0.0001; PS30 versus CPQ_056) to r = 0.938 (p < 0.0001; PS28 versus PS30). A comparison of bacteriophage-like marker concentrations with cultured GB-124 in sewage samples showed no significant correlations (r ≤ 0.215, p ≥ 0.183). Finally, novel qPCR methods for potential human sewage management applications and future research directions are discussed.