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Prevalence and abundance of traditional and host-associated fecal indicators in urban estuarine sediments: Potential implications for estuarine water quality monitoring
Abstract
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.
... To overcome the shortcomings of FIB analyses, Microbial Source Tracking (MST) approaches, have been increasingly adopted to deliver more precise information on the sources of faecal contamination in natural environments (Ahmed et al., 2020). 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. ...
... 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). ...
... However, in this drain, as well as several of the other drains experiencing high Enterococci levels during the rainfall event (specifically Drains 4, 5, 6 and 8), significant peaks in the dog faeces marker co-occurred with peaks in the human faecal markers. This indicates that both sewage and dog faeces potentially contribute to the high Enterococci levels observed in stormwater drains during rainfall at Rose Bay, which is a pattern consistent with reports from other coastal environments (Ahmed et al., 2020). We posit that this pattern of concentration in, and near to, the stormwater drains is likely indicative of dog faeces being washed into the stormwater system from the surrounding catchment, rather than significant levels of dog faeces being washed from the beach into the seawater at Rose Bay. ...
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.
... In Australia, studies of sources of contamination in swimming and other recreational water sources have been undertaken across the country. These include beaches during the dry season in Darwin where sewage outfalls were identified as the source of contamination (Neave et al. 2014), sub-tropical Brisbane where bovine (zoonotic) sources were identified (Ahmed et al. 2013), and the cooler Sydney estuary, where avian sources were the dominant animal source (Ahmed et al. 2020). Thus, molecular tools offer a complementary approach to using FIB as standard practice to more accurately assess the human health risks across the Basin. ...
Management of water resources in the Murray-Darling Basin has historically focussed on water security and the allocation of water for users with competing needs. This focus was reflected in the seminal paper on multiple risks to shared water across the basin by the Commonwealth Scientific and Industrial Research Organisation 15 years ago. That paper captured key concerns that were at the forefront for decision-makers, managers and policy-makers who were, at that time, experiencing the early impacts of the Millennium Drought. Water quality, then, was secondary to the issues of water security. Across the following years, new water quality risks have emerged along with a more nuanced understanding of the complex interplay between climate, floodplain/catchment vegetation, hydrology, and water quality. Critically, this improved understanding applies to the systemic shocks of extreme events, such as the 2020 bushfires and hypoxic blackwater events, as well as the variability, duration and volumes of natural and regulated river flows. In this paper, we explore the key water quality issues that currently face the Basin, and reframe water quality as an integral rather than incidental component of the risks to shared water in the Basin, with the associated implications for policy development that this implies.
... Microbial contamination prediction methods prove to be a feasible tool in water quality control [8,9]. Additionally, during the assessment of microbiological pollution sources, the sediment and the beach sand are only sporadically examined, although the data indicate that this habitat could serve as an important reservoir of microorganisms [10][11][12][13]. Moreover, the WHO's recent guidelines on recreational water quality recommend including the microbiological quality control of beach sand in regular monitoring [14]. ...
A comparative study of the two northeastern ports of the Adriatic Sea indicated that the port of Rijeka is microbiologically more loaded than the port of Pula and posing a greater threat to other ports through a potential transfer of pathogens by ballast water. Fecal indicator bacteria, Escherichia coli and intestinal enterococci, were investigated seasonally in 2014-2015 in the ports and during the bathing season monitoring in the two bays where ports are located in 2009-2020. In addition, the indicators and pathogens related to human health were determined in the ports' seawater and sediment. The determined factors contributing to microbiological pollution were higher number of tourists and locals, potential wastewater and ballast water discharge and enclosed port configuration, with high solar radiation and low precipitation reducing the negative effects. Our research points to the necessity of including Clostridium perfringens in monitoring beach sand during the bathing seasons and a wider list of pathogens in port monitoring due to a potential transfer by shipping ballast water.
... Metal ions in water and soil are carried by the runoff and finally deposited in sediments (Muhammad and Ahmad, 2020;Miranda et al., 2021). When the sediment-water interface environment changes, the metal pollution in the sediment may be released and cause a potential risk to the waterbody (Ahmed et al., 2020;Wang et al., 2020aWang et al., , 2020bWang et al., , 2020c. Lake is a pool of material throughout the basin and the lower edge of the entire region. ...
For the first time, background quality guidelines have been developed for lake sediments along the Yangtze River. Evolution Rules of watershed environment in Eastern China were analyzed in 1937~2017. These methods of ¹³⁷Cs and ²¹⁰Pb radionuclide, 75% cumulative frequency, and background method were applied to calculate the sediment geochemical backgrounds (GB). The average GB values of Cu, Zn, Cd, Pb, Cr, total carbon (TC), total nitrogen (TN) and total phosphorus (TP) are 45.14 mg/kg, 86.99 mg/kg, 0.29 mg/kg, 33.71 mg/kg, 110.90 mg/kg, 17.20 mg/g, 1.60 mg/g, and 665.78 mg/kg, respectively. The radionuclide methods indicated that the sediment rate of 34 cm corresponding to 1963 is 0.63 cm yr⁻¹. The risk and accumulation of the sediment metals and nutrients in Yangtze Plain were uncontaminated levels before 1960, raised since 1980, and increased significantly in 2000. The Cd, TC, and TN in lake sediment were at low to moderate pollution, and few lakes are at high pollution. Sediment background values of the plain are different from soil background values in China and Consensus-Based Sediment Quality Guidelines in Europe/America. Results of sediment quality guidelines provide an important guidance for pollution prevention, environmental management, and risk assessment, especially the formulation of environmental laws.
Viral indicators of human-fecal contamination in wastewaters and environmental waters have been getting much attention in the past decade. Cross-assembly phage (crAssphage) is the most abundant DNA virus in human feces. Recently, the usefulness of crAssphage as a microbial source tracking and water quality monitoring tool for human-fecal contamination has been highlighted. Here, we conducted a comprehensive review on crAssphage in water, focusing on detection methodology, concentration range in various waters and wastewaters, specificity to human-fecal contamination, and reduction in wastewater treatment systems. This review highlights that crAssphage is globally distributed in wastewaters and various fecal-contaminated water bodies at high concentrations without seasonal fluctuations. CrAssphage is highly specific to human-fecal contamination and is rarely found in animal feces. It also has a good potential as a performance indicator to ensure virus reduction in wastewater treatment systems. Accordingly, crAssphage could be an effective tool for monitoring of human-fecal contamination and potential presence of fecal pathogenic microbes in environmental waters. Bridging the research gaps highlighted in this review would make crAssphage a powerful tool to support the control of water-related health risks.
Agricultural soil is highly susceptible to manure contamination and thus is a potential source for the spread of pathogens and antibiotic resistance genes (ARGs). Routine monitoring fecal contamination in agricultural soil can reduce the manure-derived ARG contaminations. This study investigated the distribution of crAssphage, a highly human-specific indicator of fecal pollution, in agricultural soils in the Yangtze River Delta (YRD) of China, and its potential in serving as an indicator of soil ARGs. CrAssphage was indeed strongly correlated with the abundance of soil ARGs, and particularly tetracycline resistance gene tetW (rho = 0.55, p < 0.01). Meanwhile, with the increasing of crAssphage abundance, the frequency of multiple abundant ARGs is also increased. When the relative abundance of crAssphage in soil samples exceeded 4.94 × 10–4 copies per copy of the 16S rRNA gene, there would be more than three types of co-existing ARGs. Regional differences in crAssphage and ARGs abundances were observed for samples collected from Zhejiang, Shanghai, Jiangsu, and Anhui in the YRD, indicating different levels of fecal pollution therein. High sewage treatment capacity could contribute to the reduce of fecal pollution and the control ARG transmission in agricultural soils.
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.
Taihu Lake is one of the largest freshwater lakes in China, serving as an important source of drinking water; >60% of source water to this lake is provided by the Tiaoxi River. This river faces serious fecal contamination issues, and therefore, a comprehensive investigation to identify the sources of fecal contamination was carried out and is presented here. The performance of existing universal (BacUni and GenBac), human (HF183-Taqman, HF183-SYBR, BacHum, and Hum2), swine (Pig-2-Bac), ruminant (BacCow), and avian (AV4143 and GFD) associated microbial source tracking (MST) markers was evaluated prior to their application in this region. The specificity and sensitivity results indicated that BacUni, HF183-TaqMan, Pig-2-Bac, and GFD assays are the most suitable in identifying human and animal fecal contamination. Therefore, these markers along with marker genes specific to selected bacterial pathogens were quantified in water and sediment samples of the Tiaoxi River, collected from 15 locations over three seasons during 2014 and 2015. Total/universal Bacteroidales markers were detected in all water and sediment samples (mean concentration 6.22 log10 gene copies/100 ml and 6.11 log10 gene copies/gram, respectively), however, the detection of host-associated MST markers varied. Human and avian markers were the most frequently detected in water samples (97 and 89%, respectively), whereas in sediment samples, only human-associated markers were detected more often (86%) than swine (64%) and avian (8.8%) markers. The results indicate that several locations in the Tiaoxi River are heavily polluted by fecal contamination and this correlated well with land use patterns. Among the five bacterial pathogens tested, Shigella spp. and Campylobacter jejuni were the most frequently detected pathogens in water (60% and 62%, respectively) and sediment samples (91% and 53%, respectively). Shiga toxin-producing Escherichia coli (STEC) and pathogenic Leptospira spp. were less frequently detected in water samples (55% and 33%, respectively) and sediment samples (51% and 13%, respectively), whereas E. coli O157:H7 was only detected in sediment samples (11%). Overall, the higher prevalence and concentrations of Campylobacter jejuni, Shigella spp., and STEC, along with the MST marker detection at a number of locations in the Tiaoxi River, indicates poor water quality and a significant human health risk associated with this watercourse. GRAPHICAL ABSTRACT Tracking fecal contamination and pathogens in watersheds using molecular methods. Tracking fecal contamination and pathogens in watersheds using molecular methods.
The discharge of human-derived wastewater represents a major threat to water quality with the potential for waterborne disease outbreaks mainly associated with enteric viruses. To prevent illnesses, indicators associated with fecal contamination are monitored in polluted areas, however, their prevalence often does not correlate well with viral pathogens. In this study, we used crAssphage, a recently discovered human-specific gut-associated bacteriophage, for the surveillance of wastewater-derived viral contamination. Untreated and treated wastewater, surface water, sediment and mussel samples were collected monthly over 1 year from the Conwy River and estuary (UK) and were analyzed for crAssphage marker by quantitative PCR. This is the first long-term catchment-to-coast scale study of environmental crAssphage concentrations. CrAssphage was detected in all sample types and showed no distinct seasonal pattern. CrAssphage concentrations were 2 × 10⁵–10⁹ genome copies (gc)/L in all untreated wastewater influent and 10⁷–10⁸ gc/L in secondary treated effluent samples, 3 × 10³ gc/L–3 × 10⁷ gc/L in surface water samples (94% positive) and 2 × 10²–10⁴ gc/g sediment (68% positive) and mussel digestive tissue (79% positive). CrAssphage concentrations were 1–5 log10 higher than human enteric virus titers (norovirus, sapovirus, adenovirus, polyomavirus). Our results indicate that crAssphage is well suited to tracking human wastewater contamination and pollution risk assessment in aquatic environments.
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The HF183 marker gene, derived from the 16S rRNA gene of Bacteroides dorei, has been widely used to identify sewage pollution in environmental waters. CrAssphages are recently discovered DNA bacteriophages that are highly abundant in untreated sewage and have shown promises for tracking sewage contamination in environmental waters. In this paper, we report the development of a duplex quantitative PCR (qPCR) assay for simultaneous quantification of HF183 and crAssphage CPQ_056 marker genes in untreated sewage and sewage impacted stormwater. Same primer and probe sequences were used in the duplex qPCR assay as used in published simplex qPCR assays. The performance characteristics of the duplex qPCR assay were similar to its simplex counterparts. We validated the performance of the duplex assay in a collaborative laboratory study with the aim to evaluate reproducibility, sensitivity and concordance for field study. The concordance values between the simplex vs. duplex qPCR assays for HF183 and crAssphage CPQ_056 marker genes ranged from 96.7 to 100% and the mean concentrations of HF183 and CPQ_056 in environmental water samples were remarkably similar or in some cases slightly greater for the duplex qPCR assay suggesting the reliability of this assay for monitoring HF183 and CPQ_056 simultaneously. The newly developed duplex qPCR assay will be a valuable addition to the MST toolbox for sewage pollution monitoring and would allow rapid and comparative sample analysis.
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.
To assess fecal pollution in coastal waters, current monitoring is reliant on culture-based enumeration of bacterial indicators, which does not account for the presence of viable but non-culturable or sediment-associated micro-organisms, preventing effective quantitative microbial risk assessment (QMRA). Seasonal variability in viable but non-culturable or sediment-associated bacteria challenge the use of fecal indicator organisms (FIOs) for water monitoring. We evaluated seasonal changes in FIOs and human enteric pathogen abundance in water and sediments from the Ribble and Conwy estuaries in the UK. Sediments possessed greater bacterial abundance than the overlying water column, however, key pathogenic species (Shigella spp., Campylobacter jejuni, Salmonella spp., hepatitis A virus, hepatitis E virus and norovirus GI and GII) were not detected in sediments. Salmonella was detected in low levels in the Conwy water in spring/summer and norovirus GII was detected in the Ribble water in winter. The abundance of E. coli and Enterococcus spp. quantified by culture-based methods, rarely matched the abundance of these species when measured by qPCR. The discrepancy between these methods was greatest in winter at both estuaries, due to low CFU's, coupled with higher gene copies (GC). Temperature accounted for 60% the variability in bacterial abundance in water in autumn, whilst in winter salinity explained 15% of the variance. Relationships between bacterial indicators/pathogens and physicochemical variables were inconsistent in sediments, no single indicator adequately described occurrence of all bacterial indicators/pathogens. However, important variables included grain size, porosity, clay content and concentrations of Zn, K, and Al. Sediments with greater organic matter content and lower porosity harbored a greater proportion of non-culturable bacteria (including dead cells and extracellular DNA) in winter. Here, we show the link between physicochemical variables and season which govern culturability of human enteric pathogens and FIOs. Therefore, knowledge of these factors is critical for accurate microbial risk assessment. Future water quality management strategies could be improved through monitoring sediment-associated bacteria and non-culturable bacteria. This could facilitate source apportionment of human enteric pathogens and FIOs and direct remedial action to improve water quality.
Humans can be exposed to pathogens from poorly managed animal feces, particularly in communities where animals live in proximity to humans. This systematic review of peer-reviewed and grey literature examines the human health impacts of exposure to poorly managed animal feces transmitted via water, sanitation, and hygiene (WASH)-related pathways in low- and middle-income countries where household livestock, small-scale animal operations, and free-roaming animals are common. We identify routes of contamination by animal feces, control measures to reduce human exposure, and propose research priorities for further inquiry. Exposure to animal feces has been associated with diarrhea, soil-transmitted helminth infection, trachoma, environmental enteric dysfunction, and growth faltering. Few studies have evaluated control measures, but interventions include reducing cohabitation with animals, provision of animal feces scoops, controlling animal movement, creating safe child spaces, improving veterinary care, and hygiene promotion. Future research should evaluate: behaviors related to points of contact with animal feces; animal fecal contamination of food; cultural behaviors of animal fecal management; acute and chronic health risks associated with exposure to animal feces; and factors influencing concentrations and shedding rates of pathogens originating from animal feces.
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.
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 date, the microbiological quality of river sediments and its impact on water resources are not included in the water quality monitoring assessment. Therefore, the aim of this study was to establish genetic relatedness between faecal coliforms and enterococci isolated from the river water and riverbed sediments of Apies River to better understand the genetic similarity of microorganisms between the sediment and water phases. Indicator bacteria were subjected to a molecular study, which consisted of PCR amplification and sequence analysis of the 16S rRNA and 23S rRNA gene using specific primers for faecal coliforms and enterococci, respectively. Results revealed that the Apies River had high faecal pollution levels with enterococci showing low to moderate correlation coefficient (r² values ranged from 0.2605 to 0.7499) compared to the faecal coliforms which showed zero to low correlation (r² values ranged from 0.0027 to 0.1407) indicating that enterococci may be better indicator than faecal coliforms for detecting faecal contamination in riverbed sediments. The phylogenetic tree of faecal coliforms revealed a 98% homology among their nucleotide sequences confirming the close genetic relatedness between river water and riverbed sediment isolates. The phylogenetic tree of the enterococci showed that Enterococcus faecalis and Enterococcus faecium are the predominant species found in both river water and riverbed sediments with bootstrap values of ≥99%. A high degree of genetic relatedness between sediment and water isolates indicated a possible common ancestry and transmission pathway. We recommend the microbial monitoring of riverbed sediments as it harbours more diverse microbial community and once resuspended may cause health and environmental problems.
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A seasonal study on the occurrence of six opportunistic premise plumbing pathogens (OPPPs) in 24 roof-harvested rainwater (RHRW) tanks repeatedly sampled over six monthly sampling events (n = 144) from August 2015 to March 2016 was conducted using quantitative qPCR. Fecal indicator bacteria (FIB) Escherichia coli (E. coli) and Enterococcus spp. were enumerated using culture-based methods. All tank water samples over the six events were positive for at least one OPPP (Legionella spp., Legionella pneumophila, Mycobacterium avium, Mycobacterium intracellulare, Pseudmonas aeruginosa, or Acanthamoeba spp.) during the entire course of the study. FIB were positively but weakly correlated with P. aeruginosa (E. coli vs. P. aeruginosa τ = 0.090, p = 0.027; Enterococcus spp. vs. P. aeruginosa τ = 0.126, p = 0.002), but not the other OPPPs. FIBs were more prevalent during the wet season than the dry season, and L. pneumophila was only observed during the wet season. However, concentrations of Legionella spp., M. intracellulare, Acanthamoeba spp., and M. avium peaked during the dry season. Correlations were assessed between FIB and OPPPs with meteorological variables, and it was determined that P. aeruginosa was the only OPPP positively associated with an increased antecedent dry period, suggesting stagnation time may play a role for the occurrence of this OPPP in tank water. Infection risks may exceed commonly cited benchmarks for uses reported in the rainwater usage survey such as pool top-up, and warrant further exploration through quantitative microbial risk assessment (QMRA).
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.
Given the high numbers of deaths and the debilitating nature of diseases caused by the use of unclean water it is imperative that we have an understanding of the factors that control the dispersion of water borne pathogens and their respective indicators. This is all the more important in developing countries where significant proportions of the population often have little or no access to clean drinking water supplies. Moreover, and notwithstanding the importance of these bacteria in terms of public health, at present little work exists on the persistence, transfer and proliferation of these pathogens and their respective indicator organisms, e.g., fecal indicator bacteria (FIB) such as Escherichia coli and fecal coliforms in humid tropical systems, such as are found in South East Asia or in the tropical regions of Africa. Both FIB and the waterborne pathogens they are supposed to indicate are particularly susceptible to shifts in water flow and quality and the predicted increases in rainfall and floods due to climate change will only exacerbate the problems of contamination. This will be furthermore compounded by the increasing urbanization and agricultural intensification that developing regions are experiencing. Therefore, recognizing and understanding the link between human activities, natural process and microbial functioning and their ultimate impacts on human health are prerequisites for reducing the risks to the exposed populations. Most of the existing work in tropical systems has been based on the application of temperate indicator organisms, models and mechanisms regardless of their applicability or appropriateness for tropical environments. Here, we present a short review on the factors that control FIB dynamics in temperate systems and discuss their applicability to tropical environments. We then highlight some of the knowledge gaps in order to stimulate future research in this field in the tropics.
Tide-induced residual circulation and
dispersion in Sydney Harbour has been studied with a vertically integrated,
two-dimensional, primitive equation model. The tide-induced residual
circulation consists of a series of recirculating gyres that are due to the
interaction of the tidal current with the complex coastal geometry and
bathymetry of the harbour. Inclusion of the S2 tide and
homogeneous wind stress in the input forcing does not significantly change the
residual circulation pattern. Residuals are produced mainly by the
M2 tide and there is no significant nonlinear
interaction between the M2 and the
S2 tide. Tracer simulations show that tidal mixing is
limited in the vicinity of the entrance and the flushing rates of different
segments of the harbour vary significantly. Lack of similarity between the
Eulerian and Lagrangian residual fields demonstrates that the net displacement
of material released during a particular phase of the tide has little
connection with the mean current observed at fixed locations and is extremely
sensitive to the timing and location of release.
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.
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.
Groundwater is routinely analyzed for fecal indicators but direct comparisons of fecal indicators to the presence of bacterial and viral pathogens are rare. This study was conducted in rural Bangladesh where the human population density is high, sanitation is poor, and groundwater pumped from shallow tubewells is often contaminated with fecal bacteria. Five indicator microorganisms (E. coli, total coliform, F+RNA coliphage, Bacteroides and human-associated Bacteroides) and various environmental parameters were compared to the direct detection of waterborne pathogens by quantitative PCR in groundwater pumped from 50 tubewells. Rotavirus was detected in groundwater filtrate from the largest proportion of tubewells (40%), followed by Shigella (10%), Vibrio (10%), and pathogenic E. coli (8%). Spearman rank correlations and sensitivity-specificity calculations indicate that some, but not all, combinations of indicators and environmental parameters can predict the presence of pathogens. Culture-dependent fecal indicator bacteria measured on a single date did not predict total bacterial pathogens, but annually averaged monthly measurements of culturable E. coli did improve prediction for total bacterial pathogens. A qPCR-based E. coli assay was the best indicator for the bacterial pathogens. F+RNA coliphage were neither correlated nor sufficiently sensitive towards rotavirus, but were predictive of bacterial pathogens. Since groundwater cannot be excluded as a significant source of diarrheal disease in Bangladesh and neighboring countries with similar characteristics, the need to develop more effective methods for screening tubewells with respect to microbial contamination is necessary.
Avian feces contaminate waterways but contribute fewer human pathogens than human sources. Rapid identification and quantification
of avian contamination would therefore be useful to prevent overestimation of human health risk. We used subtractive hybridization
of PCR-amplified gull fecal 16S RNA genes to identify avian-specific fecal rRNA gene sequences. The subtracters were rRNA
genes amplified from human, dog, cat, cow, and pig feces. Recovered sequences were related to Enterobacteriaceae (47%), Helicobacter (26%), Catellicoccus (11%), Fusobacterium (11%), and Campylobacter (5%). Three PCR assays, designated GFB, GFC, and GFD, were based on recovered sequence fragments. Quantitative PCR assays
for GFC and GFD were developed using SYBR green. GFC detected down to 0.1 mg gull feces/100 ml (corresponding to 2 gull enterococci
most probable number [MPN]/100 ml). GFD detected down to 0.1 mg chicken feces/100 ml (corresponding to 13 Escherichia coli MPN/100 ml). GFB and GFC were 97% and 94% specific to gulls, respectively. GFC cross-reacted with 35% of sheep samples but
occurred at about 100,000 times lower concentrations in sheep. GFD was 100% avian specific and occurred in gulls, geese, chickens,
and ducks. In the United States, Canada, and New Zealand, the three markers differed in their geographic distributions but
were found across the range tested. These assays detected four important bird groups contributing to fecal contamination of
waterways: gulls, geese, ducks, and chickens. Marker distributions across North America and in New Zealand suggest that they
will have broad applicability in other parts of the world as well.
Escherichia coli is naturally present in the intestinal tracts of warm-blooded animals. Since E. coli is released into the environment through deposition of fecal material, this bacterium is widely used as an indicator of fecal contamination of waterways. Recently, research efforts have been directed towards the identification of potential sources of fecal contamination impacting waterways and beaches. This is often referred to as microbial source tracking. However, recent studies have reported that E. coli can become "naturalized" to soil, sand, sediments, and algae in tropical, subtropical, and temperate environments. This phenomenon raises issues concerning the continued use of this bacterium as an indicator of fecal contamination. In this review, we discuss the relationship between E. coli and fecal pollution and the use of this bacterium as an indicator of fecal contamination in freshwater systems. We also discuss recent studies showing that E. coli can become an active member of natural microbial communities in the environment, and how this bacterium is being used for microbial source tracking. We also discuss the impact of environmentally-"naturalized" E. coli populations on water quality.
Recreational water quality is currently monitored using culture-based methods that require 18 to 96 h for results. Quantitative
PCR (QPCR) methods that can be completed in less than 2 h have been developed, but they could yield different results than
the conventional methods. We present two studies in which samples were processed simultaneously for Enterococcus spp. and Escherichia coli using two culture-based methods (EPA method 1600 and Enterolert/Colilert-18) and QPCR. The proprietary QPCR assays targeted
the 23S rRNA (Enterococcus spp.) and uidA (E. coli) genes and were conducted using lyophilized beads containing all reagents. In the first study, the QPCR method developers
processed 54 blind samples that were inoculated with sewage or pure cultures or were ambient beach samples. The second study
involved 163 samples processed by water quality personnel. The correlation between results of QPCR and EPA 1600 during the
first study (r2) was 0.69 for Enterococcus spp., which was less than that observed between the culture-based methods (r2, 0.87). During the second study, the correlations were similar. No false positives occurred in either study when QPCR-based
assays were used with blank samples. Levels of reproducibility measured through coefficients of variation were similar for
results by Enterococcus QPCR and culture-based methods during both studies but were higher for E. coli QPCR results in the first study. Regarding the concentration at which beach management decisions are issued in the State
of California, the agreement between results of Enterococcus QPCR and EPA method 1600 was 88%, compared to 94% agreement between EPA method 1600 and Enterolert. The beach management
decision agreement between E. coli QPCR and Colilert-18 was 94%. The samples showing disagreement suggested an underestimation bias for QPCR.
Despite the recognized potential of long-term survival or even growth of fecal indicators bacteria (FIB) in marine sediments,
this compartment is largely ignored by health protection authorities. We conducted a large-scale study over approximately
50 km of the Marche coasts (Adriatic Sea) at depths ranging from 2 to 5 m. Total and fecal coliforms (FC) were counted by
culture-based methods. Escherichia coli was also quantified using fluorescence in situ hybridization targeting specific 16S rRNA sequences, which yielded significantly higher abundances than culture-based methods,
suggesting the potential importance of viable but nonculturable E. coli cells. Fecal coliforms displayed high abundances at most sites and showed a prevalence of E. coli. FC isolates (n = 113) were identified by API 20E, additional biochemical tests, and internal transcribed spacer-PCR. E. coli strains, representing 96% of isolates, were then characterized for genomic relatedness and phylogenetic group (A, B1, B2,
and D) of origin by randomly amplified polymorphic DNA and multiplex-PCR. The results indicated that E. coli displayed a wide genotypic diversity, also among isolates from the same station, and that 44 of the 109 E. coli isolates belonged to groups B2 and D. Further characterization of B2 and D isolates for the presence of 11 virulence factor
genes (pap, sfa/foc, afa, eaeA, ibeA, traT, hlyA, stx1, stx2, aer, and fyuA) showed that 90% of B2 and 65% of D isolates were positive for at least one of these. Most of the variance of both E. coli abundance and assemblage composition (>62%) was explained by a combination of physical-chemical and trophic variables. These
findings indicate that coastal sediments could represent a potential reservoir for commensal and pathogenic E. coli and that E. coli distribution in marine coastal sediments largely depends upon the physical and trophic status of the sediment. We conclude
that future sampling designs aimed at monitoring the microbiological quality of marine coastal areas should not further neglect
the analysis of the sediment and that monitoring of these environments can be improved by including molecular methods as a
complement of culture-based techniques.
When exposed extreme environmental conditions such as sea water, bacteria have been shown different survival strategy for continue their life. One of this strategy known as viable but nonculturable (VBNC) state which is very important for nondifferiation bacteria. VBNC cells cause serious human health problems. Little is known, however, about the genetic mechanisms underlying the VBNC state. Under different environmental conditions, porins are important in the survival strategy of bacteria. EnvZ/OmpR work together as regulators of ompF and ompC gene expression. It is known that the EnvZ system has a role in VBNC state. In this study we tried to find out the viability of EnvZ, OmpC and OmpF mutant E. coli under stress effect of osmolarity, pH and starvation. Bacteria were suspended in filtered-autoclaved sea water microcosms and numbers determined over 25 day incubation periods by plate count (PC), direct viable count (DVC) and count of cells capable of respiration (RCC). As regard to results, alkaline pH affected E. coli more than acidic pH, which led to decline in number. On the contrary glycine betaine addition to sea water protected E. coli porin mutants and also reduced the death rate of bacteria. Under the effect of pH, osmotic stress and starvation stress, wild type E. coli and porin mutants entered a dormant state or became VBNC with the exception of MSZ31 (envZ mutant) E. coli cells which did not enter the VBNC state under the three tested stress conditions. This study is the first report to demonstrate that E. coli could not enter the VBNC state in the lack of EnvZ product under the stress of osmolarity, pH and starvation and the relationship between EnvZ and VBNC state are not affected by pH, osmolarity and starvation.
Bacteria in the water-saturated sand of a relatively unpolluted sand beach were enumerated by direct microscope and viable counting. The number of interstitial bacteria was estimated to be a significant fraction of the total number of bacteria present. Three hundred sixty-two strains were isolated and submitted to cultural and biochemical tests. Fermentational abilities and the production of indole suggested that a significant number of these bacteria were symbiotically associated with resident metazoans.
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.
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.
A prospective matched case‐control study was conducted to evaluate associations between dietary histories, including consumption of bivalves, diarrhoea, and norovirus positive diarrhoea in adult ambulatory patients at an outpatient clinic of a hospital in Tokyo, Japan. Ambulatory cases with diarrhoea were matched with non‐diarrheal control patients, who visited the same clinic. A standardized questionnaire was used to obtain patients' information, including histories of food consumption and clinical information. Norovirus infection was confirmed using real‐time reverse transcription polymerase chain reaction (real‐time RT‐PCR). A total of 207 patients, including 69 diarrheal cases and 138 non‐diarrheal cases were included in the analysis. Among them, 60 (29.0%) participants reported consuming bivalves. Norovirus was detected in 35% (24/69) of diarrheal cases. Of those, 10 (41.7%) reported consumption of bivalves and of those, 6 (60.0%) consumed raw bivalves. The proportion of those who consumed raw bivalves was significantly higher in norovirus‐positive diarrheal cases than in norovirus‐negative diarrheal cases [25.0% vs. 6.7%, Odds ratio (OR) 4.67 (95% Confidence Interval (CI): 1.1－20.7)] and matched non‐diarrheal controls [25.0% vs. 6.3%, OR: 5.00 (95% CI: 1.1－22.2)]. The attributable fraction of consuming raw bivalves for norovirus‐associated diarrhoea to matched non‐diarrheal controls was 20.0%. Consuming raw bivalves was substantially attributed to norovirus‐associated diarrhoea in adult ambulatory patients and preventive measures for reducing the risk associated with consumption of raw bivalves could decrease the incidence of norovirus‐associated diarrhoea.
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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.
Estuarine and coastal waterways are commonly monitored for fecal and sewage contamination to protect recreator health and ecosystem functions. Such monitoring programs commonly rely on cultivation-based counts of fecal indicator bacteria (FIB) in water column samples. Recent studies demonstrate that sediments and beach sands can be heavily colonized by FIB, and that settling and resuspension of colonized particles may significantly influence the distribution of FIB in the water column. However, measurements of sediment FIB are rarely incorporated into monitoring programs, and geographic surveys of sediment FIB are uncommon. In this study, the distribution of FIB and the extent of benthic-pelagic FIB coupling were examined in the urbanized, lower Hudson River Estuary. Using cultivation-based enumeration, two commonly-measured FIB, enterococci and Escherichia coli, were widely distributed in both sediment and water, and were positively correlated with each other. The taxonomic identity of FIB isolates from water and sediment was confirmed by DNA sequencing. The geometric mean of FIB concentration in sediment was correlated with both the geometric mean of FIB in water samples from the same locations and with sediment organic carbon. These two positive associations likely reflect water as the FIB source for underlying sediments, and longer FIB persistence in the sediments compared to the water, respectively. The relative representation of other fecal associated bacterial genera in sediment, determined by 16S rRNA gene sequencing, increased with the sequence representation of the two FIB, supporting the value of these FIB for assessing sediment contamination. Experimental resuspension of sediment increased shoreline water column FIB concentrations, which may explain why shoreline water samples had higher average FIB concentrations than samples collected nearby but further from shore. In combination, these results demonstrate extensive benthic-pelagic coupling of FIB in an urbanized estuary and highlight the importance of sediment FIB distribution and ecology when interpreting water quality monitoring data.
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.
Fecal contamination of recreational waters with cattle manure can pose a risk to public health due to the potential presence of various zoonotic pathogens. Fecal indicator bacteria (FIB) have a long history of use in the assessment of recreational water quality, but FIB quantification provides no information about pollution sources. Microbial source tracking (MST) markers have been developed in response to a need to identify pollution sources, yet factors that influence their decay in ambient waters are often poorly understood. We investigated the influence of water type (freshwater versus marine) and select environmental parameters (indigenous microbiota, ambient sunlight) on the decay of FIB and MST markers originating from cattle manure. Experiments were conducted in situ using a submersible aquatic mesocosm containing dialysis bags filled with a mixture of cattle manure and ambient water. Culturable FIB (E. coli, enterococci) were enumerated by membrane filtration and general fecal indicator bacteria (GenBac3, Entero1a, EC23S857) and MST markers (Rum2Bac, CowM2, CowM3) were estimated by qPCR. Water type was the most significant factor influencing decay (three-way ANOVA, p: 0.006 to <0.001), although the magnitude of the effect differed among microbial targets and over time. The presence of indigenous microbiota and exposure to sunlight were significantly correlated (three-way ANOVA, p: 0.044 to <0.001) with decay of enterococci and CowM2, while E. coli, EC23S857, Rum2Bac, and CowM3 (three-way ANOVA, p: 0.044 < 0.001) were significantly impacted by sunlight or indigenous microbiota. Results indicate extended persistence of both cultivated FIB and genetic markers in marine and freshwater water types. Findings suggest that multiple environmental stressors are important determinants of FIB and MST marker persistence, but their magnitude can vary across indicators. Selective exclusion of natural aquatic microbiota and/or sunlight typically resulted in extended survival, but the effect was minor and limited to select microbial targets.
CrAssphage are recently-discovered DNA bacteriophages that are prevalent and abundant in human feces and sewage. We assessed the performance characteristics of a crAssphage quantitative PCR (qPCR) assay for quantifying sewage impacts in stormwater and surface water in subtropical Tampa, Florida. The mean concentrations of crAssphage in untreated sewage ranged from 9.08 to 9.98 log10 gene copies/L. Specificity was 0.927 against 83 non-human fecal reference samples and the sensitivity was 1.0. Cross-reactivity was observed in DNA extracted from soiled poultry litter but the concentrations were substantially lower than untreated sewage. The presence of the crAssphage marker was monitored in water samples from storm drain outfalls during dry and wet weather conditions in Tampa, Florida. In dry weather conditions, 41.6% of storm drain outfalls samples were positive for the crAssphage marker and the concentrations ranged from 3.60 to 4.65 log10 gene copies/L of water. After a significant rain event, 66.6% of stormwater outlet samples were positive for the crAssphage marker and the concentration ranged from 3.62 to 4.91 log10 gene copies/L of water. The presence of the most commonly used Bacteroides HF183 marker in storm drain outfalls was also tested along with the crAssphage. Thirteen samples (55%) were either positive (i.e., both markers were present) or negative (i.e., both markers were absent) for both the markers. Due to the observed cross-reactivity of this marker with DNA extracted from poultry litter samples, it is recommended that this marker should be used in conjunction with additional markers such as HF183. Our data indicate that the crAssphage marker is highly sensitive to sewage, is adequately specific, and will be a valuable addition to the MST toolbox.
The microbiological quality of recreational waters (RW) has been assessed through the use of microbial indicators. Usually, three typical fecal bacteria, namely fecal coliforms, Escherichia coli and Enterococcus spp., are deemed by legislation and regulatory agencies for routine monitoring of the fecal contamination in RW. However, these traditional indicators have some limitations, as they underestimate health risks and do not determine the sources of contamination. Currently, the increasing concern with the minimization of human health risks associated with recreational water use and the protection of water resources outweigh the actual normative perspective. The potentials and limitations of conventional and alternative fecal contamination detection systems must be carefully evaluated, taking into account the specificities of each recreational water body and the natural or anthropogenic sources of environmental variability. The careful choice of analysis for detection and quantification of indicators in the light of their potential and shortcomings is of major importance for the timely evaluation of risks and implementation of strategies that effectively protect users and preserve water quality. This paper presents a critical literature review of the traditional and innovative methodologies for the analysis of fecal indicators in recreational water and intends to discuss their contribution for the management of the microbiological quality and safety of leisure surface waters.
It has been known for some time that substantial populations of
fecal coliforms and E. coli are harbored in freshwater bottom sediments,
bank soils, and beach sands. However, the relative importance
of sediments as bacterial habitats and as a source of waterborne
fecal coliforms and E. coli has not been recognized until
recently, when a large number of publications have shown that in
many cases the resuspension of sediment, rather then runoff from
surrounding lands, can create elevated E. coli concentrations in
water. This review is an attempt to develop the first comprehensive
single source of existing information about fecal coliforms and
E. coli in sediments and adjacent soils and to outline the knowledge
gaps and research needs. The authors summarize available information
on variability and environmental correlations of E. coli
and FC concentrations in sediments, genetic diversity of E. coli in
sediments, survival of E. coli and FC in sediments, release with
resuspended sediment and settling of E. coli and FC, modeling of
sediment effects on fate and transport of E. coli in surface waters,
and implications for monitoring and management of microbiological
water quality. The demonstrated role of pathogenic E. coli
strains in food and water quality challenges reinforces the need
in better understanding ecological and hydrological factors that
affect functioning of sediments as E. coli reservoirs.
Bed sediments of streams and rivers may store high concentrations of fecal indicator bacteria (FIB) and pathogens. Due to resuspension events, these contaminants can be mobilized into the water column and affect overall water quality. Other bacteria indicators such as microbial source tracking (MST) markers, developed to determine potential sources of fecal contamination, can also be resuspended from bed sediments. The primary objective of this study was to predict occurrence of waterborne pathogens in water and streambed sediments using a simple statistical model that includes traditionally measured FIB, environmental parameters and source allocation, using MST markers as predictor variables. Synoptic sampling events were conducted during baseflow conditions downstream from agricultural (AG), forested (FORS), and waste water pollution control plant (WPCP) land uses. Concentrations of FIB and MST markers were measured in water and sediments, along with occurrences of the enteric pathogens Campylobacter, Listeria and Salmonella, and the virulence gene that carries Shiga toxin, stx2. Pathogens were detected in water more often than in underlying sediments. Shiga toxin was significantly related to land use, with concentrations of the ruminant marker selected as an independent variable that could correctly classify 76% and 64% of observed Shiga toxin occurrences in water and sediment, respectively. FIB concentrations and water quality parameters were also selected as independent variables that correctly classified Shiga toxin occurrences in water and sediment (54%–87%), and Salmonella occurrences in water (96%). Relationships between pathogens and indicator variables were generally inconsistent and no single indicator adequately described occurrence of all pathogens. Because of inconsistent relationships between individual pathogens and FIB/MST markers, incorporating a combination of FIB, water quality measurements, and MST markers may be the best way to assess microbial water quality in mixed land use systems.
Limited clean water supplies in urbanizing watersheds create challenges for safely sustaining irrigated agriculture and global food security. On-farm interventions, such as riverbank filtration (RBF), are used in developing countries to treat irrigation water from rivers with extensive fecal contamination. Using a Bayesian approach incorporating ethnographic data and pathogen measurements, quantitative microbial risk assessment (QMRA) methods were employed to assess the impact of RBF on consumer health burdens for Giardia, Cryptosporidium, rotavirus, norovirus, and adenovirus infections resulting from indirect wastewater reuse, with lettuce irrigation in Bolivia as a model system. Concentrations of microbial source tracking markers for pepper mild mottle virus and HF183 Bacteroides were respectively 2.9 and 5.5 log10 units lower in RBF-treated water than in the river water. Consumption of lettuce irrigated with river water caused an estimated median health burden that represents 37% of Bolivia's overall diarrheal disease burden, but RBF resulted in an estimated health burden that is only 1.1% of this overall diarrheal disease burden. Variability and uncertainty associated with environmental and cultural factors affecting exposure correlated more with QMRA-predicted health outcomes than factors related to disease vulnerability. Policies governing simple on-farm interventions like RBF can be intermediary solutions for communities in urbanizing watersheds that currently lack wastewater treatment.
Avian fecal droppings may negatively impact environmental water quality due to the presence of high concentrations of fecal indicator bacteria (FIB) and zoonotic pathogens. This study was aimed at evaluating the performance characteristics and utility of a Helicobacter spp. associated GFD marker by screening 265 fecal and wastewater samples from a range of avian and non-avian host groups from two continents (Brisbane, Australia and Florida, USA). The host-prevalence and -specificity of this marker among fecal and wastewater samples tested from Brisbane were 0.58 and 0.94 (maximum value of 1.00). These values for the Florida fecal samples were 0.30 (host-prevalence) and 1.00 (host-specificity). The concentrations of the GFD markers in avian and non-avian fecal nucleic acid samples were measured at a test concentration of 10 ng of nucleic acid at Brisbane and Florida laboratories using the quantitative PCR (qPCR) assay. The mean concentrations of the GFD marker in avian fecal nucleic acid samples (5.2 × 10(3) gene copies) were two orders of magnitude higher than non-avian fecal nucleic acid samples (8.6 × 10(1) gene copies). The utility of this marker was evaluated by testing water samples from the Brisbane River, Brisbane and a freshwater creek in Florida. Among the 18 water samples tested from the Brisbane River, 83% (n = 18) were positive for the GFD marker, and the concentrations ranged from 6.0 × 10(1)-3.2 × 10(2) gene copies per 100 mL water. In all, 92% (n = 25) water samples from the freshwater creek in Florida were also positive for the GFD marker with concentrations ranging from 2.8 × 10(1)-1.3 × 10(4) gene copies per 100 mL water. Based on the results, it can be concluded that the GFD marker is highly specific to avian host groups, and could be used as a reliable marker to detect the presence and amount of avian fecal pollution in environmental waters.
Both microbial metabolism and pathogen retention and remobilization are dependent on downstream transport of fine particles, which migrate in a series of deposition and resuspension events. All fine particles, including clay minerals, particulate organic carbon, nutrients and microbes, are often considered to be transported similarly in the environment because of a lack of specific observations comparing their relative transport. We conducted a tracer injection study to compare the transport and retention of the fecal indicator bacterium Escherichia coli, synthetic inert fluorescent fine particles, and a dissolved conservative tracer. We found that the fluorescent fine particles and bacteria were transported similarly, with both having greater retention than the solute tracer. We used a stochastic model to evaluate in-stream retention and migration of the solute, fluorescent particles, and E. coli. The best-fit model parameters indicate that different stream reaches had varied retention characteristics, but always showed greater retention of fluorescent particles and E. coli compared to the solute tracer. Direct measurements within known retention areas after the injection showed that the majority of the fluorescent particles and E. coli were retained near the sediment-water interface in macrophyte stands or filtered within the top 3 cm of the streambed sediment. Both the tracer particles and E. coli were retained within these regions for multiple months following the injection experiment. The stochastic model properly captured the wide range of storage timescales and processes we observed in the stream. Our results demonstrate the importance of the streambed sediment and in-stream macrophytes as short- and long-term reservoirs for fine organic particles and microbes in streams.
In this study, a tracer bacteria was used to investigate the resuspension and persistence of sediment-associated bacteria in a small alluvial stream. The study was conducted in Swan Creek, located within the Grand River watershed of Ontario, Canada. A 1.1-m² section of the bed was seeded with a strain of Escherichia coli resistant to nalidixic acid (E. coli NAR). The survival, transport, and redistribution of the tracer bacteria within a 1.7-km river section downstream of the source cell was assessed for a 2-mo period following the introduction of the tracer bacteria. This study has illustrated that enteric bacteria can survive in bed sediments for up 6 wk and that inactivation of the tracer bacteria resembled typical first-order decay. Critical conditions for resuspension, as well as resuspension rates, of sediment-associated bacteria were determined for several storm events. The critical shear stress for E. coli NAR resuspension in Swan Creek ranged from 1.5 to 1.7 N m⁻², which is comparable with literature values for critical shear stresses for erosion of cohesive sediments. Bacteria resuspension was primarily limited to the rising limb of storm hydrographs implying that a finite supply of sediment-associated bacteria are available for resuspension during individual storm events. The information presented in this paper will further the development of representative microbial water quality models.
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Microbial source tracking (MST) describes a suite of methods and an investigative strategy for determination of fecal pollution sources in environmental waters that rely on the association of certain fecal microorganisms with a particular host. MST is used to assess recreational water quality and associated human health risk, and total maximum daily load (TMDL) allocations. Many methods rely on signature molecules (markers) such as DNA sequences of host-associated microbes. Human sewage pollution is among the greatest concerns for human health due to 1) the known risk of exposure to human waste, and 2) the public and regulatory will to reduce sewage pollution; however, methods to identify animal sources are receiving increasing attention as our understanding of zoonotic disease potential improves. Here, we review the performance of MST methods in initial reports and field studies, with particular emphasis on quantitative PCR (qPCR). Relationships among human-associated MST markers, fecal indicator bacteria, pathogens, and human health outcomes are presented along with recommendations for future research. An integrated understanding of the advantages and drawbacks of the many MST methods targeting human sources advanced over the past several decades will benefit managers, regulators, researchers, and other users of this rapidly growing area of environmental microbiology. This article is protected by copyright. All rights reserved.
Water, sand, and sediment from a Lake Superior harbor site continuously receiving wastewater effluent was sampled monthly for June to October 2010 and from May to September 2011. Understanding the dynamics of genetic markers of fecal bacteria in these matrices is essential to accurately characterizing health risks. Genetic markers for enterococci, total Bacteroides, and human-associated Bacteroides were measured in site-water, sand, and sediment and in final effluent by quantitative PCR. The similarity between the quantity of molecular markers in the water column and effluent indicated that the abundance of genetic markers in the water column was likely controlled by effluent inputs. Effluent turbidity was positively correlated (p ≤ 0.05) with AllBac and HF183 in final effluent and AllBac in the water column. In sand and sediment, Entero1 and AllBac were most abundant in the upper 1- 3 cm depths, whereas HF183 was most abundant in the upper 1 cm of sand and at 7 cm in sediment. The AllBac and Entero1 markers were 1- and 2-orders of magnitude more abundant in sand and sediment relative to the water column per unit mass. These results indicate that sand and sediment may act as reservoirs for genetic markers of fecal pollution at some freshwater sites.
A laboratory‐based microcosm study was conducted to assess the survival of Enterococci faecalis in the estuarine sediments of Lake Pontchartrain, Louisiana, USA. The survival curves of E. faecalis were characterized by a growth phase followed by a stationary phase, a logarithmic decay period, and/or finally by a tailing region. The usual exponential decay model could not be applied to E. faecalis survival in the estuarine sediments. The estuarine sediments appeared to stimulate E. faecalis reproduction within 10 days of incubation. Furthermore, the estuarine sediments prolonged the survival of E. faecalis for a period of at least a month under the study conditions. Based on research results, the amount of organic matter and nutrients did not significantly affect the survival of E. faecalis in the estuarine sediments. This outcome may have occurred due to the small sample size. Throughout the duration of the 45 day experiment, relatively the same proportion of E. faecalis remained culturable. Therefor
The US EPA is currently investigating the use of quantitative PCR (qPCR) analysis techniques to estimate densities of fecal
indicator bacteria (FIB) in recreational waters. Present water quality guidelines, based on culturable FIB, prevent same day
water quality determination, whereas results from qPCR-based approaches are available within several hours. Epidemiological
studies at Publicly-Owned Treatment Works (POTW)-impacted freshwater beaches have also indicated correlations between qPCR
determined Enterococcus densities and swimming-related illness rates. Similar qPCR assays are now available for several other accepted or emerging
FIB groups. This study provides an initial assessment of qPCR estimated Enterococcus, Bacteroidales, E.coli and Clostridium spp. densities in marine water and sand samples collected over one summer from two POTW-impacted recreational beaches. Relative
target sequence densities of these organisms in the samples did not correspond with their relative estimated cell densities.
These observations were attributable to differences in target sequences recovered from the calibrator cells of the different
types of organisms. Comparative cycle threshold (CT) qPCR analyses of whole cell calibrator samples provide a simple and standardizable
approach for estimating both total cell and target sequence densities of different types of FIB in water. Cell density estimates
obtained by this approach are subject to uncertainty due to potential variability in absolute numbers of target sequences
in the target organisms under different physiological or environmental conditions, but still may allow for informative comparisons
with the target sequence estimates.
Intertidal sediment samples from three EU recognised bathing waters in Morecambe Bay, Lancashire, U.K. (Morecambe North, Morecambe South and Heysham) were analysed for thermophilic campylobacters, salmonellae, faecal coliforms and faecal streptococci over a 12-month period. Campylobacters show a strong seasonality. They are generally absent in the summer months but are consistently present in the winter. The main Campylobacter species isolated were C. lari and urease-positive thermophilic campylobacters (UPTC), both of which are associated with avian hosts. C. jejuni and C. coli and Salmonella were not isolated from the sediments. Faecal coliforms and faecal streptococci were isolated throughout the year with no obvious seasonal trends in their numbers. Higher numbers were found at Morecambe North and South than at Heysham. There was no discernible relationship between the numbers of campylobacters and faecal indicators. Faecal indicators were found predominantly in the surface layers of the sediments and declined in number with depth. Campylobacters were restricted to the surface layer. Experimental results for the in situ deposition of bacteria onto clean, sterile surfaces from the water column during tidal cover showed deposition rates equivalent to approximately 0.1% of the total population of faecal coliforms, 0.01% of the faecal streptococci and 1% of the campylobacters in the sediments. These rates of accretion were not high enough to be detected during routine sampling carried out before and after high water, using standard methods. The results show that the sediments act as a reservoir for bacteria, especially the faecal indicators. In rough weather resuspension can contribute significantly to bacterial numbers in the surface waters.
Elevated concentrations of fecal indicator bacteria (FIB) in aquatic sediments and vegetation have prompted concern that environmental reservoirs of FIB disrupt the correlation between indicator organisms, pathogens and human health risks. FIB numbers, however, are typically normalized to volume of water or mass of substrate. Because these reservoirs tend to differ greatly in magnitude within and between water bodies, direct comparison between water column and benthic population sizes can be problematic. Normalization to a set volume of water or mass of substrate, e.g. cfu (100 ml)(-1) or cfu(100 g)(-1), can give a false picture of the relative contributions of various reservoirs to FIB numbers across the ecosystem, and of the potential for FIBs to trigger health advisories as they pass from one reservoir to another. Here, we normalized enterococci concentrations from water, sediment and submerged aquatic vegetation (SAV) to land surface area (m(2) ) to compare their relative importance in the entire system. SAV-associated enterococci comprised only 0-18% of the entire population, even though they displayed the highest concentrations of enterococci per unit mass. The largest proportion of the enterococci population was in the water column (4-77%) or sediments (20-95%), depending on the volume of each substrate available at a site and FIB concentrations within them. Models indicated that large shifts in the relative size of FIB populations in each substrate can result from changes in per cent SAV cover, water depth and depth of sediment colonization. It follows that high concentrations of FIB in sediments or SAV do not necessarily signify large environmental reservoirs of FIB that can affect the water column. Comprehensive analyses that include FIB measurements from water, SAV and sediment normalized to land surface area offer a more balanced perspective on total FIB numbers contained in various matrices of an aquatic system.
To develop a quantitative, real-time PCR assay to detect the nifH gene of Methanobrevibacter smithii. Methanobrevibacter smithii is a methanogenic archaea found in the intestinal tract of humans that may be a useful indicator of sewage pollution in water.
Quantification standards were prepared from Meth. smithii genomic DNA dilutions, and a standard curve was used to quantify the target gene and calculate estimated genome equivalency units. A competitive internal positive control was designed and incorporated into the assay to assess inhibition in environmental extracts. Testing the assay against a panel of 23 closely related methanogen species demonstrated specificity of the assay for Meth. smithii. A set of 36 blind water samples was then used as a field test of the assay. The internal control identified varying levels of inhibition in 29 of 36 (81%) samples, and the Meth. smithii target was detected in all water samples with known sewage input.
The quantitative PCR assay developed in this study is a sensitive and rapid method for the detection of the Meth. smithii nifH gene that includes an internal control to assess inhibition. Further research is required both to better evaluate host specificity of this assay and the correlation with human health risks.
This research is the first description of the development of a rapid and sensitive quantitative assay for a methanogenic archaeal indicator of sewage pollution.
Molecular methods for quantifying defined Bacteroidales species from the human gastrointestinal tract may have important clinical and environmental applications, ranging from diagnosis of infections to fecal source tracking in surface waters. In this study, sequences from the V2 region of the small subunit ribosomal RNA gene were targeted in the development of qPCR assays to quantify DNA from six Bacteroides and one Prevotella species. In silico and experimental analyses suggested that each of the assays was highly discriminatory in detecting DNA from the intended species. Analytical sensitivity, precision and ranges of quantification were demonstrated for each assay by coefficients of variation of less than 2% for cycle threshold measurements over a range from 10 to 4×10(4) target sequence copies. The assays were applied to assess the occurrence and relative abundance of their target sequences in feces from humans and five animal groups as well as in 14 sewage samples from 13 different treatment facilities. Sequences from each of the species were detected at high levels (>10(3)copies/ng total extracted DNA) in human wastes. Sequences were also detected by each assay in all sewage samples and, with exception of the Prevotella sequences, showed highly correlated (R(2)≥0.7) variations in concentrations between samples. In contrast, the occurrence and relative abundance profiles of these sequences differed substantially in the fecal samples from each of the animal groups. These results suggest that analyses for multiple individual Bacteroidales species may be useful in identifying human fecal pollution in environmental waters.
The use of enterococci as the primary fecal indicator bacteria (FIB) for the determination of recreational water safety has been questioned, particularly in sub/tropical marine waters without known point sources of sewage. Alternative FIB (such as the Bacteroidales group) and alternative measurement methods (such as rapid molecular testing) have been proposed to supplement or replace current marine water quality testing methods which require culturing enterococci. Moreover, environmental parameters have also been proposed to supplement current monitoring programs. The objective of this study was to evaluate the health risks to humans from exposure to subtropical recreational marine waters with no known point source. The study reported symptoms between one set of human subjects randomly assigned to marine water exposure with intensive environmental monitoring compared with other subjects who did not have exposure. In addition, illness outcomes among the exposed bathers were compared to levels of traditional and alternative FIB (as measured by culture-based and molecular-based methods), and compared to easily measured environmental parameters. Results demonstrated an increase in self-reported gastrointestinal, respiratory and skin illnesses among bathers vs. non-bathers. Among the bathers, a dose-response relationship by logistic regression modeling was observed for skin illness, where illness was positively related to enterococci enumeration by membrane filtration (odds ratio = 1.46 [95% confidence interval = 0.97-2.21] per increasing log10 unit of enterococci exposure) and positively related to 24 h antecedent rain fall (1.04 [1.01-1.07] per increasing millimeters of rain). Acute febrile respiratory illness was inversely related to water temperature (0.74 [0.56-0.98] per increasing degree of water temperature). There were no significant dose-response relationships between report of human illness and any of the other FIB or environmental measures. Therefore, for non-point source subtropical recreational marine waters, this study suggests that humans may be at increased risk of reported illness, and that the currently recommended and investigational FIB may not track gastrointestinal illness under these conditions; the relationship between other human illness and environmental measures is less clear.
Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments.
The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement.
Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.