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Antimicrobial resistance (AR) is a serious global problem due to the overuse of antimicrobials in human, animal, and agriculture sectors. There is intense research to control the dissemination of AR, but little is known regarding the environmental drivers influencing its spread. Although AR genes (ARGs) are detected in many different environments, the risk associated with the spread of these genes to microbial pathogens is unknown. Recreational microbial exposure risks are likely to be greater in water bodies receiving discharge from human and animal waste in comparison to less disturbed aquatic environments. Given this scenario, research practitioners are encouraged to consider an ecological context to assess the effect of environmental ARGs on public health. Here, we use a stratified, probabilistic survey of nearly 2000 sites to determine national patterns of the anthropogenic indicator class I integron Integrase gene (

Wastewater-based SARS-CoV-2 monitoring is an effective tool for rapid management of the pandemic. Evaluating methodologies during the height of this crisis strengthens the field to ensure reproducibility across states, nations, and the globe.

Facing challenges in water demands and population size, particularly in the water-scarce regions in the United States, the reuse of treated municipal wastewater has become a viable potential to relieve the ever-increasing demands of providing water for (non-)potable use. The objectives of this study were to assess microbial quality of reclaimed water and to investigate treatability of microorganisms during different treatment processes. Raw and final treated effluent samples from three participating utilities were collected monthly for 16 months and analyzed for various microbial pathogens and fecal indicator organisms. Results revealed that the detectable levels of microbial pathogens tested were observed in the treated effluent samples from all participating utilities. Log10 reduction values (LRVs) of Cryptosporidium oocysts and Giardia cysts were at least two orders of magnitude lower than those of human adenovirus and all fecal indicator organisms except for aerobic endospores, which showed the lowest LRVs. The relatively higher LRV of the indicator organisms such as bacteriophages suggested that these microorganisms are not good candidates of viral indicators of human adenovirus during wastewater treatment processes. Overall, this study will assist municipalities considering the use of wastewater effluent as another source of drinking water by providing important data on the prevalence, occurrence, and reduction of waterborne pathogens in wastewater. More importantly, the results from this study will aid in building a richer microbial occurrence database that can be used towards evaluating reuse guidelines and disinfection practices for water reuse practices.

Wastewater monitoring of SARS-CoV-2 presents a means of tracking COVID-19 community infection dynamics on a broader geographic scale. However, accounting for environmental and sample-processing losses may be necessary for wastewater measurements to readily inform our understanding of infection prevalence. Here, we present measurements of the SARS-CoV-2 N1 and N2 gene targets from weekly wastewater samples at three sites in Hamilton County, Ohio, during an increase and subsequent decline of COVID-19 infections. The concentration of N1 or N2 RNA in wastewater, measured over the course of six months, ranged from below the detection limit to over 104 gene copies/L, and correlated with case data at two wastewater treatment plants, but not at a sub-sewershed-level sampling site. We also evaluated the utility of a broader range of variables than has been reported consistently in previous work, in improving correlations of SARS-CoV-2 concentrations with case data. These include a spiked matrix recovery control (OC43), flow-normalization, and assessment of fecal loading using endogenous fecal markers (HF183, PMMoV, CrAssphage). We found that adjusting for recovery, flow, and fecal indicators increased these correlations for samples from a larger sewershed (serving ~488,000 people) with greater industrial and stormwater inputs, but raw N1/N2 concentrations corresponded better with case data at a smaller, residential-oriented sewershed. Our results indicate that the optimal adjustment factors for correlating wastewater and clinical case data moving forward may not be generalizable to all sewersheds., Graphical abstract Unlabelled Image

Correction for ‘Evaluation of intra- and inter-lab variability in quantifying SARS-CoV-2 in a state-wide wastewater monitoring network’ by Angela Davis et al., Environ. Sci.: Water Res. Technol., 2023, https://doi.org/10.1039/D2EW00737A.

Microbiota regulate important physiologic processes during early host development. They also biotransform xenobiotics and serve as key intermediaries for chemical exposure. Antimicrobial agents in the environment may disrupt these complex interactions and alter key metabolic functions provided by host-associated microbiota. To examine the role of microbiota in xenobiotic metabolism, we exposed zebrafish larvae to the antimicrobial agent triclosan. Conventionally colonized (CC), microbe-free axenic (AX), or axenic colonized on day 1 (AC1) zebrafish were exposed to 0.16–0.30 µM triclosan or vehicle on days 1, 6, 7, 8, and 9 days post fertilization (dpf). After 6 and 10 dpf, host-associated microbial community structure and putative function were assessed by 16S rRNA gene sequencing. At 10 dpf, triclosan exposure selected for bacterial taxa, including Rheinheimera. Triclosan-selected microbes were predicted to be enriched in pathways related to mechanisms of antibiotic resistance, sulfonation, oxidative stress, and drug metabolism. Furthermore, at 10 dpf, colonized zebrafish contained 2.5–3 times more triclosan relative to AX larvae. Nontargeted chemical analysis revealed that, relative to AX larvae, both cohorts of colonized larvae showed elevations in 23 chemical features, including parent triclosan and putative triclosan sulfate. Taken together, these data suggest that triclosan exposure selects for microbes that harbor the capacity to biotransform triclosan into chemical metabolites with unknown toxicity profiles. More broadly, these data support the concept that microbiota modify the toxicokinetics of xenobiotic exposure.

Estrogenic chemicals are widespread environmental contaminants associated with diverse health and ecological effects. During early vertebrate development, estrogen receptor signaling is critical for many different physiologic responses, including nervous system function. Recently, host-associated microbiota have been shown to influence neurodevelopment. Here, we hypothesized that microbiota may biotransform exogenous 17-βestradiol (E2) and modify E2 effects on swimming behavior. Colonized zebrafish were continuously exposed to non-teratogenic E2 concentrations from 1 to 10 days post-fertilization (dpf). Changes in microbial composition and predicted metagenomic function were evaluated. Locomotor activity was assessed in colonized and axenic (microbe-free) zebrafish exposed to E2 using a standard light/dark behavioral assay. Zebrafish tissue was collected for chemistry analyses. While E2 exposure did not alter microbial composition or putative function, colonized E2-exposed larvae showed reduced locomotor activity in the light, in contrast to axenic E2-exposed larvae, which exhibited normal behavior. Measured E2 concentrations were significantly higher in axenic relative to colonized zebrafish. Integrated peak area for putative sulfonated and glucuronidated E2 metabolites showed a similar trend. These data demonstrate that E2 locomotor effects in the light phase are dependent on the presence of microbiota and suggest that microbiota influence chemical E2 toxicokinetics. More broadly, this work supports the concept that microbial colonization status may influence chemical toxicity.

Roof runoff has the potential to serve as an important local water source in regions with growing populations and limited water supply. Given the scarcity of guidance regulating the use of roof runoff, a need exists to characterize the microbial quality of roof runoff. The objective of this 2-year research effort was to examine roof runoff microbial quality in four U.S. cities: Fort Collins, CO; Tucson, AZ; Baltimore, MD; and Miami, FL. Seven participants, i.e., homeowners and schools, were recruited in each city to collect roof runoff samples across 13 precipitation events. Sample collection was done as part of a citizen science approach. The presence and concentrations of indicator organisms and potentially human-infectious pathogens in roof runoff were determined using culture methods and digital droplet polymerase chain reaction (ddPCR), respectively. The analyzed pathogens included Salmonella spp., Campylobacter spp., Giardia duodenalis, and Cryptosporidium parvum. Several factors were evaluated to study their influence on the presence of potentially human-infectious pathogens including the physicochemical characteristics (total suspended solids, volatile suspended solids, total dissolved solids, chemical oxygen demand, and turbidity) of roof runoff, concentrations of indicator organisms, presence/absence of trees, storm properties (rainfall depth and antecedent dry period), percent of impervious cover surrounding each sampling location, seasonality, and geographical location. E. coli and enterococci were detected in 73.4% and 96.2% of the analyzed samples, respectively. Concentrations of both E. coli and enterococci ranged from0 log

Greywater, the wastewater from sinks, showers, and laundry, is an understudied environment for bacterial communities. Most greywater studies focus on quantifying pathogens, often via proxies used in other wastewater, like fecal indicator bacteria; there is a need to identify more greywater-appropriate surrogates, like Staphylococcus sp. Sequencing-based studies have revealed distinct communities in different types of greywater as well as in different parts of greywater infrastructure, including biofilms on pipes, holding tanks, and filtration systems. The use of metagenomic sequencing provides high resolution on both the taxa and genes present, which may be of interest in cases like identifying pathogens and surrogates relevant to different matrices, monitoring antibiotic resistance genes, and understanding metabolic processes occurring in the system. Here we review what is known about bacterial communities in different types of greywater and its infrastructure. We suggest that wider adoption of environmental sequencing in greywater research is important because it can describe the entire bacterial community along with its metabolic capabilities, including pathways for removal of nutrients and organic materials. We briefly describe a metagenomic dataset comparing different types of greywater samples in a college dormitory building to highlight the type of questions these methods can address. Metagenomic sequencing can help further the understanding of greywater treatment for reuse because it allows for identification of new pathogens or genes of concern.

Background Across taxa, animals with depleted intestinal microbiomes show disrupted behavioral phenotypes. Axenic (i.e., microbe-free) mice, zebrafish, and fruit flies exhibit increased locomotor behavior, or hyperactivity. The mechanism through which bacteria interact with host cells to trigger normal neurobehavioral development in larval zebrafish is not well understood. Here, we monoassociated zebrafish with either one of six different zebrafish-associated bacteria, mixtures of these host-associates, or with an environmental bacterial isolate. Results As predicted, the axenic cohort was hyperactive. Monoassociation with three different host-associated bacterial species, as well as with the mixtures, resulted in control-like locomotor behavior. Monoassociation with one host-associate and the environmental isolate resulted in the hyperactive phenotype characteristic of axenic larvae, while monoassociation with two other host-associated bacteria partially blocked this phenotype. Furthermore, we found an inverse relationship between the total concentration of bacteria per larvae and locomotor behavior. Lastly, in the axenic and associated cohorts, but not in the larvae with complex communities, we detected unexpected bacteria, some of which may be present as facultative predators. Conclusions These data support a growing body of evidence that individual species of bacteria can have different effects on host behavior, potentially related to their success at intestinal colonization. Specific to the zebrafish model, our results suggest that differences in the composition of microbes in fish facilities could affect the results of behavioral assays within pharmacological and toxicological studies.

Antimicrobial resistance (AMR) in the environment is a growing global health concern, especially the dissemination of AMR into surface waters due to human and agricultural inputs. Within recent years, research has focused on trying to understand the impact of AMR in surface waters on human, agricultural and ecological health (One Health). While surface water quality assessments and surveillance of AMR have historically utilized culture-based methods, culturing bacteria has limitations due to difficulty in isolating environmental bacteria and the need for a priori information about the bacteria for selective isolation. The use of molecular techniques to analyze AMR at the genetic level has helped to overcome the difficulties with culture-based techniques since they do not require advance knowledge of the bacterial population and can analyze uncultivable environmental bacteria. The aim of this review is to provide an overview of common contemporary molecular methods available for analyzing AMR in surface waters, which include high throughput real-time polymerase chain reaction (HT-qPCR), metagenomics, and whole genome sequencing. This review will also feature how these methods may provide information on human and animal health risks. HT-qPCR works at the nanoliter scale, requires only a small amount of DNA, and can analyze numerous gene targets simultaneously, but may lack in analytical sensitivity and the ability to optimize individual assays compared to conventional qPCR. Metagenomics offers more detailed genomic information and taxonomic resolution than PCR by sequencing all the microbial genomes within a sample. Its open format allows for the discovery of new antibiotic resistance genes; however, the quantity of DNA necessary for this technique can be a limiting factor for surface water samples that typically have low numbers of bacteria per sample volume. Whole genome sequencing provides the complete genomic profile of a single environmental isolate and can identify all genetic elements that may confer AMR. However, a main disadvantage of this technique is that it only provides information about one bacterial isolate and is challenging to utilize for community analysis. While these contemporary techniques can quickly provide a vast array of information about AMR in surface waters, one technique does not fully characterize AMR nor its potential risks to human, animal, or ecological health. Rather, a combination of techniques (including both molecular- and culture-based) are necessary to fully understand AMR in surface waters from a One Health perspective.

Because disease pandemics can accelerate rapidly in areas with limited clean-water access, a portable greywater reuse system may be useful to provide wash water at emergency health care units. In this study, a novel fed-batch (hybrid continuous-batch flow) net-zero water (NZW), or nearly closed-loop, reuse system comprising screening, 5 μm filter, and ozone-UV advanced oxidation was designed, constructed, and tested for performance with simulated and actual human showers. Water quality was tested for compliance with US drinking water standards, total organic carbon < 0.5 mg/L, and pathogen inactivation including 12 log(10) virus, 10 log(10) protozoa, and 9 log(10) bacteria as has been recommended for direct potable reuse. Energy, operation, and maintenance requirements were also evaluated, along with the system’s capacity to handle shock events such as unintentional contamination with urine. Design goals were achieved without the addition of GAC point-of-use filter, except compliance with bromate and nitrate drinking water standards, which were met only for temporary use of up to three years per person. A capacity of 32 showers/day at 1920 W continuous power is projected, without generation of potentially-infectious concentrate. To avoid the further increase in system weight and energy demand needed to address urine input, future integrated urine diversion and collection, and system drain-and-fill following detection of urine in recycled water by electrical conductivity, are suggested for the field unit. Field testing is recommended. Further research should focus on potential need for bromate/nitrate mitigation, and longer-term study of microbiological inactivation.

Host-associated microbiota can biotransform xenobiotics, mediate health effects of chemical exposure, and play important roles in early development. Bisphenol A (BPA) is a widespread environmental chemical that has been associated with adverse endocrine and neurodevelopmental effects, some of which may be mediated by microbiota. Growing public concern over the safety of BPA has resulted in its replacement with structurally similar alternatives. In this study, we evaluated whether BPA and BPA alternatives alter microbiota and modulate secondary adverse behavioral effects in zebrafish. Zebrafish were developmentally exposed to BPA, Bisphenol AF (BPAF), Bisphenol B (BPB), Bisphenol F (BPF), or Bisphenol S (BPS). At 10 days post fertilization (dpf), toxicity assessments were completed and 16S rRNA gene sequencing was performed to evaluate potential chemical-dependent shifts in microbial community structure and predicted function. A standard light/dark behavioral assay was used to assess locomotor activity. Based on developmental toxicity assessments at 10 dpf, a range of potencies was observed: BPAF > BPB > BPF ∼ BPA > BPS. Analysis of 16S rRNA gene sequencing data showed significant concentration-dependent disruption of microbial community structure and enrichment of putative microbial functions with exposure to BPS, BPA, or BPF, but not BPB or BPAF. Interestingly, microbial disruption was inversely related to host developmental toxicity and estrogenicity. Exposure to BP analogs did not cause behavioral effects at 10 dpf. Our findings indicate that some BP analogs disrupt host microbiota early in life and demonstrate novel chemical-microbiota interactions that may add important context to current hazard identification strategies.

Drinking water treatment plants rely on purification of contaminated source waters to provide communities with potable water. One group of possible contaminants are enteric viruses. Measurement of viral quantities in environmental water systems are often performed using polymerase chain reaction(PCR) or quantitative PCR (qPCR). However, these results can often be underestimated due to challenges involved in a multi-step viral concentration process and PCR inhibition. In this study, water samples were concentrated from 25 drinking water treatment plants (DWTPs) across the US to study the occurrence of enteric viruses in source water and removal after treatment. The five different types of viruses studied were adenovirus, norovirus GI, norovirus GII, enterovirus, and polyomavirus. Quantitative PCR was performed on all samples to determine presence or absence of these viruses in each sample. Ten DWTPs showed presence of one or more viruses in source water, with four DWTPs having treated drinking water testing positive. Furthermore, PCR inhibition was assessed for each sample using an exogenous amplification control, which indicated that all of the DWTP samples, including source and treated water samples, had some level of inhibition, confirming that inhibition plays an important role in PCR based assessments of environmental samples. PCR inhibition measurements, viral recovery, and other assessments were incorporated into a Bayesian model to more accurately determine viral load in both source and treated water. Results of the Bayesian model documented that viruses are present in source water and treated water. By using a Bayesian framework that incorporates inhibition, as well as many other parameters that affect viral detection, this study offers an approach for more accurately estimating the occurrence of viral pathogens in environmental waters.

Antimicrobial resistance (AMR) is a global concern, pertaining not only to human health but also to the health of industry and the environment. AMR research has traditionally focused on genetic exchange mechanisms and abiotic environmental constraints, leaving important aspects of microbial ecology unresolved. The genetic and ecological aspects of AMR, however, not only contribute separately to the problem but also are interrelated. For example, mutualistic associations among microbes such as biofilms can both serve as a barrier to antibiotic penetration and a breeding ground for horizontal exchange of antimicrobial resistance genes (ARGs). In this review, we elucidate how species interactions promote and impede the establishment, maintenance, and spread of ARGs and indicate how management initiatives might benefit from leveraging the principles and tools of community ecology to better understand and manipulate the processes underlying AMR.

The efficacy of germicidal ultraviolet (UV-C) light emitting diodes (LEDs) was evaluated for inactivating human enteroviruses included on the United States Environmental Protection Agency (EPA)’s Contaminant Candidate List (CCL). A UV-C LED device, emitting at peaks of 260 nm and 280 nm and the combination of 260/280 nm together, was used to measure and compare potential synergistic effects of dual wavelengths for disinfecting viral organisms. The 260 nm LED proved to be the most effective at inactivating the CCL enteroviruses tested. To obtain 2-log10 inactivation credit for the 260 nm LED, the fluences (UV doses) required are approximately 8 mJ/cm2 for coxsackievirus A10 and poliovirus 1, 10 mJ/cm2 for enterovirus 70, and 13 mJ/cm2 for echovirus 30. No synergistic effect was detected when evaluating the log inactivation of enteroviruses irradiated by the dual-wavelength UV-C LEDs.

Somatic coliphages are alternative indicators of fecal pollution and attractive surrogates for viral pathogens. Here, we report the draft genome sequences of three replicate plaques from a novel Myoviridae bacteriophage isolated from raw wastewater. These genomes were similar to felix01virus phage and are predicted to contain up to 148 protein-coding genes.

Real-time monitoring of water reuse systems ensures the production of high quality water to protect human health at the point-of-use. In this study, several online real-time sensors were utilized to monitor effluent from a wastewater fed laboratory-scale membrane bioreactor (MBR) under natural and simulated failure conditions. These simulated failures included adding reactor mixed liquor to emulate a membrane breach, and spiking MS2 bacteriophage into the reactor to create a high viral load, which might be observed during an outbreak. The CANARY event detection software was used to analyze sensor data and report changes in water quality that might be indicative of poor system behavior. During simulated failure conditions, CANARY reported 20 alarms, accurately detecting each failure. During natural operating conditions, 219 alarms were produced and 189 were attributed to known events (e.g., system and sensor maintenance). The remaining alarms (23) during natural operating conditions were considered to have an unknown cause. However, 13 of those had signal deviations similar to known events, but could not be definitively linked to a source. The results of this study suggest that real-time monitoring in conjunction with CANARY analysis may be useful as an early warning system for monitoring the effluent of water reuse systems, and may help to quickly identify treatment malfunctions or other abnormal conditions.

Anaerobic treatment of domestic wastewater (DWW) produces dissolved methane that needs to be recovered for use as an energy product. Membrane-based recovery systems have been reported in the literature but are often limited by fouling. The objective of this study was to develop a methane producing biofilm on the shell side surface a membrane to allow for immediate recovery of methane as it was produced, negating mass transfer resistance caused by fouling. Between 89 and 96% of total methane produced was recovered via in-situ degassing without the need for fouling control or cleaning throughout 72 weeks of operation. High methane recovery efficiencies led to predictions of net positive energy yield in one reactor and a 32–61% reduction in energy demand in the others compared to the control. This research demonstrates the feasibility and usefulness of combining attached growth anaerobic wastewater treatment processes with hollow fiber membrane methane recovery systems for improved operation.

Risk-based treatment of onsite wastewaters for decentralized reuse requires information on the occurrence and density of pathogens in source waters, which differ from municipal wastewater due to scaling and dilution effects in addition to variable source contributions. In this first quantitative report of viral enteric pathogens in onsite-collected graywater and wastewater, untreated graywater (n = 50 samples) and combined wastewater (i.e., including blackwater; n = 28) from three decentralized collection systems were analyzed for two norovirus genogroups (GI/GII) and human adenoviruses using droplet digital polymerase chain reaction (ddPCR). Compared to traditional quantitative PCR (qPCR), which had insufficient sensitivity to quantify viruses in graywater, ddPCR allowed quantification of norovirus GII and adenovirus in 4% and 14% of graywater samples, respectively (none quantifiable for norovirus GI). Norovirus GII was routinely quantifiable in combined wastewater by either PCR method (96% of samples), with well-correlated results between the analyses (R2 = 0.96) indicating a density range of 5.2–7.9 log10 genome copies/L. These concentrations are greater than typically reported in centralized municipal wastewater, yet agree well with an epidemiology-based model previously used to develop pathogen log-reduction targets (LRTs) for decentralized non-potable water systems. Results emphasize the unique quality of onsite wastewaters, supporting the previous LRTs and further quantitative microbial risk assessment (QMRA) of decentralized water reuse.

A newly developed integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method and its applicability in UV disinfection studies is described. This method utilizes a singular cell culture system coupled with four RTqPCR assays to detect infectious serotypes that represent the four human-related species of enterovirus (e.g., Enterovirus A, Enterovirus B, Enterovirus C and Enterovirus D). Evaluation of the RTqPCR assays was conducted with coxsackievirus A10, echovirus 30, poliovirus 1 and enterovirus 70 and resulted in 100% specificity for the tested assays. A comparison of ICC-RTqPCR between the individual enterovirus serotypes and a mixture of all four viruses resulted in an approximate 1:1 correlation, demonstrating a lack of competition during incubation in cell culture and RTqPCR. The simultaneous detection of multiple human enterovirus species within mixed cultures is relevant to many applications, including virus disinfection studies. This high-throughput, multiplex approach delivers the practical benefits of time- and cost-efficient, and consequently will help the collection of more data leading to more statistically sound data sets for direct comparison of four enterovirus inactivation rates.

Aims Development of efficacious grey water (GW) treatment systems would benefit from detailed knowledge of the bacterial composition of GW. Thus, the aim of this study was to characterize the bacterial composition from (i) various points throughout a GW recycling system that collects shower and sink handwash (SH) water into an equalization tank (ET) prior to treatment and (ii) laundry (LA) water effluent of a commercial-scale washer. Methods and Results Bacterial composition was analysed by high-throughput pyrosequencing of the 16S rRNA gene. LA was dominated by skin-associated bacteria, with Corynebacterium, Staphylococcus, Micrococcus, Propionibacterium and Lactobacillus collectively accounting for nearly 50% of the total sequences. SH contained a more evenly distributed community than LA, with some overlap (e.g. Propionibacterium), but also contained distinct genera common to wastewater infrastructure (e.g. Zoogloea). The ET contained many of these same wastewater infrastructure-associated bacteria, but was dominated by genera adapted for anaerobic conditions. Conclusions The data indicate that a relatively consistent set of skin-associated genera are the dominant human-associated bacteria in GW, but infrastructure-associated bacteria from the GW collection system and ET used for transient storage will be the most common bacteria entering GW treatment and reuse systems. Significance and Impact of the Study This study is the first to use high-throughput sequencing to identify the bacterial composition of various GW sources.

Changes in resident microbiota may have wide-ranging effects on human health. We investigated whether early life microbial disruption alters neurodevelopment and behavior in larval zebrafish. Conventionally colonized, axenic, and axenic larvae colonized at 1 day post fertilization (dpf) were evaluated using a standard locomotor assay. At 10 dpf, axenic zebrafish exhibited hyperactivity compared to conventionalized and conventionally colonized controls. Impairment of host colonization using antibiotics also caused hyperactivity in conventionally colonized larvae. To determine whether there is a developmental requirement for microbial colonization, axenic embryos were serially colonized on 1, 3, 6, or 9 dpf and evaluated on 10 dpf. Normal activity levels were observed in axenic larvae colonized on 1–6 dpf, but not on 9 dpf. Colonization of axenic embryos at 1 dpf with individual bacterial species Aeromonas veronii or Vibrio cholerae was sufficient to block locomotor hyperactivity at 10 dpf. Exposure to heat-killed bacteria or microbe-associated molecular patterns pam3CSK4 or Poly(I:C) was not sufficient to block hyperactivity in axenic larvae. These data show that microbial colonization during early life is required for normal neurobehavioral development and support the concept that antibiotics and other environmental chemicals may exert neurobehavioral effects via disruption of host-associated microbial communities.

Enterovirus infections are often not tracked or reported to health officials. This makes it hard to know how many people in a community are infected with these viruses at any given time. Here, we explored enterovirus in municipal wastewater to look at this issue. We show that enteroviruses are present year-round in municipal wastewater at levels of up to 800,000 genomic copies per liter. We estimate that, on average, 2.8% of the people contributing to the wastewater shed enterovirus daily. Sequence analysis of the viral capsid protein 4 gene shows that 8 enterovirus types are key drivers of seasonal trends. Populations of Enterovirus A members peak in the spring, while Enterovirus B types are most prevalent during the summer and fall months and Enterovirus C members influence the winter months. Enterovirus D was observed sporadically and did not influence seasonal trends., Enteroviruses are RNA viruses that are responsible for both mild gastroenteritis and mild respiratory illnesses as well as debilitating diseases such as meningitis and myocarditis. The disease burden of enteroviruses in the United States is difficult to assess because most infections are not recorded. Since infected individuals shed enterovirus in feces and urine, surveillance of municipal wastewater can reveal the diversity of enteroviruses circulating in human populations. Therefore, monthly municipal wastewater samples were collected for 1 year and enteroviruses were quantified by reverse transcriptase quantitative PCR and identified by next-generation, high-throughput sequencing. Enterovirus concentrations ranged from 3.8 to 5.9 log10 equivalent copies/liter in monthly samples. From the mean monthly concentration, it can be estimated that 2.8% of the contributing population was shedding enterovirus daily. Sequence analysis showed that Enterovirus A and Enterovirus B alternate in predominance, with Enterovirus B comprising over 80% of the reads during the summer and fall months and Enterovirus A accounting for >45% of the reads in spring. Enterovirus C was observed throughout the year, while Enterovirus D was present intermittently. Principal-component analysis further supported the date corresponding to enterovirus seasonal trends as CVA6 (Enterovirus A) was predominant in the spring months; CVB3, CVB5, and E9 (Enterovirus B) were predominant in the summer and fall months; and CVA1, CVA19, and CVA22 (Enterovirus C) and EV97 (Enterovirus B) were predominant in winter. Rhinoviruses were also observed. Wastewater monitoring of human enterovirus provided improved insight into the seasonal patterns of enteroviruses circulating in communities and can contribute to understanding of enterovirus disease burden. IMPORTANCE Enterovirus infections are often not tracked or reported to health officials. This makes it hard to know how many people in a community are infected with these viruses at any given time. Here, we explored enterovirus in municipal wastewater to look at this issue. We show that enteroviruses are present year-round in municipal wastewater at levels of up to 800,000 genomic copies per liter. We estimate that, on average, 2.8% of the people contributing to the wastewater shed enterovirus daily. Sequence analysis of the viral capsid protein 4 gene shows that 8 enterovirus types are key drivers of seasonal trends. Populations of Enterovirus A members peak in the spring, while Enterovirus B types are most prevalent during the summer and fall months and Enterovirus C members influence the winter months. Enterovirus D was observed sporadically and did not influence seasonal trends.

Correction for ‘Ozone–UV net-zero water wash station for remote emergency response healthcare units: design, operation, and results’ by Lucien W. Gassie et al., Environ. Sci.: Water Res. Technol., 2019, 5, 1971–1984, DOI: 10.1039/C9EW00126C.

The objective of this study was to compare three nucleic acid extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) approaches for norovirus (NoV) detection in drinking water with respect to performance, costs, and analysis time. The approaches evaluated were: (A) an approach that utilizes the QIAamp DNA Blood Mini Kit and multiplex primers and probes for detection; (B) a procedure which includes the NucliSENS Magnetic Extraction Kit and other components of a proposed European Union standard method for NoV detection in foods; and (C) a commercialized assay which uses NucliSENS extraction and Cepheid SmartCycler® technologies. Each approach was evaluated by most probable number (MPN) analysis for detection of GI.1 and GII.4 NoVs from human stool. Furthermore, recoveries of spiked primary effluent in tap water concentrates were compared for each approach. Few significant differences were observed between approaches with regard to performance. However, Approach C was the most time consuming and expensive to perform. This research presents a case study of how molecular-based approaches for detection of NoVs can be compared and how various factors may play a role in which approach laboratories choose to employ.

Enteroviruses, noroviruses and adenoviruses are among the most common viruses infecting humans worldwide. These viruses are shed in the feces of infected individuals and can accumulate in wastewater, making wastewater a source of a potentially diverse group of enteric viruses. In this study, two procedures were evaluated to concentrate noroviruses, adenoviruses and enteroviruses from primary effluent of wastewater. In the first procedure, indigenous enteroviruses, noroviruses and adenoviruses were concentrated using celite (diatomaceous earth) followed by centrifugation through a 30K MWCO filter and nucleic acid extraction. The second procedure used celite concentration followed by nucleic acid extraction only. Virus quantities were measured using qPCR. A second set of primary effluent samples were seeded with Coxsackievirus A7, Coxsackievirus B1, poliovirus 1 or enterovirus 70 before concentration and processed through both procedures for recovery evaluation of enterovirus species representatives. The pairing of the single step extraction procedure with the celite concentration process resulted in 47-98% recovery of examined viruses, while the celite concentration process plus additional centrifugal concentration before nucleic acid extraction showed reduced recovery (14-47%). The celite concentration process followed by a large volume nucleic acid extraction technique proved to be an effective procedure for recovering these important human pathogens from wastewater.

The U.S. EPA developed a sample concentration and preparation assay in conjunction with the total culturable virus assay for concentrating and measuring culturable viruses in source and drinking waters as part of the Information Collection Rule (ICR) promulgated in 1996. In an effort to improve upon this method, the U.S. EPA recently developed Method 1615: Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Method 1615 uses a culturable virus assay with reduced equipment and labor costs compared to the costs associated with the ICR virus method and introduces a new molecular assay for the detection of enteroviruses and noroviruses by reverse transcription-quantitative PCR. In this study, we describe the optimization of several new components of the molecular assay and examine virus recovery from ground, reagent-grade, and surface water samples seeded with poliovirus type 3 and murine norovirus. For the culturable virus and molecular assays, mean poliovirus recovery using the complete method was 58% and 20% in groundwater samples, 122% and 39% using low-titer spikes in reagent-grade water, 42% and 48% using high-titer spikes in reagent-grade water, and 11% and 10% in surface water with high turbidity, respectively. Murine norovirus recovery by the molecular assay was 30% in groundwater samples, less than 8% in both low- and high-titer spikes in reagent-grade water, and 6% in surface water with high turbidity. This study demonstrates the effectiveness of Method 1615 for use with groundwater samples and highlights the need for further research into its effectiveness with surface water.

Water reuse, via either centralized treatment of traditional wastewater or decentralized treatment and on-site reuse, is becoming an increasingly important element of sustainable water management. Despite advances in waterborne pathogen detection methods, low and highly variable pathogen levels limit their utility for routine evaluation of health risks in water reuse systems. Therefore, there is a need to improve our understanding of the linkage between pathogens and more readily measured process indicators during treatment. This paper describes an approach for constructing spiking experiments to relate the behavior of viral, bacterial, and protozoan pathogens with relevant process indicators. General issues are reviewed, and the spiking protocol is applied as a case study example to improve microbial performance monitoring and health risk evaluation in a water reuse system. This approach provides a foundation for the development of novel approaches to improve real or near-real time performance monitoring of water recycling systems.

Metagenomics is a powerful tool for characterizing viral composition within environmental samples, but sample and molecular processing steps can bias the estimation of viral community structure. The objective of this study is to understand the inherent variability introduced when conducting viral metagenomic analyses of wastewater and provide a bioinformatic strategy to accurately analyze sequences for viral community analyses. A standard approach using a combination of ultrafiltration, membrane filtration, and DNase treatment, and multiple displacement amplification (MDA) produced DNA preparations without any bacterial derived genes. Results showed recoveries in wastewater matrix ranged between 60–100%. A bias towards small single stranded DNA (ssDNA; polyomavirus) virus types vs larger double stranded DNA (dsDNA; adenovirus) viruses was also observed with a total estimated recovery of small circular viruses to be as much as 173-fold higher. Notably, ssDNA abundance decreased with sample dilution while large dsDNA genomes (e.g., Caudovirales) initially increased in abundance with dilution before gradually decreasing with further dilution in wastewater samples. The present study revealed the inherent biases associated with different components of viral metagenomic methods applied to wastewater. Overall, these results provide a well-characterized approach for effectively conducting viral metagenomics analysis of wastewater and reveal that dilution can effectively mitigate MDA bias.

Noroviruses are the leading cause of nonbacterial gastroenteritis outbreaks in the United States, some of which are caused by the ingestion of contaminated water. Detection and genotypic characterization of noroviruses is commonly performed by reverse transcription-polymerase chain reaction (RT-PCR) followed by sequencing. However, sequencing of products amplified from environmental water samples is often hindered by the co-amplification of non-specific cDNA. To overcome this issue, a generic microarray was evaluated to genotype noroviruses by probe hybridization. RT-PCR amplicons are used in a single base extension (SBE) reaction where genotype-specific probes are labeled and then hybridized to an Affymetrix GeneChip ® GenFlex™ Tag Array for detection. Using a standardized, multiplex SBE reaction, genotyping of representative strains was accomplished through the identification of genotype-specific patterns of positive hybridization results. Furthermore, the SBE-GenFlex array method was successful in the genotype identification of noroviruses seeded into tap and Ohio River water samples. This study shows the utility of using a microarray to genotype noroviruses in complex environmental matrices.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida ( C. albicans , C. tropicalis , C. krusei , C. parapsilosis , C. glabrata and C. lusitaniae ) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

Aims A microarray was developed to simultaneously detect Cryptosporidium parvum, Cryptosporidium hominis, Enterococcus faecium, Bacillus anthracis and Francisella tularensis in water. Methods and Results A DNA microarray was designed to contain probes that specifically detected C. parvum, C. hominis, Ent. faecium, B. anthracis and F. tularensis. The microarray was then evaluated with samples containing target and nontarget DNA from near-neighbour micro-organisms, and tap water spiked with multiple organisms. Results demonstrated that the microarray consistently detected Ent. faecium, B. anthracis, F. tularensis and C. parvum when present in samples. Cryptosporidium hominis was only consistently detected through the use of shared probes between C. hominis and C. parvum. Conclusions This study successfully developed and tested a microarray-based assay that can specifically detect faecal indicator bacteria and human pathogens in tap water. Significance and Impact of the Study The use of indicator organisms has become a practical solution for monitoring for water quality. However, they do not always correlate well with the presence of many microbial pathogens, thus necessitating direct monitoring for most pathogens. This microarray can be used to simultaneously detect multiple organisms in a single sample. More importantly, it can provide occurrence information that may be used in assessing potential exposure risks to waterborne pathogens.

Three comparatively rapid methods for the extraction of DNA from fungal conidia and yeast cells in environmental (air, water and dust) samples were evaluated for use in real-time PCR (TaqMan) analyses. A simple bead milling method was developed to provide sensitive, accurate and precise quantification of target organisms in air and water (tap and surface) samples. However, quantitative analysis of dust samples required further purification of the extracted DNA by a streamlined silica adsorption procedure.