Recreational bathing waters are complex systems with diverse inputs from multiple anthropogenic and zoogenic sources of faecal contamination. Faecal contamination is a substantial threat to water ...quality and public health. Here we present a comprehensive strategy to estimate the contribution of faecal indicator bacteria (FIB) from different biological sources on two at-risk beaches in Dublin, Ireland. The daily FIB loading rate was determined for three sources of contamination: a sewage-impacted urban stream, dog and wild bird fouling. This comparative analysis determined that the stream contributed the highest daily levels of FIB, followed by dog fouling. Dog fouling may be a significant source of FIB, contributing approximately 20 % of E. coli under certain conditions, whereas wild bird fouling contributed a negligible proportion of FIB (<3 %). This study demonstrates that source-specific quantitative microbial source apportionment (QMSA) strategies are vital to identify primary public health risks and target interventions to mitigate faecal contamination.
Display omitted
•Source-specific QMSA is vital to quantify diverse biological sources of faeces.•Sewage-impacted stream contributed the highest daily FIB loading onto beaches.•Dogs were estimated to load 0.6 –h 2.2 kg of faeces and 20 % of E. coli daily.•Wild bird fouling contributed a negligible proportion of FIB.
Faecal pollution in aquatic environments is a worldwide public health concern, yet the reliability and comprehensiveness of the methods used to assess faecal contamination are still debated. We ...compared three approaches, namely a culture-based method to enumerate Faecal Indicator Bacteria (FIB), a FIB-targeting qPCR assay, and High-Throughput Sequencing (HTS) to detect faeces- and sewage-associated taxa in water and sediment samples of an impacted model lagoon and its adjacent sea across one year. Despite at different levels, all approaches agreed in showing a higher contamination in the lagoon than in the sea, and higher in sediments than water. FIB significantly correlated when considering separately sediment and water, and when using both cultivation and qPCR. Similarly, FIB correlated between cultivation and qPCR, but qPCR provided consistently higher estimates of FIB. Faeces-associated bacteria positively correlated with cultivated FIB in both compartments, whereas sewage-associated bacteria did only in water. Considering their benefits and limitations, we conclude that, in our study site, improved quali-quantitative information on contamination is provided when at least two approaches are combined (e.g., cultivation and qPCR or HTS data). Our results provide insights to move beyond the use of FIB to improve faecal pollution management in aquatic environments and to incorporate HTS analysis into routine monitoring.
Display omitted
•Three approaches to assess faecal pollution were compared.•Each approach exhibited benefits and limitations.•Combining at least two approaches provides exhaustive information on contamination.
Many wastewater treatment technologies have been shown to remove bacterial pathogens more effectively than viral pathogens and, in aquatic environments, levels of traditional faecal indicator ...bacteria (FIB) do not appear to correlate consistently with levels of human viral pathogens. There is, therefore, a need for novel viral indicators of faecal pollution and surrogates of viral pathogens, especially given the increasing importance of indirect and direct wastewater reuse. Potential candidates include bacteriophages (phages) and the study described here sought to elucidate the relationship between three groups of phages (somatic coliphages (SOMPH), F-RNA coliphages (F-RNAPH) and human-specific phages infecting B. fragilis (Bf124PH) – enumeration using double layer agar technique) and viral pathogens (human adenovirus (HuAdV) and norovirus (NoV) – enumeration using molecular methods) through full-scale municipal wastewater treatment processes. FIB (faecal coliforms (FC) and intestinal enterococci (ENT) – enumeration using membrane filtration) were also monitored. Samples were collected every fortnight, during a twelve-month period, at each stage of four full-scale wastewater treatment plants (WWTP) in southern England (two activated sludge (AS) and two trickling filter (TF) plants) (n = 360 samples). FIB and SOMPH were consistently found in all samples tested, whereas F-RNAPH, Bf124PH and HuAdV were less frequently detected, especially following AS treatment. The detection rate of NoV was low and consequently discussion of this group of viruses is limited. Concentrations of SOMPH and FIB were statistically higher (p value < 0.05) than concentrations of F-RNAPH, Bf124PH and HuAdV in raw wastewater. FIB were more effectively removed than phages in both systems. Removal rates of HuAdV were similar to those of phages at the secondary treatment stage of both systems. In TF systems, HuAdV were removed at the same rate as F-RNAPH, but at lower rates than SOMPH and Bf124PH. The findings suggest that phages (in particular SOMPH) are better indicators of the fate of viral pathogens in WWTP than existing FIB and that these organisms may have a useful role to play in future sanitation safety planning.
•FIB were more readily removed than phages and viral pathogens in all WWTP monitored.•Removal rates of phages were shown to be similar to those of human adenovirus.•Phages likely to better indicate the removal of viral pathogens in WWTP than FIB.•Phages as surrogates of viral pathogens in WWTP may support safe wastewater reuse.
Quantitative PCR (qPCR) and next generation sequencing (NGS) are nucleic acid based microbiology techniques that provide new insights into drinking water quality, but considerable uncertainty remains ...around their correct interpretation. We noticed the presence of bacterial DNA from various putative pathogens, including from faecal indicator bacteria (FIB), in disinfected water, when culturable FIB were absent. To understand these observations better we studied the effect of chlorination on conventional and DNA based microbial water quality assessments. Surface water chlorination reduced plate counts for various FIB by up to >6 log units, intact cell counts by flow cytometry by 3.3 log units, and 16S rRNA gene copies by qPCR by 1.5 and 1.6 log units for total bacteria and total coliforms, respectively. Nanopore sequencing of 16S rRNA amplicons with the portable MinION device revealed the DNA from several families containing putative pathogens appeared to be more resistant than that of other bacteria to degradation by chlorine disinfection. For instance, 16S rRNA genes assigned to the Enterobacteriaceae family, members of which are mostly the target of coliform tests, increased in relative abundance from 0.001 ± 0.0002% to 0.0036 ± 0.003% after chlorine treatment. Hence, metagenomic drinking water data needs to be interpreted with caution. Plate counts and flow cytometry in combination with DNA based analysis provide more robust insight than NGS or qPCR alone.
Display omitted
•The effects of chlorination on water microbiomes were studied.•The DNA from the inactivated cells can persist in disinfected water.•16S rRNA amplicon sequencing outcomes were altered by chlorination.•Multiple lines of evidence are required for drinking water assessment.•Frequent water quality monitoring is key element of the water security plan.
•Faecal indicators should be based on the hydrodynamic properties of water systems.•FIB are appropriate in advection dominant waterbodies.•crAssphage and PMMoV are appropriate in dispersion/advection ...dominant waterbodies.
The EU Bathing Water Directive (BWD) requires member states to assess bathing water quality according to the levels of faecal indicator bacteria (FIB) in designated bathing areas. However, this criterion has two significant limitations given that the BWD does not; (i) account for differences in hydrodynamic properties of bathing waters and, (ii) assumes that all faecal pathogens decay equally in aquatic environments. This study simulated sewage discharge events in three hypothetical aquatic environments characterised by different advection and dispersion parameters in the solute transport equation. Temporal changes in the downstream concentration of six faecal indicators were determined in simulations that utilised measured decay rates of each faecal indicator from a programme of controlled microcosm experiments in fresh and seawater environments. The results showed that the decay rates of faecal indicators are not a critical parameter in advection dominant water bodies, such as in fast-flowing rivers. Therefore, faecal indicator selection is less important in such systems and for these, FIB remains the most cost-effective faecal indicator to monitor the public health impacts of faecal contamination. In contrast, consideration of faecal indicator decay is important when assessing dispersion and advection/dispersion dominant systems, which would pertain to transitional (estuarine) and coastal waterbodies. Results suggest that the inclusion of viral indicators, such as crAssphage and PMMoV, could improve the reliability of water quality modelling and minimise the risk of waterborne illnesses from faecal contamination.
Display omitted
Monitoring of a constructed wetland receiving episodic pulses of subsurface drainage waters from grazed dairy pastures showed good nitrogen reductions, but moderate increases in median Escherichia ...coli concentrations. This unexpected increase of E. coli was initially postulated to be an artefact of the routine sampling approach, which was biased towards receding flows and likely missed high inflows of contaminated drainage. Intensive monitoring during storm events was therefore instigated using auto-samplers to quantify responses during rainfall-generated flow events. Determination of inflow and outflow E. coli fluxes and loads for ten storm events concluded that the wetland was consistently a net exporter of E. coli with increases ranging from 1.5 to 26-fold (median 10.2-fold). Whilst wildlife defecation in the wetland may account for a proportion of the rise in E. coli, it is likely that (consistent with associated genetic profiling studies) much of the increase is due to mobilisation of environmentally adapted strains of E. coli persisting and growing within the sediment, decomposing plant litter and organic detritus of the wetland. This raises interesting questions regarding the microbial ecology of E. coli and its utility as an indicator of faecal pathogens, and potential health risks from diffuse agricultural drainage after passage through wetlands.
Display omitted
•Unexpected E. coli increase in a constructed wetland treating agricultural drainage.•In and out-flows, concentrations and loads intensively monitored over 10 stormflows.•Median 10-fold increase in exported E. coli load after passage through wetland.•Credible evidence for persistence and generation of E. coli within the wetland.•Implications for the utility of E. coli as a faecal indicator for human health risk.
•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.
Display omitted
•Machine-learning is applied to mimic a process-based hydrodynamic and water quality model.•Process-based models forced by highly variable inputs are well emulated by the LSTM.•Spatio-temporal ...variations of water quality in reservoirs can be efficiently predicted by the LSTM.•Compared to process-based models, shorter simulation time is required with the LSTM.•Real-time hydrodynamic and water quality simulations can be made easier with machine-learning.
Process-based models are very efficient in simulating hydrodynamics and water quality in surface water bodies. However, their complex characteristics in terms of implementation, data requirements, and simulation time limit their application in regular drinking water source management. This study demonstrates the potential of a ML model (Long Short-Term Memory (LSTM)) as viable alternatives to process-based hydrodynamic and water quality models in water source management. Using meteorological and hydrological measurements, a hydrodynamic and water quality model was first calibrated to predict time series, profiles, and contours of water variables namely Escherichia coli (E. coli), faecal coliforms, zinc, and lead concentrations in the Brusdalsvatnet lake, which is the drinking water source for the city of Ålesund in Norway. The results obtained were combined with the input data to train a suite of LSTM models to emulate the results achieved with the process-based modelling. The results indicate that ML models can conveniently reproduce the spatio-temporal evolution of water quality in the lake that is achievable with the process-based model, particularly when specific locations within the lake are of interest. Compared to R2, NS and MSE ranges of 0.72–0.87, 0.68–0.85, and 0.21–0.44 achieved with the process-based model in the prediction of temperature in the lake, 0.78–0.95, 0.75–0.89, and 0.011–0.028 were respectively achieved in testing the LSTM models. Similar performance levels were achieved with the LSTM model in the prediction of Escherichia coli (E. coli), faecal coliforms, Zinc, and Lead concentrations at different depths in the lake. While setting up and training the LSTM model to emulate the process-based model simulations was very time-consuming, a validated model as developed in this study can offer an opportunity for real-time simulation of water quality in drinking water sources when integrated with cloud data transmission from field sensors.
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.
Display omitted
•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.
PDF
