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Background: During a period of rapid growth in our understanding of the microbiology of the built environment in recent years, the majority of research has focused on bacteria and fungi. Viruses, while probably as numerous, have received less attention. In response, the Alfred P. Sloan Foundation supported a workshop entitled "Viruses in the Built Environment (VIBE)," at which experts in environmental engineering, environmental microbiology, epidemiology, infection prevention, fluid dynamics, occupational health, metagenomics, and virology convened to synthesize recent advances and identify key research questions and knowledge gaps regarding viruses in the built environment., Results: Four primary research areas and funding priorities were identified. First, a better understanding of viral communities in the built environment is needed, specifically which viruses are present and their sources, spatial and temporal dynamics, and interactions with bacteria. Second, more information is needed about viruses and health, including viral transmission in the built environment, the relationship between virus detection and exposure, and the definition of a healthy virome. The third research priority is to identify and evaluate interventions for controlling viruses and the virome in the built environment. This encompasses interactions among viruses, buildings, and occupants. Finally, to overcome the challenge of working with viruses, workshop participants emphasized that improved sampling methods, laboratory techniques, and bioinformatics approaches are needed to advance understanding of viruses in the built environment., Conclusions: We hope that identifying these key questions and knowledge gaps will engage other investigators and funding agencies to spur future research on the highly interdisciplinary topic of viruses in the built environment. There are numerous opportunities to advance knowledge, as many topics remain underexplored compared to our understanding of bacteria and fungi. Video abstract.

Although infectious disease risk from recreational exposure to waterborne pathogens has been an active area of research for decades, beach sand is a relatively unexplored habitat for the persistence of pathogens and fecal indicator bacteria (FIB). Beach sand, biofilms, and water all present unique advantages and challenges to pathogen introduction, growth, and persistence. These dynamics are further complicated by continuous exchange between sand and water habitats. Models of FIB and pathogen fate and transport at beaches can help predict the risk of infectious disease from beach use, but knowledge gaps with respect to decay and growth rates of pathogens in beach habitats impede robust modeling. Climatic variability adds further complexity to predictive modeling because extreme weather events, warming water, and sea level change may increase human exposure to waterborne pathogens and alter relationships between FIB and pathogens. In addition, population growth and urbanization will exacerbate contamination events and increase the potential for human exposure. The cumulative effects of anthropogenic changes will alter microbial population dynamics in beach habitats and the assumptions and relationships used in quantitative microbial risk assessment (QMRA) and process-based models. Here, we review our current understanding of microbial populations and transport dynamics across the sand-water continuum at beaches, how these dynamics can be modeled, and how global change factors (e.g., climate and land use) should be integrated into more accurate beachscape-based models., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study addressed whether digital droplet PCR (ddPCR) could improve sensitivity and specificity of human-associated Bacteroidales genetic markers, BacHum and B. theta, and their quantification in environmental and fecal composite samples. Human markers were quantified by qPCR and ddPCR platforms obtained from the same manufacturer. A total of 180 samples were evaluated by each platform including human and animal feces, sewage, and environmental water. The sensitivity of ddPCR and qPCR marker assays in sewage and human stool was 0.85-1.00 with marginal reduction in human stool by ddPCR relative to qPCR (<10%). The prevalence and distribution of markers across complex sample types was similar (74-100% agreement) by both platforms with qPCR showing higher sensitivity for markers in environmental and composite samples and ddPCR showing greater reproducibility for marker detection in fecal composites. Determination of BacHum prevalence in fecal samples by ddPCR increased specificity relative to qPCR (from 0.58 to 0.88) and accuracy (from 0.77 to 0.94), while the B. theta assay performed similarly on both platforms (specificity = 0.98). In silico analysis indicated higher specificity of ddPCR for BacHum was not solely attributed to reduced sensitivity relative to qPCR. Marker concentrations measured by ddPCR for all sample types were consistently lower than those measured by qPCR, by a factor of 2.6 ± 2.8 for B. theta and 18.7 ± 10.0 for BacHum. We suggest that differences in assay performance on ddPCR and qPCR platforms may be linked to the characteristics of the assay targets (that is, genes with multiple versus single copies and encoding proteins versus ribosomal RNA) however further work is needed to validate these ideas. We conclude that ddPCR is a suitable tool for microbial source tracking, however, other factors such as cost-effectiveness and assay-specific performance should be considered., (Copyright © 2019. Published by Elsevier Ltd.)

The effects of manure application in agriculture on surface water quality has become a local to global problem because of the adverse consequences on public health and food security. This study evaluated (i) the spatial distribution of bovine (cow) and porcine (pig) genetic fecal markers, (ii) how hydrologic factors influenced these genetic markers, and (iii) their variations as a function of land use, nutrients, and other physiochemical factors. We collected 189 samples from 63 watersheds in Michigan's Lower Peninsula during baseflow, spring melt, and summer rain conditions. For each sample, we quantified the concentrations of bovine and porcine genetic markers by digital droplet polymerase chain reaction and measured , dissolved oxygen, pH, temperature, total phosphorus, total nitrogen, nitrate-nitrite (NO), ammonia (NH), soluble reactive phosphorus, streamflow, and watershed specific precipitation. Bovine and porcine manure markers were ubiquitous in rivers that drain agricultural and natural fields across the study region. This study provides baseline conditions on the state of watershed impairment, which can be used to develop best management practices that could improve water quality. Similar studies should be performed with higher spatial sampling density to elucidate detailed factors that influence the transport of manure constituents., (Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.)

The island city country of Singapore served as a model to validate the use of host-associated Bacteroidales 16S rRNA gene marker assays for identifying sources of fecal pollution in the urban tropical environment of Southeast Asia. A total of 295 samples were collected from sewage, humans, domesticated animals (cats, dogs, rabbits and chicken), and wild animals (birds, monkeys and wild boars). Samples were analyzed by real time PCR using five human-associated assays (HF183-SYBR Green, HF183, BacHum, BacH and B. thetaiotaomicron α-1-6, mannanase (B. theta), one canine-associated assay (BacCan), and a total Bacteroidales assay (BacUni). The best performing human-associated assay was B. theta with a diagnostic sensitivity of 69% and 100% in human stool and sewage, respectively, and a specificity of 98%. BacHum achieved the second highest sensitivity and specificity for human stool at 65% and 91%, respectively. The canine-associated Bacteroidales assay (BacCan) had a sensitivity and specificity above 80% and was validated for tracking fecal pollution from dogs. BacUni demonstrated a sensitivity and specificity of 100% for mammals, thus BacUni was confirmed for total Bacteroidales detection in the region. We showed for the first time that rabbit fecal samples cross-react with human-associated assays (HF183-SYBR Green, HF183, BacHum and BacH) and with BacCan. Our findings regarding the best performing human-associated assays differ from those reported in Bangladesh and India, which are geographically close to Southeast Asia, and where HF183 and BacHum were the preferred assays, respectively., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A major challenge for assessment of water quality in tropical environments is the natural occurrence and potential growth of Fecal Indicator Bacteria (FIB). To gain a better understanding of the relationship between measured levels of FIB and the distribution of sewage-associated bacteria, including potential pathogens, in the tropics this study compared the abundance of FIB (Total coliforms and E. coli) and the Bacteroidales (HF183 marker) with bacterial community structure determined by next-generation amplicon sequencing. Water was sampled twice over 6 months from 18 sites within a tropical urban catchment and reservoir, followed by extraction of DNA from microorganisms, and sequencing targeting the V3-V4 region of the 16S rRNA gene. Multivariate statistical analyses indicated that bacterial community composition (BCC) varied between reservoir and catchment, within catchment land-uses, and with E. coli concentration. Beta-regression indicated that the proportion of sequences from sewage-associated taxa (SAT) or pathogen-like sequences (PLS) were predicted most significantly by measured levels of E. coli(log MPN/100 mL) (χ 2 > 8.7; p < 0.003). In addition, SAT were significantly predicted by log HF183 levels (χ 2 =13.1; p = 0.0003) while PLS were not. Our study suggests that measurements of E. coli concentration could be useful in predicting samples enriched in sewage-associated and pathogen-like bacteria in tropical environments despite the potential for nonconservative behavior.

Water quality is an emergent property of a complex system comprised of interacting microbial populations and introduced microbial and chemical contaminants. Studies leveraging next-generation sequencing (NGS) technologies are providing new insights into the ecology of microbially mediated processes that influence fresh water quality such as algal blooms, contaminant biodegradation, and pathogen dissemination. In addition, sequencing methods targeting small subunit (SSU) rRNA hypervariable regions have allowed identification of signature microbial species that serve as bioindicators for sewage contamination in these environments. Beyond amplicon sequencing, metagenomic and metatranscriptomic analyses of microbial communities in fresh water environments reveal the genetic capabilities and interplay of waterborne microorganisms, shedding light on the mechanisms for production and biodegradation of toxins and other contaminants. This review discusses the challenges and benefits of applying NGS-based methods to water quality research and assessment. We will consider the suitability and biases inherent in the application of NGS as a screening tool for assessment of biological risks and discuss the potential and limitations for direct quantitative interpretation of NGS data. Secondly, we will examine case studies from recent literature where NGS based methods have been applied to topics in water quality assessment, including development of bioindicators for sewage pollution and microbial source tracking, characterizing the distribution of toxin and antibiotic resistance genes in water samples, and investigating mechanisms of biodegradation of harmful pollutants that threaten water quality. Finally, we provide a short review of emerging NGS platforms and their potential applications to the next generation of water quality assessment tools.

Aims: The study goals were to determine the relationship between faecal indicator bacteria (FIB), the HF183 marker and land use, and the phylogenetic diversity of HF183 marker sequences in a tropical urban watershed., Methods and Results: Total coliforms, Escherichia coli, and HF183 were quantified in 81 samples categorized as undeveloped, residential and horticultural from the Kranji Reservoir and Catchment in Singapore. Quantitative-PCR for HF183 followed by analysis of variance indicated that horticultural areas had significantly higher geometric means for marker levels (4·3 × 10(4) HF183-GE 100 ml(-1)) than nonhorticultural areas (3·07 × 10(3) HF183-GE 100 ml(-1)). E. coli and HF183 were moderately correlated in horticultural areas (R = 0·59, P = 0·0077), but not elsewhere in the catchment. Initial upstream surveys of candidate sources revealed elevated HF183 in a wastewater treatment effluent but not in aquaculture ponds. The HF183 marker was cloned, sequenced and determined by phylogenetic analysis to match the original marker description., Conclusion: We show that quantification of the HF183 marker is a useful tool for mapping the spatial distribution and potential sources of human sewage contamination in tropical environments such as Singapore., Significance and Impact: A major challenge for assessment of water quality in tropical environments is the natural occurrence and nonconservative behaviour of FIB. The HF183 marker has been employed in temperate environments as an alternative indicator for human sewage contamination. Our study supports the use of the HF183 marker as an indicator for human sewage in Singapore and motivates further work to determine HF183 marker levels that correspond to public health risk in tropical environments., (© 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)