Publications by authors named "Warish Ahmed"

63 Publications

Variability in RT-qPCR assay parameters indicates unreliable SARS-CoV-2 RNA quantification for wastewater surveillance.

Water Res 2021 Sep 5;203:117516. Epub 2021 Aug 5.

CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, Dutton Park 4102, QLD, Australia. Electronic address:

Due to the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to generate large datasets aimed at detecting and quantifying SARS-CoV-2 RNA in wastewater. Although RT-qPCR is rapid and sensitive, there is no standard method yet, there are no certified quantification standards, and experiments are conducted using different assays, reagents, instruments, and data analysis protocols. These variations can induce errors in quantitative data reports, thereby potentially misleading interpretations, and conclusions. We review the SARS-CoV-2 wastewater surveillance literature focusing on variability of RT-qPCR data as revealed by inconsistent standard curves and associated parameters. We find that variation in these parameters and deviations from best practices, as described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines suggest a frequent lack of reproducibility and reliability in quantitative measurements of SARS-CoV-2 RNA in wastewater.
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http://dx.doi.org/10.1016/j.watres.2021.117516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341816PMC
September 2021

Comparative analysis of rapid concentration methods for the recovery of SARS-CoV-2 and quantification of human enteric viruses and a sewage-associated marker gene in untreated wastewater.

Sci Total Environ 2021 Jul 31;799:149386. Epub 2021 Jul 31.

United States Environmental Protection Agency, Office of Research and Development, 26W Martin Luther King Jr. Drive, Cincinnati, OH 45268, USA.

To support public-health-related disease surveillance and monitoring, it is crucial to concentrate both enveloped and non-enveloped viruses from domestic wastewater. To date, most concentration methods were developed for non-enveloped viruses, and limited studies have directly compared the recovery efficiency of both types of viruses. In this study, the effectiveness of two different concentration methods (Concentrating pipette (CP) method and an adsorption-extraction (AE) method amended with MgCl) were evaluated for untreated wastewater matrices using three different viruses (SARS-CoV-2 (seeded), human adenovirus 40/41 (HAdV 40/41), and enterovirus (EV)) and a wastewater-associated bacterial marker gene targeting Lachnospiraceae (Lachno3). For SARS-CoV-2, the estimated mean recovery efficiencies were significantly greater by as much as 5.46 times, using the CP method than the AE method amended with MgCl. SARS-CoV-2 RNA recovery was greater for samples with higher titer seeds regardless of the method, and the estimated mean recovery efficiencies using the CP method were 25.1 ± 11% across ten WWTPs when wastewater samples were seeded with 5 × 10 gene copies (GC) of SARS-CoV-2. Meanwhile, the AE method yielded significantly greater concentrations of indigenous HAdV 40/41 and Lachno3 from wastewater compared to the CP method. Finally, no significant differences in indigenous EV concentrations were identified in comparing the AE and CP methods. These data indicate that the most effective concentration method varies by microbial analyte and that the priorities of the surveillance or monitoring program should be considered when choosing the concentration method.
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http://dx.doi.org/10.1016/j.scitotenv.2021.149386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8325557PMC
July 2021

Occurrence of SARS-CoV-2 RNA in Six Municipal Wastewater Treatment Plants at the Early Stage of COVID-19 Pandemic in The United States.

Pathogens 2021 Jun 23;10(7). Epub 2021 Jun 23.

Institute of Environmental Science and Research Ltd., Porirua 5240, New Zealand.

In this study, we investigated the occurrence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) RNA in primary influent ( = 42), secondary effluent ( = 24) and tertiary treated effluent ( = 34) collected from six wastewater treatment plants (WWTPs A-F) in Virginia (WWTP A), Florida (WWTPs B, C, and D), and Georgia (WWTPs E and F) in the United States during April-July 2020. Of the 100 wastewater samples analyzed, eight (19%) untreated wastewater samples collected from the primary influents contained SARS-CoV-2 RNA as measured by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays. SARS-CoV-2 RNA were detected in influent wastewater samples collected from WWTP A (Virginia), WWTPs E and F (Georgia) and WWTP D (Florida). Secondary and tertiary effluent samples were not positive for SARS-CoV-2 RNA indicating the treatment processes in these WWTPs potentially removed SARS-CoV-2 RNA during the secondary and tertiary treatment processes. However, further studies are needed to understand the log removal values (LRVs) and transmission risks of SARS-CoV-2 RNA through analyzing wastewater samples from a wider range of WWTPs.
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http://dx.doi.org/10.3390/pathogens10070798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8308538PMC
June 2021

Virological Characterization of Roof-Harvested Rainwater of Densely Urbanized Low-Income Region.

Food Environ Virol 2021 Sep 29;13(3):412-420. Epub 2021 Jun 29.

Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute (IOC), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ, Brazil.

Roof-harvested rainwater (RHRW) is considered relatively clean water, even though the possible presence of pathogens in the water may pose human health risks. In this study, we investigated the occurrence of enteric viruses in the first flush (10 mm) of RHRW from a densely populated and low-income urbanized region of Rio de Janeiro. One hundred samples (5 L) were collected from 10 rainfall events between April 2015 and March 2017. RNA and DNA viruses were concentrated using the skimmed milk flocculation method and analyzed using the TaqMan® quantitative RT-qPCR and qPCR. Human adenoviruses, noroviruses, rotaviruses A, and avian parvoviruses were detected in 54%, 31%, 12%, and 12% of the positive samples. JC polyomavirus, also targeted, was not detected. Virus concentrations ranged from 1.09 × 10 to 2.58 × 10 genome copies/Liter (GC/L). Partial nucleotide sequence confirmed the presence of HAdV type 41, norovirus genotype GII.4, and avian parvovirus 1. The results suggest that the first flush diversion devices may not adequately remove enteric virus from the rainwater. Additional treatment of RHRW is required to mitigate potential health risks from potable use of captured water.
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http://dx.doi.org/10.1007/s12560-021-09484-yDOI Listing
September 2021

Antibiotic Resistance and Sewage-Associated Marker Genes in Untreated Sewage and a River Characterized During Baseflow and Stormflow.

Front Microbiol 2021 11;12:632850. Epub 2021 Jun 11.

College of Marine Science, University of South Florida, St. Petersburg, St. Petersburg, FL, United States.

Since sewage is a hotspot for antibiotic resistance genes (ARGs), the identification of ARGs in environmental waters impacted by sewage, and their correlation to fecal indicators, is necessary to implement management strategies. In this study, sewage treatment plant (STP) influent samples were collected and analyzed using quantitative polymerase chain reaction (qPCR) to investigate the abundance and correlations between sewage-associated markers (i.e., HF183, Lachno3, crAssphage) and ARGs indicating resistance to nine antibiotics (belonging to aminoglycosides, beta-lactams, sulfonamides, macrolides, and tetracyclines). All ARGs, except , and sewage-associated marker genes were always detected in untreated sewage, and and were detected in the greatest abundances. was also highly abundant in untreated sewage samples. Significant correlations were identified between sewage-associated marker genes, ARGs and the in untreated sewage (τ = 0.488, = 0.0125). Of the three sewage-associated marker genes, the BIO-ENV procedure identified that HF183 alone best maximized correlations to ARGs and (τ = 0.590). Additionally, grab samples were collected from peri-urban and urban sites along the Brisbane River system during base and stormflow conditions, and analyzed for , ARGs, the , and sewage-associated marker genes using quantitative polymerase chain reaction (qPCR). Significant correlations were identified between , ARGs, and (τ = 0.0893, = 0.0032), as well as with sewage-associated marker genes in water samples from the Brisbane River system (τ = 0.3229, = 0.0001). Of the sewage-associated marker genes and , the BIO-ENV procedure identified that crAssphage alone maximized correlations with ARGs and in river samples (τ = 0.4148). Significant differences in , ARGs, , and sewage-associated marker genes, and by flow condition (i.e., base vs. storm), and site types (peri-urban vs. urban) combined were identified ( = 0.3668, = 0.0001), where percent dissimilarities between the multi-factorial groups ranged between 20.8 and 11.2%. Results from this study suggest increased levels of certain ARGs and sewage-associated marker genes in stormflow river water samples compared to base flow conditions. , HF183 and crAssphage may serve as potential indicators of sewage-derived ARGs under stormflow conditions, and this merits further investigation. Data presented in this study will be valuable to water quality managers to understand the links between sewage pollution and ARGs in urban environments.
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http://dx.doi.org/10.3389/fmicb.2021.632850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226142PMC
June 2021

Data-driven estimation of COVID-19 community prevalence through wastewater-based epidemiology.

Sci Total Environ 2021 Oct 23;789:147947. Epub 2021 May 23.

School of Civil, Mining and Environmental Engineering, University of Wollongong, Australia; Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, Australia. Electronic address:

Wastewater-based epidemiology (WBE) has been regarded as a potential tool for the prevalence estimation of coronavirus disease 2019 (COVID-19) in the community. However, the application of the conventional back-estimation approach is currently limited due to the methodological challenges and various uncertainties. This study systematically performed meta-analysis for WBE datasets and investigated the use of data-driven models for the COVID-19 community prevalence in lieu of the conventional WBE back-estimation approach. Three different data-driven models, i.e. multiple linear regression (MLR), artificial neural network (ANN), and adaptive neuro fuzzy inference system (ANFIS) were applied to the multi-national WBE dataset. To evaluate the robustness of these models, predictions for sixteen scenarios with partial inputs were compared against the actual prevalence reports from clinical testing. The performance of models was further validated using unseen data (data sets not included for establishing the model) from different stages of the COVID-19 outbreak. Generally, ANN and ANFIS models showed better accuracy and robustness over MLR models. Air and wastewater temperature played a critical role in the prevalence estimation by data-driven models, especially MLR models. With unseen datasets, ANN model reasonably estimated the prevalence of COVID-19 (cumulative cases) at the initial phase and forecasted the upcoming new cases in 2-4 days at the post-peak phase of the COVID-19 outbreak. This study provided essential information about the feasibility and accuracy of data-driven estimation of COVID-19 prevalence through the WBE approach.
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http://dx.doi.org/10.1016/j.scitotenv.2021.147947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8141262PMC
October 2021

Development of a large volume concentration method for recovery of coronavirus from wastewater.

Sci Total Environ 2021 Jun 9;774:145727. Epub 2021 Feb 9.

Office of Research and Development, United States Environmental Protection Agency, 26 West Martin Luther King Drive, Cincinnati, OH 45268, United States.

Levels of severe acute respiratory coronavirus type 2 (SARS CoV 2) RNA in wastewater could act as an effective means to monitor coronavirus disease 2019 (COVID-19) within communities. However, current methods used to detect SARS CoV 2 RNA in wastewater are limited in their ability to process sufficient volumes of source material, inhibiting our ability to assess viral load. Typically, viruses are concentrated from large liquid volumes using two stage concentration, primary and secondary. Here, we evaluated a dead-end hollow fiber ultrafilter (D-HFUF) for primary concentration, followed by the CP Select™ for secondary concentration from 2 L volumes of primary treated wastewater. Various amendments to each concentration procedure were investigated to optimally recover seeded OC43 (betacoronavirus) from wastewater. During primary concentration, the D-HFUF recovered 69 ± 18% (n = 29) of spiked OC43 from 2 L of wastewater. For secondary concentration, the CP Select™ system using the Wastewater Application settings was capable of processing 100 mL volumes of primary filter eluates in <25 min. A hand-driven syringe elution proved to be significantly superior (p = 0.0299) to the CP Select™ elution for recovering OC43 from filter eluates, 48 ± 2% compared to 31 ± 3%, respectively. For the complete method (primary and secondary concentration combined), the D-HFUF and CP select/syringe elution achieved overall 22 ± 4% recovery of spiked OC43 through (n = 8) replicate filters. Given the lack of available standardized methodology confounded by the inherent limitations of relying on viral RNA for wastewater surveillance of SARS CoV 2, it is important to acknowledge these challenges when interpreting this data to estimate community infection rates. However, the development of methods that can substantially increase sample volumes will likely allow for reporting of quantifiable viral data for wastewater surveillance, equipping public health officials with information necessary to better estimate community infection rates.
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http://dx.doi.org/10.1016/j.scitotenv.2021.145727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870434PMC
June 2021

SARS-CoV-2 RNA monitoring in wastewater as a potential early warning system for COVID-19 transmission in the community: A temporal case study.

Sci Total Environ 2021 Mar 5;761:144216. Epub 2020 Dec 5.

Queensland Alliance for Environmental Health Sciences (QAEHS), The University of Queensland, 20 Cornwall Street, Woolloongabba, QLD 4103, Australia.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus which causes coronavirus disease (COVID-19), has spread rapidly across the globe infecting millions of people and causing significant health and economic impacts. Authorities are exploring complimentary approaches to monitor this infectious disease at the community level. Wastewater-based epidemiology (WBE) approaches to detect SARS-CoV-2 RNA in municipal wastewater are being implemented worldwide as an environmental surveillance approach to inform health authority decision-making. Owing to the extended excretion of SARS-CoV-2 RNA in stool, WBE can surveil large populated areas with a longer detection window providing unique information on the presence of pre-symptomatic and asymptomatic cases that are unlikely to be screened by clinical testing. Herein, we analysed SARS-CoV-2 RNA in 24-h composite wastewater samples (n = 63) from three wastewater treatment plants (WWTPs) in Brisbane, Queensland, Australia from 24th of February to 1st of May 2020. A total of 21 samples were positive for SARS-CoV-2, ranging from 135 to 11,992 gene copies (GC)/100 mL of wastewater. Detections were made in a Southern Brisbane WWTP in late February 2020, up to three weeks before the first clininal case was reported there. Wastewater samples were generally positive during the period with highest caseload data. The positive SARS-CoV-2 RNA detection in wastewater while there were limited clinical reported cases demonstrates the potential of WBE as an early warning system to identify hotspots and target localised public health responses, such as increased individual testing and the provision of health warnings.
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http://dx.doi.org/10.1016/j.scitotenv.2020.144216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718102PMC
March 2021

Performance of viral and bacterial genetic markers for sewage pollution tracking in tropical Thailand.

Water Res 2021 Feb 28;190:116706. Epub 2020 Nov 28.

Research Laboratory of Biotechnology, Chulabhorn Research Institute, 54 Kampangpetch 6 Road, Laksi, Bangkok, 10210, Thailand; Center of Excellence on Environmental Health and Toxicology, CHE, Ministry of Education, 272 Rama 6 Road, Ratchathevi, Bangkok, 10400, Thailand. Electronic address:

Identifying sewage contamination via microbial source tracking (MST) marker genes has proven useful for effective water quality management worldwide; however, performance evaluations for these marker genes in tropical areas are limited. Therefore, this research evaluated four human-associated MST marker genes (human polyomaviruses (JC and BK viruses [HPyVs]), bacteriophage crAssphage (CPQ_056), Lachnospiraceae Lachno3, and Bacteroides BacV6-21) for tracking sewage pollution in aquatic environments of Thailand. The viral marker genes, HPyV and crAssphage were highly sensitive and specific to sewage from onsite wastewater treatment plants (OWTPs; n = 19), with no cross-detection in 120 composite swine, cattle, chicken, duck, goat, sheep, and buffalo fecal samples. The bacterial marker genes, Lachno3 and BacV6-21, demonstrated high sensitivity but moderate specificity; however, using both markers could improve specificity to >0.80 (max value of 1.00). The most abundant markers in OWTP samples were Lachno3 and BacV6-21 (5.42-8.02 and nondetect-8.05 log copies/100 mL), crAssphage (5.28-7.38 log copies/100 mL), and HPyVs (3.66-6.53 log copies/100 mL), respectively. Due to their increased specificity, the abundance of viral markers were further investigated in environmental waters, in which HPyVs showed greater levels (up to 4.33 log copies/100 mL) and greater detection rates (92.7%) in two coastal beaches (n = 41) than crAssphage (up to 3.51 log copies/100 mL and 56.1%). HPyVs were also found at slightly lower levels (up to 5.10 log copies/100 mL), but at higher detection rates (92.6%), in a freshwater canal (n = 27) than crAssphage (up to 5.21 log copies/100 mL and 88.9%). HPyVs and crAssphage marker genes were identified as highly sensitive and specific for tracking sewage pollution in aquatic environments of Thailand. This study underlines the importance of characterizing and validating MST markers in host groups and environmental waters before including them in a water quality management toolbox.
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http://dx.doi.org/10.1016/j.watres.2020.116706DOI Listing
February 2021

Intraday variability of indicator and pathogenic viruses in 1-h and 24-h composite wastewater samples: Implications for wastewater-based epidemiology.

Environ Res 2021 02 26;193:110531. Epub 2020 Nov 26.

United States Environmental Protection Agency, Office of Research and Development, 26W Martin Luther King Jr. Drive, Cincinnati, OH, 45268, USA.

We monitored the concentration of indicator viruses crAssphage and pepper mild mottle virus (PMMoV) and human pathogen adenovirus (HAdV) in influent from a wastewater treatment plant in Brisbane, Australia in 1-h and 24-h composite samples. Over three days of sampling, the mean concentration of crAssphage gene copies (GC)/mL in 24-h composite samples did not differ significantly (p = 0.72-0.92), while for PMMoV GC/mL (p value range: 0.0002-0.0321) and HAdV GC/mL (p value range: 0.0028-0.0068) significant differences in concentrations were observed on one day of sampling compared to the other two. For all three viruses, the variation observed in 1-h composite samples was greater than the variation observed in 24-h composite samples. For crAssphage, in 54.1% of 1-h composite samples, the concentration was less than that observed in 24-h composite samples; whereas for PMMoV and HAdV the concentration was less in 79.2 and 70.9% of 1-h composite samples, respectively, compared to the relevant 24-h composite samples. Similarly, the concentration of crAssphage in 1-h compared to 24-h composite samples did not differ (p = 0.1082) while the concentrations of PMMoV (p < 0.0001) and HAdV (p < 0.0001) in 1-h composite samples were significantly different from 24-h composite samples. These results suggest that 24-h composite samples offer increased analytical sensitivity and decreased variability compared to 1-h composite samples when monitoring wastewater, especially for pathogenic viruses with low infection rates within a community. Thus, for wastewater-based epidemiology applications, 24-h composite samples are less likely to produce false negative results and erroneous public health information.
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http://dx.doi.org/10.1016/j.envres.2020.110531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8267967PMC
February 2021

Antibiotic-Resistant Species in Marine Habitats: A Review.

Curr Opin Environ Sci Health 2020 Aug;19:92-100

University of South Florida, Tampa, FL, USA.

Antibiotic-resistant (ARE) are among leading causes of nosocomial infections worldwide. spp. are ubiquitous in sewage, which can contaminate surface waters via many pathways, providing a route of exposure for humans. This review focuses on ARE in marine and estuarine habitats, including marine animals. Phylogenetic confirmation of the genus and intermediate or full resistance to clinically relevant antibiotics were inclusion criteria. The proportion of resistant isolates varied greatly among antibiotics, for example, 24.2% for ampicillin and 2.4% for vancomycin. The water column contained the highest proportion of ARE observations (18.8%), followed by animal feces and tissues (14.8%), sediment (9.4%), and sand (2.0%). The proportion of multidrug-resistant isolates was the greatest in animal tissue and fecal samples, followed by water and sediments. This review indicates that clinically relevant ARE are present in marine/estuarine habitats and that animals may be important reservoirs.
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http://dx.doi.org/10.1016/j.coesh.2020.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7592714PMC
August 2020

Surveillance of SARS-CoV-2 RNA in wastewater: Methods optimisation and quality control are crucial for generating reliable public health information.

Curr Opin Environ Sci Health 2020 Sep 30. Epub 2020 Sep 30.

Queensland Alliance for Environmental Health Sciences, The University of Queensland, Woolloongabba, QLD, 4102.

Monitoring for SARS-CoV-2 RNA in wastewater through the process of wastewater-based epidemiology (WBE) provides an additional surveillance tool, contributing to community-based screening and prevention efforts as these measurements have preceded disease cases in some instances. Numerous detections of SARS-CoV-2 RNA have been reported globally using various methods, demonstrating the technical feasibility of routine monitoring. However, in order to reliably interpret data produced from these efforts for informing public health interventions, additional quality control information and standardization in sampling design, sample processing, and data interpretation and reporting is needed. This review summarizes published studies of SARS-CoV-2 RNA detection in wastewater as well as available information regarding concentration, extraction, and detection methods. The review highlights areas for potential standardization including considerations related to sampling timing and frequency relative to peak fecal loading times; inclusion of appropriate information on sample volume collected; sample collection points; transport and storage conditions; sample concentration and processing; RNA extraction process and performance; effective volumes; PCR inhibition; process controls throughout sample collection and processing; PCR standard curve performance; and recovery efficiency testing. Researchers are recommended to follow the Minimum Information for Publication of Quantitative Real-Time PCR (MIQE) guidelines. Adhering to these recommendations will enable robust interpretation of wastewater monitoring results and improved inferences regarding the relationship between monitoring results and disease cases.
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http://dx.doi.org/10.1016/j.coesh.2020.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544017PMC
September 2020

Comparative decay of culturable faecal indicator bacteria, microbial source tracking marker genes, and enteric pathogens in laboratory microcosms that mimic a sub-tropical environment.

Sci Total Environ 2021 Jan 9;751:141475. Epub 2020 Aug 9.

Department of Soil, Water, and Climate, and the BioTechnology Institute, University of Minnesota, St. Paul, MN 55108, USA.

Enteric pathogens can be present in drinking water catchments due to several point and non-point sources of faecal contamination. Pathogen and contaminant signatures will decay due to environmental stresses, such as temperature, Ultra Violet (UV) radiation, salinity, and predation. In this study, we determined the decay of the culturable faecal indicator bacterium (FIB) Escherichia coli (E. coli), two sewage-associated marker genes (Bacteroides HF183 and crAssphage CPQ_056), and enteric pathogens (Campylobacter spp., human adenovirus 40/41, and Cryptosporidium parvum) in two freshwater laboratory microcosms using culture-based, quantitative PCR (qPCR) and vital dye (determine the fraction of viable Cryptosporidium oocysts) assays. Freshwater samples from the Lake Wappa and Lake Wivenhoe (Australia) were seeded with untreated sewage and C. parvum oocysts, and their declining concentrations were measured over a 28-day period. Moreover, 16S rRNA amplicon sequencing was also undertaken to determine the change/shift in sewage-associated bacterial communities using SourceTracker. Overall, culturable E. coli and the HF183 marker gene decayed significantly (p < 0.05) faster than did the qPCR measured enteric pathogens suggesting that the absence of culturable FIB or qPCR HF183 in water samples may not indicate the absence of pathogens. The decay of crAssphage was similar to that of HAdV 40/41 and other pathogens tested, suggesting crAssphage may be a better surrogate for enteric viruses in sub-tropical catchment waters. The decay rates were greater at 25 °C compared to 15 °C, suggesting that FIB and pathogens persist longer in the winter season compared to summer. Overall decay rates of the tested microorganisms in this microcosm study suggest that sub-tropical conditions, especially temperature, have a negative impact on the persistence of tested microorganisms. Sewage-associated bacterial communities also showed similar patterns. Based on the results, which showed differences in simulated summer and winter temperatures for pathogen decay, corresponding management options and treatment need to be adjusted accordingly to minimize human health risks effectively.
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http://dx.doi.org/10.1016/j.scitotenv.2020.141475DOI Listing
January 2021

Interlaboratory accuracy and precision among results of three sewage-associated marker genes in urban environmental estuarine waters and freshwater streams.

Sci Total Environ 2020 Nov 9;741:140071. Epub 2020 Jun 9.

Sydney Water, 1 Smith Street, Parramatta, NSW 2150, Australia.

The application of quantitative polymerase chain reaction (qPCR) based microbial source tracking (MST) marker genes are increasingly being used to identify contaminating sources and inform management decisions. In this study, we assessed interlaboratory agreement on duplicate environmental water samples collected from estuarine and freshwater locations, by comparing results of qPCR based testing for Bacteroides HF183, crAssphage CPQ_056, and pepper mild mottle virus (PMMoV). The overall agreements (co-detection and non-co-detection) between CSIRO Land and Water (CLW) laboratory and Sydney Water (SW) laboratory for the HF183, crAssphage CPQ_056 and PMMoV marker genes for duplicate water samples were 74, 75 and 74%, respectively. Cohene's kappa (k) revealed fair to moderate agreements and acceptable relative percent difference (RPD) values of <15% for duplicate samples. The pooled mean abundances of HF183, CPQ_056, and PMMoV in measurable samples at the CLW laboratory were 5.19 ± 0.93, 5.12 ± 0.82, and 4.42 ± 0.65 log copies/L, respectively. However, the pooled mean abundances were significantly lower at the SW laboratory, HF183 (4.58 ± 0.84 log copies/L), crAssphage CPQ_056 (4.20 ± 0.63 log copies/L), and PMMoV (3.89 ± 0.41 log copies/L). At individual sample level, most of the paired samples had <1 log difference. Significant positive Spearman rank correlations were obtained between two laboratories for the HF183 (R = 0.65; p < 0.05), CPQ_056 (R = 0.79; p < 0.05), and PMMoV (R = 0.54; p < 0.05) marker genes. Several factors such as standards, qPCR platforms, PCR inhibitors, nucleic acid extraction efficiency and low levels of targets in some samples may have contributed to the observed discrepancies. Results presented in this study highlight the importance of standardized protocol, laboratory equipment (such as digital PCR), sample processing strategies and appropriate quality controls that may need implementation to further improve accuracy and precision of results between laboratories.
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http://dx.doi.org/10.1016/j.scitotenv.2020.140071DOI Listing
November 2020

Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology.

Environ Res 2020 12 27;191:110092. Epub 2020 Aug 27.

Department of Civil & Environmental Engineering & Earth Sciences, 156 Fitzpatrick Hall, University of Notre Dame, Notre Dame, IN, 46656, USA.

Wastewater-based epidemiology (WBE) demonstrates potential for COVID-19 community transmission monitoring; however, data on the stability of SARS-CoV-2 RNA in wastewater are needed to interpret WBE results. The decay rates of RNA from SARS-CoV-2 and a potential surrogate, murine hepatitis virus (MHV), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in untreated wastewater, autoclaved wastewater, and dechlorinated tap water stored at 4, 15, 25, and 37 °C. Temperature, followed by matrix type, most greatly influenced SARS-CoV-2 RNA first-order decay rates (k). The average T (time required for 1-log reduction) of SARS-CoV-2 RNA ranged from 8.04 to 27.8 days in untreated wastewater, 5.71 to 43.2 days in autoclaved wastewater, and 9.40 to 58.6 days in tap water. The average T for RNA of MHV at 4 to 37 °C ranged from 7.44 to 56.6 days in untreated wastewater, 5.58-43.1 days in autoclaved wastewater, and 10.9 to 43.9 days in tap water. There was no statistically significant difference between RNA decay of SARS-CoV-2 and MHV; thus, MHV is suggested as a suitable persistence surrogate. Decay rate constants for all temperatures were comparable across all matrices for both viral RNAs, except in untreated wastewater for SARS-CoV-2, which showed less sensitivity to elevated temperatures. Therefore, SARS-CoV-2 RNA is likely to persist long enough in untreated wastewater to permit reliable detection for WBE application.
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http://dx.doi.org/10.1016/j.envres.2020.110092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7451058PMC
December 2020

Prevalence and abundance of traditional and host-associated fecal indicators in urban estuarine sediments: Potential implications for estuarine water quality monitoring.

Water Res 2020 Oct 10;184:116109. Epub 2020 Jul 10.

Sydney Water, 1 Smith Street, Parramatta, NSW, 2150, Australia.

This study aimed to determine the prevalence and abundance of sewage and animal fecal contamination of sediment at seven estuarine locations in Sydney, NSW, Australia. Sediment samples were tested for the occurrence of microbial targets including molecular marker genes of enterococci (ENT), Bacteroides HF183 (HF183), Methanobrevibacter smithii (nifH), human adenovirus (HAdV) and emerging sewage-associated marker genes crAssphage (CPQ_056) and Lachnospiraceae (Lachno3) and animal feces-associated marker genes, including avian feces-associated Helicobacter spp. (GFD), canine-feces associated Bacteroides (DogBact), cattle-feces associated (cowM2) and horse feces-associated Bacteroides (HoF597). Results from this study showed that urban estuarine sediment can act as a reservoir of fecal indicator bacteria (FIB) and several microbial source tracking (MST) marker genes, including previously unreported Lachno3. The sewage-associated marker gene CPQ_056 was most prevalent, in 63.8% of sediment samples, while the avian associated marker gene GFD had the highest mean abundance. The GFD marker gene was highly abundant and widely detected in sediment samples from all seven locations compared to the other animal feces-associated marker genes. In all, 31 (44.9%) sediment samples were positive for at least two sewage-associated marker genes. However, the non-quantifiable detection of the HAdV marker gene did not always align with the detection of two or more sewage-associated marker genes. In addition, the most frequent wet weather overflow exposure occurred at locations that did not have a consistent pattern of detection of the sewage-associated marker genes, suggesting sediments may not be a suitable measure of recent sewage contamination. To assist water quality and public health managers better understand past microbial contamination of estuarine sediment, further studies seem justified to explore the role of decay of MST marker genes in sediment. Further work is also needed on the role of resuspension of MST marker genes from sediment during storm events to the water column as a source of contamination for both the GFD and sewage-associated marker genes.
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http://dx.doi.org/10.1016/j.watres.2020.116109DOI Listing
October 2020

Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater.

Sci Total Environ 2020 Oct 5;739:139960. Epub 2020 Jun 5.

Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, North 13 West 8, Kita-ku, Sapporo, Hokkaido 060-0032, Japan.

There is currently a clear benefit for many countries to utilize wastewater-based epidemiology (WBE) as part of ongoing measures to manage the coronavirus disease 2019 (COVID-19) global pandemic. Since most wastewater virus concentration methods were developed and validated for nonenveloped viruses, it is imperative to determine the efficiency of the most commonly used methods for the enveloped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Municipal wastewater seeded with a human coronavirus (CoV) surrogate, murine hepatitis virus (MHV), was used to test the efficiency of seven wastewater virus concentration methods: (A-C) adsorption-extraction with three different pre-treatment options, (D-E) centrifugal filter device methods with two different devices, (F) polyethylene glycol (PEG 8000) precipitation, and (G) ultracentrifugation. MHV was quantified by reverse-transcription quantitative polymerase chain reaction and the recovery efficiency was calculated for each method. The mean MHV recoveries ranged from 26.7 to 65.7%. The most efficient methods were adsorption-extraction methods with MgCl pre-treatment (Method C), and without pre-treatment (Method B). The third most efficient method used the Amicon® Ultra-15 centrifugal filter device (Method D) and its recovery efficiency was not statistically different from the most efficient methods. The methods with the worst recovery efficiency included the adsorption-extraction method with acidification (A), followed by PEG precipitation (F). Our results suggest that absorption-extraction methods with minimal or without pre-treatment can provide suitably rapid, cost-effective and relatively straightforward recovery of enveloped viruses in wastewater. The MHV is a promising process control for SARS-CoV-2 surveillance and can be used as a quality control measure to support community-level epidemic mitigation and risk assessment.
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http://dx.doi.org/10.1016/j.scitotenv.2020.139960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273154PMC
October 2020

SARS-CoV-2 in wastewater: State of the knowledge and research needs.

Sci Total Environ 2020 Oct 30;739:139076. Epub 2020 Apr 30.

Department of Fisheries and Wildlife, Michigan State University, 480 Wilson Road, East Lansing, MI 48824, USA.

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a Public Health Emergency of International Concern, which was officially declared by the World Health Organization. SARS-CoV-2 is a member of the family Coronaviridae that consists of a group of enveloped viruses with single-stranded RNA genome, which cause diseases ranging from common colds to acute respiratory distress syndrome. Although the major transmission routes of SARS-CoV-2 are inhalation of aerosol/droplet and person-to-person contact, currently available evidence indicates that the viral RNA is present in wastewater, suggesting the need to better understand wastewater as potential sources of epidemiological data and human health risks. Here, we review the current knowledge related to the potential of wastewater surveillance to understand the epidemiology of COVID-19, methodologies for the detection and quantification of SARS-CoV-2 in wastewater, and information relevant for human health risk assessment of SARS-CoV-2. There has been growing evidence of gastrointestinal symptoms caused by SARS-CoV-2 infections and the presence of viral RNA not only in feces of infected individuals but also in wastewater. One of the major challenges in SARS-CoV-2 detection/quantification in wastewater samples is the lack of an optimized and standardized protocol. Currently available data are also limited for conducting a quantitative microbial risk assessment (QMRA) for SARS-CoV-2 exposure pathways. However, modeling-based approaches have a potential role to play in reducing the impact of the ongoing COVID-19 outbreak. Furthermore, QMRA parameters obtained from previous studies on relevant respiratory viruses help to inform risk assessments of SARS-CoV-2. Our understanding on the potential role of wastewater in SARS-CoV-2 transmission is largely limited by knowledge gaps in its occurrence, persistence, and removal in wastewater. There is an urgent need for further research to establish methodologies for wastewater surveillance and understand the implications of the presence of SARS-CoV-2 in wastewater.
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http://dx.doi.org/10.1016/j.scitotenv.2020.139076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7191289PMC
October 2020

First detection of SARS-CoV-2 RNA in wastewater in North America: A study in Louisiana, USA.

Sci Total Environ 2020 Nov 30;743:140621. Epub 2020 Jun 30.

Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, North 13 West 8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan.

We investigated the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater samples in southern Louisiana, USA. Untreated and treated wastewater samples were collected on five occasions over a four-month period from January to April 2020. The wastewater samples were concentrated via ultrafiltration (Method A), and an adsorption-elution method using electronegative membranes (Method B). SARS-CoV-2 RNA was detected in 2 out of 15 wastewater samples using two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays (CDC N1 and N2). None of the secondary treated and final effluent samples tested positive for SARS-CoV-2 RNA. To our knowledge, this is the first study reporting the detection of SARS-CoV-2 RNA in wastewater in North America, including the USA. However, concentration methods and RT-qPCR assays need to be refined and validated to increase the sensitivity of SARS-CoV-2 RNA detection in wastewater.
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http://dx.doi.org/10.1016/j.scitotenv.2020.140621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833249PMC
November 2020

Detection of SARS-CoV-2 RNA in commercial passenger aircraft and cruise ship wastewater: a surveillance tool for assessing the presence of COVID-19 infected travellers.

J Travel Med 2020 08;27(5)

Queensland Alliance for Environmental Health Sciences (QAEHS), The University of Queensland, 20 Cornwall Street, Woolloongabba, QLD 4103, Australia.

Background: Wastewater-based epidemiology (WBE) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be an important source of information for coronavirus disease 2019 (COVID-19) management during and after the pandemic. Currently, governments and transportation industries around the world are developing strategies to minimize SARS-CoV-2 transmission associated with resuming activity. This study investigated the possible use of SARS-CoV-2 RNA wastewater surveillance from airline and cruise ship sanitation systems and its potential use as a COVID-19 public health management tool.

Methods: Aircraft and cruise ship wastewater samples (n = 21) were tested for SARS-CoV-2 using two virus concentration methods, adsorption-extraction by electronegative membrane (n = 13) and ultrafiltration by Amicon (n = 8), and five assays using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR). Representative qPCR amplicons from positive samples were sequenced to confirm assay specificity.

Results: SARS-CoV-2 RNA was detected in samples from both aircraft and cruise ship wastewater; however concentrations were near the assay limit of detection. The analysis of multiple replicate samples and use of multiple RT-qPCR and/or RT-ddPCR assays increased detection sensitivity and minimized false-negative results. Representative qPCR amplicons were confirmed for the correct PCR product by sequencing. However, differences in sensitivity were observed among molecular assays and concentration methods.

Conclusions: The study indicates that surveillance of wastewater from large transport vessels with their own sanitation systems has potential as a complementary data source to prioritize clinical testing and contact tracing among disembarking passengers. Importantly, sampling methods and molecular assays must be further optimized to maximize detection sensitivity. The potential for false negatives by both wastewater testing and clinical swab testing suggests that the two strategies could be employed together to maximize the probability of detecting SARS-CoV-2 infections amongst passengers.
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http://dx.doi.org/10.1093/jtm/taaa116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454825PMC
August 2020

First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community.

Sci Total Environ 2020 Aug 18;728:138764. Epub 2020 Apr 18.

Queensland Alliance for Environmental Health Sciences (QAEHS), The University of Queensland, 20 Cornwall Street, Woolloongabba, QLD 4103, Australia.

Infection with SARS-CoV-2, the etiologic agent of the ongoing COVID-19 pandemic, is accompanied by the shedding of the virus in stool. Therefore, the quantification of SARS-CoV-2 in wastewater affords the ability to monitor the prevalence of infections among the population via wastewater-based epidemiology (WBE). In the current work, SARS-CoV-2 RNA was concentrated from wastewater in a catchment in Australia and viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) resulting in two positive detections within a six day period from the same wastewater treatment plant (WWTP). The estimated viral RNA copy numbers observed in the wastewater were then used to estimate the number of infected individuals in the catchment via Monte Carlo simulation. Given the uncertainty and variation in the input parameters, the model estimated a median range of 171 to 1,090 infected persons in the catchment, which is in reasonable agreement with clinical observations. This work highlights the viability of WBE for monitoring infectious diseases, such as COVID-19, in communities. The work also draws attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater.
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http://dx.doi.org/10.1016/j.scitotenv.2020.138764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7165106PMC
August 2020

Identification of reliable marker genes for the detection of canine fecal contamination in sub-tropical Australia.

Sci Total Environ 2020 May 13;718:137246. Epub 2020 Feb 13.

CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, Dutton Park, QLD 4102, Australia. Electronic address:

Animal fecal contamination in aquatic environments is a major source of zoonotic diseases in humans. While concerns are focused on livestock, companion animals such as dogs can also be a source of a wide range of zoonotic pathogens. Therefore, detection of dog or canine fecal contamination in aquatic environments is important for mitigating risks. In this study, host-sensitivity and specificity of four canine fecal-associated marker genes were evaluated by analyzing 30 canine and 240 non-canine fecal samples. The application of these markers was also tested in water from an urban river under dry weather conditions. The host sensitivity values of the Bacteroides BacCan-UCD, DogBact, DF113 and DF418 were 1.00, 0.90, 0.83, and 0.90, respectively. The host specificity value of the BacCan-UCD, DogBact, DF113 and DF418 were 0.87, 0.98, 0.83, and 0.41, respectively. The mean concentrations of DF418 were highest (7.82 ± 1.13 log gene copies (GC)/g of feces) followed by BacCan-UCD (7.61 ± 1.06 log GC/g) and DogBact (7.15 ± 0.92 log GC/g). The mean concentration of DF113 (5.80 ± 1.25 log GC/g) was 1.5 to 2.5 orders of magnitude lower than the other marker genes. The DogBact marker gene was not detected in any other animal feces other than a small number of untreated sewage samples. The BacCan-UCD marker gene cross-reacted with cat, chicken, and pig fecal samples, while the DF113 marker gene cross-reacted with cat, chicken, cattle fecal and untreated sewage samples. The DF418 marker gene was detected in all sewage and animal feces and deemed not suitable for canine fecal contamination tracking in sub-tropical Australia. Canine fecal contamination was infrequently detected in environmental water samples. Based on the results obtained in this study, we recommend that at least two canine feces-associated marker genes should be used in field studies.
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http://dx.doi.org/10.1016/j.scitotenv.2020.137246DOI Listing
May 2020

Expression of attack and growth phase genes of Bdellovibrio bacteriovorus in the presence of Gram-negative and Gram-positive prey.

Microbiol Res 2020 May 13;235:126437. Epub 2020 Feb 13.

Department of Microbiology, Faculty of Science, Stellenbosch University, Private Bag X1, Stellenbosch, 7602, South Africa. Electronic address:

The expression of attack phase (AP) and growth phase (GP) genes of Bdellovibrio bacteriovorus (B. bacteriovorus) was compared in the presence of Gram-negative [Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae)] and Gram-positive [Enterococcus faecium (E. faecium)] prey, using relative quantitative polymerase chain reaction (relative qPCR) assays. The genes bd0108 (pili retraction/extrusion) and merRNA (massively expressed riboswitch RNA) were highly expressed in the AP cells [3.99- to 6.06-fold (E. coli), 3.91- to 7.05-fold (K. pneumoniae) and 2.91- to 7.30-fold (E. faecium)]. The fliC1 gene (flagella filament) was also expressed at a high level in the AP cells however, after 240 min of co-culture with E. faecium the expression of fliC1 remained low (at 0.759-fold), while in the presence of the Gram-negative prey fliC1 expression increased. Additionally, the GP genes bd0816 (peptidoglycan-modifying enzyme) and groES1 (chaperone protein) were not induced in the presence of E. faecium. However, they were expressed in the early GP and GP of B. bacteriovorus after exposure to the Gram-negative prey. It can thus be concluded that B. bacteriovorus senses the presence of potential prey when exposed to Gram-positive and Gram-negative bacteria, however the GP genes are not induced in co-culture with E. faecium. The results from this study thus indicate that B. bacteriovorus does not actively grow in the presence of E. faecium and the second predatory cue (induces active growth of B. bacteriovorus) is lacking when B. bacteriovorus is co-cultured with the Gram-positive prey.
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http://dx.doi.org/10.1016/j.micres.2020.126437DOI Listing
May 2020

Ecological and Technical Mechanisms for Cross-Reaction of Human Fecal Indicators with Animal Hosts.

Appl Environ Microbiol 2020 02 18;86(5). Epub 2020 Feb 18.

School of Freshwater Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, USA

Quantitative PCR (qPCR) assays for human/sewage marker genes have demonstrated sporadic positive results in animal feces despite their high specificities to sewage and human feces. It is unclear whether these positive reactions are caused by true occurrences of microorganisms containing the marker gene (i.e., indicator organisms) or nonspecific amplification (false positive). The distribution patterns of human/sewage indicator organisms in animals have not been explored in depth, which is crucial for evaluating a marker gene's true- or false-positive reactions. Here, we analyzed V6 region 16S rRNA gene sequences from 257 animal fecal samples and tested a subset of 184 using qPCR for human/sewage marker genes. Overall, specificities of human/sewage marker genes within sequencing data were 99.6% (BacV6-21), 96.9% (Lachno3), and 96.1% (HF183, indexed by its inferred V6 sequence). Occurrence of some true cross-reactions was associated with atypical compositions of organisms within the genera or For human/sewage marker qPCR assays, specificities were 96.7% (HF183/Bac287R), 96.2% (BacV6-21), 95.6% (human [HB]), and 94.0% (Lachno3). Select assays duplexed with either or spp. were also validated. Most of the positive qPCR results in animals were low level and, on average, 2 orders of magnitude lower than the copy numbers of and spp. The lower specificity in qPCR assays compared to sequencing data was mainly caused by amplification of sequences highly similar to the marker gene and not the occurrence of the exact marker sequence in animal fecal samples. Identifying human sources of fecal pollution is critical to remediate sanitation concerns. Large financial investments are required to address these concerns; therefore, a high level of confidence in testing results is needed. Human fecal marker genes validated in this study showed high specificity in both sequencing data and qPCR results. Human marker sequences were rarely found in individual animals, and in most cases, the animals had atypical microbial communities. Sequencing also revealed the presence of closely related organisms that could account for nonspecific amplification in certain assays. Both the true cross-reactions and the nonspecific amplification had low signals well below or levels and likely would not impact the assay's ability to reliably detect human fecal pollution. No animal source had multiple human/sewage marker genes present; therefore, using a combination of marker genes would increase the confidence of human fecal pollution detection.
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http://dx.doi.org/10.1128/AEM.02319-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028960PMC
February 2020

Sewage-associated marker genes illustrate the impact of wet weather overflows and dry weather leakage in urban estuarine waters of Sydney, Australia.

Sci Total Environ 2020 Feb 23;705:135390. Epub 2019 Nov 23.

Sydney Water, 1 Smith Street, Parramatta, NSW 2150, Australia.

This study investigates the impact of wet weather overflows (WWOs) at three estuarine locations in Sydney, NSW, Australia. WWOs can occur when infiltration of stormwater leads to an excess volume of flow within the sewerage system, resulting in the release of diluted sewage into the environment. Sewage contamination poses a risk to human health due to the presence of pathogens. The magnitude of sewage contamination was monitored using established and novel sewage-associated marker genes, Bacteroides HF183, pepper mild mottle virus (PMMoV), crAssphage CPQ_056, Lachnospiraceae (Lachno3) marker genes along with culturable fecal indicator bacteria (FIB) Escherichia coli (E. coli) and enterococci. Water samples were collected at two water depths (0.5 m below the water surface and 1 m above the bottom surface) during one dry weather and two storm events. Analysis of sewage-associated marker genes showed greater (i.e., 3-5 orders of magnitude) concentrations in water samples collected during the storm events compared to dry weather event. Water samples were also analysed for four animal feces-associated marker genes targeting avian (GFD), dog (BacCan-UCD), cow (cowM2) and horse (HoF597) species to determine the extent of animal fecal contamination. Among the four marker genes, cowM2 and HoF597 could not be detected, while GFD marker gene was consistently present and BacCan-UCD was occasionally detected. Overall results suggested that after rainfall, untreated sewage from WWOs was present at sampling locations. In addition, microbial source tracking (MST) monitoring was able to distinguish the presence of a leaking sewer impacting on the recreational area during dry weather condition. This study demonstrated the capability of the MST monitoring approach to understand sources (sewage or animal) of fecal contamination. This capability will greatly enhance management decisions assisting in the prioritisation of remediation efforts of the sewerage system to improve estuarine bathing water quality and diminish human health risk.
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http://dx.doi.org/10.1016/j.scitotenv.2019.135390DOI Listing
February 2020

A review on microbial contaminants in stormwater runoff and outfalls: Potential health risks and mitigation strategies.

Sci Total Environ 2019 Nov 5;692:1304-1321. Epub 2019 Jul 5.

CSIRO Land and Water, Waite Laboratories, Waite Rd., Urrbrae, SA 5064, Australia.

Demands on global water supplies are increasing in response to the need to provide more food, water, and energy for a rapidly growing population. These water stressors are exacerbated by climate change, as well as the growth and urbanisation of industry and commerce. Consequently, urban water authorities around the globe are exploring alternative water sources to meet ever-increasing demands. These alternative sources are primarily treated sewage, stormwater, and groundwater. Stormwater including roof-harvested rainwater has been considered as an alternative water source for both potable and non-potable uses. One of the most significant issues concerning alternative water reuse is the public health risk associated with chemical and microbial contaminants. Several studies to date have quantified fecal indicators and pathogens in stormwater. Microbial source tracking (MST) approaches have also been used to determine the sources of fecal contamination in stormwater and receiving waters. This review paper summarizes occurrence and concentrations of fecal indicators, pathogens, and MST marker genes in urban stormwater. A section of the review highlights the removal of fecal indicators and pathogens through water sensitive urban design (WSUD) or Best Management Practices (BMPs). We also discuss approaches for assessing and mitigating health risks associated with stormwater, including a summary of existing quantitative microbial risk assessment (QMRA) models for potable and non-potable reuse of stormwater. Finally, the most critical research gaps are identified for formulating risk management strategies.
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http://dx.doi.org/10.1016/j.scitotenv.2019.07.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7126443PMC
November 2019

Enhanced insights from human and animal host-associated molecular marker genes in a freshwater lake receiving wet weather overflows.

Sci Rep 2019 08 29;9(1):12503. Epub 2019 Aug 29.

Sydney Water, 1 Smith Street, Parramatta, NSW, 2150, Australia.

This study investigated the magnitude of wet weather overflow (WWO)-driven sewage pollution in an urban lake (Lake Parramatta) located in Sydney, New South Wales, Australia. Water samples were collected during a dry weather period and after two storm events, and tested for a range of novel and established sewage- [Bacteroides HF183, crAssphage CPQ_056 and pepper mild mottle virus (PMMoV)] and animal feces-associated (Bacteroides BacCan-UCD, cowM2 and Helicobacter spp. associated GFD) microbial source tracking marker genes along with the enumeration of culturable fecal indicator bacteria (FIB), namely Escherichia coli (E. coli) and Enterococcus spp. The magnitude of general and source-specific fecal pollution was low in water samples collected during dry weather compared to storm events. The levels of HF183, crAssphage and PMMoV in water samples collected during storm events were as high as 6.39, 6.33 and 5.27 log GC/L of water, respectively. Moderate to strong positive correlations were observed among the quantitative occurrence of sewage-associated marker genes. The concentrations of HF183 and PMMoV in most storm water samples exceeded the risk benchmark threshold values established in the literature for primary contact recreators. None of the samples tested was positive for the cowM2 (cow) marker gene, while BacCan-UCD (dog) and GFD (avian) animal-associated markers were sporadically detected in water samples collected from both dry weather and storm events. Based on the results, the ongoing advice that swimming should be avoided for several days after storm events appears appropriate. Further research to determine the decay rates of sewage-associated marker genes in relation to each other and enteric viruses would help refine current advice. Microbial source tracking approaches employed in this study provided insights into sources of contamination over currently used FIB.
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http://dx.doi.org/10.1038/s41598-019-48682-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715810PMC
August 2019

Compositional and temporal stability of fecal taxon libraries for use with SourceTracker in sub-tropical catchments.

Water Res 2019 Nov 13;165:114967. Epub 2019 Aug 13.

Genecology Research Centre, School of Health and Sport Sciences, University of the Sunshine Coast, Maroochydore, QLD, 4558, Australia. Electronic address:

Characterization of microbial communities using high-throughput amplicon sequencing is an emerging approach for microbial source tracking of fecal pollution. This study used SourceTracker software to examine temporal and geographical variability of fecal bacterial community profiles to identify pollutant sources in three freshwater catchments in sub-tropical Australia. Fecal bacterial communities from 10 animal species, humans, and composite wastewater samples from six sewage treatment plants were characterized and compared to freshwater samples using Illumina amplicon sequencing of the V5-V6 regions of the 16S rRNA gene. Source contributions were calculated in SourceTracker using new fecal taxon libraries as well as previously generated libraries to determine the effects of geographic and temporal variability on source assignments. SourceTracker determined 16S rRNA bacterial communites within freshwater samples, shared taxonomic similarities to that of wastewater at low levels (typically <3%). SourceTraker also predicted occasional fecal detection of deer and flying fox sources in the water samples. No significant differences in source contributions were observed within sequences from current and previously characterized fecal samples (P ≥ 0.107). However, significant differences were observed between previously characterized and newly characterized source communities (ANOSIM P ≤ 0.001), which shared <15% community composition. Results suggest temporal instability of fecal taxon libraries among tested sources and highlight continual evaluation of community-based MST using confirmatory qPCR analyses of marker genes.
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http://dx.doi.org/10.1016/j.watres.2019.114967DOI Listing
November 2019

Impacts of a changing earth on microbial dynamics and human health risks in the continuum between beach water and sand.

Water Res 2019 Oct 3;162:456-470. Epub 2019 Jul 3.

Department of Integrative Biology, University of South Florida, Tampa, FL, USA.

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.
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http://dx.doi.org/10.1016/j.watres.2019.07.006DOI Listing
October 2019
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