Environmental Science: Water Research & Technology

Metagenomic sequencing is increasingly applied to wastewater to characterize the diversity, dynamics, and relative abundance of human and animal viruses. Among these sequencing approaches are those that enrich viral nucleic acids from the wastewater matrix, aiming to increase the viral read fraction for analysis. However, the feasibility of scaling targeted viral sequencing to diverse sewersheds across large geographic scales is currently unknown. In this study, we apply hybrid capture metagenomic sequencing to nearly 450 weekly wastewater samples collected during the respiratory virus season in the United States and evaluate sequencing performance for generating public health-relevant data. Analysis of data from 15 wastewater treatment plants demonstrates that our approach enabled efficient capture of pathogens of interest, achieving a median viral read fraction over 19%. Importantly, relative abundance estimates of common pathogens correlated with direct quantification of viral targets using RT-ddPCR. Together, our results demonstrate that hybrid capture sequencing of wastewater is a viable tool to monitor both common and rare pathogens across geographically diverse sewersheds.

Power-to-Gas technologies are emerging as a key strategy to integrate surplus renewable electricity into energy systems, through the conversion of green hydrogen into methane. However, the practical implementation of biological in situ biomethanation is still constrained by operational and design requirements that are incompatible with most existing anaerobic digestion infrastructures. This study demonstrates a stable and efficient mesophilic (37{degrees}C) in situ biomethanation process driven by substrate-induced microbial selection rather than relying on continuous hydrogen supply. Anaerobic digesters co-digesting sewage sludge from a wastewater treatment plant with lipid-rich greases recovered from dairy wastewater developed a pre-adapted hydrogenotrophic consortium capable of effective CO2-H2 conversion under mesophilic conditions. Long-term operation confirmed the robustness and persistence of this microbial structure. Upon H2 addition, methane concentrations up to 82 % were achieved under atmospheric pressure, without biogas recirculation, with hydrogen-to-methane conversion efficiencies up to 90% and methane productivities of 1.64 NLCH4.L-1d-1. 16SrRNA-based microbial community analysis revealed that dairy grease co-digestion selectively enriched hydrogenotrophic methanogens, particularly Methanospirillum, together with syntrophic fatty-acid-degrading bacteria such as Syntrophomonas, promoting efficient interspecies hydrogen transfer. Importantly, the lipid co-substrate enabled the establishment and long-term stability of the hydrogenotrophic pathway independently of hydrogen availability, mitigating challenges associated with intermittent renewable energy supply. Overall, these findings challenge the common reliance on thermophilic conditions, continuous hydrogen input, pressurization, and gas recirculation in in situ biomethanation, demonstrating that substrate-driven microbial selection can replace conventional engineering requirements such as thermophilic operation or reactor modifications, providing a simpler and scalable strategy for mesophilic in situ biomethanation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/731101v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@c62086org.highwire.dtl.DTLVardef@1813ed6org.highwire.dtl.DTLVardef@4462bcorg.highwire.dtl.DTLVardef@1ae2cfb_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG Highlights- Lipid-assisted co-digestion promotes stable biogas and biomethane production - Dairy wastewater greases enable mesophilic in situ biomethanation - An enriched hydrogenotrophic methanogenic consortium yields >82% CH4 - 70-90% H2-to-CH4 conversion efficiency under mesophilic, unpressurized conditions - Substrate-driven microbial selection enables in situ biomethanation in WWTP digesters

To understand the utility of healthcare facility-level wastewater surveillance (WWS) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is important to correlate wastewater SARS-CoV-2 RNA detection with the number of clinical infections. WWS for SARS-CoV-2 was performed at three skilled nursing facilities (SNFs) over 25 weeks. Electronegative membrane filtration (enMF) and Nanotrap(R) Magnetic Virus Particles (NP) virus concentration methods were compared. Extracts were tested by droplet digital polymerase chain reaction. Spearman's correlations ({rho}) between wastewater virus RNA concentrations and infection counts were calculated. From split wastewater samples, enMF recovered higher SARS-CoV-2 RNA concentrations than NP. Combining data from all facilities, the median concentrations were 53.0 versus 38.6 gc/100 mL for enMF and NP, respectively (p=0.001). Using enMF, correlations were moderate to strong at SNF A ({rho} ranged 0.67 to 0.86, all p-values <0.001). Weak to moderate correlations can be explained by the sampled manhole not representing the entire facility (SNF B, {rho} ranged 0.47 to 0.72, p-values ranged <0.001 to 0.12) and longitudinal data gaps from summer heat and equipment maintenance (SNF C, {rho} ranged 0.14 to 0.59, p-values ranged 0.52 to <0.01). WWS can be a valuable tool for tracking dynamics of SARS-CoV-2 infections in healthcare facilities.

As the United Kingdom (UK) targets net-zero emissions by 2050, anaerobic digestion (AD) has become a cornerstone of renewable energy infrastructure. However, mathematical models, such as the Anaerobic Digestion Model No. 1 (ADM1), often struggle with high-solids agricultural feedstocks because they rely on Chemical Oxygen Demand (COD), a metric that introduces significant experimental error. To overcome this, this study applies an established mass-based ADM1 framework tailored for the co-digestion of maize silage and cow manure sourced from a UK AD site. This study uses a parallel reactor framework, using two identical laboratory-scale reactors to physically replicate the dynamic conditions of the full-scale site. A Global Sensitivity Analysis was first conducted, identifying biomass decay and carbohydrate breakdown rates as the most influential factors affecting system stability and model accuracy. The model was calibrated using data from the first reactor and then tested against an independent second reactor subjected to significant organic loading stress. Results show high predictive capabilities, with the model achieving a R2 of 0.81 for biogas production during calibration. The model maintained high predictive accuracy during the validation test of the second physical twin, achieving an R2 of 0.85, proving that the framework is robust and not overfitted to a single dataset. While predicting rapid fluctuations in pH and alkalinity remains challenging, the mass-based approach effectively forecasts gas yields and process stability. This methodology provides a reliable foundation for robust process modelling, offering a scalable tool for the UK biogas sector to optimise AD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/721061v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@92c7e2org.highwire.dtl.DTLVardef@80d723org.highwire.dtl.DTLVardef@ac3d24org.highwire.dtl.DTLVardef@1e21a51_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sewer biofilms represent dynamic interfaces for exchange of bacteria and antibiotic resistance genes between biofilms and the overlying wastewater. Using inline, biofilm reactors, the movement of bacteria and 16S rRNA and carbapenemase genes (blaKPC, blaVIM, blaNDM, blaOXA-48-like, and blaIMP) between wastewater and sewer biofilms was investigated. Established, complex biofilms without these {beta}-lactamase (bla) genes, absorbed resistant bacteria within two minutes of exposure to high concentrations of resistant cultures in lab settings. Carbapenem-resistant organisms from these high-concentration source biofilms transferred to downstream biofilms over 60 minutes of representative sewer shear flows. Mass balances of bacteria and genes in biofilms versus wastewater under representative shear flow showed that biofilms exposed to resistant cultures contributed more to the wastewater than to the downstream biofilms. In field studies, established, complex biofilms without target carbapenem-resistant bacteria and genes from wastewater within hours and then stabilized between 2 to 15 days, not varying by more than 0.5 MPN/cm2 or 0.5 log gene copies (GC)/cm2. In contrast, metagenomic profiles of the bacterial community species continued to change up to 21 days. Established biofilms with resistant bacteria and genes exposed to tertiary-treated wastewater without target carbapenemase genes or meropenem antibiotics did not lose resistant genes or bacteria over nine days of exposure (i.e., < 1 log GC/cm2 reduction). Results show that sewer biofilms contribute to the resistance-gene signal found in sewer wastewater by absorbing and releasing bacteria and genes. Consideration of sewer biofilm dynamics is essential for more accurately interpreting wastewater bacterial concentrations in wastewater-based epidemiology studies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/726639v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@19f6ce0org.highwire.dtl.DTLVardef@1a507c8org.highwire.dtl.DTLVardef@1a2013dorg.highwire.dtl.DTLVardef@ff8613_HPS_FORMAT_FIGEXP M_FIG C_FIG

Municipal wastewater constitutes a major reservoir of unutilized reactive nitrogen, representing a significant opportunity for biological valorization. The biopolymer cyanophycin is promising as a means of nitrogen capture and recovery, but current production strategies are not optimized for the physicochemical constraints of municipal wastewater systems. Here, we engineered the naturally competent soil bacterium Acinetobacter baylyi ADP1 ISx to synthesize cyanophycin from carbon and nitrogen sources prevalent in municipal wastewater and over a range of wastewater-relevant temperatures. To overcome the recurring problem of arginine availability limiting cyanophycin synthesis, we engineered an arginine-producing strain (AP1) which accumulated cyanophycin when grown on acetate and ammonium (19% CDW), nitrate (9% CDW), or urea (29% CDW) and without arginine supplementation. During this work, we observed that conditions associated with reduced cell fitness correlated with increased intracellular cyanophycin content. As temperature strongly influences cell growth but cannot be realistically modulated in wastewater contexts, we investigated the potential of induced fructose-auxotrophy to modulate cell growth independently from temperature. This intervention, accomplished with a single knockout (gap), expanded the effective range of cyanophycin accumulation from 12 C up to 30 C. Collectively, these results establish the relevance of arginine-producing strains for cyanophycin biosynthesis and position A. baylyi as a promising chassis for continued development under real-world wastewater conditions.

Wastewater metagenomic sequencing (WW-MGS) enables simultaneous detection of hundreds of pathogens, but its use for quantitative pathogen tracking has not been robustly validated. Like wastewater PCR (WW-PCR), WW-MGS is affected by biases from variable fecal dilution and sample processing, but must additionally contend with the compositional structure of sequencing data, where a taxon's apparent abundance depends on the abundance of every other taxon in the sample. Simple summaries such as a pathogen's fraction of total reads may therefore be poorly suited to quantitative use. We retrospectively evaluated seven normalization approaches that attempt to control for these sources of bias against a baseline of total read relative abundance, using 1,425 samples from the CASPER consortium spanning 25 U.S. sites. Each approach was compared against WW-PCR and clinical data across eight total pathogens. Among the normalization strategies we evaluated, tobamovirus markers, diet-derived plant viruses abundant in human stool, performed best. Normalizing WW-MGS data by tobamovirus-genus counts improved median site concordance for 18 of 19 pathogen and comparison-source combinations. Gains were largest for year-round-circulating SARS-CoV-2 and norovirus and smaller for sharply seasonal pathogens such as influenza and respiratory syncytial virus, where baseline concordance was already high. Tobamovirus normalization rarely degraded concordance, with median gains roughly five times larger than median losses. Tobamovirus-normalized WW-MGS reached clinical concordance comparable to targeted WW-PCR, supporting its use as a quantitative trend-monitoring tool alongside pathogen-agnostic detection.

Simultaneous removal of organic waste and industrial gypsum was assessed in continuous flow-through bioreactors that treat sulfate-rich sewage sludge. Metabolic fluxes, the composition of microbial communities, and profiles of organic matter in the presence of different organic loads were tracked over [~]190 days. The addition of a pre-enriched microbial community enhanced the rates of sulfate reduction during the establishment of the sludge blanket, but microbial diversity in established reactors depended primarily on organic loading. Organic removal rates were comparable to those in standard anaerobic digesters, but methane production accounted for [~]1% of electron flow compared to >70% in traditional systems. Stoichiometric analyses revealed that molar COD: sulfate ratios below [~]1 favored complete oxidation of acetate by sulfate-reducing bacteria (SRB) and those above [~]2.1 permitted either complete or incomplete oxidation, allowing sulfate reduction and methanogenesis to co-occur. Sequencing of the 16S rRNA confirmed these trends by revealing that the faster-growing SRB that do not oxidize acetate were more abundant at higher organic loads and during the establishment of the sludge blanket, whereas complete oxidizers became more abundant when the molar COD: sulfate ratio was [&le;]3.2. In reactors that had been seeded with the pre-enriched communities, acetate-oxidizing SRB became prevalent over the incomplete oxidizers 25-50 days earlier. These results enable targeted design and control of microbial processes and bioreactors that remove waste organics and gypsum while producing less methane due to the competition for acetate between methanogenic archaea and SRB that oxidize acetate.

Enteroviruses (EVs) are ubiquitous contaminants of surface waters, where they can remain infectious for long periods of time. Most methods used for EV monitoring are unable to distinguish between infectious and non-infectious particles or between EV types. Because different types exhibit both distinct environmental persistence and health implications, there is a need for type-resolved infectivity measurements. Here we developed Integrated Cell Culture-Nanopore Sequencing (ICC-NanoporeSeq), a method combining short-term cell culture amplification with Nanopore sequencing of the VP1 gene. The ICC approach was adapted from a previously described ICC-RTqPCR protocol, while the NanoporeSeq workflow was derived from a clinical EV typing protocol and optimized for environmentally circulating EV types. Using samples containing known concentrations of ten EV types, the NanoporeSeq method accurately and reproducibly recovered the original proportions of all EV types after correction of biases. Furthermore, type-specific calibration curves generated with ICC-NanoporeSeq enabled quantification of the infectious concentrations of six EV types, allowing a simultaneous and type-resolved assessment of infectivity in mixed samples. Overall, ICC-NanoporeSeq provides a scalable approach for the parallel analysis of multiple EV types. Compared with the predecessor ICC-RTqPCR method, it eliminates the need for multiple type-specific PCR primers and can therefore be readily expanded to include additional EV types. IMPORTANCECurrent methods used to detect EVs in environmental samples generally measure viral genome copies without determining whether viruses remain infectious, limiting their use in public health risk assessment or water quality monitoring. At the same time, available infectivity assays are often labor-intensive and cannot distinguish between different EV types. Here, we developed ICC-NanoporeSeq, a method combining cell culture and Nanopore sequencing to simultaneously quantify the infectious concentrations of multiple EV types in samples containing mixed EV populations. The method provides an efficient and scalable approach for studying EVs in complex environmental matrices. ICC-NanoporeSeq has potential applications in wastewater-based epidemiology, environmental surveillance, and disinfection studies, where understanding the persistence of different EV types simultaneously is crucial.

Wastewater surveillance (WS) has been widely adopted as a cost-effective and population-representative infectious disease monitoring tool and is increasingly being applied to bacterial and antimicrobial resistance gene (ARG) targets. However, some of these targets may persist in pipe biofilms and detach into wastewater, complicating accurate WS interpretation. To investigate biofilm contributions to wastewater pathogen and ARG signals, paired sink-drain biofilm, branch-drain-plumbing biofilm (sewer biofilm), and wastewater were collected from five hospital sites over a four-month period and analyzed using 16S rRNA gene amplicon sequencing and probe-capture metagenomics. Overall, sewer biofilm bacterial communities were as diverse as wastewater. Across sites, a mean of 9% (0.9 to 23.3%) of wastewater bacterial communities could be attributed to sewer biofilm communities. Many clinically relevant pathogens were consistently detected both in sewer biofilm and wastewater, including environmentally persistent and/or biofilm-associated taxa (e.g., Pseudomonas aeruginosa, Klebsiella pneumoniae). While many ARGs overlapped between wastewater and biofilms (e.g., tetA, sul1, blaCTX-M, vanA), others were significantly enriched in sewer biofilms (e.g., qacL, van-operon and OXA genes). Together, these findings confirm that wastewater pathogen and resistome profiles integrate inputs from both human shedding and pipe-resident communities and therefore need to be considered when selecting WS targets and interpreting signal.

Wastewater genomic surveillance provides an opportunity to detect human and animal influenza A virus (IAV). We aimed to implement an IAV genomic surveillance framework agnostic to subtype, which enables recovery of IAV from multiple hosts and estimation of proportions across subtypes. We conducted IAV genomic surveillance in wastewater during the 2024-2025 flu season at multiple sites in California and compared these data with available human clinical IAV sequences and test positivity. We applied a custom whole-genome, multi-host IAV probe enrichment panel and adapted our custom expectation-maximization (EM) algorithm to deconvolute IAV mixtures in wastewater and infer subtype relative abundances. Absolute IAV concentrations were quantified using RT-PCR-based assays. H5N1 wastewater and clinical sequences were further characterized by constructing a whole-genome maximum-likelihood phylogenetic tree. Finally, we performed variant analysis to examine amino acid substitutions detected in wastewater. Our IAV probe enrichment method and EM algorithm successfully enriched all eight segments of three circulating IAV subtypes and accurately estimated subclade relative abundances for mixed IAV samples. Seasonal human H1N1pdm09 and H3N2 were detected throughout the study period from both wastewater and clinical sequencing data, with H1N1 subclades 6B.1A.5a.2a.1 and 6B.1A.5a.2a co-circulating, and H3N2 dominated by subclade 3C.2a1b.2a.2a.3a.1. Wastewater surveillance consistently detected H5N1 clade 2.3.4.4b across three monitored wastewater sites, while clinical H5N1 detections, from anywhere in CA, were sporadic and rare. Whole-genome phylogenetic analysis revealed that wastewater H5N1 sequences clustered with reference sequences associated with dairy cow and avian infections, while all human clinical H5N1 sequences clustered exclusively with reference sequences associated with dairy cow infections. Amino acid substitutions were identified across viral segments, and no mutations associated with mammalian adaptation were observed from wastewater samples.

Oil sands process-affected water (OSPW) contains complex mixtures of naphthenic acids (NA) that are the central targets for water treatment and reclamation. Here, we compared three whole-cell bacterial NA biosensors with Orbitrap mass spectrometry (MS) for quantifying NA remediation in greenhouse mesocosms and a pilot-scale constructed wetland. Solid-phase extracts from both systems were analyzed in parallel by Orbitrap MS and biosensor assays, enabling direct comparison of biosensor-derived NA estimates with MS-derived naphthenic acid fraction compounds (NAFC) concentrations. Across both treatment systems, biosensor outputs broadly tracked declines in NAFC measured by Orbitrap MS, and positive linear relationships were observed between methods. Biosensor 2 (3680-lux) and biosensor 3 (atuA-lux) showed, early rapid decreases in NA, whereas biosensor 1 (marR-lux) frequently remained elevated later in treatment. These differences are consistent with the distinct chemical response profiles of the biosensor panel and suggest that biosensor outputs reflect shifts in NA mixture composition as remediation proceeds. This interpretation is supported by the published Orbitrap analysis of the constructed wetland, which showed decreasing O2-NAFCs and increasing O3/O4-containing species, consistent with oxidative degradation. Together, these results support the use of NA-responsive biosensors as rapid and scalable complementary tools for tracking remediation trends, while Orbitrap MS remains the reference method for molecular-level characterization.

Nutrient-rich food-processing wastewaters represent valuable yet under-utilised side streams for sustainable protein production in the form of microbial biomass. Here we present an integrated dual-loop bioprocess that converts soybean-processing wastewater into microbial single-cell protein (SCP) while achieving substantial nutrient removal and product refinement. In the first loop, previously enriched microbial consortia were inoculated and cultivated in four parallel sequencing batch reactors (SBRs) for 44days at a hydraulic retention time (HRT) of 3days. This bioprocess configuration demonstrated features that support future scale-up while maintaining process stability, achieving a protein content of 33.3{+/-}3.2%, doubling the protein yield (15.32{+/-}3.49g dry weight per g soluble TKN) and quadrupling the production rate (0.29{+/-}0.06g dry weight L-1 d-1) compared to operating reactors without inoculation (HRT: 7.2days). Effluent treatment was stable, with 84% carbon and 78% nitrogen removal efficiencies, demonstrating efficient nutrient recovery. The SCP biomass was enriched in functional taxa, including Acidipropionibacterium, Lactococcus, Megasphaera, and Azospirillum, suggesting that reactor conditions and inoculum selection promoted a stable, protein-productive microbial community with potential probiotic benefits. In the second loop, bioreactor effluent was reused as aqueous matrix for heat treatment (60{degrees}C) of the SCP biomass, reducing the RNA content from 8.6% to 2.6%, with a 39% biomass loss accompanied by a 30% increase in total amino acid concentration. Hence, our valorisation approach integrates microbial biomass production, effluent reuse, and product refinement within a circular framework. The system provides a resource-efficient pathway for converting food-sector side streams into high-quality microbial community-based SCP, highlighting its potential scalability for sustainable nutrient and water management.

Surface waters in urban watersheds receive episodic inputs of wastewater, runoff, and road-salt residues during spring, yet the contribution of these short hydrological windows to antibiotic resistance gene (ARG) loading remains poorly resolved. Weekly samples were collected from offshore and nearshore sites in the Detroit River, a Great Lakes transboundary connecting channel, from February to December 2025. Five clinically relevant ARGs encoding resistance to carbapenems, methicillin, and colistin, alongside the fecal marker pepper mild mottle virus (PMMoV), were quantified by qPCR and paired with ten conventional water-quality variables. blaNDM, mcr-1, and blaVIM-7 were not detected while blaKPC occurred as discrete pulses. One week (5 May) accounted for 37.2% of annual offshore blaKPC loading, and three weeks in late April to early May accounted for 72.8%, with peak concentrations reaching 5.4 x 10E3 and 7.2 x 10E3 copies/L. mecA was detected in nearly all samples without a dominant pulse. PMMoV normalization showed blaKPC did not vary seasonally (Kruskal-Wallis p = 0.198), consistent with diluted wastewater during spring precipitation events rather than an emergent source. mecA/PMMoV varied seasonally (p = 0.003) and was lowest in spring, implicating non-wastewater inputs in summer and autumn. Seven water-quality variables were significantly elevated during blaKPC pulse weeks. PCA distinguished pulse from background conditions, explaining 81.3% of variance. A random forest classifier achieved leave-one-out AUC of 0.917; ROC AUC reached 0.943 for total phosphorus, 0.924 for chloride, and 0.974 for the multivariate model. These results demonstrate that blaKPC and mecA operate through distinct source pathways and that routine water-quality monitoring can flag elevated blaKPC risk without additional sampling infrastructure.

Groundwater contamination presents challenges across world, yet remediation solutions in variably oxidized regions are limited and many co-interactions between contaminant metals and microbial reactions occur. Here we present a genomic and metabolic study into the biogeochemistry of a uranium-contaminated surficial aquifer site in Riverton, WY. We identified unique communities that varied based on geochemistry, geography, and compartment, matching microbial subsurface studies. Cross-site metabolism tests showed communities had functional capabilities of nitrogen respiration, manganese reduction, iron reduction, and sulfide oxidization. No sites showed evidence of microbial U-bioreduction nor ammonium oxidation. Only former tailings area groundwater and ditch surface water sites nearest a retention pond, and a downgradient oxbow lake exhibited sulfate reduction metabolisms. This was contrary to our hypothesis of near-river downgradient groundwater sites having U and S reduction capability. Most communities which showed S reduction capacity exhibited Fe oxidation capacity. Modeling demonstrated U as calcium uranyl carbonates. Based on our metabolism tests and known mineral and microbial metabolism reduction potentials, this suggests U reduction could only be achieved via abiotic reaction with biogenic sulfide. Of eleven sites tested, it is possible in four. This has impact on future site-specific remediation plans and understanding of microbial reactions in variably reduced zones. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/729369v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1926923org.highwire.dtl.DTLVardef@13486f9org.highwire.dtl.DTLVardef@1895a91org.highwire.dtl.DTLVardef@990213_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Abstract TextWe performed microbial membership and metabolism measurements across a uranium-contaminated sites surface and ground waters, then performed analyses relating these metrics to geochemistry at the site. Findings showed variations in the membership, yet mainly similar functional capabilities. Metabolic differences were explained in relationship to uranium cycling and remediation implications.

Bacterial membrane vesicles (BMVs) have emerged as important contributors to the dissemination of antibiotic resistance genes (ARGs) in the environment. Here, we developed a high-performance immunomagnetic isolation method that improves the purity and selectivity of BMV recovery from wastewater, minimizes contamination from eDNA and viruses, and enables differentiation of BMVs originating from Gram-positive versus Gram-negative bacteria with minimal cross-reactivity. Using this approach, we found that ARGs such as the kanamycin resistance gene (kanR) was highly abundant in BMVs from both raw and treated wastewater, exhibited persistence following treatment, and retained the ability to generate antibiotic-resistant bacteria via transformation. Metagenomic sequencing further revealed that tetracycline resistance genes were the most abundant ARG class across all wastewater samples, while the composition of BMV-associated ARGs differed from the bulk ARG profile. These findings highlight the critical yet underrecognized role of BMVs in the spread of antimicrobial resistance and underscore the need to address BMV-mediated pathways within a One Health framework linking environmental and human health.

Combined sewer overflows (CSOs) are a major pathway for untreated wastewater into urban streams, yet their role in shaping antimicrobial resistance (AMR) dynamics remains poorly understood. Here, we used high-frequency, time-resolved sampling during two storm-triggered CSO events across two monitoring locations and one stormwater-only control site in an urban stream to quantify how these disturbances affect microbial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) in an urban stream. CSO events caused rapid, up to two orders of magnitude, increases in bacterial, pathogen, and ARG abundance, with multiple transient peaks occurring within single overflow episodes. However, these increases were largely proportional to the total bacterial load, and most ARGs and MGEs did not change in relative abundance, indicating that CSOs primarily act as mass-transfer events rather than drivers of in situ selection. Downstream attenuation was governed by hydrological dilution despite additional CSO inputs: Both microbial and resistance signals largely returned to baseline within short time frames. This demonstrates that CSOs function as hydrologically driven pulse disturbances that generate acute but transient AMR exposure. Because CSO events lack the sustained pressure associated with continuous wastewater discharges, rapid washout prevents the long-term establishment of sewage-derived resistance. These findings highlight that AMR risk in CSO-impacted systems is driven primarily by short-term exposure rather than by persistent ecological transformation, with important implications for urban water management under increasingly extreme rainfall conditions.

Fresh produce encounters pathogens at various stages of production and supply, with the harvesting process serving as one of these stages. To evaluate contamination associated with harvesting, we systematically swabbed zone 1 harvester surfaces and quantified Bacteroidales as a fecal biomarker using quantitative polymerase chain reaction (qPCR). Baseline contamination was dominated by non-detects, with occasional low-level detections (<25 copies/cm2) near the assay limit of detection (LoD). Detection occurred more frequently post-harvest (overall [~]4% pre-harvest and 10% post-harvest), while microbial loads remained low, indicating that harvesting primarily affected the likelihood of low-level contamination rather than increasing contamination abundance. Additionally, we developed and field-deployed a portable loop- mediated isothermal amplification (LAMP) assay for rapid harvester hygiene assessment and benchmarked its field performance against qPCR. Together, these results support a practical molecular tool for monitoring fecal contamination and informing cleaning and sanitization decisions.

The selection of appropriate viral indicators for evaluating wastewater treatment performance remains challenging because candidate markers have rarely been compared systematically within a unified analytical framework. Here, we collected influent and effluent samples monthly for one year from two wastewater treatment plants in Japan and conducted, to our knowledge, the first comprehensive comparison of 19 viral targets and one protozoan target using high-throughput quantitative PCR. Pepper mild mottle virus (PMMoV) was consistently detected at high concentrations, showed limited seasonal variability, and exhibited an approximately 1.0 log10 reduction, comparable to those observed for pathogenic viruses. In contrast, Carjivirus, formerly known as crAssphage, was present at the highest concentrations but showed significantly greater reduction than pathogenic viruses. Tomato brown rugose fruit virus (ToBRFV), despite its high abundance and emerging recognition as a potential marker, exhibited pronounced seasonal fluctuations. Other Tobamovirus species, such as cucumber green mottle mosaic virus and tobacco mild green mosaic virus, exhibited similar removal but lower prevalence compared with PMMoV. Overall, PMMoV demonstrated the most balanced performance in terms of abundance, stability, and removal behavior, supporting its use as a robust indicator for monitoring virus removal in wastewater treatment.

Quaternary ammonium compounds (QACs) are high production volume biocidal compounds increasingly scrutinized for their potential to promote antimicrobial resistance spread. This study compared the release of QACs, QAC resistance indicator genes (qacE/qacE{Delta}1), and QAC tolerant bacteria from livestock and human waste streams into the environment. Five livestock farms with on-farm biogas plants (BGPs), a rural and an urban municipal wastewater treatment plant (WWTP) were studied in parallel. In WWTPs, <1% of incoming QACs were discharged with treated wastewater but 10-20% were transferred to sewage sludge. QAC concentrations in sewage sludge far exceeded those in raw and digested manure. The qacE/qacE{Delta}1 genes were detected in all samples with a higher relative abundance in solid than liquid samples. Relative abundances of QAC tolerant fast growing heterotrophic bacteria cultivated under high nutrient conditions at 37{degrees}C were higher in human than livestock waste streams. Providencia and Pseudomonas dominated the cultivated QAC tolerant bacteria in both systems but showed higher QAC tolerance when originating from human waste streams. Additionally, Enterobacteriaceae with higher QAC tolerance were cultivated from human waste streams. Most QAC tolerant strains carried antibiotic resistances without strong system differences. Only few strains carried the qacE/qacE{Delta}1 gene indicating that other mechanisms must be responsible for the increased QAC tolerance. In conclusion, QACs, qacE/qacE{Delta}1, and viable QAC tolerant bacteria including potential pathogenic bacteria were released from livestock and human waste streams into the environment with highest abundances in a post-pandemic sewage sludge sample. Highlights- QACs most abundant in human waste streams, especially biosolids - Higher relative abundance of QAC tolerant bacteria in human waste streams - Pseudomonas and Providencia dominated QAC tolerant bacteria in both waste streams - Enterobacteriaceae with higher QAC tolerance abundant in human waste streams - Most QAC tolerant strains carried additional antibiotic resistances Environmental implicationMunicipal wastewater treatment plants (WWTPs) and livestock farms are hotspots for antimicrobial resistance (AMR) propagation. We compared the simultaneous occurrence of quaternary ammonium compounds (QACs), resistance genes (RGs), QAC-tolerant bacteria, and their multidrug-resistance status in livestock and human waste streams. QACs, indicators of QAC tolerance and AMR occurred in both systems but were higher in WWTPs, especially sewage sludge. Our findings highlight the need for prudent disinfectant use and enhanced waste treatments to reduce the risks of spreading micropollutants, pathogens, and AMR via organic fertilizers or treated wastewater recycled in circular agricultural practice.