•PMA could deplete relic DNA signal while preserving viable microbial composition•Absolute quantification could be accurately achieved using cellular spike-ins•High agreement between PMA-enabled ...viable cell absolute quantification and CFU•Improved human health risk assessment by PMA in metagenomics through QMRA•High proportions of relic DNA (avg. 40%) found in beach waters
The majority of the current regulatory practices for routine monitoring of beach water quality rely on the culture-based enumeration of faecal indicator bacteria (FIB) to develop criteria for promoting the general public's health. To address the limitations of culture methods and the arguable reliability of FIB in indicating health risks, we developed a Nanopore metagenomic sequencing-based viable cell absolute quantification workflow to rapidly and accurately estimate a broad range of microbes in beach waters by a combination of propidium monoazide (PMA) and cellular spike-ins. Using the simple synthetic bacterial communities mixed with viable and heat-killed cells, we observed near-complete relic DNA removal by PMA with minimal disturbance to the composition of viable cells, demonstrating the feasibility of PMA treatment in profiling viable cells by Nanopore sequencing. On a simple mock community comprised of 15 prokaryotic species, our results showed high accordance between the expected and estimated concentrations, suggesting the accuracy of our method in absolute quantification. We then further assessed the accuracy of our method for counting viable Escherichia coli and Vibrio spp. in beach waters by comparing to culture-based method, which were also in high agreement. Furthermore, we demonstrated that 1 Gb sequences obtained within 2 h would be sufficient to quantify a species having a concentration of ≥ 10 cells/mL in beach waters. Using our viability-resolved quantification workflow to assess the microbial risk of the beach water, we conducted (1) screening-level quantitative microbial risk assessment (QMRA) to investigate human illness risk and site-specific risk patterns that might guide risk management efforts and (2) metagenomics-based resistome risk assessment to evaluate another layer of risk caused by difficult illness treatment due to antimicrobial resistance (AMR). In summary, our metagenomic workflow for the rapid absolute quantification of viable bacteria demonstrated its great potential in paving new avenues toward holistic microbial risk assessment.
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Faecal pollution of water and the resulting potential presence of human enteric pathogens is a predominant threat to public health. Microbiological water quality can be assessed by the detection of ...standard faecal indicator bacteria (SFIB) such as E. coli or certain Enterococcus species. In recent years, isothermal amplification methods have become a useful alternative to polymerase chain reaction (PCR), allowing molecular diagnostics with simple or no instrumentation. In this study, a novel screening method for the molecular detection of Enterococcus spp. by loop-mediated isothermal amplification (LAMP) is described. A set of six specific LAMP primers was designed to amplify a diagnostic fragment of the Enterococcus 23S rRNA gene, which is present in several enterococcal species targeted by quantitative PCR (qPCR), which is the standard technique recommended by the US Environmental Protection Agency. Sensitivity and specificity tests were performed using a set of 30 Enterococcus and non-target bacterial reference strains. It is shown that LAMP is equally sensitive and even more specific than the qPCR assay. A dilution series of Enterococcus faecalis DNA revealed that the LAMP method can reliably detect 130 DNA target copies per reaction within 45 min. Additionally, enterococci isolated from Austrian surface waterbodies, as well as a set of DNA extracts from environmental waters, were tested. Contingency analysis demonstrated a highly significant correlation between the results of the developed LAMP assay and the reference qPCR method. Furthermore, a simple staining procedure with a fluorescence dye demonstrated the identification of amplified products by eye. In conclusion, this method is an important component for the efficient screening and testing of water samples in low-resource settings lacking sophisticated laboratory equipment and highly trained personnel, requiring only a simple heating block.
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•A rapid method for the molecular detection of enterococci in water is presented.•The developed LAMP assay is equally sensitive and specific as the reference qPCR.•The reaction takes place at a constant temperature provided by a heating block.•Results can be visualised within 1 min.
The microbial faecal pollution of rivers has wide-ranging impacts on a variety of human activities that rely on appropriate river water quality. Thus, detailed knowledge of the extent and origin of ...microbial faecal pollution is crucial for watershed management activities to maintain safe water use. In this study, the microbial faecal pollution levels were monitored by standard faecal indicator bacteria (SFIB) along a 2580 km stretch of the Danube, the world's most international river, as well as the Danube's most important tributaries. To track the origin of faecal pollution, host-associated Bacteroidetes genetic faecal marker qPCR assays for different host groups were applied in concert with SFIB. The spatial resolution analysis was followed by a time resolution analysis of faecal pollution patterns over 1 year at three selected sites. In this way, a comprehensive faecal pollution map of the total length of the Danube was created, combining substantiated information on both the extent and origin of microbial faecal pollution. Within the environmental data matrix for the river, microbial faecal pollution constituted an independent component and did not cluster with any other measured environmental parameters. Generally, midstream samples representatively depicted the microbial pollution levels at the respective river sites. However, at a few, somewhat unexpected sites, high pollution levels occurred in the lateral zones of the river while the midstream zone had good water quality. Human faecal pollution was demonstrated as the primary pollution source along the whole river, while animal faecal pollution was of minor importance. This study demonstrates that the application of host-associated genetic microbial source tracking markers in concert with the traditional concept of microbial faecal pollution monitoring based on SFIB significantly enhances the knowledge of the extent and origin of microbial faecal pollution patterns in large rivers. It constitutes a powerful tool to guide target-oriented water quality management in large river basins.
Longitudinal development of E. coli and Enterococci concentrations along the increasing discharge (grey shaded area) of the Danube in the midstream, at the left (upper line) and the right river-side (lower line) and in the tributaries sampled during JDS 2013. Not-sampled tribtaries are also indiacted on the x-axes. Symbol size varies according to log-transformed E. coli and Enterococci concentrations. Colours depict the faecal pollution levels: blue - little, green - moderate, yellow - crtitical, orange - strong and red excessive pollution. Grey-shaded triangles indicate discharge contributions from the main tributaries. Display omitted
•Faecal pollution patterns along the whole Danube river are shown.•Combination of standard parameters and microbial source tracking markers used.•Dominance of anthropogenic pollution along the whole Danube River.•Midstream samples generally depict microbial faecal pollution levels.•High pollution levels of the lateral zones at specific, partly unexpected sites.
This study looked at the survival, mobilization, and transport of Escherichia coli from sheep faeces over an extended period, which was then repeated for all four seasons.
Rain simulation was used to ...measure E. coli mobilization directly from faecal pats and subsequent transport across a soil surface. The rain simulation experiments were conducted over the same 90-day period and for the four seasons as per the survival experiments conducted to determine E. coli survival in faecal pats. The survival experiments demonstrated up to three orders of magnitude increase of E. coli in sheep faeces. Peak E. coli concentrations in the faeces were observed on Day 2 through to Day 30, with no significant effect of seasons or weather patterns. The E. coli concentrations in the runoff followed the survival pattern of E. coli concentrations in the faeces.
There was a statistically significant relationship between the E. coli concentrations in the faeces and the runoff.
The data generated on the relationship between Escherichia coli concentrations in the faeces and in the runoff can support the development of runoff risk models for sheep grazing pastureland to support catchment modelling, land use decisions, and public health.
Faecal contamination of bivalve molluscan shellfish (BMS) can lead to infections from enteric pathogens if consumed. Across Europe, the faecal indicator bacteria Escherichia coli, is used to ...determine contamination of BMS harvesting areas. The reference most probable number (MPN) method for E. coli in BMS takes around 48 h from sample receipt to result. In this study, an alternative method was developed in which the final, E. coli confirmation step in the MPN method (usually carried out on chromogenic TBX agar) was replaced by presence/absence real-time PCR (qPCR). This qPCR-MPN method was directly compared with the reference TBX-MPN method using 194 BMS samples consisting of mussels (Mytilus spp.), Pacific oysters (Crassostrea gigas) and common cockles (Cerastoderma edule). The qPCR-MPN method correlated positively with the TBX-MPN method (Kendall's tau coefficient = 0.812). However, the strength of this correlation varied between BMS species, with mussels having the poorest correlation (0.677) followed by Pacific oysters (0.795) and common cockles (0.890). There were some samples for which the difference between the two methods was higher than might be expected by statistical probability alone. Variations in the way in which the two confirmation methods work may account for much of this variation. This method may serve as an ad hoc, rapid assessment method that is complementary to the official reference method and could be easily implemented in many official control laboratories.
•Rapid, qPCR-based MPN method for E. coli enumeration in bivalves was developed.•An internal amplification control was developed and was vital for reliable results.•The qPCR-MPN method compares well with the reference MPN method.•Some improvements to the method may be required and are discussed.
Abstract
The contamination of water catchments by nonpoint source faecal pollution is a major issue affecting the microbial quality of receiving waters and is associated with the occurrence of a ...range of enteric illnesses in humans. The potential sources of faecal pollution in surface waters are diverse, including urban sewage leaks, surface runoff and wildlife contamination originating from a range of hosts. The major contributing hosts require identification to allow targeted management of this public health concern. In this study, two high-performing Microbial Source Tracking (MST) assays, HF183/Bac242 and BacCan-UCDmodif, were used for their ability to detect host-specific Bacteroides 16Sr RNA markers for faecal pollution in a 12-month study on an urban coastal lagoon in Sydney, Australia. The lagoon was found to contain year-round high numbers of human and canine faecal markers, as well as faecal indicator bacteria counts, suggesting considerable human and animal faecal pollution. The high sensitivity and specificity of the HF183/Bac242 and BacCan-UCDmodif assays, together with the manageable levels of PCR inhibition and high level DNA extraction efficiency obtained from lagoon water samples make these markers candidates for inclusion in an MST ‘toolbox’ for investigating host origins of faecal pollution in urban surface waters.
Emerging evidence suggests close domestic proximity of livestock and humans may lead to microbiological contamination of hands, objects, food and water supplies within domestic environments, ...adversely impacting public health. However, evidence quantifying the relationship between livestock, domestic animals, humans and microbiological contamination of household stored water remains limited.
This longitudinal study aimed to examine the relationship between domestic contact with livestock and domestic animals on microbiological contamination of household Point-of-Use (POU) stored drinking water in rural Kenya and assess the influence of choice of faecal indicator on such associations.
A survey was performed in 234 households in Siaya county, Kenya, to observe presence of livestock (cattle, goats, poultry) and domestic animals (cats, dogs) in household compounds, alongside other risk factors for contamination of POU stored drinking water such as sanitation, storage conditions and hygiene practices. Samples from water sources (e.g. piped, spring/wells, boreholes, surface and rainwater) and from POU storage containers were tested for E. coli and intestinal enterococci. Livestock-related risk factors for water contamination were examined through multinomial regression, controlling for confounders.
Rainwater was the main POU water source and was found to be highly susceptible to contamination. Multivariate analysis showed greater risk of gross (>100 CFU/100 mL) water contamination (with E. coli) for households where goats were observed, and/or where poultry roosted in proximity to stored household water (relative risk RR = 2.71; p = 0.001 and RR = 2.02; p = 0.012 respectively). Presence of a poultry coop was also associated with elevated intestinal enterococci densities (RR = 4.46; p = 0.001). Associations between contamination and livestock risk factors were thus similar for both bacteria groups, but E. coli counts declined more rapidly following collection from surface waters than enterococci counts (p = 0.024).
The presence of livestock (particularly goats) and poultry within household compounds increases POU water contamination risk, suggesting the need for improved interventions to address cross-contamination within rural domestic settings. Within Siaya county, more effective community education is needed to raise awareness of POU water quality protection, particularly of rainwater.
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•Poultry and goats are risk factors for household stored water contamination.•Poultry are risk factors for both enterococci and E. coli contamination.•Attenuation of enterococci in household stored water is lower than for E. coli.•Residual free chlorine is mostly too low to prevent stored water recontamination.
Microbial surface water quality is important, as it is related to health risk when the population is exposed through drinking, recreation or consumption of irrigated vegetables. The microbial surface ...water quality is expected to change with socio-economic development and climate change. This study explores the combined impacts of future socio-economic and climate change scenarios on microbial water quality using a coupled hydrodynamic and water quality model (MIKE21FM-ECOLab). The model was applied to simulate the baseline (2014–2015) and future (2040s and 2090s) faecal indicator bacteria (FIB: E. coli and enterococci) concentrations in the Betna river in Bangladesh. The scenarios comprise changes in socio-economic variables (e.g. population, urbanization, land use, sanitation and sewage treatment) and climate variables (temperature, precipitation and sea-level rise). Scenarios have been developed building on the most recent Shared Socio-economic Pathways: SSP1 and SSP3 and Representative Concentration Pathways: RCP4.5 and RCP8.5 in a matrix. An uncontrolled future results in a deterioration of the microbial water quality (+75% by the 2090s) due to socio-economic changes, such as higher population growth, and changes in rainfall patterns. However, microbial water quality improves under a sustainable scenario with improved sewage treatment (-98% by the 2090s). Contaminant loads were more influenced by changes in socio-economic factors than by climatic change. To our knowledge, this is the first study that combines climate change and socio-economic development scenarios to simulate the future microbial water quality of a river. This approach can also be used to assess future consequences for health risks.
Presently, concentrations of fecal indicator bacteria (FIB) in raw water sources are not known before water undergoes treatment, since analysis takes approximately 24h to produce results. Using data ...on water quality and environmental variables, models can be used to predict real time concentrations of FIB in raw water. This study evaluates the potentials of zero-inflated regression models (ZI), Random Forest regression model (RF) and adaptive neuro-fuzzy inference system (ANFIS) to predict the concentration of FIB in the raw water source of a water treatment plant in Norway. The ZI, RF and ANFIS faecal indicator bacteria predictive models were built using physico-chemical (pH, temperature, electrical conductivity, turbidity, color, and alkalinity) and catchment precipitation data from 2009 to 2015. The study revealed that pH, temperature, turbidity, and electrical conductivity in the raw water were the most significant factors associated with the concentration of FIB in the raw water source. Compared to the other models, the ANFIS model was superior (Mean Square Error=39.49, 0.35, 0.09, 0.23CFU/100ml respectively for coliform bacteria, E. coli, Intestinal enterococci and Clostridium perfringens) in predicting the variations of FIB in the raw water during model testing. However, the model was not capable of predicting low counts of FIB during both training and testing stages of the models. The ZI and RF models were more consistent when applied to testing data, and they predicted FIB concentrations that characterized the observed FIB concentrations. While these models might need further improvement, results of this study indicate that ZI and RF regression models have high prospects as tools for the real-time prediction of FIB in raw water sources for proactive microbial risk management in water treatment plants.
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•Zero-inflated (ZI) regression accommodate typical variations in faecal indicator bacteria observed in water.•ZI models predict zero counts of faecal indicator bacteria better than high positive counts.•Adaptive neuro-fuzzy inference system may not be very efficient for predicting count variables such as indicator bacteria.•Random Forest and ZI regression models have high potentials for real time prediction of indicator bacteria in water supply.
The aim of this study is to investigate the occurrence of faecal indicator and microbial pathogens (bacteria and virus) in the shallow urban aquifer of the Besòs River Delta (NE Spain). To this end, ...human adenovirus (HAdV) and Norovirus of genogroups I and II (NoV GI and NoV GII) as well as the faecal indicator bacteria (FIB) Escherichia coli (EC) and faecal enterococci (FE) were monitored in groundwater and in the River Besòs in December 2013 and in July 2104. None of the targeted pathogens were detected in groundwater in December 2013 but contamination of human origin was observed in approximately 50% of the points sampled in July 2014 reaching concentrations up to 99 GC/100 mL for HAdV. Generally, microbial concentrations in river water were higher than those detected in groundwater. This observation indicates that pathogens are naturally attenuated when river water infiltrates and flows through the aquifer, however HAdV were detected at a sampling point located at 380 m from the river in the absence of FIB. The presence of human viral contamination may represent a risk for the use of groundwater as a drinking water source. Further research is needed to understand the dynamics of pathogens in river–groundwater interface over long time periods and a wide range of flow conditions (wet and dry periods) since the urban groundwater of this aquifer might be a valuable drinking water resource in Barcelona especially during drought periods. The methodology followed in this research can be applied to other urban aquifers with similar purposes since the scarcity and contamination of freshwater resources are worldwide issues.
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•Urban groundwater (UGW) contaminated by pathogens•Human viruses were more frequently detected than faecal indicator bacteria in UGW.•The main source of groundwater contamination is a polluted river.•Natural attenuation of pathogens occurred when river water infiltrates the aquifer.•Chemical and microbial tracers used to track pathogen pollution sources in UGW
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