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Agreement on the percentage of positive/negative co-occurrence among the Bacteroides HF183, crAssphage CPQ_056 and pepper mild mottle virus (PMMoV) marker genes in water samples collected from Lake Parramatta during dry and wet weather events.
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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...
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... were highly prevalent (95-100%), whereas the prevalence of the PMMoV marker gene was relatively lower (80%). The agreements (co-presence or co-absence) between the occurrence of sewage-associated marker genes in samples collected during dry and storm events were high, ranging from 80-90% (for dry weather) and 75-95% (for storm events) events (Fig. 2). During the two storm events combined, 75% of samples were positive for all three markers, whereas, 95% (HF183 and CPQ_056), 85% (HF183 and PMMoV) and 75% (CPQ_056 and PMMoV) were positive for at least two marker ...
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Clam Beach is located in Northern California, USA, and is listed as an impaired waterway by the federal government. The scope of this study was to investigate this beach and surrounding watershed to determine, if possible, the source of the impairment by conducting an 11-h beach study and 8-week watershed study. We used traditional fecal indicator...
Abstract
Faecal indicator bacteria (FIB) are used for the assessment of faecal pollution and possible water quality deterioration. There is growing evidence that FIB used in temperate regions are not adequate and reliable to detect faecal pollution in tropical regions. Hence, this study evaluated the adequacy of FIB, including total coliforms (TC),...
Faecal indicator bacteria (FIB) are used for the assessment of faecal pollution and possible water quality deterioration. There is growing evidence that FIB used in temperate regions are not adequate and reliable to detect faecal pollution in tropical regions. Hence, this study evaluated the adequacy of FIB, including total coliforms (TC), Escheric...
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 wild...
Fecal indicator bacteria (FIB) (e.g., fecal coliforms, Escherichia coli, and enterococci) have been used for decades to monitor for and protect the public from waterborne pathogens from fecal contamination. However, FIB may not perform well at predicting the presence of waterborne pathogens or human health outcomes from recreational exposure to fec...
Citations
... Following fresh sewage pollution episodes, PMMoV correlates well with bacterial fecal markers such as Bacteroides HF183 in sewage wastewaters, and E. coli and enterococci in sewage waters [214]. In surface waters affected by sewage, PMMoV was present more frequently and in higher concentrations than adenovirus, polyomavirus and norovirus. ...
Global water scarcity has led to significant dependence on reclaimed or recycled water for potable uses. Effluents arising from human and animal gut microbiomes highly influence water quality. Wastewater pollution is, therefore, frequently monitored using bacterial indicators (BI). However, threats to public health arise from the frequent incidence of wastewater-mediated viral infections–undetected by BI. Moreover, the enteric viromes contaminating wastewater are characterized by high abundance, genetic diversity and persistence in various water environments. Furthermore, humans usually suffer a minimum of a single acute diarrheal episode over their lifetime arising from extraneously acquired enteric microbiomes. A wide range of management methods are employed—in particular, microbial source tracking (MST) approaches to confront infections arising from exposure to contaminated wastewater. This review elaborates the viral contamination of treated wastewater and associated public health issues. Latterly, we discuss the various management strategies of wastewater pollution using conventional fecal indicators, viral indicators and human viral surrogates, with particular interest in the pepper mild mottle virus (PMMoV). Globally, PMMoV has been detected in rivers, aquifers, irrigation systems, and coastal and marine waters at high prevalence rates and concentrations greater than 105 genome copies per liter (gc/L). PMMoV was also found in almost all untreated wastewater environments. PMMoV concentrations in wastewater vary from 103 to 107 gc/L. These values are more than the maximum recorded viral indicator concentrations in wastewater for other proposed indicators. Limited variability in the daily concentrations of PMMoV in fecal wastewater has been studied, with an estimated average concentration of 105 gc/L with insignificant seasonal variability. The information summarized in this article offers fundamental knowledge for decision making in terms of defining the suitability criteria of candidate fecal indicators, risk assessment application and efficient wastewater management.
... 16S rRNA genes, Enterococcus esp gene, and Lachnospiraceae Lachno3 marker have been used as indicators for human fecal contamination [36][37][38][39]. There are also MST markers derived from human enteric viruses, such as adenovirus (AdV), polyomavirus (PyV), norovirus (NoV), and pepper mild mottle virus (PMMoV), which have been used to link pollution with human and/or domestic WW [40,41]. Recently, a novel human bacteriophage marker, crAssphage, has shown great potential for pinpointing anthropogenic/sewage-associated pollution [42][43][44]. ...
... This study emphasized the importance of applying MST for the routine and rapid resolution of variable fecal pollution sources across a vast spatial range, enabling the prioritized remediation of disparate sources. Urban and suburban lakes (e.g., Lake Parramatta in Sydney, Australia, and Lake Carroll in Florida, USA) were found to be vulnerable to human fecal pollution, as revealed by investigations using human-and sewage-associated MST markers, e.g., HF183, crAssphage, Enterococcus faecium esp gene, and human PyV and PMMoV [40,42,59]. Moreover, these recreational lakes were found to be directly fecally polluted by both anthropogenic and zoogenic sources, humans (e.g., swimmers and beach visitors) and animals (e.g., dogs and gulls), respectively. ...
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.
... MST typically employs molecular microbiological methods, such as quantitative PCR (qPCR) (Feng et al., 2018;Green et al., 2014aGreen et al., , 2014bGreen et al., , 2012Templar et al., 2016), and more recently DNA sequencing approaches (Brumfield et al., 2021;Newton et al., 2013a), to quantify specific microbial marker genes. Whilst one potential caveat of DNA-based approaches is that they may sometimes detect a signal from unviable cells, potentially leading to over-estimates of impact, they have continuously been able to unambiguously identify sources of faecal contamination within an environment (Ahmed et al., 2020(Ahmed et al., , 2019Alm et al., 2018;Green et al., 2019;Li et al., 2021b;Shrestha et al., 2020). ...
Urbanised beaches are regularly impacted by faecal pollution, but management actions to resolve the causes of contamination are often obfuscated by the inability of standard Faecal Indicator Bacteria (FIB) analyses to discriminate sources of faecal material or detect other microbial hazards, including antibiotic resistance genes (ARGs). We aimed to determine the causes, spatial extent, and point sources of faecal contamination within Rose Bay, a highly urbanised beach within Sydney, Australia's largest city, using molecular microbiological approaches. Sampling was performed across a network of transects originating at 9 stormwater drains located on Rose Bay beach over the course of a significant (67.5 mm) rainfall event, whereby samples were taken 6 days prior to any rain, on the day of initial rainfall (3.8mm), three days later after 43mm of rain and then four days after any rain. Quantitative PCR (qPCR) was used to target marker genes from bacteria (i.e., Lachnospiraceae and Bacteroides) that have been demonstrated to be specific to human faeces (sewage), along with gene sequences from Heliobacter and Bacteriodes that are specific to bird and dog faeces respectively, and ARGs (sulI, tetA, qnrS, dfrA1 and vanB). 16S rRNA gene amplicon sequencing was also used to discriminate microbial signatures of faecal contamination. Prior to the rain event, low FIB levels (mean: 2.4 CFU/100ml) were accompanied by generally low levels of the human and animal faecal markers, with the exception of one transect, potentially indicative of a dry weather sewage leak . Following 43 mm of rain, levels of both human faecal markers increased significantly in stormwater drain and seawater samples, with highest levels of these markers pinpointing several stormwater drains as sources of sewage contamination. During this time, sewage contamination was observed up to 1000 m from shore and was significantly and positively correlated with often highly elevated levels of the ARGs dfrA1, qnrS, sulI and vanB. Significantly elevated levels of the dog faecal marker in stormwater drains at this time also indicated that rainfall led to increased input of dog faecal material from the surrounding catchment. Using 16S rRNA gene amplicon sequencing, several indicator taxa for stormwater contamination such as Arcobacter spp. and Comamonadaceae spp. were identified and the Bayesian SourceTracker tool was used to model the relative impact of specific stormwater drains on the surrounding environment, revealing a heterogeneous contribution of discrete stormwater drains during different periods of the rainfall event, with the microbial signature of one particular drain contributing up to 50% of bacterial community in the seawater directly adjacent. By applying a suite of molecular microbiological approaches, we have precisely pinpointed the causes and point-sources of faecal contamination and other associated microbiological hazards (e.g., ARGs) at an urbanised beach, which has helped to identify the most suitable locations for targeted management of water quality at the beach.
... Later technologies employed species specific markers (e.g. human specific HF183) to directly attribute pollution sources, providing an advantage over fecal indicator organisms assessments alone (Harwood et al. 2014;Ahmed et al. 2019). Human-associated methods for viruses, such as the novel bacteriophage crAssphage, have also been targeted as another human-associated method for environmental water quality testing (Stachler et al. 2018;Korajkic et al. 2020), and the EPA has recently approved two standardized quantitative polymerase chain reaction (qPCR) methods for the characterization of human fecal pollution in water (Methods 1696 and 1697), which have advanced the standardization of MST methods. ...
... identify sewage contamination of surface waters (55,62). HF183 levels of 6.31 Â 10 5 to 6.15 Â 10 6 GC/100 mL have been measured in sewage diluted 100-fold (1%) (63)(64)(65) which is within the range of 1.80 Â 10 3 to 6.30 Â 10 7 GC/100 mL observed in moderately to severely impacted surface waters (15,(65)(66)(67)(68). ...
Vibrio vulnificus is a naturally occurring, potentially lethal pathogen found in coastal waters, fish, and shellfish. Sewage spills in coastal waters occur when infrastructure fails due to severe storms or age, and may affect bacterial populations by altering nutrient levels. This study investigated effects of sewage on clonal and natural V. vulnificus populations in microcosms. Addition of 1% sewage to estuarine water caused the density of a pure culture of V. vulnificus CMCP6 and a natural V. vulnificus population to increase significantly, by two to three orders of magnitude, whether measured by quantitative PCR (qPCR) or culture and in batch and continuous cultures. Changes in the transcription of six virulence- and survival-associated genes in response to sewage were assessed using continuous culture. Exposure to sewage affected transcription of genes that may be associated with virulence, i.e., it modulated the oxidative stress response by altering superoxide dismutase transcription, significantly increasing sodB transcription while repressing sodA. Sewage also repressed transcription of nptA, which encodes a sodium-phosphate cotransporter. Sewage had no effect on sodC transcription or the putative virulence-associated genes hupA or wza. The effects of environmentally relevant levels of sewage on V. vulnificus populations and gene transcription suggest that sewage spills that impact warm coastal waters could lead to an increased risk of V. vulnificus infections. IMPORTANCE Vibrio vulnificus infections have profound impacts such as limb amputation and death for individuals with predisposing conditions. The warming climate is contributing to rising V. vulnificus prevalence in waters that were previously too cold to support high levels of the pathogen. Climate change is also expected to increase precipitation in many regions, which puts more pressure on wastewater infrastructure and will result in more frequent sewage spills. The finding that 1% wastewater in estuarine water leads to 100 to over 1,000-fold greater V. vulnificus concentrations suggests that human exposure to oysters and estuarine water could have greater health impacts in the future. Further, wastewater had a significant effect on gene transcription and has the potential to affect virulence during the initial environment-to-host transition.
... This is owing to their inclusive distribution, associated with ever higher densities in sewage, than other detected viruses and the fact they follow a similar pattern to enteric viruses prevalence in different water bodies. Consequently, they are efficient as indicators of virus-mediated fecal pollution in lakes, rivers, and recreational waters [297][298][299][300][301]. Thus, traceability procedures provide a mandate to determine fecal contamination sources so that the risk can be assessed to initiate a proper water management to counteract it at its source. ...
Recycled wastewater is widely used owing to the potential shortage of water resources for drinking purposes, recreational activities, and irrigation. However, gut microbiomes of both human beings and animals negatively affect this water quality. Wastewater contamination is continuously monitored, using fecal contamination indicators or microbial source tracking approaches, to oppose arising enteric infections. Viral gastroenteritis is considered a principal manifestation of waterborne pathogenic virome-mediated infections, which are mainly transmitted via the fecal-oral route. Furthermore, acquired enteric viromes are the common cause of infantile acute diarrhea. Moreover, public exposure to wastewater via wastewater discharge or treated wastewater reuse has led to a significant surge of public health concerns. In this review, we discussed the etiology of waterborne enteric viromes, notably gastrointestinal virus infections, and public exposure to municipal wastewater. Conclusively, the early human virome is affected mainly by birth mode, dietary behavior, and maternal health, and could provide a signature of disease incidence, however, more virome diversification is acquired in adulthood. A multi-phase treatment approach offered an effective means for the elimination of wastewater reuse mediated public risks. The insights highlighted in this paper offer essential information for defining probable etiologies and assessing risks related to exposure to discharged or reused wastewater.
... Sewage treatment plants (STPs) collect sewage from various sources, including households, hospitals, commercial and industrial sites followed by treatment processes to remove biological and chemical contaminants before the treated water is discharged back into the environment or recycled for commercial enterprises. Human and animal fecal waste contamination is a global problem and can occur from wet/dry weather overflows, septic tanks, faulty sewer lines, illicit sewer connections, lift stations, and in the event of natural disasters such as earthquakes and flooding (Olds et al., 2018;Ahmed et al., 2019a). Sewage contamination results in the dissemination of pathogens, nutrients, toxicants, endocrine disruptors, antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) into the environment (Rodriguez-Manzano et al., 2010). ...
... The presence of fecal contamination is typically determined by monitoring fecal indicator bacteria (FIB), such as Escherichia coli for freshwater and enterococci for marine and estuarine waters (Rochelle-Newall et al., 2015;Ahmed et al., 2019a). However, limitations of FIB monitoring include factors such as differential decay rates and poor correlations to pathogens in aquatic environments (Harwood et al., 2005;Signor et al., 2005;Wade et al., 2008;Ferguson et al., 2012;Korajkic et al., 2019), potential environmental, non-fecal sources (Badgley et al., 2011;Byappanahalli et al., 2012), and most importantly, their inability to identify the source of contamination (Harwood et al., 2014). ...
... An experiment was conducted to determine the presence of PCR inhibitors in DNA samples from untreated sewage and water samples collected from the Brisbane River system using a Sketa22 qPCR assay (Haugland et al., 2005). DNA samples with a 2-quantification cycle (Cq) delay were considered to have potential PCR inhibitors (Ahmed et al., 2019a). Samples with PCR inhibitors were subjected to a 10-fold dilution with TE buffer and reanalyzed with the Sketa22 assay. ...
Since sewage is a hotspot for antibiotic resistance genes (ARGs), the identification of ARGs in environmental waters impacted by sewage, and their correlation to fecal indicators, is necessary to implement management strategies. In this study, sewage treatment plant (STP) influent samples were collected and analysed using quantitative polymerase chain reaction (qPCR) to investigate the abundance and correlations between sewage-associated markers (i.e., Bacteroides HF183, Lachnospiraceae Lachno3, crAssphage) and ARGs indicating resistance to nine antibiotics (belonging to aminoglycosides, beta-lactams, class 1 integrase, sulfonamides, macrolides, and tetracycline). All ARGs, except blaVIM, and sewage-associated marker genes were always detected in untreated sewage, and ermF and sul1 were detected in the greatest abundances. Class 1 integrase integron intl1 element was also highly abundant in untreated sewage samples. Significant correlations were identified between sewage-associated marker genes, ARGs and intl1 element in untreated sewage ( = 0.488, p = 0.0125). Of the three sewage-associated marker genes, the BIO-ENV procedure identified that HF183 alone best maximized correlations to ARGs and intl1 element ( = 0.590). Additionally, grab samples were collected from peri-urban and urban sites along the Brisbane River system during base and stormflow conditions, and analysed for E. coli, ARGs, intl1 element, and sewage-associated marker genes using qPCR. Significant correlations were identified between E. coli, ARGs, and intl1 element ( = 0.0893, p = 0.0032), as well as with sewage-associated marker genes in water samples from the Brisbane River system ( = 0.3229, p = 0.0001). Of the sewage-associated marker genes and E. coli, the BIO-ENV procedure identified that crAssphage alone maximized correlations with ARGs and intl1 element in river samples ( = 0.4148). Significant differences in E. coli, ARGs, intl1 element, and sewage-associated marker genes, and by flow condition (i.e., base vs. storm), and site types (peri-urban vs. urban) combined were identified (R = 0.3668, p = 0.0001), where percent dissimilarities between the multi-factorial groups ranged between 20.8% and 11.2%. Results from this study suggest increased levels of certain ARGs and sewage-associated marker genes in stormflow river water samples compared to base flow conditions. E. coli, HF183 and crAssphage may serve as potential indicators of sewage derived ARGs under stormflow conditions, and this merits further investigation. Data presented in this study will be valuable to water quality managers to understand the links between sewage pollution and ARGs in urban environments.
... Therefore, the role of FIB in determining effective management of water quality can significantly improve by employing new methodologies that allow for the discrimination of the sources of these microorganisms. Source tracking (ST) methods have been shown to provide rapid and efficient fecal source discrimination in order to expedite cost-effective remediation actions (Ahmed et al., 2019;Kongprajug et al., 2019). Several ST methods have been tested previously including anaerobic bacteria (Bifidobacterium and Bacteroides), enteric viruses and bacteriophages and mitochondrial DNA (mtDNA). ...
Determining the source of fecal contamination in a water body is important for the application of appropriate remediation measures. However, it has been suggested in the extant literature that this can best be achieved using a ‘toolbox’ of molecular- and culture-based methods. In response, this study deployed three indicators (Escherichia coli (EC), intestinal enterococci (IE) and somatic coliphages (SC)), one culture-dependent human marker (Bacteroides (GB-124) bacteriophage) and five culture-independent markers (human adenovirus (HAdV), human (HMMit), cattle (CWMit), pig (PGMit) and poultry (PLMit) mitochondrial DNA markers (mtDNA)) within the River Tagus catchment (n = 105). Water samples were collected monthly over a 13-month sampling campaign at four sites (impacted by significant specific human and non-human inputs and influenced by differing degrees of marine and freshwater mixing) to determine the dominant fecal inputs and assess geographical, temporal, and meteorological (precipitation, UV, temperature) fluctuations. Our results revealed that all sampling sites were not only highly impacted by fecal contamination but that this contamination originated from human and from a range of agricultural animal sources. HMMit was present in a higher percentage (83%) and concentration (4.20 log GC/100 mL) than HAdV (32%, 2.23 log GC/100 mL) and GB-124 bacteriophage with the latter being detected once. Animal mtDNA markers were detected, with CWMit found in 73% of samples with mean concentration of 3.74 log GC/100 mL. Correlation was found between concentrations of fecal indicators (EC, IE and SC), CWMit and season. Levels of CWMit were found to be related to physico-chemical parameters, such as temperature and UV radiation, possibly as a result of the increasing presence of livestock outside in warmer months. This study provides the first evaluation of such a source-associated ‘toolbox’ for monitoring surface water in Portugal, and the conclusions may inform future implementation of surveillance and remediation strategies for improving water quality.
... Sewage treatment processes are capable of decreasing the concentration of faecal pathogens 14,27,28 . However, studies also showed the public health risk of streams impacted by STP effluent as metagenome analyses showed pathogenic bacteria can escape STP treatment processes [29][30][31][32] . These metagenomic studies found nucleic acid indicators of pathogens such as Bacteroides HF183, Helicobacterspp, E. coli, Enterococci, and Acinetobacter baumannii. ...
The purpose of the sewage treatment process is to decrease the concentration of contaminants, including pathogens, before discharging into the receiving streams. And the standard operating procedure of STP in Malaysia is to discharge the treated wastewater with low nutrient and low organic materials into the streams but the bacterial content of the discharge and its risk to the stream’s natural microbial verity or health is unknown. However, studies reported that pathogens could escape sewage treatment plants (STPs) processes and showed health risk of streams impacted by STP effluent. On the other hand, majority of these studies relied on metagenomic strategy, without assessing changes to culturable bacteria. Isolation of living microbes provides realistic risk assessment compared to metagenome survey alone. Therefore, this study aims to determine the presence of culturable pathogenic bacteria from water impacted by STP effluent to establish justifiable public health risk. For that, the presence of bile resistant bacteria was determined from water taken from surface water receiving effluent from STP-1 (Kolej 9, UTM) in Malaysia. Enumeration and isolation of bacteria were done on MacConkey agar through membrane filtration method, followed by partial identification, using Triple Sugar Iron agar (TSI). The result showed that STP effluent changes the diversity, and abundance, of bile resistant bacteria (specifically Enterobacteriaceae family) of receiving streams. Most of the isolated bile resistant bacteria are opportunistic pathogens for human. Findings from this study provide a snapshot of the bigger picture of microbial changes in a stream impacted by STP effluent painted initially by metagenome studies. And shows that despite of treatment, some contaminants (microbes) remained and released into surface waters, which contribute to the water pollutions.
... Pollution of coastal waters and rivers by sewage or by faecal contamination from non-point animal sources therefore poses a serious public health risk due to ingestion of contaminated water during recreational activities or contamination for of shellfish for human consumption ( Ballesté et al., 2020 ;Bellou et al., 2013 ;Soller et al., 2010 ;Yamahara et al., 2007 ). Human faecal contamination originating from sewerage misconnections, combined sewage overflows or discharges from wastewater treatment plants may occur frequently in urban areas ( Ahmed et al., 2019 ;Kay et al., 2008 ;Panasiuk et al., 2015 ;Reynolds et al., 2020 ). * Corresponding author. ...
The introduction of SARS-CoV-2 containing human stool and sewage into water bodies may raise public health concerns. However, assessment of public health risks by faecally contaminated water is limited by a lack of knowledge regarding the persistence of infectious SARS-CoV-2 in water. In the present study the decay rates of viable infectious SARS-CoV-2 and SARS-CoV-2 RNA were determined in river and seawater at 4 and 20°C. These decay rates were compared to S. typhimurium bacteriophage MS2 and pepper mild mottle virus (PMMoV). Persistence of viable SARS-CoV-2 was temperature dependent, remaining infectious for significantly longer periods of time in both freshwater and seawater at 4°C than at 20°C. T90 for infectious SARS-CoV-2 in river water was 2.3 days and 3.8 days at 20°C and 4°C, respectively. The T90 values were 1.1 days and 2.2 days in seawater at 20°C and 4°C, respectively. In contrast to the rapid inactivation of infectious SARS-CoV-2 in river and sea water, viral RNA was relatively stable. The RNA decay rates were increased in non-sterilised river and seawater, presumably due to the presence of microbiota. The decay rates of infectious MS2, MS2 RNA and PMMoV RNA differed significantly from the decay rate of SARS-CoV-2 RNA, suggesting that their use as surrogate markers for the persistence of SARS-CoV-2 in the environment is limited.
