ACS ES&T Water

Mass gatherings pose significant public health risks by facilitating the spread of infectious diseases. While wastewater-based surveillance (WBS) has been widely used to monitor pathogens in high-income settings, its use as a practical, multi-pathogen surveillance tool during mass gatherings in low- and middle-income countries remains limited. This study aimed to assess the operational feasibility, epidemiological significance, and public health utility of multi-pathogen WBS during the African Nations Championship (CHAN) football tournament in Uganda. Wastewater surveillance was conducted at Mandela National Stadium during eight match days in August 2025. Moore swabs were deployed at 38 manholes receiving wastewater from different toilet facilities across the stadium to capture representative wastewater samples. Samples were processed using Nanotrap(R) microbiome virus particles to concentrate pathogens, followed by nucleic acid extraction. Samples were analyzed for multiple enteric and respiratory pathogens, including Mpox, using quantitative PCR (qPCR). Descriptive analyses were performed to characterize pathogen detection patterns, positivity rates, and temporal distribution across surveillance sites. A total of 304 wastewater samples were collected and analyzed, of which 259 (85.2%) tested positive for at least one pathogen. Multiple pathogens were consistently detected across sampling days, with enteric pathogens predominating, particularly Shigella spp. (53.6%), Rotavirus A (35.9%) and Enterovirus (32.2%). The mpox virus was also detected in a notable proportion of samples (28.6%) across several sampling days. Respiratory pathogens, including SARS-CoV-2 (11.8%) and Influenza B (8.2%), were identified intermittently at lower frequencies. Pathogen diversity varied over time, with up to eight pathogens detected on a single day, and co-detection of multiple pathogens observed in the majority of positive samples. Cq value distributions further demonstrated variability in detected signal patterns across pathogens. Surveillance findings informed real-time public health interventions, including sanitation reinforcement, intensified hygiene promotion, environmental disinfection, and targeted risk communication, strengthened syndromic surveillance with on-site triage, and targeted environmental health assessments of food handling and wastewater infrastructure. These findings demonstrate the operational feasibility and public health utility of integrating multi-pathogen wastewater-based surveillance into mass-gathering preparedness and response frameworks in low-resource settings. By capturing diverse pathogen signals and informing targeted interventions during the CHAN football tournament, WBS can provide actionable population-level insights that can support outbreak preparedness and response. Scaling WBS within national preparedness systems could strengthen epidemic intelligence, enhance early warning capacity, and support data-driven public health decision-making during future mass gatherings and emerging infectious disease threats.

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.

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.

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

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.

O_LIHeavy metals are pervasive environmental contaminants that can impair the health of organisms globally. As the largest anthropogenic source of the potent neurotoxin mercury (Hg), gold mining has amplified these threats throughout the tropics. Consequently, there is a mounting need to monitor Hg contamination of the richest biological communities on Earth. Venous whole blood provides a reliable, nonlethal measurement of recent dietary and site-derived contamination, but collecting and cold-storing samples can be impractical in field conditions. C_LIO_LITo overcome these challenges, we developed and evaluated a method to assay Hg exposure in vascular organisms by measuring the volume of dried blood spots (DBS) in the field, which can be stored at ambient temperatures until analysis. We explored the methods precision and accuracy in estimating whole blood Hg concentrations by collecting paired whole blood and DBS aliquots from birds (n = 527 individuals, 140 species) along a trophic gradient (i.e., granivores to piscivores) in Belize and Peru. C_LIO_LIUsing a Bayesian linear mixed-effects model, we found a highly precise and unbiased relationship between DBS and whole blood total Hg concentrations that was centered at perfect unity (R2 = 0.99; {beta} = 1.00 {+/-} 0.03; 95% CrI: 0.95-1.05). Agreement between individual paired aliquots was more variable, in which approximately 12% of DBS containing at least 1 ng THg differed from whole blood by more than {+/-}20%. However, DBS accuracy increased at higher THg concentrations, suggesting that disagreement at low concentrations is an expected consequence of higher measurement error near the analytical limit of detection of our instruments. C_LIO_LICompared to whole-blood collection and analysis workflows, DBS offer substantial logistical advantages by eliminating cold-chain dependence and reducing transport burden, laboratory handling time, and overall operational costs. Consequently, volume-measured DBS provide a practical and highly reliable alternative for monitoring Hg contamination in both humans and wildlife, particularly for ecological and population-level applications in remote and resource-limited environments. C_LI

Wastewater-based surveillance (WBS) is increasingly used to monitor infectious disease dynamics, yet most evaluations focus on correlation or forecasting - neither of which directly assesses whether wastewater signals can identify the epidemiological events most relevant to public health decision-making. We argue that outbreak onset and epidemic peak detection are the operationally critical use cases of WBS, requiring a fundamentally different evaluation framework. We introduce a classification-based framework that treats WBS as an event-detection problem, defining outbreaks and peaks as discrete events, establishing detection intervals to account for timing uncertainty, and incorporating censoring and data completeness criteria for valid comparisons against imperfect clinical reference outcomes. Within this framework, we apply a Bayesian exponential growth model for outbreak detection - benchmarked against a standard reproductive number (Rt)-based method - and a rule-based algorithm for peak detection, evaluating performance via sensitivity and positive predictive value (PPV). Applied to county-level SARS-CoV-2 wastewater data from 281 U.S. counties (Biobot, 2021-2024), the exponential growth approach substantially outperforms the Rt-based baseline: sensitivity 0.82 and PPV 0.64 versus sensitivity 0.58 and PPV 0.19 for the best-performing Rt variant. Peak detection achieves sensitivity 0.84 and PPV 0.70 at the county level. Both peak and outbreak detection achieve strong and consistent performance against hospitalizations and deaths at the state level. Spatial aggregation yields a statistically significant improvement in peak detection PPV against a curated reference standard ($p < 0.001$), while outbreak detection improvements under aggregation are directionally consistent but not statistically significant. Wastewater leads case-defined outbreaks by 4-6 days but minimally leads epidemic peaks, consistent with wastewater approximating prevalence rather than incidence. These findings demonstrate that wastewater signals can reliably detect outbreak onset and epidemic peaks across spatial scales and clinical outcomes, and that the choice of detection method matters substantially in practice. The classification framework developed here provides a reusable and principled tool for evaluating any surveillance signal as an event-detection system, with direct relevance to how WBS is actually used in public health decision-making.

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.

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.

Dietary exposure to heavy metals (HMs) via animal-source foods is a critical environmental health pathway. In rapidly industrializing Bangladesh, contamination of the bovine food chain from agricultural feeds and industrial emissions poses an unquantified public health burden. This study evaluated exposure pathways, spatial distribution, mass-transfer dynamics, and health risks of six HMs (Cr, Cu, Cd, Pb, As, and Hg) across the fodder-cattle-human continuum. Samples of beef (n = 76), raw milk (n = 76), commercial cattle feed (n = 40), and fodder (n = 88) were collected from eight sites across industrial and non-industrial zones in Bangladesh and analysed by atomic absorption spectroscopy. Probabilistic Monte Carlo simulations (10,000 iterations) quantified estimated daily intake, target hazard quotients (THQ), cumulative hazard index (HI), and lifetime carcinogenic risk (CR) for adult and pediatric receptors. Copper (Cu) was the dominant contaminant across all matrices, peaking in beef (103.89 {+/-} 15.87 mg/kg) and milk (13.67 {+/-} 1.53 mg/L). Spatial analysis revealed distinct contamination profiles: Pb burden peaked in industrial zones while Cr was elevated in non-industrial sectors. Monte Carlo modelling identified commercial feed as the most efficient transfer vector into beef. Pediatric THQ for Cu significantly exceeded the safety threshold (THQ > 1), and upper-bound lifetime carcinogenic risk from As approached the critical USEPA 10- regulatory ceiling. These findings demonstrate that industrial and agricultural externalities efficiently contaminate the bovine food supply chain in Bangladesh, with copper and arsenic representing the most critical non-carcinogenic and carcinogenic dietary hazards, respectively. Children are disproportionately vulnerable due to lower body weight. The results underscore the need for targeted upstream interventions in commercial feed production and provide evidence to support feed-quality regulation and environmental monitoring in rapidly industrializing settings.

Geothermal springs are unique environments that harbor diverse populations of microorganisms. As a result of their environmental and geochemical variability, different springs can support distinct heterogeneous communities of organisms with unique functional adaptations and metabolic capabilities. A recent molecular survey of Aotearoa-New Zealand hot springs indicated that these springs may support thermophilic microorganisms be able to degrade plastics. To test this, we applied a cultivation-centered approach via an in situ enrichment of putative plastic-degraders using high surface are polyethylene terephthalate (PET), polylactic acid (PLA), and polyhydroxybutyrate (PHB) substrates in a diversity of New Zealand hot springs. The plastic associated microbial communities were characterized via marker gene and analyses. Finally, plastic-associated biofilms were used as inoculum to isolate thermophilic plastic degraders. Via this process, we confirm that there are plastic degrading bacteria are present in springs across Aotearoa-New Zealand. Moreover, we isolated two PHB degrading strains (Cuprividus sp. and Rubrobacter sp.) and demonstrated their capability to metabolize plastic under thermophilic conditions in vitro. While the pathways identified in our plastic degrading isolates suggest they may be able to metabolize plastics for carbon, the primary use of plastics by geothermal microbial communities does not appear to be as an energy source. Instead, they appear to mainly serve as surfaces for microbial attachment, composed primarily of non-plastic degrading taxa.

Antibiotic resistance genes (ARGs) are an emerging class of environmental contaminants with significant implications for public health. Previous studies have linked fungicide exposure to elevated levels of ARGs in soil microbiomes, but research investigating the impacts of fungicide on ARGs within phyllosphere bacterial communities is limited. To address this, creeping bentgrass was treated with the fungicide active ingredients chlorothalonil, fluxapyroxad, and propiconazole and sampled at 4 hours, 96 hours, and one week post-application. Quantitative PCR (qPCR) was performed to quantify the abundance of ARGs and a metal resistance genes (MRG) abundance relative to 16s. Additionally, 16S rRNA gene sequencing was performed to characterize bacterial community composition. Results indicated that fungicide treatments did not significantly alter the relative abundance of ARGs or an MRG within bent grass bacterial communities. However, significant changes were observed over time, with changes in ARG and MRG abundance mirroring temporal shifts in bacterial beta diversity. ARGs and the MRG relative abundance had significant correlations with Proteobacteria, Actinobacteria, Bacteriadota, and Firmicutes, including with genera Pseudoxanthomas, and Dyandobacter, which contain opportunistic human pathogens. This study demonstrates that fungicides have limited influence on the abundance of ARGs and MRGs in the phyllosphere and helps guide further investigations aiming to mitigate the spread of antibiotic resistance. ImportanceAntibiotic resistance makes it harder to treat bacterial infections. Recent evidence suggests that fungicides may increase the abundance of antibiotic resistance genes (ARGs) in soil. Leaves are also treated with fungicides, but it is unclear if there will be increases of antibiotic resistance in this environment because bacteria on leaves face different physiological stresses. Additionally, plants may serve as a route of infection to humans or animals with antibiotic resistant bacteria making it a critical micro-environment to investigate. The purpose of this study was to determine whether the prevalence of antibiotic resistance on plant surfaces changes after short-term exposure to fungicides. This study shows that over short-term application periods, time has a greater effect on the abundance of genes that cause antibiotic resistance than treatment with the fungicides chlorothalonil, fluxapyroxad, and propiconazole on plants. This work helps inform future efforts to mitigate the spread of antibiotic resistance.

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.

The foodborne mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) have been associated with several adverse effects, including cytotoxicity, genotoxicity, endocrine disruption, and immunomodulation. As these endpoints are typically observed in vitro at micromolar concentrations, the question arises whether such levels are attainable in exposed humans. To address this data gap in chemical risk assessment, a physiologically based kinetic (PBK) model was developed to predict internal exposure doses to AOH and AME in humans. As input parameters, kinetic constants for hepatic glucuronidation were obtained in vitro by incubating Sprague Dawley rat and human liver S9 fractions with 0.5-50 M AOH and 0.5-20 M AME, demonstrating rapid biotransformation in both species. Intestinal absorption of AME and physicochemical parameters were estimated using quantitative structure-activity relationship (QSAR) models. Sensitivity analysis identified parameters describing hepatic glucuronidation and gastrointestinal uptake as among the most influential, confirming the importance of their reliable estimation. The PBK model was evaluated against available rodent toxicokinetic data and subsequently extrapolated to humans. Ultimately, the currently available exposure estimates published by EFSA in 2016 were applied to predict target tissue concentrations, which were compared to points of departure (PoDs) for relevant toxicological endpoints. Even in the most susceptible group of male toddlers, predicted internal concentrations (10-4 M range) were approximately four orders of magnitude below the respective PoDs. Consequently, under the applied exposure assumptions and considering the compounds as isolated chemicals, AOH and AME are not expected to reach systemic or tissue concentrations associated with the investigated effects.

Agricultural fields in the Mezquital Valley, Mexico, were irrigated with untreated wastewater over several decades. Following the construction of a wastewater treatment plant (WWTP) in Atotonilco de Tula, WWTP effluent is used for irrigation. To evaluate the effects of changed irrigation, a soil incubation experiment was performed. Soils of the Mezquital Valley long-term irrigated with untreated wastewater were irrigated with WWTP influent or effluent, both unspiked and spiked with antibiotics and biocidal compounds and incubated four weeks. We investigated the effects of shifted irrigation on the abundance of cultivable total heterotrophic and resistant bacteria (RB). Additionally, RB were cultivated from Coriandrum sativum (cilantro) sown in soil of the incubation experiment. While wastewater treatment significantly reduced the bacterial abundance in effluent, spiking increased RB abundance in both wastewater types including ciprofloxacin (CIP) RB. Before wastewater addition, all soils contained cultivable RB. Irrigation increased the relative abundance of RB cultivated on Mueller Hinton (MH) agar in Leptosols and Phaeozems, compared to soils prior to wastewater addition irrespective of the water type, but not in Vertisols, suggesting the soil type rather than water qualities influenced the RB abundance. Diverse CIP RB were cultivated including strains of 14 genera of three phyla. Among those, Achromobacter spp. strains related to potentially pathogenic A. spanius originating from soil were abundant in both leaves and roots of cilantro. Our results showed that the implementation of wastewater treatment does not reduce the abundance of cultivable RB in Mezquital Valley soils and cilantro plants. Health risk associated monitoring should include long-term persistent RB colonizing plants cultivated in wastewater irrigated soils.

Riverine ecosystems particularly in industrialized environment are subjected to chronic press disturbances, resulting from the decadal release of synthetic organic compounds and other xenobiotics. While indigenous microbial communities are highly sensitive to such stressors, the resulting metabolic restructuring and functional reshaping of the microbiome, driven by these long-term anthropogenic pressures remains poorly characterized. In this study, a microbial ecology of Bhadar River flowing across the Jetpur Industrial Estate, (Jetpur) were studied. Using a cross-sectional comparative approach, soil/sediment samples were collected from the diverse polluted and non-polluted sites from the estate. The taxonomic profiling using 16S rRNA gene amplicon sequencing, taxo-phenomic shifts (through metaphenomics) was studied, while the functional potential of metabolic pathways was validated using high-resolution shot-gun metagenomic study. Due to prolong pollution, the samples were rich in sulphur (9809 to 12391 mg/L), where polluted samples were having elevated COD (2432 to 4150 mg/L) as well as BOD (1000 to 1420 mg/L) values, along with the presence of heavy metals (e.g., Fe, Mg). Results revealed a distinct taxonomic shift at both the bacterial and archaeal levels. In non-polluted sites Proteobacteria (33 to 57%) dominated along with Acidobacteria and Actinobacteria, with diverse genera like Alcaligenes and Serratia. Whereas, polluted sites exhibited marked increase in Bacteroidetes (13 to 29%), Firmicutes, and Synergistetes and genera like Alkalitalea, Mesotoga and Desulfomicrobium, reflecting anaerobic, fermentative, and sulfate-reducing phenotypes. The archaeal communities at polluted sites were dominated by Euryarchaeota (78 to 99%), specifically methanogenic genera of Methanosaeta and Methanocalculus, contrasting with the Methanomassiliicoccus dominance in non-polluted areas. The alpha-diversity was marginally higher in polluted sites (Shannon: 4.11 to 4.81 vs. 3.81 to 5.39 (non-polluted)), but beta-diversity underscored clear separation (94% variance explained by pollution). The shot-gun metagenomic analysis indicated a substantial enhancement in anaerobic metabolic capacities within the polluted microbiome, primarily in sulphur respiration (dissimilatory sulfate reduction), methanogenesis (elucidating biogenic pathways), along with nitrogen cycling (identifying key denitrification and ammonification genes). The polluted microbiome have developed the potential to metabolise/degrade complex aromatic compounds (pcaK for benzoate/protocatechuate transport) and heavy metal resistance. The strong positive co-occurrences among anaerobic phyla (Thermotogae, Synergistetes, Bacteroidetes) in polluted sites was established, indicating syntrophic interactions for xenobiotic metabolism. These findings provide a theoretical ecological model for perturbed industrial ecosystems, emphasizing the role of habitat selection in shaping microbial functional diversity and demonstrate the remarkable adaptation of autochthonous communities to persistent press disturbances.

Coastal concrete structures and drainage pipes are prone to microbially influenced deterioration. However, differences in microbial communities and their corrosion/healing potentials between these habitats remain unclear. Here, we compared bacterial(16S), fungal (ITS) and algal(18S) communities on coastal concrete(C) and drainage pipe(P) surfaces. Fungal and algal -diversity were significantly higher in P than in C, while bacterial diversity did not differ. {beta}-Diversity strongly separated bacterial and algal communities between habitats, but not fungi. A shared core "seed bank" of 575 bacterial, 520 fungal and 40 algal ASVs was identified. Students t-test revealed that P enriched oligotrophic degraders (Sphingomonas) and acid-producing fungi (Arxiella, Bisifusarium), whereas C selected for halotolerant EPS-producing bacteria (Tunicatimonas, Muricauda) and the extremotolerant alga Coelastrella. db-RDA linked these differences to salinity, NH4+-N, NO3--N, and COD. Functional prediction indicated a shift from metabolism pathways in C to signaling in P. Co-occurrence networks revealed cross-kingdom competition and within-kingdom cooperation, especially among algae. Importantly, both habitats harbored microorganisms with documented corrosion and healing potentials, but under natural conditions, net deterioration dominated, microbial healing is hardly to counteract the negative effects. This study provides a functional taxonomic framework for understanding and managing concrete microbiomes in coastal and sewer infrastructure. ImportanceConcrete is the most widely used construction material, and its deterioration in coastal and sewer environments poses significant economic and safety challenges. Microorganisms play a dual role in concrete durability -- they can both corrode and heal concrete, but the net outcome under natural conditions is poorly understood. Most studies have focused on bacteria alone, overlooking the contributions of fungi and algae, and few reports on the of coastal concrete which also exist the microbially influenced concrete corrosion (MICC) similar to sewer. Here, we simultaneously analyzed all three microbial kingdoms on coastal concrete and drainage pipes. We found that while concrete surfaces harbor a diverse "seed bank" of microorganisms potentially involved in both corrosion and healing, under natural conditions, net deterioration dominated, indicating microbial healing is hard to counteract the negative effects from the environment and microbial. Our work provides a functional framework to guide the development of microbiome-based strategies for enhancing concrete durability, such as activating rare healing taxa in drainage pipes or selecting for endogenous healers in marine environments.

The slightly-alkaline (pH [~]8.5), boiling ([~]90{degrees}C) vent-water of a Trans-Himalayan geothermal spring, moderately-rich in dissolved solids ([~]1500 ppm), was explored six times over a year. 11 archaeal and 46 bacterial species were detected consistently, while nine bacteria occurred intermittently, in the vent-epicenter featuring a largely-stable physicochemical milieu. All 11 archaea were detected as metagenome-assembled genomes ascribable to Thermoproteota. Of the total 55 bacteria detected, 32 were retrieved as MAGs, 20 as isolates, and three in both forms. Four bacteria could not be classified below the domain-level; three and four belonged to hyperthermophilic (Aquificia) and thermophilic (Thermaceae and Thermoflexaceae) taxa respectively; 27 belonged to taxa having some moderately-thermophilic members; 17 belonged to mesophilic taxa. According to metagenomics, an Aquificia, followed by two Thermoprotei and one Thermoproteales, dominated the microbiome overwhelmingly. Metatranscriptomically, however, the Thermoproteales was most active. Metatranscriptomic signatures envisaged the in situ metabolic status of the 66 species discovered as follows. Among the 18 putative hyperthermophiles and thermophiles identified, 17 rendered wide-ranging activities including growth; one Thermoproteota species had considerable activities sans growth. One new-phylum-level bacterium rendered wide-ranging activities including growth, while three such entities had considerable/minimal activities sans growth. Among the 27 potential moderate-thermophiles discovered, two Armatimonadota and one Thermosynechococcus species rendered wide-ranging activities including growth, 20 had considerable/minimal activities sans growth, whereas four had zero activities. Among the 17 mesophiles identified, 16 rendered considerable/minimal activities sans growth, whereas one had zero activity. Molecular drivers were envisaged from the metatranscriptomic data to explain the trends of inequitable population ecology.

Wastewater treatment plants (WWTPs) are known reservoirs of antibiotic resistance genes (ARGs). Non-antibiotic compounds such as antidepressants may further promote ARG acquisition through horizontal gene transfer (HGT). Desvenlafaxine, a serotonin-norepinephrine reuptake inhibitor (SNRI) listed on the EU Surface Water Watch Lists, is among the most frequently detected antidepressants in WWTP effluents, yet its role in HGT has not been examined. Here, we detected desvenlafaxine at the highest concentrations among four antidepressants monitored across three municipal WWTPs in western New York. Using Acinetobacter baylyi ADP1 as a model recipient in natural transformation assays (n = 6), we found that desvenlafaxine significantly increased transformation frequency at 10 mg/L (1.74 {+/-} 0.33-fold) and 50 mg/L (1.49 {+/-} 0.19-fold; Padj < 0.05). Effects were independent of reactive oxygen species or membrane permeability stress, consistent with its very low toxicity (IC20 ~1353 mg/L). Instead, desvenlafaxine induced dose-dependent increases in membrane fluidity and shifts to less negative zeta potentials, suggesting that electrostatic interactions between its cationic amine group and the negatively charged membrane reduce surface repulsion and facilitate plasmid proximity during uptake. Non-targeted proteomics revealed a biphasic response: at 10 mg/L, competence-associated proteins (PilB, ComM) were upregulated and STRING analysis identified networks linked to membrane transport, transcriptional regulation, and envelope remodeling, while no connected network was recovered at 50 mg/L. Electron microscopy confirmed higher pili frequency at both doses. Together, these findings reveal an overlooked role of this non-antibiotic pharmaceutical in promoting ARG spread from wastewater environments.