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

Jessica R Willis; Mano Sivaganesan; Brian McMinn; Asja Korajkic; Christopher Staley; Orin C Shanks

doi:10.1016/j.envpol.2025.126806

Home/Archive/PubMed

PubMed · 40645262

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

Jessica R Willis·Mano Sivaganesan·Brian McMinn·Asja Korajkic·Christopher Staley·Orin C Shanks

View original source ↗Export metadata ↓

Abstract

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).

Explore related subjects

Sewage Humans Environmental Monitoring Bacteriophages Bacteroides Real-Time Polymerase Chain Reaction Feces Water Pollution Journal Article

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Jessica R Willis, Mano Sivaganesan, Brian McMinn, Asja Korajkic, Christopher Staley, Orin C Shanks. 2025-07-09. Novel Bacteroides bacteriophage-like quantitative real-time PCR assays for human sewage pollution measurement.. https://doi.org/10.1016/j.envpol.2025.126806

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2026

Genome-Resolved Metagenomics Revealed the Functional Potential of Core Novel and Known Genera Key to Processes in Full-Scale Aerobic Granular Sludge Plants.

Microbial communities are critical for nutrient removal in aerobic granular sludge (AGS) wastewater treatment plants (WWTPs). Despite the stable long-term operation of full-scale AGS WWTPs, the microbial populations and functional traits sustaining stable long-term performance remain poorly resolved. To address this gap, the recovered MAG catalog from nine full-scale AGS WWTPs across five countries was analyzed. From this catalog, 74 high-quality core MAGs were identified and used for downstream taxonomic characterization and functional analyses. These high-quality core MAGs spanned 48 established and 7 novel genera, representing 31 known and 43 novel species. Functional analysis linked core MAGs to key WWTP processes: polyphosphate accumulation (9), glycogen accumulation (12), denitrification (62), and nitrification (1). These included four novel MAGs with glycogen-accumulating (3) and polyphosphate-accumulating (1) potential and 11 capable of nitrous oxide reduction, critical for mitigating greenhouse gas emissions. Ca. Phosphoribacter was the most abundant genus, highlighting its underestimated role caused by misclassification as Tetrasphaera in 16S rRNA surveys. Specifically, Ca. P. hodrii was the dominant species, exhibiting enhanced sugar uptake and amino acid synthesis as likely drivers of its enrichment in the AGS WWTPs. Overall, this study resolves for the first time the taxa and functional traits consistently enriched in full-scale AGS systems, enabling a shift from an empirical performance assessment toward biologically informed process interpretation.

Sewage↗

PubMed2026

Metagenomic insights into the mechanisms of heteroatom-doped, iron-loaded biochar in enhancing anaerobic digestion of waste activated sludge.

Anaerobic digestion is a crucial technology for resource recovery from waste activated sludge. Enhancing its methane production efficiency using conductive materials is a key research objective. This study aimed to elucidate the mechanisms by which conductive materials promote this process. Three types of biochar(FeS@BC300, FeP@BC600, and FeP@BC900) were prepared by doping bamboo powder with N, P, S and iron salts under pyrolysis conditions at 300-900 °C, and their physical and chemical properties were characterized, including surface functional groups, specific surface area, capacitance, electrical resistance, electron-accepting capacity (EAC), and electron-donating capacity (EDC). These analyses assessed the influence of synthesis parameters. These materials were subsequently introduced into the anaerobic digestion of thermally hydrolyzed sludge to evaluate their impacts on methanogenic performance, microbial community structure, and metabolic pathways. The results show that the FeP@BC600 material, which exhibited the highest EDC, substantially increased microbial cytochrome c production (by 29.2 % compared to the control). This enhancement improved interspecies electron transfer, stimulated ATP synthesis (increased by 41.5 %), and reinforced both hydrogenotrophic and acetoclastic methanogenic pathways, ultimately elevating methane production by 55 %. Integrated analysis of metagenomic data, material properties, and performance metrics revealed that the key mechanism by which FeP@BC600 promotes methanogenesis is through the enrichment of cytochrome c-encoding genes, thereby facilitating direct interspecies electron transfer (DIET) and augmenting ATP synthesis. This study provides a foundation for the subsequent application of conductive materials to enhance anaerobic digestion and offers guidance for the optimized design of such materials.

Sewage↗

PubMed2025

Diversity of Salmonella enterica isolates from urban river and sewage water in Blantyre, Malawi.

BACKGROUND: Salmonella enterica encompasses over 2,600 serovars, including several commonly associated with severe infection in humans. Salmonella is a major cause of sepsis in Africa; however, diagnosis requires clinical microbiology facilities. Environmental surveillance has the potential to play a role in Salmonella surveillance. METHODS: We undertook water-based environmental surveillance in Blantyre, Malawi, from 2018-2020, taking samples from rivers (87.9%), a sewage plant (8.85%) and other water sources (3.24%), isolating and storing 1,042 non-typhoidal Salmonella (NTS) isolates in this period. Of these, 341 NTS isolates were whole genome sequenced, genome quality was checked, duplicate genomes from any given sample were removed and core genome phylogeny was reconstructed. AMRFinder, PathogenWatch and SISTR were used to further investigate serovar, sequence type and antimicrobial resistance determinants. RESULTS: After quality checks, and removal of duplicate genomes, 270 NTS genomes remained for further analysis. Multiple Salmonella serovars associated with human infection were detected, of which S. Typhimurium (55/270 isolates) was the most common, including 44 of Sequence Type (ST) 313, a serovar commonly associated with severe invasive disease (iNTS). Six lineage 2 ST313 genomes possessed AMR genes predicting multidrug resistance (MDR), while 29 lineage 3 isolates contained no AMR predictive genes. PCR based detection of staG has been proposed as a diagnostic marker of S. Typhi; however, all eight genomes that contained staG identified as Salmonella enterica serovar Orion, raising concerns about the specificity of this marker as a monoplex for environmental surveillance of S. Typhi. DISCUSSION: The study identified diverse Salmonella serovars in the environment, including those reported to cause invasive disease, emphasizing the complex but potentially valuable contribution of implementing environmental surveillance for Salmonella in high burden areas lacking diagnostic microbiology capacity.

Sewage↗