Human Gut-Associated ΦB124-14 Bacteriophage-Like Quantitative Real-Time PCR Assays for Sewage Measurement
Citation:
Willis, J., M. Sivaganesan, B. McMinn, A. Korajkic, C. Staley, AND O. Shanks. Human Gut-Associated ΦB124-14 Bacteriophage-Like Quantitative Real-Time PCR Assays for Sewage Measurement. ASM Microbe 2024, Atlanta, GA, June 13 - 17, 2024.
Impact/Purpose:
Many U.S. coastline beaches experience elevated levels of fecal pollution potentially exposing swimmers, boaters, and fishermen to disease-causing organisms. As a result, beaches must be closed to the public, which can reduce revenue and morale in the local communities. Most importantly, beach closures do not resolve the issue of fecal contamination. Fecal indicator bacteria (E. coli and enterococci) serve as a monitoring method to measure the total amount of fecal pollution accumulated across various sources such as birds, dogs, and human waste. As a result, this practice does not provide source information often necessary to mitigate polluted beaches and take steps to prevent future occurrences. The use of host-associated qPCR technologies designed to characterize fecal pollution from a particular animal group could dramatically improve water quality management. In response, the U.S. EPA ORD maintains an active research program to develop, validate, implement, and provide technical support for tools to characterize fecal pollution sources. Information covered in this abstract was prepared based on research priorities defined in the U.S. EPA Strategic Research Action Plan (SSWR 403.1.1).
Description:
The ability to identify human sewage in environmental, municipal storm system drains, and other waste flows 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 source identification DNA-based monitoring applications, the genome was screened for potential human-associated genetic targets. To assess bioinformatic predictions, a total of 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 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 (qPCR) assays, 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 sequences in sewage impacted environmental water samples. PS28 and PS30 performance was then compared to top performing viral and bacterial human-associated technologies including CPQ_056, CPQ_064, HF183/BacR287, and HumM2 qPCR assays. Findings indicate ΦB124-14-like qPCR assays exhibit superior specificity, but target sequences consistently occur at lower concentrations in primary influent sewage samples. Furthermore, paired measurements of ΦB124-14-like and crAssphage-like sequences in high volume (10 L) primary influent sewage samples (n = 38) indicate significant correlations (p < 0.0001) of r = 0.593 (PS30 versus CPQ_056), r = 0.646 (PS28 versus CPQ_056), and r = 0.938 (PS28 versus PS30). These new qPCR methods could serve as potential future viral-based human sewage monitoring tools.