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Sewage-associated marker genes illustrate the impact of wet weather overflows and dry weather leakage in urban estuarine waters of Sydney, Australia
Abstract
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.
... Human faecal contamination, originating from discharge of contaminated rivers and streams, wastewater treatment plants and combined sewer overflows can be a major source of contamination in urban bathing waters (Ahmed et al., 2020;Bedri et al., 2015;Brown et al., 2004;Reynolds et al., 2020;Unc and Goss, 2004). Pollution episodes in this regard can be most acute during severe weather events that trigger the activation of combined sewer and stormwater overflows but which also drive the release and transport of faecal matter from catchments such that faecal indicator concentrations from both point and diffuse sources are increased (Ahmed et al., 2019a;Panasiuk et al., 2015). ...
... The use of crAssphage as a human faeces marker has been demonstrated in the field, predominantly in rivers, lakes and estuaries (Ahmed et al., 2020(Ahmed et al., , 2018b(Ahmed et al., , 2018aBallesté et al., 2019;Kongprajug et al., 2019;Malla et al., 2019). This study evaluates the use of crAssphage as a tool to differentiate between human and animal pollution in spatial and temporal transects as well as in routine compliance sampling in a complex marine environment that is impacted by multiple sources of faecal pollution, including by seabirds, dogs and contaminated urban streams. ...
... The two markers displayed a high degree of co-occurrence, with 76% of samples testing either both positive or both negative for the HF183 and crAss_2 marker. This percentage is slightly lower than that reported by Ahmed et al. (2020) in which both markers showed 85% agreement on the presence or absence of the two markers in estuarine waters (Ahmed et al., 2020). This discrepancy may be attributed to the different persistence rates of these host-specific markers in the environment. ...
Bathing water quality may be negatively impacted by diffuse pollution arising from urban and agricultural activities and wildlife, it is therefore important to be able to differentiate between biological and geographical sources of faecal pollution. crAssphage was recently described as a novel human-associated microbial source tracking marker. This study aimed to evaluate the performance of the crAssphage marker in designated bathing waters. The sensitivity and specificity of the crAss_2 marker was evaluated using faecal samples from herring gulls, dogs, sewage and a stream impacted by human pollution (n=80), which showed that all human impacted samples tested positive for the marker while none of the animal samples did. The crAss_2 marker was field tested in an urban marine bathing water close to the discharge point of human impacted streams. In addition, the bathing water is affected by dog and gull fouling. Analysis of water samples taken at the compliance point every 30 minutes during a tidal cycle following a rain event showed that the crAss_2 and HF183 markers performed equally well (Spearman correlation ρ=0.84). The levels of these marker and faecal indicators (Escherichia coli, intestinal enterococci, somatic coliphages) varied by up to 2.5 log10 during the day. Analysis of a high-tide transect perpendicular to the shoreline revealed high levels of localised faecal contamination 1km offshore, with a concomitant spike in the gull marker. In contrast, both the crAss_2 and HF183 markers remained at a constant level, showing that human faecal contamination is homogenously distributed, while gull pollution is localised. Performance of the crAss_2 and HF183 assay was further evaluated in bimonthly compliance point samples over an 18-month period. The co-occurrence between the crAss_2 and HF183 markers in compliance sampling was 76%. A combination of both markers should be applied in low pollution impacted environments to obtain a high confidence level.
... In addition, increases in hydraulic loads on WWTPs due to stormwater infiltration to sewerage networks during heavy rainfall can cause overloading of WWTPs. The overloading could reduce the treatment efficiencies and allow the release of microbial pathogens to receiving water environments (Ahmed et al., 2020;Okoh et al., 2007). Heavy rainfall can also result in surcharging of sewerage networks leading to the release of untreated sewage/stormwater mixtures to the environment (Al Aukidy and Verlicchi, 2017). ...
... The low occurrence of FIBs in the estuarine waters was likely due to a combination of factors. Firstly, the lower reaches of the river are estuarine therefore contaminants delivered by freshwater inflows are significantly diluted by mixing with cleaner oceanic water (Ahmed et al., 2020). Secondly, as FIBs are derived from live material and survive longer in freshwaters (Korajkic et al., 2013), the proportion of live cells can be expected to decline with increasing salinity and distance from the upstream freshwater sources. ...
... In one of the sampled industrial sites (Industrial 01), only one genetic marker (HF183) was above the threshold concentration, with abundances similar to those reported in urban stormwater samples elsewhere (Ahmed et al., 2020;Staley and Sadowsky, 2016;Steele et al., 2018). Given that the recovery efficiency is similar between both the HF183 and Lachno3 markers (Ahmed et al., 2020), it is also possible that the measured HF183 response was due to a cross reaction with avian fauna; for example, Ahmed et al. (2020) detected avian faeces in >86% of water samples obtained from Sydney estuaries during dry weather events. ...
The Hawkesbury-Nepean River (HNR) is the largest catchment in the Sydney region and is undergoing unprecedented population growth. The HNR system receives a mix of anthropogenic inputs such as treated sewage, stormwater and agricultural runoff. Combined, these can diminish the ecological system health and pose potential concerns to human health. Of particular concern are inputs of untreated sewage, that can occur due to a range of different reasons including illegal point source discharges, failure of the sewerage network, and overloading of wastewater treatment plants during storm events. Here, we present findings of an intensive assessment across the HNR catchment where we used a weight-of-evidence (WOE) approach to identify untreated sewage contamination in surface waters against the background of treated effluent and diffuse inputs during post high flow conditions. Total nitrogen and phosphorus concentrations were used to assess treated effluent and diffuse inputs, and microbial analysis, including both culture-based traditional methods for E. coli and enterococci and qPCR analysis of Bacteroides and Lachnospiraceae, were used to assess raw sewage contamination. Despite a background of diffuse inputs from recent high flow events and the influence of treated wastewater, we found no gradient of faecal contamination along the HNR system or its tributaries. However, we observed two sites with evidence of untreated sewage contamination, where the human markers Bacteroides and Lachnospiraceae qPCR copy numbers were high. The biological and chemical approaches suggested these latter two hotspots originate from an industrial runoff source and possibly from a dry weather sewage leak. Our findings demonstrate the potential of a WOE approach in the assessment of human faecal signal in an urban river that can also pinpoint small sources of contamination as a strategy that can reshape the way monitoring is performed and the chemical end-points chosen to provide pertinent information on the potential risks to aquatic system health.
... In the SIPP study, reference fecal materials were seeded in an artificial freshwater matrix, which is different than actual environmental samples such as estuarine or marine waters that are prone to event based fecal contamination. This could affect PCR amplification due to the presence of different levels of inhibitors (Steele et al., 2018;Ahmed et al., 2020). Also, a maximum of two sources of contamination was seeded; therefore, determining the source in blind samples was relatively easy compared to real-world environmental samples, which may contain fecal contamination from multiple sources at low levels, making accurate identification challenging. ...
... During periods of excessive rain, the sewerage system can become overloaded, and wet weather overflows (WWOs) will discharge to environmental waters to alleviate sewer surcharge into households and business premises. Our recent study has established a link between WWOs and sewage contamination levels of estuarine waters in Australia (Ahmed et al., 2020). This study also demonstrated the capability of the MST monitoring approach to understanding sources (sewage or animal) of fecal contamination. ...
... These were DP1-DP6 in DP, GB1-GB6 in GB, and HCB1-HCB6 in HCB and LP1-LP5 in LP. Site characteristics and locations have been described in more detail in our recent publications (Ahmed et al., 2019a(Ahmed et al., , 2019bAhmed et al., 2020). Results obtained from the CLW laboratory have been published (Ahmed et al., 2019a(Ahmed et al., , 2019bAhmed et al., 2020) and used in this study to compare with the unpublished results obtained by the SW laboratory. ...
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, 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 log10 copies/L, respectively. However, the pooled mean abundances were significantly lower at the SW laboratory, HF183 (4.58 ± 0.84 log10 copies/L), CPQ_056 (4.20 ± 0.63 log10 copies/L), and PMMoV (3.89 ± 0.41 log10 copies/L). At individual sample level, most of the paired samples had <1 log10 difference. Significant positive Spearman rank correlations were obtained between two laboratories for the HF183 (Rs = 0.65; p < 0.05), CPQ_056 (Rs = 0.79; p < 0.05), and PMMoV (Rs = 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 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.
... The used modeling techniques are quite diverse, varying from purely simulation to the hybrid Artificial Intelligence (AI)-based techniques (Even et al., 2004;Itaquy et al., 2017). Similarly, numerous studies have been conducted, so as to observe the associate between SO and its impact on the environment using different types of experiments (Ahmed et al., 2020;Phillips et al., 2012;Hajj-Mohamad et al., 2014;Soriano and Rubió, 2019;Passerat et al., 2010;Cao et al., 2019). ...
... Viviana et al., 2017;Simon et al., 1994;Montserrat et al., 2015;Ahmed et al., 2020; and ...
Sewer networks play a pivotal role in our everyday lives by transporting the stormwater and urban sewage away from the urban areas. In this regard, Sewer Overflow (SO) has been considered as a detrimental threat to our environment and health, which results from the wastewater discharge into the environment. In order to grapple with such deleterious phenomenon, numerous studies have been conducted; however, there has not been any review paper that provides the researchers undertaking research in this area with the following inclusive picture: (1) detailed-scientometric analysis of the research undertaken hitherto, (2) the types of methodologies used in the previous studies, (3) the aspects of environment impacted by the SO occurrence, and (4) the gaps existing in the relative literature together with the potential future works to be undertaken. Based on the comprehensive review undertaken, it is observed that simulation and artificial intelligence-based methods have been the most popular approaches. In addition, it has come to the attention that the detrimental impacts associated with the SO are fourfold as follows: air, quality of water, soil, and business and structure. Among these, the majority of the studies' focus have been tilted towards the impact of SO on the quality of ground water. The outcomes of this state-of-the-art review provides the researchers and environmental engineers with inclusive hindsight in dealing with such serious issue, which in turn, this culminates in a significant improvement in our environment as well as humans’ well-beings.
... As the viral RNA is usually present at low concentrations in wastewater, the samples need to be concentrated prior to nucleic acid extraction and the quantification of the viral target using quantitative or digital PCR (19). Many methods have been used for wastewater concentration for SARS-CoV-2 RNA quantification, including ultracentrifugation, filtration, ultrafiltration, adsorption and precipitation-based approaches (20)(21)(22). However, the viral recovery using these methods is usually not assessed (23)(24)(25). ...
Wastewater-based epidemiology (WBE) has been widely used to track levels of SARS-CoV-2 infection in the community during the COVID-19 pandemic. Due to the rapid expansion of WBE, many methods have been used and developed for virus concentration and detection in wastewater. However, very little information is available on the relative performance of these approaches. In this study, we compared the performance of five commonly used wastewater concentration methods for the detection and quantification of pathogenic viruses (SARS-CoV-2, norovirus, rotavirus, influenza, and measles viruses), fecal indicator viruses (crAssphage, adenovirus, pepper mild mottle virus), and process control viruses (murine norovirus and bacteriophage Phi6) in laboratory spiking experiments. The methods evaluated included those based on either ultrafiltration (Amicon centrifugation units and InnovaPrep device) or precipitation (using polyethylene glycol [PEG], beef extract-enhanced PEG, and ammonium sulfate). The two best methods were further tested on 115 unspiked wastewater samples. We found that the volume and composition of the wastewater and the characteristics of the target viruses greatly affected virus recovery, regardless of the method used for concentration. All tested methods are suitable for routine virus concentration; however, the Amicon ultrafiltration method and the beef extract-enhanced PEG precipitation methods yielded the best recoveries. We recommend the use of ultrafiltration-based concentration for low sample volumes with high virus titers and ammonium levels and the use of precipitation-based concentration for rare pathogen detection in high-volume samples.
... In addition, several of these pharmaceuticals are administered to pets and livestock (Plumb, 2018), and thiabendazole is also used as a fungicide in agricultural applications (Lombardi et al., 2003). Runoff of pet waste, barnyards, and land application of manure are well-recognized sources of contamination to surface water (Ahmed et al., 2020;Clarke & Cummins, 2015;Ervin et al., 2014;Feng et al., 2018;Khaleel et al., 1980;Müller et al., 2020). All eight of these pharmaceuticals have been detected in surface waters from multiple regions of the world at concentrations similar to or exceeding the concentrations observed in the present study (Bradley et al., 2017;Choi et al., 2008;Fick et al., 2009;Furlong et al., 2017;González Alonso et al., 2010;Hughes et al., 2013;Johnson et al., 2015;Jux et al., 2002;Matongo et al., 2015;Ramaswamy et al., 2011;Schultz & Furlong, 2008;Sun et al., 2015;Zhou et al., 2009). ...
In a study of 44 diverse sampling sites across 16 Great Lakes tributaries, 110 pharmaceuticals were detected of 257 monitored. This study evaluated the ecological relevance of detected chemicals and identified heavily impacted areas to help inform resource managers and guide future investigations. Ten pharmaceuticals (caffeine, nicotine, albuterol, sulfamethoxazole, venlafaxine, acetaminophen, carbamazepine, gemfibrozil, metoprolol, and thiabendazole) were distinguished as having the greatest potential for biological effects based on comparison to screening‐level benchmarks derived using information from two biological effects databases, the ECOTOX Knowledgebase and the ToxCast database. Available evidence did not suggest substantial concern for 75% of the monitored pharmaceuticals, including 147 undetected pharmaceuticals and 49 pharmaceuticals with screening‐level alternative benchmarks. However, due to a lack of biological effects information, screening values were not available for 51 detected pharmaceuticals. Samples containing the greatest pharmaceutical concentrations and highest detection frequencies were from Lake Erie, southern Lake Michigan, and Lake Huron tributaries. Samples collected during low‐flow periods had higher pharmaceutical concentrations than those collected during increased‐flow periods. The wastewater treatment plant (WWTP) effluent content in streams correlated positively with pharmaceutical concentrations. However, deviation from this correlation demonstrated that secondary factors, such as multiple pharmaceutical sources, were likely present at some sites. Further research could investigate high‐priority pharmaceuticals as well as those for which alternative benchmarks could not be developed. This article is protected by copyright. All rights reserved.
... In a recent sanitary survey, a shallow coastal lagoon in Manly Beach, Sydney (Australia), was found to be contaminated mainly by sewage overflows and dog feces, as disclosed by MST using human HF183/Bac242 and dog BacCan-UCDmodif markers [102]. Similar findings were reported in urban estuarine waters where sewer overflows originated from combined sources, such as human wastes from peri-urban settlements, and animal feces (e.g., avian and dogs) transported by stormwater runoff were detected using respective human (e.g., HF183, crAssphage CPQ_056, Lachno3), avian GFD, and dog BacCan-UCD host-specific markers [103,104]. Additionally, coastal waters in estuarine regions used for shellfish harvesting and aquafarming have been quality examined. Fecal contaminations in these waters are closely related to land-use patterns; thus, impacts from various polluting sources can be expected. ...
Fecal contamination of water constitutes a serious health risk to humans and environmental ecosystems. This is mainly due to the fact that fecal material carries a variety of enteropathogens, which can enter and circulate in water bodies through fecal pollution. In this respect, the prompt identification of the polluting source(s) is pivotal to guiding appropriate target-specific remediation actions. Notably, microbial source tracking (MST) is widely applied to determine the host origin(s) contributing to fecal water pollution through the identification of zoogenic and/or anthropogenic sources of fecal environmental DNA (eDNA). A wide array of host-associated molecular markers have been developed and exploited for polluting source attribution in various aquatic ecosystems. This review is intended to provide the most up-to-date overview of genetic marker-based MST studies carried out in different water types, such as freshwaters (including surface and groundwaters) and seawaters (from coasts, beaches, lagoons, and estuaries), as well as drinking water systems. Focusing on the latest scientific progress/achievements, this work aims to gain updated knowledge on the applicability and robustness of using MST for water quality surveillance. Moreover, it also provides a future perspective on advancing MST applications for environmental research.
... Sydney Harbour, Australia, is an urban estuary with a history of sewage and industry pollution that resulted in a legacy of metallic and organic contamination in the sediments that remain affected by stormwater runoff and sewage overflows (Ahmed et al., 2020;Birch, 2017). In particular, Sydney Harbour sediments have levels of total nitrogen and total phosphorus exceeding Australian water quality guidelines for the protection of environmental value of estuaries (Birch et al., 2010). ...
Many important ecosystem functions are underpinned by belowground biodiversity and processes. Marine sediments, one of the most abundant habitats on earth, are essential to the mineralisation of organic matter. However, they are increasingly polluted by urban activities leading to the loss of biodiversity and the functions they provide. While traditional sediment remediation strategies are focussed on microbial and engineering solutions, we propose that the reintroduction of belowground ecosystem engineers (bioturbators) is important to rehabilitate polluted sediments and drive recovery of their functions in urban coastal ecosystems. We tested this notion by introducing bioturbators to nutrient polluted sediments to assess their survival, as well as their capacity to drive biodiversity and oxygenation and their potential to remediate nutrient pollution. Polychaete worms Diopatra aciculata and clams Katelysia sp. were added to mesocosms (ex‐situ), and the worms also added to experimental plots in‐situ. Potential for remediation was assessed with measures of nutrient content. All animals survived when introduced to polluted sediments and showed no evidence of sub‐lethal effects. Worms oxygenated sediments and reduced organic matter content by up to 50% in‐situ. The worms also drove shifts in the receiving communities at all locations and increased the number of taxa at one location. On the other hand, the effects of clams were variable, showing opposite effects in organic matter content at different sites and levels of pollution. Synthesis and applications. Global seafloor habitats are becoming increasingly degraded and novel strategies that combine biodiversity restoration with remediation are urgently needed to return function. Tube‐building bioturbators can stimulate nutrient processing in sediments proving multiple functional outcomes, but these effects are dependent on the receiving environment. In areas with medium levels of pollution, they can kick‐start recovery in a feedback loop whereby bioturbation increases oxygenation and nutrient remediation, shifting sediment biodiversity and contributing to further recovery. This can drive long‐term changes in sediment communities, particularly in urban areas where unvegetated sediments are conspicuous.
... Socio-economic risks are very much connected to the evolution of the COVID-19 pandemic. The rapid increase in the number of infections and deaths in many regions around the world has led states to declare a state of emergency [15]. This included quarantine for infected people and isolation for their contacts, social distancing, but also a series of restrictive measures regarding traffic and public events, along with new programs and regulations for carrying out economic activities. ...
n the event of any crisis, such as, in this case, the COVID-19 pandemic, new challenges arise, ranging from social and environmental phenomena to economic issues. One of the most affected economic sectors was the cultural one, especially independent artists, whose financial stability is usually inconsistent. The aim of this article was to test the immediate reactions of the cultural sector, both public and private, to the pandemic shock and, implicitly, to the restrictions imposed during the state of emergency in Romania (27 February–14 May 2020). By using grounded theory, 36 public documents of cultural stakeholders were coded and analyzed. All documents were identified in the Romanian online environment during the state of emergency. Based on the identified interrelationships, it was found that the independent contractors, self-employed workers in the creative-cultural sector, whether or not associated with NGOs or employees of public institutions, need financial and community support. However, the resilience of the cultural sector is conditioned by the creation of new multi-level policies for crisis management.
... Furthermore, agricultural land run-off may also negatively impact water quality and introduce ARGs into waterbodies (Unc and Goss, 2004;Ballesté et al., 2020). Watercourses discharging into the marine environment, especially close to designated bathing waters, may expose the users to fecal pollution and therefore may increase the likelihood that they will be exposed to ARGs (Molina et al., 2014;Leonard et al., 2018;Ahmed et al., 2020;Reynolds et al., 2020;Sala-Comorera et al., 2021b). ...
... Infiltration and inflow between the sanitary and storm sewer via the 'urban karst' phenomenon (Bonneau et al., 2017;Shepley et al., 2020), fecal matter from wild animals, and accidental or illicit wastewater connections to the storm sewer may result in the transport of SARS-CoV-2 by stormwater. Wastewater sewage leaking into storm sewers is an established issue where aging infrastructure conveying human sewage may leak into the storm sewer, discharging the contaminated stormwater with minimal treatment to surface waters, potentially at a location where the public may interact with the contaminated water (Ahmed et al., 2020b). Human and animal fecal contamination in stormwater collected from urban areas has already been confirmed, possibly making stormwater a new SARS-CoV-2 transmission pathway . ...
While wastewater has been found to harbor SARS-CoV-2, the persistence of SARSCoV-2 in stormwater and potential transmission is poorly understood. It is plausible that the virus is detectable in stormwater samples where human-originated fecal contamination may have occurred from sources like sanitary sewer overflows, leaky wastewater pipes, and non-human animal waste. Because of these potential contamination pathways, it is possible that stormwater could serve as an environmental reservoir and transmission pathway for SARS-CoV-2. The objectives of this study are: 1) determine whether the presence of SARS-CoV-2 could be detected in stormwater via RT-ddPCR (reverse transcription-digital droplet PCR); 2) quantify human-specific fecal contamination using microbial source tracking; and 3) examine whether rainfall characteristics influence virus concentrations. To accomplish these objectives, we investigated whether SARS-CoV-2 could be detected from 10 storm sewer outfalls each draining a single, dominant land use in Columbus, Xenia, and Springboro, Ohio. Of the 25 samples collected in 2020, at minimum one SARS-CoV-2 target gene (N2 [US-CDC and CN-CDC], and E) was detected in 22 samples (88%). A single significant correlation (p = 0.001), between antecedent dry period and the USCDC N2 gene, was found between target gene concentrations and rainfall characteristics. Grouped by city, two significant relationships emerged showing cities had different levels of the SARS-CoV-2 E gene. Given the differences in scale, the county-level COVID-19 confirmed cases COVID-19 rates were not significantly correlated with stormwater outfall-scale SARS-CoV-2 gene concentrations. Countywide COVID-19 data did not accurately portray neighborhood-scale confirmed COVID-19 case rates. Potential hazards may arise when human fecal contamination is present in stormwater and facilitates future investigation on the threat of viral outbreaks via surfaces waters where fecal contamination may have occurred. Future studies should investigate whether humans are able to contract SARS-CoV-2 from surface waters and the factors that may affect viral longevity and transmission.
... The copy numbers of these markers were higher for HF183/Bac242, potentially suggesting that sewage was the main contributor of faecal pollution. Consistent with Water Science & Technology Vol 84 No 7, 1741 this, a recent study of three Sydney estuarine waters found sewage-associated MSTs, including the HF183 marker (Green et al. 2014), to be present in concentrations 3-5 times higher following rainfall compared with dry samples (Ahmed et al. 2020). ...
The contamination of water catchments by nonpoint source faecal pollution is a major issue affecting the microbial quality of receiving waters and is associated with the occurrence of a range of enteric illnesses in humans. The potential sources of faecal pollution in surface waters are diverse, including urban sewage leaks, surface runoff and wildlife contamination originating from a range of hosts. The major contributing hosts require identification to allow targeted management of this public health concern. In this study, two high-performing Microbial Source Tracking (MST) assays, HF183/Bac242 and BacCan-UCDmodif, were used for their ability to detect host-specific Bacteroides 16Sr RNA markers for faecal pollution in a 12-month study on an urban coastal lagoon in Sydney, Australia. The lagoon was found to contain year-round high numbers of human and canine faecal markers, as well as faecal indicator bacteria counts, suggesting considerable human and animal faecal pollution. The high sensitivity and specificity of the HF183/Bac242 and BacCan-UCDmodif assays, together with the manageable levels of PCR inhibition and high level DNA extraction efficiency obtained from lagoon water samples make these markers candidates for inclusion in an MST ‘toolbox’ for investigating host origins of faecal pollution in urban surface waters. HIGHLIGHTS
This long-term study (twelve months with bi-monthly sampling) validated the use of HF183/BacCan242 and BacCan-UCDmodif to quantify host-associated faecal inputs in urban surface waters.;
Correlations between MST markers, traditional FIB measurements, and physiochemical parameters indicate that the latter may be used to predict the likelihood of human- and canine-associated faecal inputs at this site.;
... In Australia, the COVID-19 RNA was identified from catchment area with wastewater using RT-qPCR assay and later by Monte Carlo simulation model projected a mean value of infections varies between 171 and 1090. The projected simulation was found to match with experimental observations (Ahmed et al., 2020). Air quality parameters like NO 2 , SO 2 and PM 10 monitored before and after lockdown period ...
This study aims to explore the state-wise assessment of SARS-CoV-2 (COVID-19) pandemic spread in Malaysia with focus on influence of meteorological parameters and air quality. In this study, state-wise COVID-19 data, meteorological parameters and air quality index (AQI) were collected from March 13 to April 30, 2020, which encompass three movement control order (MCO) periods in the country. Overall, total infected cases were observed to be higher in MCO phase 1 and 2 and significantly reduced in MCO phase 3. Due to the variation in the spatial interval of population density and individual immunity, the relationship of these parameters to pandemic spread could not be achieved. The study infers that temperature (T) between 23 and 25 °C and relative humidity (RH) (70-80%) triggered the pandemic spread by increase in the infected cases in northern and central Peninsular Malaysia. Selangor, WP Kuala Lumpur and WP Putrajaya show significantly high infected cases and a definite trend was not observed with respect to a particular meteorological factor. It is identified that high precipitation (PPT), RH and good air quality have reduced the spread in East Malaysia. A negative correlation of T and AQI and positive correlation of RH with total infected cases were found during MCO phase 3. Principal component analysis (PCA) indicated that T, RH, PPT, dew point (DP) and AQI are the main controlling factors for the spread across the country apart from social distancing. Vulnerability zones were identified based on the spatial analysis of T, RH, PPT and AQI with reference to total infected cases. Based on time series analysis, it was determined that higher RH and T in Peninsular Malaysia and high amount of PPT, RH and good air quality in East Malaysia have controlled the spreading during MCO phase 3. The predominance of D614 mutant was observed prior to March and decreases at the end of March, coinciding with the fluctuation of meteorological factors and air quality. The outcome of this study gives a general awareness to the public on COVID-19 and the influence of meteorological factors. It will also help the policymakers to enhance the management plans against the pandemic spreading apart from social distancing in the next wave of COVID-19.
Supplementary information:
The online version contains supplementary material available at 10.1007/s10668-021-01719-z.
... Furthermore, agricultural land run-off may also negatively impact water quality and introduce ARGs into waterbodies (Unc and Goss, 2004;Ballesté et al., 2020). Watercourses discharging into the marine environment, especially close to designated bathing waters, may expose the users to fecal pollution and therefore may increase the likelihood that they will be exposed to ARGs (Molina et al., 2014;Leonard et al., 2018;Ahmed et al., 2020;Reynolds et al., 2020;Sala-Comorera et al., 2021b). ...
Fecal pollution of surface water may introduce bacteria and bacteriophages harboring antibiotic resistance genes (ARGs) into the aquatic environment. Watercourses discharging into the marine environment, especially close to designated bathing waters, may expose recreational users to fecal pollution and therefore may increase the likelihood that they will be exposed to ARGs. This study compares the bacterial and bacteriophage ARG profiles of two rivers (River Tolka and Liffey) and two small urban streams (Elm Park and Trimleston Streams) that discharge close to two marine bathing waters in Dublin Bay. Despite the potential differences in pollution pressures experienced by these waterways, microbial source tracking analysis showed that the main source of pollution in both rivers and streams in the urban environment is human contamination. All ARGs included in this study, bla TEM , bla SHV , qnrS , and sul1 , were present in all four waterways in both the bacterial and bacteriophage fractions, displaying a similar ARG profile. We show that nearshore marine bathing waters are strongly influenced by urban rivers and streams discharging into these, since they shared a similar ARG profile. In comparison to rivers and streams, the levels of bacterial ARGs were significantly reduced in the marine environment. In contrast, the bacteriophage ARG levels in freshwater and the marine were not significantly different. Nearshore marine bathing waters could therefore be a potential reservoir of bacteriophages carrying ARGs. In addition to being considered potential additional fecal indicators organism, bacteriophages may also be viewed as indicators of the spread of antimicrobial resistance.
... Figure 1b. Recommendations on the window of infectivity and detection potential were based on studies by Wu et al. [23] and Ahmed et al. [6,24]. The first confirmed case of COVID-19 in Curitiba was on 11 March 2020, with an average increase from 100 to 120 new cases/week between April and May. ...
SARS-CoV-2 environmental monitoring can track the rate of viral contamination and can be used to establish preventive measures. This study aimed to detect by RT-PCR the presence of SARS-CoV-2 from inert surface samples in public health settings with a literature review about surface contamination and its burden on spread virus. Samples were collected from health settings in Curitiba, Brazil, between July and December 2020. A literature review was conducted using PRISMA. A total of 711 environmental surface samples were collected from outpatient areas, dental units, doctors’ offices, COVID-19 evaluation areas, and hospital units, of which 35 (4.9%) were positive for SARS-CoV-2 RNA. The frequency of environmental contamination was higher in primary care units than in hospital settings. The virus was detected on doctors’ personal items. Remarkably, the previously disinfected dental chair samples tested positive. These findings agree with those of other studies in which SARS-CoV-2 was found on inanimate surfaces. Detection of SARS-CoV-2 RNA on surfaces in public health settings, including those not meant to treat COVID-19, indicates widespread environmental contamination. Therefore, the intensification of disinfection measures for external hospital areas may be important for controlling community COVID-19 dissemination.
... In site 1, HAV was not detected, and in site 4, the HAV concentration was significantly higher than HAV concentration in P2 and P3 (p = 0.0001) (Fig. 2). The highest precipitation (6 mm) occurred during collection at sites 1 and 2, which might have influenced the dilution of viral particles (Ahmed et al. 2020), probably because these sites received more discharge of urban drainage systems than the other ones. Site 3 has a greater presence of wild animals, such as ducks and capybaras, and consequently, they increase the sediment movement. ...
The present study reports the monitoring of viruses indicating fecal contamination in two distinct regions affected by poor management of wastewater located above the Guarani Aquifer, which is one of the biggest freshwater reservoirs in the world. In the city of Três Lagoas (located in the Midwest region, in the state of Mato Grosso do Sul), water samples were collected from Lagoa Maior, a lake used for recreation, and in Concórdia (located in the South region, in the state of Santa Catarina), from the Queimados River, which crosses the urban area. Four sampling sites were monitored from March to July 2018 in Lagoa Maior, and four sampling sites were monitored along the urban part of the Queimados River area over two periods (rainy and dry). Water samples were analyzed by concentration of Human adenovirus (HAdV), Norovirus (NoV), Rotavirus A (RAV), and Hepatitis A virus (HAV) for the Lagoa Maior samples and RVA, HAV, and Porcine circovirus 2 (PCV2) for the Queimados River samples. All sampling sites presented enteric viruses, demonstrating fecal input and potential contamination of groundwater. Results highlight the need for wastewater management to improve environmental health quality.
... The sewage water becomes the major pathway for transmission the novel corona virus is detected in feces and urine of infected individuals (Quilliam et al., 2020). In Netherland the presence of SARS COV-2 in waste water is reported by binary RT-q PCR data (Ahmed et al., 2020). ...
Viruses are biologically active parasites that only exist inside a host they are submicroscopic level. The novel coronavirus disease, or COVID-19, is generally caused by the SARS-CoV-2 virus and is comparable to severe acute respiratory syndrome (SARS). As a result of globalization, natural alterations or changes in the SARS-CoV-2 have created significant risks to human health over time. These viruses can live and survive in different ways in the atmosphere unless they reach another host body. At this stage, we will discuss the details of the transmission and detection of this deadly SARS-CoV-2 virus via certain environmental media, such as the atmosphere, water, air, sewage water, soil, temperature, relative humidity, and bioaerosol, to better understand the diffusion, survival, infection potential and diagnosis of COVID-19.
... A 20 mL water sample from each dialysis tube was filtered through HA electronegative membranes with a 0.45 μm pore size (47 mm diameter) (Merck Millipore, Tokyo, Japan). Prior to filtration, the pH of the sample was adjusted to 3.5 by adding 2.0 N HCl (Ahmed et al., 2020). To obtain information on the intrinsic levels of target microorganisms present in the sewage and ambient freshwater, DNA was directly extracted in triplicate from 500 μL of untreated sewage, and 500 mL aliquots of ambient freshwater taken from the Wappa and Wivenhoe lakes. ...
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.
... Storm runoff causing flooded sewers has been long thought to be the major source of wastewater pollution to Sydney coastal waters . Recent research, however, has detected pharmaceuticals, artificial sweeteners and intermittent enterococci in surface waters apparently unrelated to storm runoff during dry conditions (NHMRC, 2008;Birch et al., 2015;Ahmed et al., 2020), suggesting wastewater sources other than surface runoff, perhaps SGD. We hypothesize that (1) SGD is a source of wastewater and industrial runoff to coastal surface waters, and (2) CEC distribution will be related to a land-use gradient and water residence times. ...
Submarine groundwater discharge (SGD) is rarely considered as a pathway for contaminants of emerging concern (CECs). Here, we investigated SGD as a source of CECs in Sydney Harbour, Australia. CEC detection frequencies based on presence/absence of a specific compound were >90% for caffeine, carbamazepine, and dioxins, and overall ranged from 25 to 100% in five studied embayments. SGD rates estimated from radium isotopes explained >80% of observed CEC inventories for one or more compounds (caffeine, carbamazepine, dioxins, sulfamethoxazole, fluoroquinolones and ibuprofen) in four out of the five embayments. Radium-derived residence times imply mixing is also an important process for driving coastal inventories of these persistent chemicals. Two compounds (ibuprofen and dioxins) were in concentrations deemed a high risk to the ecosystem. Overall, we demonstrate that SGD can act as a vector for CECs negatively impacting coastal water quality.
... The contributions of land-based sewage discharge, surface runoff, and aquaculture wastewater resulting from human feces to virus contamination in coastal water have been determined (Ahmed et al., 2020;Lees, 2000;Steele et al., 2018;Viau et al., 2011;WHO, 2019). Beaches often receive virus contamination from more than one source, and different contamination sources are expected to pose different health risks to swimmers (Ahmed et al., 2018;Brown et al., 2017;Walters et al., 2009). ...
Human enteric virus occurrence in bathing beaches poses a potential health risk to swimmers. They may come from several sources, but the understanding of the seasonal contribution of contamination sources to virus occurrence is still lacking. Here, the surveillance of human enteric viruses at the First Bathing Beach in Qingdao was performed January–December 2018. The occurrence of Enteric viruses, assayed with quantitative polymerase chain reaction (qPCR), was analyzed at temporal and spatial levels to determine the viral contamination sources. The results showed that only Astroviruses (AstVs) and Adenoviruses (HAdVs) were found in the swimming area. Their occurrence correlated significantly with the sewage-polluted area, but HAdVs were only found in autumn and AstVs in spring. Meanwhile, enteric viruses in the swimming area showed significantly higher levels than the surrounding area, particularly AstVs in summer with the swimmer crowd. All these data imply that sewage discharge and swimmers co-contribute to the viral occurrence in a seasonal pattern, with the former being more focused in warm seasons (spring and autumn) and the latter in hot seasons (summer). These results indicate that sewage discharge and crowd swimmers, as unsafe swimming conditions, should be avoided to improve public health at the bathing beaches.
... Whereas the qPCR analytical protocol is the end-of-pipe step for the qPCR pipeline, other factors could affect qPCR absolute gene quantification, for example, sample collection duration and protocol, 85 laboratory protocols and instruments, 72 and recovery efficiencies. 12,86 Consequently, this study underscores the need for a standardized analytical protocol for qPCR MST assays, which should include the analytical pipeline, for consistency and comparability of results from different laboratories. ...
A widely used microbial source tracking (MST) technique, quantitative polymerase chain reaction (qPCR), quantifies host-specific gene abundance in polluted water to identify and prioritize contamination sources. This study characterized the effects of a qPCR data analysis using the sample PCR efficiencies (the LinRegPCR model) on gene abundance and compared it with the standard curve-based method (the mixed model). Five qPCR assays were evaluated: the universal GenBac3, human-specific HF183/BFDrev and CPQ_056, swine-specific Pig-2-Bac, and cattle-specific Bac3qPCR assays. The LinRegPCR model increased low-copy amplification, especially in the HF183/BFDrev assay, thus lowering the specificity to less than the recommended value of 0.80. Up to 1.41 log10 copies/g and 0.41 log10 copies/100 mL differences were observed for composite fecal and sewage samples (n=147) by the LinRegPCR approach, corresponding to an 18.2% increase and 6.4% decrease, respectively. Freshwater samples (n=48) demonstrated a maximum of 1.95 log10 copies/100 mL difference between the two models. Identical attributing sources by both models were shown in 54.55% of environmental samples; meanwhile, the LinRegPCR approach improved the inability to identify sources by the mixed model in 29.55% of the samples. This study emphasizes the need for a standardized data analysis protocol for qPCR MST assays for interlaboratory consistency and comparability.
... In developing countries, many areas lack adequate sanitary infrastructure and wastewater treatment facilities and hence faecal matter contaminates the environment and drinking water sources (Bain et al., 2014). Furthermore, large volumes of untreated wastewater may also be discharged via combined sewer overflows (CSOs) during heavy rainfall events and via dry water overflows for example during snowmelt, tidal infiltration or system failures and blockages (Ahmed et al., 2020). These events enable the direct entry of enteric pathogens into the environment (Fong et al., 2010), where people in direct or indirect contact with contaminated waters may be at risk of acquiring viral infections (Sinclair et al., 2009). ...
Waterborne enteric viruses are an emerging cause of disease outbreaks and represent a major threat to global public health. Enteric viruses may originate from human wastewater and can undergo rapid transport through aquatic environments with minimal decay. Surveillance and source apportionment of enteric viruses in environmental waters is therefore essential for accurate risk management. However, individual monitoring of the >100 enteric viral strains that have been identified as aquatic contaminants is unfeasible. Instead, viral indicators are often used for quantitative assessments of wastewater contamination, viral decay and transport in water. An ideal indicator for tracking wastewater contamination should be (i) easy to detect and quantify, (ii) source-specific, (iii) resistant to wastewater treatment processes, and (iv) persistent in the aquatic environment, with similar behaviour to viral pathogens. Here, we conducted a comprehensive review of 127 peer-reviewed publications, to critically evaluate the effectiveness of several viral indicators of wastewater pollution, including common enteric viruses (mastadenoviruses, polyomaviruses, and Aichi viruses), the pepper mild mottle virus (PMMoV), and gut-associated bacteriophages (Type II/III FRNA phages and phages infecting human Bacteroides species, including crAssphage). Our analysis suggests that overall, human mastadenoviruses have the greatest potential to indicate contamination by domestic wastewater due to their easy detection, culturability, and high prevalence in wastewater and in the polluted environment. Aichi virus, crAssphage and PMMoV are also widely detected in wastewater and in the environment, and may be used as molecular markers for human-derived contamination. We conclude that viral indicators are suitable for the long-term monitoring of viral contamination in freshwater and marine environments and that these should be implemented within monitoring programmes to provide a holistic assessment of microbiological water quality and wastewater-based epidemiology, improve current risk management strategies and protect global human health.
... We used two virus concentration methods because limited information is available on the effectiveness of enveloped virus recovery from wastewater matrices using existing virus concentration methods. The electronegative membrane used in this study is typically used for concentrating enteric viruses from wastewater and environmental waters with modest recovery (Rigotto et al., 2009;Ahmed et al., 2020). The rationale for using the electronegative membrane is that greater adsorption of enveloped viruses such as mouse hepatitis virus and Pseudomonas phage Φ6 to the solid fraction of wastewater compared to nonenveloped viruses (Ye et al., 2016). ...
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 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 1090 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.
Fecal pollution of surface waters in karst-dominated Edwards aquifer is a serious concern as contaminated waters can rapidly transmit to groundwaters, which are used for domestic purposes. Although microbial source...
In this study, two virus concentration methods, namely Adsorption-extraction (AE) and Nanotrap® Magnetic Virus Particles (NMVP) along with commercially available extraction kits were used quantify endogenous pepper mild mottle virus (PMMoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in nucleic acid extracted from 48 wastewater samples collected over six events from eight wastewater treatment plants (WWTPs). The main aim was to determine which workflow (i.e., concentration and extraction methods) produces greater concentrations of PMMoV and SARS-CoV-2 gene copies (GC) in comparison with each other. Turbidity and total suspended solids (TSS) of wastewater samples within and among the eight WWTPs were highly variable (41–385 NTU and 77–668 mg/L TSS). In 58 % of individual wastewater samples the log10 GC concentrations of PMMoV were greater by NMVP workflow compared to AE workflow. Paired measurements of PMMoV GC/10 mL from AE and NMVP across all 48 wastewater samples were weakly correlated (r = 0.455, p = 0.001) and demonstrated a poor linear relationship (r² = 0.207). The log10 GC concentrations of SARS-CoV-2 in 69 % of individual samples were greater by AE workflow compared to NMVP workflow. In contrast to PMMoV, the AE and NMVP derived SARS-CoV-2 GC counts were strongly correlated (r = 0.859, p < 0.001) and demonstrated a strong linear relationship (r² = 0.738). In general, the PMMoV GC achieved by the NMVP workflow decreased with increasing turbidity, but the PMMoV GC by the AE workflow did not appear to be sensitive to either turbidity or TSS levels. These findings suggest that suspended solids concentration, and the intended target for analysis should be considered when validating an optimal workflow for wastewater surveillance.
The present study proposes a novel hybrid methodological approach for meticulously investigating the factors causing Sewer Overflow (SO). The proposed framework is based on a systematic literature review, experts’ interviews, PLS-SEM statistical technique, and system dynamic modeling. Based on a large number of data collected from experts having rich experience in developed countries, three major findings are obtained: (1) three main factors and eighteen sub-factors are the main culprit of SO occurrence, (2) under design pipe diameter, blockages, and infiltration and inflow are the most significant sub-factors within the respective clusters, and (3) physical-related sub-factors are the most influential causes of SO occurrence under a dynamic environment. The findings attained in this study offer an insightful account for the concerned environmental decision-makers on coming up with further fecund measures towards reducing the magnitude of SO, preserving our environment from the occurrence of such harmful incidents.
Quantitative Microbial Risk Assessment (QMRA) of human health risks using human fecal marker genes (HFMGs) is an attractive water quality management tool. To inform accurate QMRA analysis, generation of probability distribution functions for HFMGs, and reference pathogenic viruses can be improved by input of correlation and ratios based upon measurement of HFMGs and gene copies (GC) of pathogenic viruses in untreated wastewater. The concentrations of four HFMGs (Bacteroides HF183, Lachnospiraceae Lachno3, CrAssphage CPQ_056 and pepper mild mottle virus (PMMoV)), and GC of three reference pathogenic viruses (human adenovirus HAdV 40/41, human norovirus HNoV GI + GII and enterovirus EV) were measured in untreated wastewater samples collected over a period of 12 months from two wastewater treatment plants in Sydney, Australia using quantitative polymerase chain reaction (qPCR) and reverse transcriptase qPCR. Over the course of the study the GC of potential pathogenic viruses were 3-5 orders of magnitude lower than HFMGs in untreated wastewater. The GC of enteric viruses were highly variable over the course of the study, which contrasted with the concentrations of HFMGs that were quite stable with little variation observed within and between WWTPs. Among the HFMGs, HF183, CrAssphage and PMMoV correlated well with pathogenic virus GC, whereas poor or negative correlations were observed between Lachno3 and HFMGs. While the two assessed WWTPs had dissimilar population service sizes the ratios between log10 transformed pathogenic virus GC and HFMGs demonstrated similar central tendency and variability for the same combinations between WWTP A and WWTP B with no difference between the WWTPs. This suggests the widespread presence of these HFMGs in both populations serviced by these two WWTPs. This correlation and ratios of HFMGs and GC of potential reference pathogenic viruses should contribute to improved QMRA of human health risks in environmental waters subject to fresh sewer overflows.
The waste of ethidium bromide (EtBr) used in the laboratory will bring a great burden to the environment, which need to be solved urgently. In the present paper, an efficient and inexpensive method for EtBr removal using chromatin extracted from common carp testis was investigated. The observation of fluorescence microscopy showed that chromatin had similar property to DNA for selective adsorption of EtBr. The results of batch adsorption showed that the removal efficiency of EtBr by chromatin exceeded 99% at pH 7.4 and 30 °C for 3 min with the EtBr concentration of 2 mg·L⁻¹ and the chromatin dosage of 0.5 g·L⁻¹, and the maximum adsorption amount of chromatin was 45.73 mg·g⁻¹. Further, the analysis of kinetic and isotherm suggested that the adsorption followed Pseudo-second-order kinetics and Langmuir isotherm model, and the calculated maximum theoretical adsorption amount of chromatin to EtBr was 48.08 mg·g⁻¹. According to thermodynamic analysis, chromatin adsorption of EtBr was a spontaneous process dominated by hydrogen bonding and van der Waals forces. This work will not only offer an adsorbent for EtBr decontamination, also provide a possibility for EtBr analogs removal.
Concurrence of pharmaceuticals and personal care products (PPCPs), pathogenic viruses, metals and microbial pollution along with their seasonal variations in the water environment are overarching in the context of existing pandemic, especially for tropical countries. The present study focuses on the seasonal influence on the vulnerability of urban water in Guwahati, the largest city in North-eastern India, through examining the concurrence of seven PPCPs, five viruses, faecal bacteria and nine metals in surface waters during monsoon (Summer-July 2017) and pre-monsoon (Winter-March 2018). Surface water sampling was carried out at different locations of the Brahmaputra River, its tributary Bharalu River (an unlined urban drain), and Dipor Bill Lake (Ramsar recognized wetland). Both PPCPs and viruses were at high concentrations (e.g. up to 970 ng L⁻¹ caffeine, 2.5 × 10³ copies/mL pepper mild mottle virus (PMMoV)) at the confluence points of urban drains and the river, while they were mostly undetectable at both upstream and downstream locations, implying strong self-purification ability of the river. All the analysed PPCPs and viruses were at much higher concentration during pre-monsoon i.e., winter with respect to monsoon, implying heavy dilution and temperature effect during the monsoon. Overall, PPCPs and viruses were more correlated in monsoon but the risk quotient in the urban tributary was higher in pre-monsoon (e.g. 5061 in pre-monsoon and 1515 in monsoon for caffeine). PMMoV was found to be an excellent faecal pollution indicator due to its prevalence, detectability and specificity in all seasons. Overall, the seasonal fluctuations of the non-enveloped viruses monitored in this study is likely to be relevant for SARS-CoV-2. We contribute to the literature scarcity pertaining to seasonal variations in the prevalence of viruses and their concurrences with contaminants of emerging concern.
Sewer overflow (SO), which has attracted global attention, poses serious threat to public health and ecosystem. SO impacts public health via consumption of contaminated drinking water, aerosolization of pathogens, food-chain transmission, and direct contact with fecally-polluted rivers and beach sediments during recreation. However, no study has attempted to map the linkage between SO and public health including Covid-19 using scientometric analysis and systematic review of literature. Results showed that only few countries were actively involved in SO research in relation to public health. Furthermore, there are renewed calls to scale up environmental surveillance to safeguard public health. To safeguard public health, it is important for public health authorities to optimize water and wastewater treatment plants and improve building ventilation and plumbing systems to minimize pathogen transmission within buildings and transportation systems. In addition, health authorities should formulate appropriate policies that can enhance environmental surveillance and facilitate real-time monitoring of sewer overflow. Increased public awareness on strict personal hygiene and point-of-use-water-treatment such as boiling drinking water will go a long way to safeguard public health. Ecotoxicological studies and health risk assessment of exposure to pathogens via different transmission routes is also required to appropriately inform the use of lockdowns, minimize their socio-economic impact and guide evidence-based welfare/social policy interventions. Soft infrastructures, optimized sewer maintenance and prescreening of sewer overflow are recommended to reduce stormwater burden on wastewater treatment plant, curtail pathogen transmission and marine plastic pollution. Comprehensive, integrated surveillance and global collaborative efforts are important to curtail on-going Covid-19 pandemic and improve resilience against future pandemics.
Shellfish growing waters contaminated with inadequately treated human wastewater is a major source of norovirus in shellfish and poses a significant human health risk to consumers. Microbial source tracking (MST) markers have been widely used to identify the source (s) of faecal contamination in water but data are limited on their use for shellfish safety. This study evaluated the source specificity, sensitivity, occurrence and concentration of three viral MST markers i.e. cross-assembly phage (crAssphage), F-specific RNA bacteriophage genogroup II (F-RNA phage GII) and pepper mild mottle virus (PMMoV) using animal faeces (n = 119; 16 animal groups), influent wastewater (n = 12), effluent wastewater (n = 16) and shellfish (n = 33). CrAssphage, F-RNA phage GII and PMMoV had source specific values of 0.97, 0.99 and 0.91, respectively. The sensitivity of MST markers was confirmed by their 100% detection frequency in influent wastewaters. The frequency of detection in effluent wastewater ranged from 81.3% (F-RNA phage GII) to 100 % (PMMoV). Concentration of F-RNA phage GII was one log10 (influent wastewater) and 2-3 log10 (effluent wastewater) lower than crAssphage and PMMoV, respectively. Despite lower prevalence of F-RNA phage GII in oysters and mussels compared to crAssphage and PMMoV, concentrations of the three MST markers were similar in mussels. As an indicator of human norovirus contamination in shellfish, crAssphage and PMMoV had greater predictive sensitivity (100%; [95% CI; 81.5%-100%)]) and F-RNA phage GII had greater predictive specificity (93.3%; [95% CI; 68.1%-99.8%]). In contrast, crAssphage and F-RNA phage GII have similar accuracy for predicting norovirus in shellfish, however, PMMoV significantly overestimated its presence. Therefore, a combination of crAssphage and F-RNA phage GII analysis of shellfish could provide a robust estimation of the presence of human faecal and norovirus contamination.
Enteric viruses are a diverse group of human pathogens which are primarily transmitted by the faecal–oral route and are a major cause of non-bacterial diarrhoeal disease in both developed and developing countries. Because they are shed in high numbers by infected individuals and can persist for a long time in the environment, they pose a serious threat to human health globally. Enteric viruses end up in the environment mainly through discharge or leakage of raw or inadequately treated sewage into water sources such as springs, rivers, dams, or marine estuaries. Human exposure then follows when contaminated water is used for drinking, cooking, or recreation and, importantly, when filter-feeding bivalve shellfish are consumed. The human health hazard posed by enteric viruses is particularly serious in Africa where rapid urbanisation in a relatively short period of time has led to the expansion of informal settlements with poor sanitation and failing or non-existent wastewater treatment infrastructure, and where rural communities with limited or no access to municipal water are dependent on nearby open water sources for their subsistence. The role of sewage-contaminated water and bivalve shellfish as vehicles for transmission of enteric viruses is well documented but, to our knowledge, has not been comprehensively reviewed in the African context. Here we provide an overview of enteric viruses and then review the growing body of research where these viruses have been detected in association with sewage-contaminated water or food in several African countries. These studies highlight the need for more research into the prevalence, molecular epidemiology and circulation of these viruses in Africa, as well as for development and application of innovative wastewater treatment approaches to reduce environmental pollution and its impact on human health on the continent.
This study describes microbial and chemical source tracking approaches for water pollution measures in rural and urban catchments. Culturable faecal indicator bacteria, represented by Escherichia coli, were quantified. Microbial source tracking (MST) using host-specific DNA markers was applied to identify the origins of faecal contamination. Chemical source tracking (CST) was conducted to determine contaminants of emerging concern (CEC) specific for human/anthropogenic origin, including pharmaceuticals and personal care products (PPCPs) and endocrine-disrupting chemicals (EDCs). In addition, some macropollutants, including eutrophication-limiting macronutrients (nitrogen and phosphorus), were studied. MST tests revealed both anthropogenic and zoogenic faecal origins, as represented by a dominance of human sources in the urban stream; non-human/environmental sources were prevalent in the rural creek. CST analyses revealed a higher number of CECs in the urban stream than in the rural watercourse. Positive correlations between PPCPs and both E. coli and human DNA marker were uncovered in the urban stream, while in the rural creek, PPCPs were only highly correlated with the anthropogenic marker. Interestingly, macronutrients were greatly associated with primary faecal pollution origins in both watercourses. This correlation pattern determines the main pollutant contributors (anthropogenic or zoogenic) to eutrophication.
Identifying sewage contamination via microbial source tracking (MST) marker genes has proven useful for effective water quality management worldwide; however, performance 3 evaluations for these marker genes in tropical areas are limited. Therefore, this research evaluated four human-associated MST marker genes for tracking sewage pollution in aquatic environments of Thailand: human polyomaviruses (JC and BK viruses [HPyVs]), bacteriophage crAssphage (CPQ_056), Lachnospiraceae Lachno3, and Bacteroides BacV6-21. The viral markers, HPyV and crAssphage, assays were highly sensitive and specific to sewage from onsite wastewater treatment plants (OWTPs; n = 19), with no cross-detection in 120 swine, cattle, chicken, duck, goat, sheep, and buffalo composite fecal samples. The bacterial markers, 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 non-detected-8.05 log10 copies/100 mL), crAssphage (5.28-7.38 log10 copies/100 mL), and HPyVs (3.66-6.53 log10 copies/100 mL), respectively. Due to their increased specificity, the abundance of viral markers were further investigated in environmental waters, in which HPyVs showed higher levels (up to 4.33 log10 copies/100 mL) and higher detection rates (92.7%) in two coastal beaches (n = 41) than crAssphage (up to 3.51 log10 copies/100 mL and 56.1%). HPyVs were also found at slightly lower levels (up to 5.10 log10 copies/100 mL), but at higher detection rates (92.6%), in a freshwater canal (n = 27) than crAssphage (up to 5.21 log10 copies/100 mL and 88.9%). Overall, both HPyVs and crAssphage are suggested as sewage-associated MST markers 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.
Identifying human sewage contamination via microbial source tracking (MST) marker genes has proven useful for effective water quality management worldwide; however, performance evaluations for these genes in tropical areas are limited. Therefore, this research assessed four human-associated MST marker genes in aquatic environments of Central Thailand: human polyomaviruses (JC and BK viruses [HPyVs]), bacteriophage crAssphage (CPQ_056), Lachnospiraceae Lachno3, and Bacteroides BacV6-21. HPyV and crAssphage assays were highly sensitive and specific to sewage (n = 19), with no cross-detection in 120 swine, cattle, chicken, duck, goat, sheep, and buffalo composite fecal samples. 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 sewage were Lachno3 and BacV6-21 (5.42–8.02 and non-detected–8.05 log10 copies/100 mL), crAssphage (5.28–7.38 log10 copies/100 mL), and HPyVs (3.66–6.53 log10 copies/100 mL), respectively. HPyVs showed higher levels (up to 4.33 log10 copies/100 mL) and higher detection rates (92.7%) in two coastal beaches (n = 41) than crAssphage (up to 3.51 log10 copies/100 mL and 56.1%). HPyVs were also found at slightly lower levels (up to 5.10 log10 copies/100 mL), but at higher detection rates (92.6%), in a freshwater canal (n = 27) than crAssphage (up to 5.21 log10 copies/100 mL and 88.9%). Overall, both HPyVs and crAssphage are suggested as human sewage-associated MST markers in aquatic environments of Central 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.
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 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.
Microbial water quality is currently assessed by fecal indicator bacteria that are poor representatives of viral pathogens in the environment. Viruses are predicted to account for the majority of infectious risk from exposure to sewage contaminated water. Previously developed viral indicators suffer from a lack of human-specificity, low concentrations in sewage, or both. In this commentary review, we discuss recent advances in developing Cross-Assembly Phage (crAssphage) and Pepper Mild Mottle Virus (PMMoV) as viral water quality indicators. CrAssphage and PMMoV are abundant in and highly associated with human sewage, correlate with viral pathogens in sewage contaminated environments, and globally present. Future work is necessary to describe crAssphage and PMMoV fate in the environment, local variation in abundance and genetic makeup, and relationship between molecular detections and pathogen viability, among other areas. These developments will allow the integration of crAssphage and PMMoV into quantitative microbial risk assessment and water quality regulation.
Consumption of bivalve molluscan shellfish (BMS) grown in faecally contaminated waters is a public health risk. While faecal contamination in BMS and water can originate from multiple sources (i.e. human, livestock, wildlife and crop production), accurate identification of the source (s) is important to establish an appropriate, cost-effective plan to minimise the potential public health risks. Microbial source tracking (MST) targets (genes) are more source-specific than faecal indicator bacteria. Application of MST methods could eliminate the requirement of multiple testing regime for bacterial and viral pathogen in BMS. This approach therefore, can assist to develop an accurate, reliable faecal pollution management plan that reduces the cost for BMS farmers as well as public health risks associate with BMS consumption. However, validation of MST targets, their recovery from BMS and correlation with other bioaccumulated pathogens in BMS is required prior to its field application.
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 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 (MST) DNA and RNA 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 log10 GC/L of water, respectively. Moderate to strong positive correlations were observed among the quantitative occurrence of sewage-associated markers. 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.
Global burden of acute viral gastroenteritis remains high, particularly in developing countries including Bangladesh. Sewage water (SW) is an important node to monitor enteric pathogens both in the environment and among the population. Analysis of SW in Dhaka city deems crucially important because a large number of urban-city dwellers live in Dhaka city, the capital of Bangladesh, under a constant threat of precarious sewerage system. In this study, we collected raw SW from five locations of Dhaka city every month from June 2016 to May 2017. It was concentrated with polyethylene glycol (PEG) and investigated for three major enteric viruses, rotavirus A (RVA), norovirus GII (NoV GII) and adenovirus (AdV) using polymerase chain reaction (PCR). Most of these SW samples collected from both hospitals and non-hospital areas yielded enteric viruses: 76% samples were positive for AdV, followed by 53% NoV GII and 38% RVA. Viral load was determined as much as 1 × 10⁷ copies/ml for RVA and 3.5 × 10³ copies/ml for NoV GII. Importantly, NoV GII and AdV that can affect people of all ages were predominated during monsoon also when SW overflows and spreads over a wide and crowded area. Genotypes G1, G2, G3, G8, and G9 for RVA, GII.4 for NoV, and type 41 for AdV were detected representing the current profile of circulating genotypes in the population. This study provides the first evidence of distribution of major diarrheal viruses in SW in Dhaka city which is alarming showing grave risk of impending outbreaks through exposure.
Campylobacter jejuni is a major foodborne pathogen that is increasingly found worldwide and that is transmitted to humans through meat or dairy products. A detailed understanding of the prevalence and characteristics of C. jejuni in dairy cattle farms, which are likely to become sources of contamination, is imperative and is currently lacking. In this study, a total of 295 dairy cattle farm samples from 15 farms (24 visits) in Korea were collected. C. jejuni prevalence at the farm level was 60% (9/15) and at the animal level was 23.8% (68/266). Using the multivariable generalized estimating equation (GEE) model based on farm-environmental factors, we estimated that a high density of cattle and average environmental temperature (7 days prior to sampling) below 24°C affects the presence and survival of C. jejuni in the farm environment. Cattle isolates, together with C. jejuni from other sources (chicken and human), were genetically characterized based on analysis of 10 virulence and survival genes. A total of 19 virulence profile types were identified, with type 01 carrying eight genes (all except hcp and virB11) being the most prevalent. The prevalence of virB11 and hcp was significantly higher in isolates from cattle than in those from other sources (p < 0.05). Multilocus sequence typing (MLST) of C. jejuni isolates from three different sources mainly clustered in the CC-21 and CC-48. Within the CC-21 and CC-48 clusters, cattle isolates shared an indistinguishable pattern with human isolates according to pulsed-field gel electrophoresis (PFGE) and flaA-restriction fragment length polymorphism (RFLP) typing. This suggests that CC-21 and CC-48 C. jejuni from dairy cattle are genetically related to clinical campylobacteriosis isolates. In conclusion, the farm environment influences the presence and survival of C. jejuni, which may play an important role in cycles of cattle re-infection, and dairy cattle represent potential reservoirs of human campylobacteriosis. Thus, environmental management practices could be implemented on cattle farms to reduce the shedding of C. jejuni from cattle, subsequently reducing the potential risk of the spread of cattle-derived C. jejuni to humans through the food chain.
Despite modern sewer system infrastructure, the release of sewage from deteriorating pipes and sewer overflows is a major water pollution problem in US cities, particularly in coastal watersheds that are highly developed with large human populations. We quantified fecal pollution sources and loads entering Lake Michigan from a large watershed of mixed land use using host-associated indicators. Wastewater treatment plant influent had stable concentrations of human 'Bacteroides' and human 'Lachnospiraceae' with geometric mean concentrations of 2.77 × 107 and 5.94 × 107 copy number (by quantitative PCR) per 100 ml, respectively. Human-associated indicator levels were four orders of magnitude higher than norovirus concentrations, suggesting that these human-associated bacteria could be sensitive indicators of pathogen risk. Norovirus concentrations in these same samples were used in calculations for quantitative microbial risk assessment. Assuming a typical recreational exposure to untreated sewage in water, concentrations of 7,800 copy number of human 'Bacteroides' per 100 mL or 14,000 copy number of human 'Lachnospiraceae' per 100 mL corresponded to an illness risk of 0.03. These levels were exceeded in estuarine waters during storm events with greater than 5 cm of rainfall. Following overflows from combined sewer systems (which must accommodate both sewage and stormwater), concentrations were 10-fold higher than under rainfall conditions. Automated high frequency sampling allowed for loads of human-associated markers to be determined, which could then be related back to equivalent volumes of untreated sewage that were released. Evidence of sewage contamination decreased as ruminant-associated indicators increased approximately one day post-storm, demonstrating the delayed impact of upstream agricultural sources on the estuary. These results demonstrate that urban areas are a diffuse source of sewage contamination to urban waters and that storm-driven release of sewage, particularly when sewage overflows occur, creates a serious though transient human health risk.
Traditional fecal indicator bacteria do not distinguish animal from human fecal pollution, which is necessary to evaluate health risks and mitigate pollution sources. Assessing water in urban areas is challenging, since the water can be impacted by sewage, which has a high likelihood of carrying human pathogens, as well as pet and urban wildlife waste. We demonstrate that the Lachno3 and Lachno12 markers are human associated and highly specific for the detection of human fecal pollution from urban sources, offering reliable identification of fecal pollution sources in urban waters.
Background
Fecal indicator bacteria used to assess illness risks in recreational waters (e.g., Escherichia coli, Enterococci) cannot discriminate among pollution sources. To address this limitation, human-associated Bacteroides markers have been proposed, but the risk of illness associated with the presence of these markers in recreational waters is unclear. Our objective was to estimate associations between human-associated Bacteroides markers in water and self-reported illness among swimmers at 6 U.S. beaches spanning 2003–2007. Methods
We used data from a prospectively-enrolled cohort of 12,060 swimmers surveyed about beach activities and water exposure on the day of their beach visit. Ten to twelve days later, participants reported gastroinestinal, diarrheal, and respiratory illnesses experienced since the visit. Daily water samples were analyzed for the presence of human-associated Bacteroides genetic markers: HF183, BsteriF1, BuniF2, HumM2. We used model-based standardization to estimate risk differences (RD) and 95% confidence intervals (CI). We assessed whether the presence of Bacteroides markers were modifiers of the association between general Enterococcus and illness among swimmers using interaction contrast. ResultsOverall we observed inconsistent associations between the presence of Bacteroides markers and illness. There was a pattern of increased risks of gastrointestinal (RD = 1.9%; 95% CI: 0.1%, 3.7%), diarrheal (RD = 1.3%; 95% CI: -0.2%, 2.7%), and respiratory illnesses (RD = 1.1%; 95% CI: -0.2%, 2.5%) associated with BsteriF1. There was no evidence that Bacteroides markers acted as modifiers of Enterococcus and illness. Patterns were similar when stratified by water matrix. Conclusions
Quantitative measures of fecal pollution using Bacteroides, rather than presence-absence indicators, may be necessary to accurately assess human risk specific to the presence of human fecal pollution.
Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal indicator bacteria are used; however, they cannot distinguish human fecal waste from other animal pollution sources. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of the crAssphage genome. Based upon initial screening, two novel crAssphage qPCR assays (CPQ_056 and CPQ_064) were designed and evaluated in reference fecal samples and water matrices. The assays exhibited high specificities (98.6%) when tested against an animal fecal reference library and crAssphage genetic markers were highly abundant in raw sewage and sewage impacted water samples. In addition, CPQ_056 and CPQ_064 performance was compared to HF183/BacR287 and HumM2 assays in paired experiments. Findings confirm viral crAssphage qPCR assays perform at a similar level to well established bacterial human-associated fecal source identification approaches. These new viral based assays could become important water quality management and research tools.
Understanding the environmental pathways of Cryptosporidium is essential for effective management of human and animal cryptosporidiosis. In this paper we aim to quantify livestock Cryptosporidium spp. loads to land on a global scale using spatially explicit process-based modelling, and to explore the effect of manure storage and treatment on oocyst loads using scenario analysis. Our model GloWPa-Crypto L1 calculates a total global Cryptosporidium spp. load from livestock manure of 3.2x10(23) oocysts per year. Cattle, especially calves, are the largest contributors, followed by chickens and pigs. Spatial differences are linked to animal spatial distributions. North America, Europe and Oceania together account for nearly a quarter of the total oocyst load, meaning that the developing world accounts for the largest share. GloWPa-Crypto L1 is most sensitive to oocyst excretion rates, due to large variation reported in literature. We compared the current situation to four alternative management scenarios. We find that although manure storage halves oocyst loads, manure treatment, especially of cattle manure and particularly at elevated temperatures, has a larger load reduction potential than manure storage (up to 4.6 log units). Regions with high reduction potential include India, Bangladesh, western Europe, China, several countries in Africa, and New Zealand.
Importance:
MST is an effective tool to help utilities and regulators improve the recreational water quality around the globe. Human fecal pollution poses significant public health risks compared to animal faecal pollution. Several human wastewater associated markers have been developed and used for MST field studies. However, a head to head comparison in terms of their performance to detect diluted human fecal pollution in recreational water is lacking. In this study, we cross compared the performance of six human wastewater associated markers in relation to FIB and enteric viruses in beach water samples seeded with raw and secondary treated wastewater. The results of this study will provide guidance to regulators and utlities on the appropriate application of MST markers for tracking the sources of human faecal pollution in environmental waters and confer human health risks.
To probe the overflow pollution of separate storm drains with inappropriate sewage entries, in terms of the relationship between sewage entries and the corresponding dry-weather and wet-weather overflow, the monitoring activities were conducted in a storm drainage system in the Shanghai downtown area (374 ha). In this study site, samples from inappropriately entered dry-weather sewage and the overflow due to storm pumps operation on dry-weather and wet-weather days were collected and then monitored for six water quality constituents. It was found that overflow concentrations of dry-weather period could be higher than those of wet-weather period; under wet-weather period, the overflow concentrations of storm drains were close to or even higher than that of combined sewers. Relatively strong first flush mostly occurred under heavy rain that satisfied critical rainfall amount, maximum rainfall intensity, and maximum pumping discharge, while almost no first flush effect or only weak first flush effect was found for the other rainfall events. Such phenomenon was attributed to lower in-line pipe storage as compared to that of the combined sewers, and serious sediment accumulation within the storm pipes due to sewage entry. For this kind of system, treating a continuous overflow rate is a better strategy than treating the maximum amount of early part of the overflow. Correcting the key inappropriate sewage entries into storm drains should also be focused.
Importance:
Stormwater runoff is one of the most prominent nonpoint sources of biological and chemical contaminants which can potentially degrade and pose risks to human and ecosystem health. Therefore, identifying fecal contamination in stormwater runoff and outfalls is essential for remediation efforts to reduce risks to public health. This study employed multiple methods of identifying levels and sources of fecal contamination in both river and stormwater outfall sites, evaluating the efficacy of using culture-based enumeration of E. coli, molecular methods of determining the source(s) of contamination, and using CST markers as indicators of fecal contamination. The results identified pervasive human sewage contamination in stormwater outfalls, and throughout an urban watershed and highlight the utility of using both MST and CST to identify raw sewage contamination.
Microbial source tracking (MST) endeavors to determine sources of fecal pollution in environmental waters by capitalizing on the association of certain microorganisms with the gastrointestinal tract and feces of specific animal groups. Several decades of research have shown that bacteria belonging to the gut-associated order Bacteroidales, and particularly the genus Bacteroides, tend to co-evolve with the host, and are, therefore, particularly suitable candidates for MST applications. This review summarizes the current research on MST methods that employ genes belonging to Bacteroidales/Bacteroides as tracers or “markers” of sewage pollution, including known advantages and deficiencies of the many polymerase chain reaction (PCR)-based methods that have been published since 2000. Host specificity is a paramount criterion for confidence that detection of a marker is a true indicator of the target host. Host sensitivity, or the prevalence of the marker in feces/waste from the target host, is necessary for confidence that absence of the marker is indicative of the absence of the pollution source. Each of these parameters can vary widely depending on the type of waste assessed and the geographic location. Differential decay characteristics of bacterial targets and their associated DNA contribute to challenges in interpreting MST results in the context of human health risks. The HF183 marker, derived from the 16S rRNA gene of Bacteroides dorei and closely related taxa, has been used for almost two decades in MST studies, and is well characterized regarding host sensitivity and specificity, and in prevalence and concentration in sewage in many countries. Other markers such as HumM2 and HumM3 show promise, but require further performance testing to demonstrate their widespread utility. An important limitation of the one-marker-one-assay approach commonly used for MST is that given the complexities of microbial persistence in environmental waters, and the methodological challenges of quantitative PCR (qPCR) in such samples, the absence of a given marker does not ensure the absence of fecal pollution in the source water. Approaches under development, such as microarray and community analysis, have the potential to improve MST practices, thereby increasing our ability to protect human and ecosystem health.
For stormwater harvesting to achieve its full potential in mitigating water scarcity problems and restoring stream health, it is necessary to evaluate the human and environmental health risks and benefits associated with it. Stormwater harbors large amounts of pollutants and has traditionally been viewed as a leading cause of water-quality degradation of receiving waters. Harvesting stormwater for household use raises questions of human exposure to pollutants, especially human pathogens, which have the potential to cause large-scale disease outbreaks. These issues are compounded by uncertainties relating to the performance of stormwater treatment technologies in pathogen removal. Quantitative microbial risk assessment provides an objective risk estimate based on scientific data and the best assumptions, which can be used to educate and instil confidence in stakeholders of the practice. Although limited, human health risk studies have positively supported the use of minimally treated rainwater and stormwater for some non-potable applications. In addition to the well-known benefit of preserving the stream hydrology and ecology, wetlands used for harvesting stormwater can also provide new habitats for wildlife that benefit environmental health. A fundamental change from viewing stormwater as waste to resource requires the coordinated efforts in research, education, and effective communication.For further resources related to this article, please visit the WIREs website.
Pathogenic human viruses cause over half of gastroenteritis cases associated with recreational water use worldwide. They are relatively difficult to concentrate from environmental waters due to typically low concentrations and their small size. Although rapid enumeration of viruses by quantitative PCR (qPCR) has the potential to greatly improve water quality analysis and risk assessment, the upstream steps of capturing and recovering viruses from environmental water sources along with removing PCR inhibitors from extracted nucleic acids remain formidable barriers to routine use. Here, we compared the efficiency of virus recovery for three rapid methods of concentrating two microbial source tracking (MST) viral markers human adenoviruses (HAdVs) and polyomaviruses (HPyVs) from one liter tap water and river water samples on HA membranes (90 mm diameter). Samples were spiked with raw sewage, and viral adsorption to membranes was promoted by acidification (Method A) or addition of MgCl2 (Methods B and C). Viral nucleic acid was extracted directly from membranes (Method A), or viruses were eluted with NaOH and concentrated by centrifugal ultrafiltration (Methods B and C). No inhibition of qPCR was observed for samples processed by Method A, but inhibition occurred in river samples processed by B and C. Recovery efficiencies of HAdVs and HPyVs were approximately ten-fold greater for Method A (31-78%) than B and C (2.4-12%). Further analysis of membranes from Method B revealed that the majority of viruses were not eluted from the membrane, resulting in poor recovery. The modification of the originally published Method A to include a larger diameter membrane and a nucleic acid extraction kit that could accommodate the membrane resulted in a rapid virus concentration method with good recovery and lack of inhibitory compounds. The frequently-used strategy of viral absorption with added cations (Mg(2+)) and elution with acid were inefficient and more prone to inhibition, and will result in underestimation of the prevalence and concentrations of HAdVs and HPyVs markers in environmental waters.
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The consumption of crops fertilized with human waste represents a potential route of exposure to antibiotic resistant fecal bacteria. The present study evaluated the abundance of bacteria and antibiotic resistance genes using both culture-dependent and molecular methods. Various vegetables (lettuce, carrots, radish and tomatoes) were sown into field plots fertilized inorganically or with Class B biosolids, or with untreated municipal sewage sludge and harvested when of marketable quality. Analysis of viable pathogenic bacteria or antibiotic-resistant coliform bacteria on plate counts did not reveal significant treatment effects of fertilization with Class B biosolids or untreated sewage sludge on the vegetables. Numerous targeted genes associated with antibiotic resistance and mobile genetic elements were detected by PCR in soil and on vegetables at harvest from plots that received no organic amendment. However, in the season of application, vegetables harvested from plots treated with either material carried gene targets not detected in the absence of amendment. Several gene targets evaluated using qPCR were considerably more abundant on vegetables harvested from sewage sludge-treated plots compared to controls in the season of application, whereas vegetables harvested the following year revealed no treatment effect. Overall, results of the present study suggest that producing vegetable crops in ground fertilized with human waste without appropriate delay or pre-treatment will result in an additional burden of antibiotic resistance genes on the harvested crops. Managing human exposure to antibiotic resistance genes carried in human waste must be undertaken through judicious agricultural practice.
To examine the occurrence, hydrologic variability, and seasonal variability of human and bovine viruses in surface water, three stream locations were monitored in the Milwaukee River watershed in Wisconsin, USA, from February 2007 through June 2008. Monitoring sites included an urban subwatershed, a rural subwatershed, and the Milwaukee River at the mouth. To collect samples that characterize variability throughout changing hydrologic periods, a process control system was developed for unattended, large-volume (56-2800L) filtration over extended durations. This system provided flow-weighted mean concentrations during runoff and extended (24-h) low-flow periods. Human viruses and bovine viruses were detected by real-time qPCR in 49% and 41% of samples (n=63), respectively. All human viruses analyzed were detected at least once including adenovirus (40% of samples), GI norovirus (10%), enterovirus (8%), rotavirus (6%), GII norovirus (1.6%) and hepatitis A virus (1.6%). Three of seven bovine viruses analyzed were detected including bovine polyomavirus (32%), bovine rotavirus (19%), and bovine viral diarrhea virus type 1 (5%). Human viruses were present in 63% of runoff samples resulting from precipitation and snowmelt, and 20% of low-flow samples. Maximum human virus concentrations exceeded 300genomiccopies/L. Bovine viruses were present in 46% of runoff samples resulting from precipitation and snowmelt and 14% of low-flow samples. The maximum bovine virus concentration was 11genomiccopies/L. Statistical modeling indicated that stream flow, precipitation, and season explained the variability of human viruses in the watershed, and hydrologic condition (runoff event or low-flow) and season explained the variability of the sum of human and bovine viruses; however, no model was identified that could explain the variability of bovine viruses alone. Understanding the factors that affect virus fate and transport in rivers will aid watershed management for minimizing human exposure and disease transmission.
Human fecal contamination of surface waters and drains is difficult to diagnose. DNA-based and chemical analyses of water samples can be used to specifically quantify human waste contamination, but their expense precludes routine use. We evaluated canine scent tracking, using two dogs trained to respond to the scent of municipal wastewater, as a field approach for surveying human fecal contamination. Fecal indicator bacteria, as well as DNA-based and chemical markers of human waste, were analyzed in waters sampled from canine scent-evaluated sites (urban storm drains and creeks). In the field, the dogs responded positively (70% and 100%) at sites for which sampled waters were then confirmed as contaminated with human waste. When both dogs indicated a negative response, human waste markers were absent. Overall, canine scent tracking appears useful for prioritizing sampling sites for which DNA-based and similarly expensive assays can confirm and quantify human waste contamination.
Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus
Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this
study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and
introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared
in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene
sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the
modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry
designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the
expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR
and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants
in ambient surface waters.
Pepper mild mottle virus (PMMoV) is a plant virus that has been recently proposed as a potential indicator of human fecal
contamination of environmental waters; however, information on its geographical occurrence in surface water is still limited.
We aimed to determine the seasonal and geographic occurrence of PMMoV in drinking water sources all over Japan. Between July
2008 and February 2011, 184 source water samples were collected from 30 drinking water treatment plants (DWTPs); viruses from
1 to 2 liters of each sample were concentrated by using an electronegative membrane, followed by RNA extraction and reverse
transcription. Using quantitative PCR, PMMoV was detected in 140 (76%) samples, with a concentration ranging from 2.03 × 103 to 2.90 × 106 copies/liter. At least one of the samples from 27 DWTPs (n = 4 or 8) was positive for PMMoV; samples from 10 of these DWTPs were always contaminated. There was a significant difference
in the occurrence of PMMoV among geographical regions but not a seasonal difference. PMMoV was frequently detected in samples
that were negative for human enteric virus or Escherichia coli. A phylogenetic analysis based on the partial nucleotide sequences of the PMMoV coat protein gene in 12 water samples from
9 DWTPs indicated that there are genetically diverse PMMoV strains present in drinking water sources in Japan. To our knowledge,
this is the first study to demonstrate the occurrence of PMMoV in environmental waters across wide geographical regions.
Before new, rapid quantitative PCR (qPCR) methods for assessment of recreational water quality and microbial source tracking (MST) can be useful in a regulatory context, an understanding of the ability of the method to detect a DNA target (marker) when the contaminant source has been diluted in environmental waters is needed. This study determined the limits of detection and quantification of the human-associated Bacteroides sp. (HF183) and human polyomavirus (HPyV) qPCR methods for sewage diluted in buffer and in five ambient, Florida water types (estuarine, marine, tannic, lake, and river). HF183 was quantifiable in sewage diluted up to 10(-6) in 500-ml ambient-water samples, but HPyVs were not quantifiable in dilutions of >10(-4). Specificity, which was assessed using fecal composites from dogs, birds, and cattle, was 100% for HPyVs and 81% for HF183. Quantitative microbial risk assessment (QMRA) estimated the possible norovirus levels in sewage and the human health risk at various sewage dilutions. When juxtaposed with the MST marker detection limits, the QMRA analysis revealed that HF183 was detectable when the modeled risk of gastrointestinal (GI) illness was at or below the benchmark of 10 illnesses per 1,000 exposures, but the HPyV method was generally not sensitive enough to detect potential health risks at the 0.01 threshold for frequency of illness. The tradeoff between sensitivity and specificity in the MST methods indicates that HF183 data should be interpreted judiciously, preferably in conjunction with a more host-specific marker, and that better methods of concentrating HPyVs from environmental waters are needed if this method is to be useful in a watershed management or monitoring context.
Fecal indicator bacteria currently employed for microbial water quality management are poor representatives of viruses. Viral water quality indicators have recently been proposed based on the human gut bacteriophage crAssphage and the food virus pepper mild mottle virus (PMMoV) due to their high abundance in sewage and association to human waste. Here, we develop a model relating crAssphage and PMMoV abundance to risk of swimmer illness in a recreational water contaminated with fresh, untreated domestic sewage. This model, entitled QMRAswim, is available via a web-based user interface and is generalizable to any indicator or pathogen. The majority of predicted illnesses from exposure to untreated domestic sewage-contaminated water were attributable to viruses, primarily norovirus. The mean crAssphage and PMMoV concentrations correlating with 30 illnesses per 1000 bathers were 4648 GC/100mL and 5054 GC/100mL, respectively, approximately fifty times their standard detection limit. This study reaffirms the importance of monitoring viral water quality to adequately protect public health, suggests the high potential of both crAssphage and PMMoV for this application, and establishes a basis to relate viral indicator abundance with probability of illness due to viral pathogens.
Escherichia coli (E. coli) is frequently used in assessment and regulation of recreational water quality, but it is a general fecal indicator that provides no information about fecal contamination source. Sewage-associated microorganisms and related marker genes have proven useful for microbial source tracking (MST) applications that link fecal contamination to host sources, but many MST marker genes are carried in taxa not used in regulatory contexts. A more direct connection with regulatory concerns, including human health risk and total maximum daily load (TMDL) assessments, could be accomplished with tools such as the human-associated marker genes of E. coli. We evaluated the performance of E. coli H8, H12, H14, and H24 marker genes for detection of domestic sewage at the isolate level in Florida. E. coli isolates (n = 1,380) from reference fecal and wastewater samples were first tested by binary PCR for the presence of each H marker gene. H8 and H12 were > 90% specific and sensitive for domestic sewage, while H14 and H24 were ≤ 86% specific. Therefore, H8 and H12 quantitative PCR (qPCR) assays were developed to quantify two marker genes at the sample level. Specificity values for the H8 and H12 qPCR assays were 96 and 93%, respectively, while both marker genes showed 100% sensitivity. H12 concentrations were tenfold lower in wastewater than H8 (~6-7 log10 gene copies (GC)/100 mL). H8 concentrations in wastewater and contaminated environmental water samples were correlated with the sewage-associated Bacteroides HF183 marker gene. This study suggests that E. coli H genes, and H8 in particular, can be useful in risk assessment and TMDL development in subtropical waters.
Large financial investments are required to remediate fecal contamination sources in waterways, and accurate results from field studies are crucial to build confidence in MST approaches. Host specificity and sensitivity are two main performance characteristics for consideration when choosing MST assays. Ongoing efforts for marker assay validation will improve interpretation of results and could shed light on patterns of occurrence in nontarget hosts that might explain the underlying drivers of cross-reaction of certain markers. For field applications, caution should be taken to choose appropriate MST marker genes and assays based on available host specificity and sensitivity data and background knowledge of the contaminating sources in the study area. Since many waterborne pathogens are viruses, employing both viral and bacterial markers in investigations could provide insight into contamination dynamics and ecological behavior in the environment. Therefore, combined usage of marker assays is recommended for more accurate and informative sewage contamination detection and fecal source resolution.
Bivalve molluscan shellfish grown in areas impacted by human faecal pollution are at risk of being contaminated with multiple enteric viruses. To minimise the public health risks associated with shellfish consumption, determining the presence of faecal contamination in shellfish and their growing waters is crucial. In this study, we evaluated the use of pepper mild mottle virus (PMMoV) as an indicator of human faecal contamination in oysters, mussels, cockles and shellfish growing waters in New Zealand. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR) the presence, and where applicable, the concentration of PMMoV was determined in faeces from 11 different animal species, influent (untreated) wastewater, shellfish and shellfish growing waters. Non-human faecal samples (from seagull, Canada goose, black swan and dog) were RT-qPCR positive for PMMoV. The faecal source specificity of PMMoV was 0.83 (maximum value of 1) when ‘detected but not quantifiable’ (DNQ) values were used. However, when ‘lower limit of quantification’ (LLOQ) values were used, the specificity increased to 0.92. The PMMoV concentration in influent wastewater (n = 10) ranged from 6.3 - 7.7 log10 genome copies (GC)/L with a mean (± standard deviation) of 7.1 ± 0.5 log10 GC/L. The overall occurrence of PMMoV in shellfish and shellfish growing waters from four different areas was 46/51 (90%) and 29/52 (56%) respectively. Of the cockles collected from an area known to be impacted by effluent wastewater, 14/14 (100%) contained PMMoV concentrations above the LLOQ. In contrast, only 13/37 (35%) shellfish and 6/52 (11.5%) growing water samples collected from three areas with low anthropogenic impact contained PMMoV concentrations above the LLOQ. The high concentration of PMMoV in influent wastewater indicates that PMMoV may be a promising indicator of human faecal contamination. The presence of PMMoV in shellfish and growing waters with a low anthropogenic impact may be of avian origin, and this needs to be considered if using PMMoV for monitoring shellfish and shellfish growing water quality.
Coliphage have been proposed as indicators of fecal contamination in recreational waters because they better reflect the persistence of pathogenic viruses in the environment and through wastewater treatment than traditional fecal indicator bacteria. Herein, we conducted a systematic literature search of peer-reviewed publications to identify coliphage density data (somatic and male-specific, or MSC) in raw wastewater and ambient waters. The literature review inclusion criteria included scope, study quality, and data availability. A non-parametric two-stage bootstrap analysis was used to estimate the coliphage distributions in raw wastewater and account for geographic region and season. Additionally, two statistical methodologies were explored for developing coliphage density distributions in ambient waters, to account for the nondetects in the datasets. In raw wastewater, the analysis resulted in seasonal density distributions of somatic coliphage (SC) (mean 6.5 log10 plaque forming units (PFU)/L; 95% confidence interval (CI): 6.2–6.8) and MSC (mean 5.9 log10 PFU/L; 95% CI: 5.5–6.1). In ambient waters, 49% of MSC samples were nondetects, compared with less than 5% for SC. Overall distributional estimates of ambient densities of coliphage were statistically higher for SC than for MSC (mean 3.4 and 1.0 log10 PFU/L, respectively). Distributions of coliphage in raw wastewater and ambient water will be useful for future microbial risk assessments.
Fecal indicator bacteria, such as Escherichia coli (E.coli) and Enterococcus, have been widely used to indicate the presence of pathogens. However, the suitability of fecal indicator bacteria to represent health risks is still being challenged, particularly in tropical aquatic environments. The objective of this study is to understand the occurrence and prevalence of indicators and pathogens in areas with contrasting land use, as well as to identify the major correlations between indicators, pathogens and environmental parameters. The spatial and temporal variation of indicators and pathogens was studied to examine the distribution patterns for areas with different land use, and the impact of seasonal changes on microbial populations. A total of 234 water samples were sampled for two years from reservoirs and their tributaries, and tested for fecal indicator bacteria, coliphages, human specific markers, pathogenic bacteria and viruses. The prevalence of indicators and pathogens in reservoirs were generally low, while relatively high concentrations were observed in tributaries to varying degrees. Of the enteric viruses, norovirus GII was among the most prevalent and had the highest concentration. Although strong correlations were found between indicators, only relatively weak correlations were found between indicators and pathogens. The results in this study showed that none of the bacteria/phage indicators were universal predictors for pathogens. Inclusion of the alternative indicators, Methanobrevibacter smithii, Bacteroides and human polyomaviruses (HPyVs) to monitoring programs could help to determine whether the fecal source was human. The microbial distribution patterns allow the classification of sampling sites to different clusters and thus, help to identify sites which have poor water quality. This approach will be useful for water quality management to pinpoint factors that influence water quality and help to prioritize sites for restoration of water quality.
There is a growing move toward using the quantitative polymerase chain (qPCR)-based sewage-associated marker genes to assess surface water quality. However, a lack of understanding about the persistence of many sewage-associated markers creates uncertainty for those tasked with investigating microbial water quality. In this study, we investigated the decay of two qPCR FIB [E. coli (EC), and Enterococcus spp. (ENT) 23S rRNA genes] and four sewage-associated microbial source tracking (MST) marker genes [human Bacteroides HF183 16S rRNA, adenovirus (HAdV), and polyomavirus (HPyV), and crAssphage, a recently described bacteriophage in feces], in outdoor mesocosms containing fresh and marine waters and their corresponding sediments. Decay rates of EC 23S rRNA, ENT 23S rRNA and HF183 16S rRNA were significantly (p <0.05) faster than the HAdV, HPyV and crAssphage markers in water samples from all mesocosms. In general, decay rates of bacterial targets were similar in the water columns of the studied mesocosms. Similarly, decay rates of viral targets were also alike in mesocosm water columns in relation to each other. The decay rates of FIB and sewage-associated markers were significantly faster in water samples compared to sediments in all three mesocosms. In the event of resuspension, FIB and marker genes from sediments can potentially recontaminate overlying waters. Thus, care should be taken when interpreting the occurrence of FIB and sewage-associated MST markers in water, which may have originated from sediments. The differential decay of these targets may also influence health outcomes and need to be considered in risk assessment models.
Considerable efforts have been made in recent years in developing novel marker genes for fecal pollution tracking in environmental waters. CrAssphage are recently discovered DNA bacteriophage that are highly abundant in human feces and untreated sewage. In this study, we evaluated the host-sensitivity and -specificity of the newly designed crAssphage qPCR assays (Stachler et al., 2017) CPQ_056 and CPQ_064 (i.e., marker genes) in fecal samples collected from various human and several animal host groups in Australia. We also investigated the utility of these marker genes to detect sewage pollution in an urban recreational lake (i.e., Lake Parramatta) in Sydney, NSW. The mean concentrations of CPQ_056 and CPQ_064 marker genes in untreated sewage were 9.43 ± 0.14 log10 GC/L and 8.91 ± 0.17 log10 GC/L, respectively, 2 to 3 orders of magnitude higher than other sewage-associated viruses used in microbial source tracking studies. Among 177 animal fecal samples tested from 11 species, the host-specificity values for CPQ_056 and CPQ_064 marker genes were 0.95 and 0.93, respectively. Limited cross-reactivity was observed with cat fecal and cattle wastewater samples. Abundance of crAssphage markers were monitored in an urban lake that receives stormwater runoff. The concentrations of both markers were higher (CPQ_056 ranging from 3.40 to 6.04 log10 GC/L and CPQ_064 ranging from 2.90 to 5.47 log10 GC/L) in 20 of 20 (for CPQ_056) and 18 of 20 (for CPQ_064) samples collected after storm events with gauged sewer overflows compared to dry weather event (10 of 10 samples were qPCR negative for the CPQ_056 and 8 of 10 were negative for the CPQ_064 marker genes) suggesting sewage pollution was transported by urban stormwater runoff to Lake Parramatta. The results of the study may provide context for management of sewage pollution from gauged overflow points of the sewerage system in the catchment.
This study investigated the risk of gastrointestinal illness associated with swimming in surface waters with aged sewage contamination. First, a systematic review compiled 333 first order decay rate constants (k) for human norovirus and its surrogates feline calicivirus and murine norovirus, Salmonella, Campylobacter, Escherichia coli O157:H7, Giardia, and Cryptosporidium, and human-associated indicators in surface water. A meta-analysis investigated effects of sunlight, temperature, and water matrix on k. There was a relatively large number of k for bacterial pathogens and some human-associated indicators (n>40), fewer for protozoan and (n=14-22), and few for human norovirus and its Caliciviridae surrogates (n=2-4). Average k ranked: Campylobacter>human-associated markers>Salmonella> E. coli O157:H7 > norovirus and its surrogates >Giardia>Cryptosporidium. Compiled k values were used in a quantitative microbial risk assessment (QMRA) to simulate gastrointestinal illness risk associated with swimming in water with aged sewage contamination. The QMRA used human-associated fecal indicator HF183 as an index for the amount of sewage present and thereby provided insight into how risk relates to HF183 concentrations in surface water. Because exposure to norovirus contributed the majority of risk, and HF183 k is greater than norovirus k, the risk associated with exposure to a fixed HF183 concentration increases with the age of contamination. Swimmer exposure to sewage after it has aged ~3 days results in median risks less than 30/1000. A risk-based water quality threshold for HF183 in surface waters that takes into account uncertainty in contamination age is derived.
Description
The main purpose of this study is to assess the sediment quality and potential ecological risk in marine sediments in Gymea Bay located in south Sydney, Australia. A total of 32 surface sediment samples were collected from the bay. Current track trajectories and velocities have also been measured in the bay. The resultant trace elements were compared with the adverse biological effect values Effect Range Low (ERL) and Effect Range Median (ERM) classifications. The results indicate that the average values of chromium, arsenic, copper, zinc, and lead in surface sediments all reveal low pollution levels and are below ERL and ERM values. The highest concentrations of trace elements were found close to discharge points and in the inner bay, and were linked with high percentages of clay minerals, pyrite and organic matter, which can play a significant role in trapping and accumulating these elements. The lowest concentrations of trace elements were found to be on the shoreline of the bay, which contained high percentages of sand fractions. It is postulated that the fine particles and trace elements are disturbed by currents and tides, then transported and deposited in deeper areas. The current track velocities recorded in Gymea Bay had the capability to transport fine particles and trace element pollution within the bay. As a result, hydrodynamic measurements were able to provide useful information and to help explain the distribution of sedimentary particles and geochemical properties. This may lead to knowledge transfer to other bay systems, including those in remote areas. These activities can be conducted at a low cost, and are therefore also transferrable to developing countries. The advent of portable instruments to measure trace elements in the field has …
Fecal indicator bacteria (FIB) are used to assess fecal pollution levels in surface water and are among the criteria used by regulatory agencies to determine water body impairment status. While FIB provide no information about pollution source, microbial source tracking (MST) does, which contributes to more direct and cost effective remediation efforts. We studied a watershed in Florida managed for wildlife conservation that historically exceeded the state regulatory guideline for fecal coliforms. We measured fecal coliforms, enterococci, a marker gene for avian feces (GFD), and a marker gene for human-associated Bacteroides (HF183) in sediment, vegetation, and water samples collected monthly from six sites over two years to: 1) assess the influence of site, temporal factors, and habitat (sediment, vegetation, and water) on FIB and MST marker concentrations, 2) test for correlations among FIB and MST markers, and 3) determine if avian feces and/or human sewage contributed to FIB levels. Sediment and vegetation had significantly higher concentrations of FIB and GFD compared to water and thus may serve as microbial reservoirs, providing unreliable indications of recent contamination. HF183 concentrations were greatest in water samples but were generally near the assay limit of detection. HF183-positive results were attributed to white-tailed deer (Odocoileus virginianus) feces, which provided a false indication of human sewage in this water body. FIB and GFD were positively correlated while FIB and HF183 were negatively correlated. We demonstrated that birds, not sewage, were the main source of FIB, thus avoiding implementation of a total maximum daily load program (TMDL). Our results demonstrate that the concomitant use of FIB and MST can improve decision-making and provide direction when water bodies are impaired, and provides a strategy for natural source exclusion in water bodies impacted by wild animal feces.
Environmental waters are monitored for fecal pollution to protect public health. Many previously developed human-specific fecal pollution indicators lack adequate sensitivity to be reliably detected in environmental waters or do not correlate well with viral pathogens. Recently, two novel human sewage-associated source tracking qPCR markers were developed based on the bacteriophage crAssphage, CPQ_056 and CPQ_064. These assays are highly human specific, abundant in sewage, and are viral-based, suggesting great promise for environmental application as human fecal pollution indicators. A 30-day sampling study was conducted in an urban stream impacted by combined sewer overflows to evaluate the crAssphage markers' performance in an environmental system. The crAssphage markers were present at concentrations of 4.02-6.04 log10 copies/100 mL throughout the study period, indicating their high abundance and ease of detection in polluted environmental waters. In addition, the crAssphage assays were correlated with rain events, molecular markers for human polyomavirus and HF183, as well as culturable E. coli, enterococci, and somatic coliphage. The CPQ_064 assay correlated strongly to a greater number of biological indicators than the CPQ_056 assay. This study is the first to evaluate both crAssphage qPCR assays in an extended environmental application of crAssphage markers for monitoring of environmental waters. It is also the first study to compare crAssphage marker concentration with other viral-based indicators.
To investigate the deteriorating bacteriological water quality of Hanauma Bay, Hawaii, samples of shoreline water, sand, land runoff, and mongoose and pigeon droppings were analysed for fecal coliforms, E. coli and enterococci. The results indicate that the major sources contributing to periodic high levels of bacteria in the waters of the Bay are contaminants of the beach sand, such as pigeon feces.
Quantitative polymerase chain reaction (qPCR) is now commonly used to detect fecal indicator bacteria (FIB) as well as pathogens in water samples. However, DNA loss during sample processing can cause underestimation of target genes. In this study, we created a sample process control strain (SPC) by genetically engineering a non-pathogenic, Gram-negative bacterium Pseudogulbenkiania sp. strain NH8B. The SPC strain, named NH8B-1D2, has a kanamycin-resistance gene inserted to one of the 23S rRNA genes. To specifically quantify the SPC strain, a new TaqMan qPCR assay was developed. To obtain the relationship between the DNA recovery efficiencies of various pathogens and those of the SPC strain, known amount of E. coli O157:H7, Salmonella Typhimurium, Campylobacter jejuni, or Listeria monocytogenes cells were co-spiked with the SPC strain to environmental water samples. The DNA recovery efficiencies were calculated by comparing the quantity of bacterial cells inoculated to water samples prior to filtration and DNA extraction, and those measured by qPCR. We then obtained the ratios in the recovery efficiencies between pathogens and SPC strain (RRPATH/SPC). The RRPATH/SPCvalues obtained using Oono pond water collected in Japan were used as a pathogen-specific constant to estimate the accurate concentrations of pathogens in water samples collected from Mississippi River in Minnesota. Estimated pathogen concentrations were not significantly different from the inoculated pathogen concentration, suggesting our normalization approach is useful to estimate the accurate concentrations of pathogens in environmental water samples. The qPCR assay targeting the SPC strains and FIB were incorporated into the microfluidic qPCR chip format (PBQ chip ver. 2); therefore, we can simultaneously quantify multiple pathogens, FIB, and the SPC strain in high throughput from many water samples. This new tool can be useful for water quality monitoring and risk assessment.