Leave No Trace? Ecological and anthropogenic determinants of antibiotic resistant bacteria in a recreational alpine environment.

Antibiotic resistant bacteria (ARB) have been detected in remote environments, but the degree to which their presence is due to anthropogenic contamination remains unclear. Here, anthropogenic and ecological determinants of ARB were characterized in remote and highly visited areas of Rocky Mountain National Park in the United States. Soil and water samples were collected from 29 sites once a month for three months and measured for bacteria resistant to seven antibiotics with flow cytometry. A novel index of the likelihood of human presence (HPI) was generated for estimating human impact on ARB abundance. The HPI accounted for 44% of variation in ARB abundance in water samples (p < 0.0001) and 51% of variation in soil samples (p < 0.00001). Human presence index was illustrated as a reliable predictor of ARB abundance despite a tendency to underpredict at higher levels of human impact. Ecological determinants such as temperature, elevation, slope, and aspect were also found to be significantly associated with ARB abundance. These findings suggest that human presence drives the abundance of ARB in Rocky Mountain National Park, but ecological variables play a significant role in their presence and dispersal.

Targeted wastewater surveillance of SARS-CoV-2 on a university campus for COVID-19 outbreak detection and mitigation.

Targeted wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proposed by the United States Centers for Disease Control and Prevention's National Wastewater Surveillance System as a complementary approach to clinical surveillance to detect the presence of Coronavirus Disease 2019 (COVID-19) at high-density facilities and institutions such as university campuses, nursing homes, and correctional facilities. In this study we evaluated the efficacy of targeted wastewater surveillance of SARS-CoV-2 RNA together with individual-level testing for outbreak mitigation on a university campus during Fall 2020 semester. Wastewater samples (n = 117) were collected weekly from manholes or sewer cleanouts that receive wastewater inputs from dormitories, community-use buildings, and a COVID-19 isolation dormitory. Quantitative RT-PCR N1 and N2 assays were used to measure SARS-CoV-2 nucleocapsid genes in wastewater. Due to varying human waste input in different buildings, pepper mild mottle virus (PMMV) RNA was also measured in all samples and used to normalize SARS-CoV-2 N1 and N2 RNA wastewater concentrations. In this study, temporal trends of SARS-CoV-2 in wastewater samples mirrored trends in COVID-19 cases detected on campus. Normalizing SARS-CoV-2 RNA concentrations using human fecal indicator, PMMV enhanced the correlation between N1 and N2 gene abundances in wastewater with COVID-19 cases. N1 and N2 genes were significant predictors of COVID-19 cases in dormitories, and the N2 gene was significantly correlated with the number of detected COVID-19 cases in dormitories. By implementing several public health surveillance programs include targeted wastewater surveillance, individual-level testing, contact tracing, and quarantine/isolation facilities, university health administrators could act decisively during an outbreak on campus, resulting in rapid decline of newly detected COVID-19 cases. Wastewater surveillance of SARS-CoV-2 is a proactive outbreak monitoring tool for university campuses seeking to continue higher education practices in person during the COVID-19 pandemic.

Impacts of Municipal Water-Rainwater Source Transitions on Microbial and Chemical Water Quality Dynamics at the Tap.

When rainwater harvesting is utilized as an alternative water resource in buildings, a combination of municipal water and rainwater is typically required to meet water demands. Altering source water chemistry can disrupt pipe scale and biofilm and negatively impact water quality at the distribution level. Still, it is unknown if similar reactions occur within building plumbing following a transition in source water quality. The goal of this study was to investigate changes in water chemistry and microbiology at a green building following a transition between municipal water and rainwater. We monitored water chemistry (metals, alkalinity, and disinfectant byproducts) and microbiology (total cell counts, plate counts, and opportunistic pathogen gene markers) throughout two source water transitions. Several constituents including alkalinity and disinfectant byproducts served as indicators of municipal water remaining in the system since the rainwater source does not contain these constituents. In the treated rainwater, microbial proliferation and Legionella spp. gene copy numbers were often three logs higher than those in municipal water. Because of differences in source water chemistry, rainwater and municipal water uniquely interacted with building plumbing and generated distinctively different drinking water chemical and microbial quality profiles.

Antibacterial and antiviral effectiveness of two household water treatment devices that use monobrominated hydantoinylated polystyrene.

Many different household water treatment (HWT) methods have been researched and promoted to mitigate the serious burden of diarrheal disease in developing countries. However, HWT methods using bromine have not been extensively evaluated. Two gravity-fed HWT devices (AquaSure™ and Waterbird™) were used to test the antimicrobial effectiveness of HaloPure® Br beads (monobrominated hydantoinylated polystyrene) that deliver bromine. As water flows over the beads, reactive bromine species are eluted, which inactivate microorganisms. To assess log10 reduction values (LRVs) for Vibrio cholerae, Salmonella enterica Typhimurium, bacteriophage MS2, human adenovirus 2 (HAdV2), and murine norovirus (MN), these organisms were added to potable water and sewage-contaminated water. These organisms were quantified before and after water treatment by the HWT devices. On average, 6 LRVs against Vibrio were attained, as well as 5 LRVs against Salmonella, 4 LRVs against MS2, 5 LRVs against HAdV2, and 3 LRVs against MN. Disinfection was similar regardless of whether sewage was present. Polymer beads delivering bromine to drinking water are a potentially effective and useful component of HWT methods in developing countries.

Well water quality in rural Nicaragua using a low-cost bacterial test and microbial source tracking.

Water-related diseases, particularly diarrhea, are major contributors to morbidity and mortality in developing countries. Monitoring water quality on a global scale is crucial to making progress in terms of population health. Traditional analytical methods are difficult to use in many regions of the world in low-resource settings that face severe water quality issues due to the inaccessibility of laboratories. This study aimed to evaluate a new low-cost method (the compartment bag test (CBT)) in rural Nicaragua. The CBT was used to quantify the presence of Escherichia coli in drinking water wells and aimed to determine the source(s) of any microbial contamination. Results indicate that the CBT is a viable method for use in remote rural regions. The overall quality of well water in Pueblo Nuevo, Nicaragua was deemed unsafe, and results led to the conclusion that animal fecal wastes may be one of the leading causes of well contamination. Elevation and depth of wells were not found to impact overall water quality. However rope-pump wells had a 64.1% reduction in contamination when compared with simple wells.

Metagenomic Investigation of Viral Communities in Ballast Water.

Ballast water is one of the most important vectors for the transport of non-native species to new aquatic environments. Due to the development of new ballast water quality standards for viruses, this study aimed to determine the taxonomic diversity and composition of viral communities (viromes) in ballast and harbor waters using metagenomics approaches. Ballast waters from different sources within the North America Great Lakes and paired harbor waters were collected around the Port of Duluth-Superior. Bioinformatics analysis of over 550 million sequences showed that a majority of the viral sequences could not be assigned to any taxa associated with reference sequences, indicating the lack of knowledge on viruses in ballast and harbor waters. However, the assigned viruses were dominated by double-stranded DNA phages, and sequences associated with potentially emerging viral pathogens of fish and shrimp were detected with low amino acid similarity in both ballast and harbor waters. Annotation-independent comparisons showed that viromes were distinct among the Great Lakes, and the Great Lakes viromes were closely related to viromes of other cold natural freshwater systems but distant from viromes of marine and human designed/managed freshwater systems. These results represent the most detailed characterization to date of viruses in ballast water, demonstrating their diversity and the potential significance of the ship-mediated spread of viruses.

Evaluation of an Automated Ultrafiltration System for Concentrating a Range of Viruses from Saline Waters.

Pathogenic viruses in environmental water are usually present in levels too low for direct detection and thus, a concentration step is often required to increase the analytical sensitivity. The objective of this study was to evaluate an automated filtration device, the Innovaprep Concentrating Pipette Select (CP Select) for the rapid concentration of viruses in saline water samples, while considering duration of process and ease of use. Four bacteriophages (MS2, P22, Phi6, and PhiX174) and three animal viruses (adenovirus, coronavirus OC43, and canine distemper virus) were seeded in artificial seawater, aquarium water, and bay water samples, and processed using the CP Select. The recovery efficiencies of viruses were determined either using a plaque assay or droplet digital PCR (ddPCR). Using plaque assays, the average recovery efficiencies for bacteriophages ranged from 4.84 ± 3.8% to 82.73 ± 27.3%, with highest recovery for P22 phage. The average recovery efficiencies for the CP Select were 39.31 ± 26.6% for adenovirus, 19.04 ± 11.6% for coronavirus OC43, and 19.84 ± 13.6% for canine distemper virus, as determined by ddPCR. Overall, viral genome composition, not the size of the virus, affected the recovery efficiencies for the CP Select. The small sample volume size used for the ultrafilter pipette of the system hinders the use of this method as a primary concentration step for viruses in marine waters. However, the ease of use and rapid processing time of the CP Select are especially beneficial when rapid detection of viruses in highly contaminated water, such as wastewater or sewage-polluted surface water, is needed.

A systematic review on global zoonotic virus-associated mortality events in marine mammals.

Marine mammals play a critical role as sentinels for tracking the spread of zoonotic diseases, with viruses being the primary causative factor behind infectious disease induced mortality events. A systematic review was conducted to document marine mammal mortality events attributed to zoonotic viral infections in published literature across the globe. This rigorous search strategy yielded 2883 studies with 88 meeting inclusion criteria. The studies spanned from 1989 to 2023, with a peak in publications observed in 2020. Most of the included studies were retrospective, providing valuable insights into historical trends. The United States (U.S.) reported the highest number of mortality events followed by Spain, Italy, Brazil and the United Kingdom. Harbor seals were the most impacted species, particularly in regions like Anholt, Denmark and the New England Coast, U.S. Analysis revealed six main viruses responsible for mortality events, with Morbillivirus causing the highest proportion of deaths. Notably, the occurrence of these viral events varied geographically, with distinct patterns observed in different regions. Immunohistochemistry emerged as the most employed detection method. This study underscores the importance of global surveillance efforts in understanding and mitigating the impact of viral infections on marine mammal populations, thereby emphasizing the necessity of collaborative One Health approaches to address emerging threats at the human-animal-environment interface. Additionally, the potential transfer of zoonotic viruses to aquatic organisms used in food production, such as fish and shellfish, highlights the broader implications for food safety, food security and public health.

Long-term monitoring of SARS-CoV-2 variants in wastewater using a coordinated workflow of droplet digital PCR and nanopore sequencing.

Quantitative polymerase chain reaction (PCR) and genome sequencing are important methods for wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The reverse transcription-droplet digital PCR (RT-ddPCR) is a highly sensitive method for quantifying SARS-CoV-2 RNA in wastewater samples to track the trends of viral activity levels but cannot identify new variants. It also takes time to develop new PCR-based assays targeting variants of interest. Whole genome sequencing (WGS) can be used to monitor known and new SARS-CoV-2 variants, but it is generally not quantitative. Several short-read sequencing techniques can be expensive and might experience delayed turnaround times when outsourced due to inadequate in-house resources. Recently, a portable nanopore sequencing system offers an affordable and real-time method for sequencing SARS-CoV-2 variants in wastewater. This technology has the potential to enable swift response to disease outbreaks without relying on clinical sequencing results. In addressing concerns related to rapid turnaround time and accurate variant analysis, both RT-ddPCR and nanopore sequencing methods were employed to monitor the emergence of SARS-CoV-2 variants in wastewater. This surveillance was conducted at 23 sewer maintenance hole sites and five wastewater treatment plants in Michigan from 2020 to 2022. In 2020, the wastewater samples were dominated by the parental variants (20A, 20C and 20 G), followed by 20I (Alpha, B.1.1.7) in early 2021 and the Delta variant of concern (VOC) in late 2021. For the year 2022, Omicron variants dominated. Nanopore sequencing has the potential to validate suspected variant cases that were initially undetermined by RT-ddPCR assays. The concordance rate between nanopore sequencing and RT-ddPCR assays in identifying SARS-CoV-2 variants to the clade-level was 76.9%. Notably, instances of disagreement between the two methods were most prominent in the identification of the parental and Omicron variants. We also showed that sequencing wastewater samples with SARS-CoV-2 N gene concentrations of >104 GC/100 ml as measured by RT-ddPCR improve genome recovery and coverage depth using MinION device. RT-ddPCR was better at detecting key spike protein mutations A67V, del69-70, K417N, L452R, N501Y, N679K, and R408S (p-value <0.05) as compared to nanopore sequencing. It is suggested that RT-ddPCR and nanopore sequencing should be coordinated in wastewater surveillance where RT-ddPCR can be used as a preliminary quantification method and nanopore sequencing as the confirmatory method for the detection of variants or identification of new variants. The RT-ddPCR and nanopore sequencing methods reported here can be adopted as a reliable in-house analysis of SARS-CoV-2 in wastewater for rapid community level surveillance and public health response.

Detection and characterization of human pathogenic viruses circulating in community wastewater using multi target microarrays and polymerase chain reaction.

Sewage pollution remains the most significant source of human waterborne pathogens. This study describes the detection and characterization of human enteric viruses in community wastewaters using cell culture coupled with multiple target microarrays (with a total of 780 unique probes targeting 27 different groups of both DNA and RNA viruses) and polymerase chain reaction (PCR) assays. Over a 13-month sampling period, RNA viruses (astroviruses and enteroviruses) were more frequently detected compared to DNA viruses (adenoviruses, particularly type 41 and BK polyomavirus). Overall, many more viruses were shed during the winter months (December-February) compared to the summer months. Exploration of the multiple types of enteric viruses particularly in winter months identified much more significant prevalence of key viral pathogens associated with sewage pollution of the water environment than previously realized and seasonal disinfection used in some parts of the world may lead to a seeding of ambient waters. Molecular characterization of pathogenic viruses in community wastewater will improve the understanding of the potential risk of waterborne disease transmission of viral pathogens.

Comparison of de novo assembly using long-read shotgun metagenomic sequencing of viruses in fecal and serum samples from marine mammals.

Introduction: Viral diseases of marine mammals are difficult to study, and this has led to a limited knowledge on emerging known and unknown viruses which are ongoing threats to animal health. Viruses are the leading cause of infectious disease-induced mass mortality events among marine mammals. Methods: In this study, we performed viral metagenomics in stool and serum samples from California sea lions (Zalophus californianus) and bottlenose dolphins (Tursiops truncates) using long-read nanopore sequencing. Two widely used long-read de novo assemblers, Canu and Metaflye, were evaluated to assemble viral metagenomic sequencing reads from marine mammals. Results: Both Metaflye and Canu assembled similar viral contigs of vertebrates, such as Parvoviridae, and Poxviridae. Metaflye assembled viral contigs that aligned with one viral family that was not reproduced by Canu, while Canu assembled viral contigs that aligned with seven viral families that was not reproduced by Metaflye. Only Canu assembled viral contigs from dolphin and sea lion fecal samples that matched both protein and nucleotide RefSeq viral databases using BLASTx and BLASTn for Anelloviridae, Parvoviridae and Circoviridae families. Viral contigs assembled with Canu aligned with torque teno viruses and anelloviruses from vertebrate hosts. Viruses associated with invertebrate hosts including densoviruses, Ambidensovirus, and various Circoviridae isolates were also aligned. Some of the invertebrate and vertebrate viruses reported here are known to potentially cause mortality events and/or disease in different seals, sea stars, fish, and bivalve species. Discussion: Canu performed better by producing the most viral contigs as compared to Metaflye with assemblies aligning to both protein and nucleotide databases. This study suggests that marine mammals can be used as important sentinels to surveil marine viruses that can potentially cause diseases in vertebrate and invertebrate hosts.

Temporal and spatial relationships of CrAssphage and enteric viral and bacterial pathogens in wastewater in North Carolina.

Enteric disease remains one of the most common concerns for public health, particularly when it results from human exposure to surface and recreational waters contaminated with wastewater. Characterizing the temporal and spatial variation of enteric pathogens prevalent in wastewater is critical to develop approaches to mitigate their distribution in the environment. In this study, we aim to characterize pathogen variability and test the applicability of the human-associated wastewater indicator crAssphage as an indicator of enteric viral and bacterial pathogens. We conducted weekly samplings for 14 months from four wastewater treatment plants in North Carolina, USA. Untreated wastewater samples were processed using hollow fiber ultrafiltration, followed by secondary concentration methods. Adenovirus, norovirus, enterovirus, Salmonella, Shiga toxin 2 (stx2), Campylobacter, and crAssphage were measured by quantitative polymerase chain reaction (qPCR) and reverse transcriptase (rt)-qPCR. Our results revealed significant correlations between crAssphage and human adenovirus, enterovirus, norovirus, Salmonella, and Campylobacter (p<0.01). Pathogens and crAssphage concentrations in untreated wastewater showed distinct seasonal patterns, with peak concentrations of crAssphage and viral pathogens in fall and winter, while bacterial pathogens showed peaked concentrations in either winter (Campylobacter), fall (Salmonella), or summer (stx2). This study enhances the understanding of crAssphage as an alternative molecular indicator for both bacterial and viral pathogens. The findings of this study can also inform microbial modeling efforts for the prediction of the impact of wastewater pathogens on surface waters due to increased flooding events and wastewater overflows associated with climate change.

Prevalence of opportunistic pathogens in a school building plumbing during periods of low water use and a transition to normal use.

The spread of opportunistic pathogens via building water supply and plumbing is of public health concern. This study was conducted to better understand microbial water quality changes in a LEED-certified school building during low water use (Summer) and normal water use (Autumn). The copper plumbed building contained water saving devices, a hot water recirculation system, and received chloraminated drinking water from a public water system. Three separate sampling events were conducted during the summer break inside the building and another three sampling events were conducted after the school returned to session. Using quantitative PCR, Legionella spp. were detected in all water samples, followed by Mycobacterium spp. (99%). Mycobacterium avium (75%) and Acanthamoeba spp. (17.5%) throughout the building water system. Legionella pneumophila and Naegleria fowleri were not detected in any of the samples. The mean concentrations of Legionella spp., Mycobacterium spp., Mycobacterium avium, and Acanthamoeba spp. detected in water samples were 3.9, 5.7, 4.7, and 2.8 log10 gene copies per 100 ml, respectively. There was a statistically significantly difference in the mean concentrations of Legionella spp., Mycobacterium spp. and M. avium gene markers in water samples between school breaks and when school was in session. Cultivable Legionella were also detected in water samples collected during periods of low water use. This study highlights the need for routine proactive water quality testing in school buildings to determine the extent of drinking water quality problems associated with plumbing and direct action to remediate microbial colonization.

Methods Evaluation for Rapid Concentration and Quantification of SARS-CoV-2 in Raw Wastewater Using Droplet Digital and Quantitative RT-PCR.

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging public health tool to understand the spread of Coronavirus Disease 2019 (COVID-19) in communities. The performance of different virus concentration methods and PCR methods needs to be evaluated to ascertain their suitability for use in the detection of SARS-CoV-2 in wastewater. We evaluated ultrafiltration and polyethylene glycol (PEG) precipitation methods to concentrate SARS-CoV-2 from sewage in wastewater treatment plants and upstream in the wastewater network (e.g., manholes, lift stations). Recovery of viruses by different concentration methods was determined using Phi6 bacteriophage as a surrogate for enveloped viruses. Additionally, the presence of SARS-CoV-2 in all wastewater samples was determined using reverse transcription quantitative PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR), targeting three genetic markers (N1, N2 and E). Using spiked samples, the Phi6 recoveries were estimated at 2.6-11.6% using ultrafiltration-based methods and 22.2-51.5% using PEG precipitation. There was no significant difference in recovery efficiencies (p < 0.05) between the PEG procedure with and without a 16 h overnight incubation, demonstrating the feasibility of obtaining same day results. The SARS-CoV-2 genetic markers were more often detected by RT-ddPCR than RT-qPCR with higher sensitivity and precision. While all three SARS-CoV-2 genetic markers were detected using RT-ddPCR, the levels of E gene were almost below the limit of detection using RT-qPCR. Collectively, our study suggested PEG precipitation is an effective low-cost procedure which allows a large number of samples to be processed simultaneously in a routine wastewater monitoring for SARS-CoV-2. RT-ddPCR can be implemented for the absolute quantification of SARS-CoV-2 genetic markers in different wastewater matrices.

Quantification and Trends of Rotavirus and Enterovirus in Untreated Sewage Using Reverse Transcription Droplet Digital PCR.

The quantification and trends in concentrations for naturally occurring rotaviruses (RV) and enteroviruses (EV) in untreated sewage in various wastewater systems have not often been compared. There is now greater interest in monitoring the infections in the community including live vaccine efficacy by evaluating untreated sewage. The goals of this study were to 1) survey the concentrations of naturally occurring RV and EV in untreated sewage using a reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR) and 2) investigate the use of a new adsorption elution (bag-mediated filtration system (BMFS) using ViroCap filters) against more traditional polyethylene glycol (PEG) precipitation for virus concentration. Sewage samples were collected from lagoons in Kenya and Michigan (MI), the United States (USA) and from wastewater treatment plants (WWTPs) in the USA. RVs were detected at geometric mean concentrations in various locations, California (CA) 1.31 × 105 genome copies/L (gc/L), Kenya (KE) 2.71 × 104 gc/L and Virginia (VA) 1.48 × 105 gc/L, and EVs geometric means were 3.72 × 106 gc/L (CA), 1.18 × 104 gc/L (Kenya), and 6.18 × 103 gc/L (VA). The mean RV concentrations using BMFS-ViroCap in split samples compared to PEG precipitation methods demonstrated that the levels were only 9% (#s BMFS/PEG) in the Michigan lagoons which was significantly different (p < 0.01). This suggests that RV concentrations in Kenya are around 1.69 × 106 gc/L. Overall, there was no difference in concentrations for the other sampling locations across the methods of virus recovery (i.e., PEG precipitation and HA filters) using one-way ANOVA (F = 1.7, p = 0.2739) or Tukey-Kramer pairwise comparisons (p > 0.05). This study provides useful data on RV and EV concentrations in untreated sewage in Kenya and the USA. It also highlights on the usefulness of the RT-ddPCR for absolute quantification of RV and EV in sewage samples. The BMFS using ViroCap filters while less efficient compared to the more traditional PEG precipitation method was able to recover RVs and EVs in untreated sewage and may be useful in poor resource settings while underestimating viruses by 1 to 1.5 logs.

Assessing visitor use impact on antibiotic resistant bacteria and antibiotic resistance genes in soil and water environments of Rocky Mountain National Park.

Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have been detected in soil and water in close proximity to anthropogenic sources, but the extent to which human impact plays into ARB and ARGs entering the environment is not well described. This study aimed to determine the impact of visitor use on ARB and ARGs in a national park environment. Soil (n = 240) and water (n = 210) samples were collected across a gradient of human activity in Rocky Mountain National Park and analyzed for bacteria resistant to doxycycline, levofloxacin, and vancomycin. Amount of physical effort required to access a sampling site was used as a metric for the likelihood of human presence. A subset of samples was analyzed for the presence and abundance of six ARGs using quantitative polymerase chain reaction. Linear regression analysis demonstrated that anthropogenic factors including hiking effort and proximity to a toilet significantly contributed to the variance of the abundance of ARB for multiple antibiotics in soil and water. Additionally, ecological factors such as water movement, soil texture, and season may play a role in the detection of ARB and ARGs. Predictive analysis suggests that both human presence and human activities, such as waste elimination, significantly contributed to the abundance of ARB in soil and water. The results of this work evidence that the ecology of antibiotic resistance in remote environments is more complex than anthropogenic impact alone, necessitating further environmental characterization of ARB and ARGs.

Antibiotic Resistance in Minimally Human-Impacted Environments.

Antibiotic resistant bacteria (ARB) have become contaminants of concern in environmental systems. Studies investigating environmental ARB have primarily focused on environments that are greatly impacted by anthropogenic activity. Background concentrations of ARB in natural environments is not well understood. This review summarizes the current literature on the monitoring of ARB and antibiotic resistance genes (ARGs) in environments less impacted by human activity. Both ARB and ARGs have been detected on the Antarctic continent, on isolated glaciers, and in remote alpine environments. The methods for detecting and quantifying ARB and ARGs from the environment are not standardized and warrant optimization. Further research should be focused on the detection and quantification of ARB and ARGs along human gradients to better characterize the factors leading to their dissemination in remote environments.

Evaluation of a portable nanopore-based sequencer for detection of viruses in water.

The newly emerged nanopore sequencing technology such as MinION™ allows for real-time detection of long DNA/RNA fragments on a portable device, yet few have examined its performance for environmental viromes. Here we seeded one RNA virus bacteriophage MS2 and one DNA virus bacteriophage PhiX174 into 10 L well water at three levels ranging from 1 to 21,100 plaque-forming units (PFU)/mL. Two workflows were established to maximize the number of sequencing reads of RNA and DNA viruses using MinION™. With dead-end ultrafiltration, PEG precipitation, and random amplification, MinION™ was capable of detecting MS2 at 155 PFU/mL and PhiX174 at 1-2 PFU/mL. While the DNA workflow only detected PhiX174, the RNA workflow detected both MS2 and PhiX174. The virus concentration, or relative abundance of viral nucleic acids in total nucleic acids, is critical to the proportion of viral reads in sequencing results. Our findings also highlight the importance of including control samples in sequencing runs for environmental water samples with low virus abundance.

Prevalence and genotypes of human noroviruses in tropical urban surface waters and clinical samples in Singapore.

The prevalence and genotypes of norovirus genogroup I (GI) and GII in tropical urban catchment waters and an estuarine bay were studied. A comparative analysis was performed with environmental isolates of noroviruses and concurrently identified clinical isolates in Singapore during gastroenteritis outbreaks between August 2006 to January 2007. Noroviruses in environmental water samples were concentrated by using ultrafiltration techniques and then analyzed by reverse transcription-seminested PCR assay targeting the partial capsid region of noroviruses and DNA sequencing. Among the 60 water samples collected, noroviruses were detected in 43 (71.7%) of these samples. Of these 43 norovirus-positive samples, the coexistence of both GI and GII strains was identified in 23 (53.5%) water samples. The phylogenetic analysis revealed multiple genotypes of noroviruses GI and GII in environmental water samples. GI and GII strains were clustered into seven and nine (including two unclassified) genotypes, respectively. The major norovirus genotypes in environmental water samples were GI/2 and GI/4 and GII/4. Genotyping of the 21 norovirus-positive clinical samples showed that all of the strains belonged to the GII/4 cluster. The environmental and clinical norovirus GII/4 isolates showed high levels of nucleotide sequence identity to each other and to the novel GII/4 variant associated with global epidemics of gastroenteritis during 2006. This study suggests the emergence and circulation of multiple novel norovirus GI and GII genotypes in water environments. Further comprehensive surveillance of water environments for noroviruses and routine clinical reporting is warranted.

Detection of coliphages and human adenoviruses in a subtropical estuarine lake.

Fecal indicator bacteria (FIB) have been used to assess fecal contamination in recreational water. However, enteric viruses have been shown to be more persistent in the environment and resistant to wastewater treatment than bacteria. Recently, U.S Environmental Protection Agency has proposed the use of coliphages as viral indicators to better protect against viral waterborne outbreaks. This study aimed to detect and determine correlation between coliphages (F-specific and somatic), fecal indicator bacteria (enterococci and fecal coliforms), and human enteric viruses (human adenovirus) in a subtropical brackish estuarine lake. Water samples were collected from 9 estuarine recreation sites on Lake Pontchartrain in southeast Louisiana. Water samples (n = 222, collected weekly) were analyzed for coliphages and fecal indicator bacteria using culture-based methods and large volume water samples (n = 54, collected monthly) were analyzed for human adenovirus using quantitative PCR. Somatic coliphage and F-specific coliphage were found in 93.7 and 65.2% of samples with geometric mean concentrations of 30 and 3 plaque forming units (PFU) per 100 mL, respectively. Enterococci, fecal coliforms, and adenovirus were found in all samples with geometric mean concentrations of 27 most probable number (MPN), 77 MPN, and 3.0 × 104 gene copies per 100 mL, respectively. Watersheds in suburban areas exhibited significantly higher concentrations of coliphages and fecal indicator bacteria, indicating potential fecal contamination from septic systems. There was no significant correlation (p > 0.05) observed between the presence of adenoviruses and fecal indicator bacteria and coliphages. The presence of human adenovirus in Lake Pontchartrain poses a significant public health problem for both recreational use and seafood harvesting as it increases exposure risks. This study demonstrated the lack of relationship between fecal indicators and human viral pathogen in Lake Pontchartrain supporting an alternative microbial surveillance system such as direct pathogen detection.