Novel Bacteroides bacteriophage-like quantitative real-time PCR assays for human sewage pollution measurement

A recent study reports the 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 source identification applications, the genome was interrogated for human-associated genetic regions using bioinformatic and laboratory approaches. Fifty-three primer sets were tested using 100 fecal samples from ten animal sources, primary influent sewage from 36 geographical locations across the 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 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 established DNA-based viral (CPQ_056 and CPQ_064) and bacterial (HF183/BacR287 and HumM2) human-associated qPCR assays. Findings indicated ΦB124-14 bacteriophage-like qPCR assays exhibited superior specificity, but markers consistently occurred at lower concentrations in United States primary influent sewage. Reduced sensitivity may be insufficient for application in U.S. environmental waters, especially when trace levels of sewage are present. Furthermore, paired measurements of ΦB124-14 and crAssphage bacteriophage-like sequences in high volume (10 L) primary influent sewage samples (n = 38) indicated 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).