Relationship between F-specific RNA phage genogroups, faecal pollution indicators and human adenoviruses in river water

Recent studies have shown the increasing interest of F-specific RNA phage genotyping to identify major sources of faecal contamination in waters. This study, conducted in a river located in an urbanized watershed with recognized anthropogenic influences, was aimed at evaluating the relevance of direct phage genotyping by real-time RT-PCR. One hundred percent of positive results were obtained with a 5 mL aliquot of river water (n = 31). Phage distribution was modified after cultivation, since the ratio of the two most abundant genogroups (II and I) reached 1.51 log₁₀ by direct RT-PCR-based method versus 0.30 log₁₀ after cultivation (n = 8). For the first time, positive correlations between the concentrations of genogroup II, bacterial indicators and human adenoviruses were observed, which may indicate a human faecal pollution. No correlation between genogroups II and I has been revealed. The concentration of genogroup I was only correlated with water turbidity, suggesting an animal pollution coming from upstream after rainfall events. Among the microbiological parameters studied, only genogroup II/genogroup I ratio shows variations occurring in the major sources of faecal pollution.     

Occurrence and persistence of enteroviruses, noroviruses and F-specific RNA phages in natural wastewater biofilms

Enteroviruses and noroviruses are pathogenic viruses excreted by infected individuals. Discharged in wastewaters, some of these viruses can be captured by biofilms. In the present study, we assessed the occurrence and persistence of these viruses in wastewaters and in corresponding biofilms. Natural wastewaters and biofilms were analyzed monthly from January to July using real-time RT-PCR. Enterovirus RNA was detected in wastewater in June while norovirus RNA was detected from January to March. In contrast, biofilm analysis revealed the presence of both enterovirus and norovirus genomes throughout the study period. For instance, enterovirus and norovirus genogroups (GG) I and II were detected in 50, 46 and 37% of the biofilm samples, respectively (n = 24). In a laboratory experiment, persistence of norovirus GGI RNA (quantified using molecular techniques) and F-specific bacteriophages (quantified using both culture and molecular techniques) was assessed in wastewater and corresponding naturally-contaminated biofilms at both 4 and 20 °C. The concentrations of viral genomes (norovirus GGI and F-specific RNA phage) were very stable in biofilms. Indeed, no significant decrease was observed during the persistence experiment that lasted 49 days. Furthermore, regardless of our experimental conditions, viral genome and infectious F-specific bacteriophages persisted longer in biofilm than in wastewater. According to our results, wastewater biofilms may contribute to the persistence and dispersal of pathogenic viruses outside of epidemic periods.     

Validation and field testing of library-independent microbial source tracking methods in the Gulf of Mexico

Water quality is frequently impacted by microbial pollution from human and animal feces. Microbial source tracking (MST) can identify dominant pollution sources and improve assessment of health risk compared to indicator bacteria alone. This study aims to standardize and validate MST methods across laboratories in coastal Gulf of Mexico states. Three laboratories evaluated library-independent MST methods for human sewage detection via conventional PCR: (1) human-associated Bacteroidales, (2) human polyomaviruses (HPyVs), and (3) Methanobrevibacter smithii. All methods detected targets in human sewage seeded into buffer, freshwater or marine water (100% sensitivity). The limit of detection (LOD) for human sewage was lowest for the Bacteroidales assay (10⁻⁵-10⁻⁶ dilution). LODs for HPyVs and M. smithii assays were similar to each other (10⁻³-10⁻⁴), but were higher than Bacteroidales. The HPyVs assay was 100% specific, showing no cross-reactivity to dog, cow, cat, bird, or wild animal feces among >300 samples from three Gulf Coast regions. The human Bacteroidales assay was 96% specific, but cross-reacted with 10% of dog and some chicken samples. The M. smithii assay was 98% specific with limited cross-reactivity with cow, dog and seagull samples. An experts’ workshop concluded that all methods showed sufficient accuracy and reliability to move forward. SOPs will be distributed to collaborating laboratories for further inter-laboratory comparison, and field validation will occur in year 2.     

Confirmation of putative stormwater impact on water quality at a Florida beach by microbial source tracking methods and structure of indicator organism populations

The effect of a stormwater conveyance system on indicator bacteria levels at a Florida beach was assessed using microbial source tracking methods, and by investigating indicator bacteria population structure in water and sediments. During a rain event, regulatory standards for both fecal coliforms and Enterococcus spp. were exceeded, contrasting with significantly lower levels under dry conditions. Indicator bacteria levels were high in sediments under all conditions. The involvement of human sewage in the contamination was investigated using polymerase chain reaction (PCR) assays for the esp gene of Enterococcus faecium and for the conserved T antigen of human polyomaviruses, all of which were negative. BOX-PCR subtyping of Escherichia coli and Enterococcus showed higher population diversity during the rain event; and higher population similarity during dry conditions, suggesting that without fresh inputs, only a subset of the population survives the selective pressure of the secondary habitat. These data indicate that high indicator bacteria levels were attributable to a stormwater system that acted as a reservoir and conduit, flushing high levels of indicator bacteria to the beach during a rain event. Such environmental reservoirs of indicator bacteria further complicate the already questionable relationship between indicator organisms and human pathogens, and call for a better understanding of the ecology, fate and persistence of indicator bacteria.     

Integrated analysis of water quality parameters for cost-effective faecal pollution management in river catchments

In many parts of the world, microbial contamination of surface waters used for drinking, recreation, and shellfishery remains a pervasive risk to human health, especially in Less Economically Developed Countries (LEDC). However, the capacity to provide effective management strategies to break the waterborne route to human infection is often thwarted by our inability to identify the source of microbial contamination. Microbial Source Tracking (MST) has potential to improve water quality management in complex river catchments that are either routinely, or intermittently contaminated by faecal material from one or more sources, by attributing faecal loads to their human or non-human sources, and thereby supporting more rational approaches to microbial risk assessment. The River Ouse catchment in southeast England (U.K.) was used as a model with which to investigate the integration and application of a novel and simple MST approach to monitor microbial water quality over one calendar year, thereby encompassing a range of meteorological conditions. A key objective of the work was to develop simple low-cost protocols that could be easily replicated. Bacteriophages (viruses) capable of infecting a human specific strain of Bacteroides GB-124, and their correlation with presumptive Escherichia coli, were used to distinguish sources of faecal pollution. The results reported here suggest that in this river catchment the principal source of faecal pollution in most instances was non-human in origin. During storm events, presumptive E. coli and presumptive intestinal enterococci levels were 1.1-1.2 logs higher than during dry weather conditions, and levels of the faecal indicator organisms (FIOs) were closely associated with increased turbidity levels (presumptive E. coli and turbidity, r = 0.43). Spatio-temporal variation in microbial water quality parameters was accounted for by three principal components (67.6%). Cluster Analysis, reduced the fourteen monitoring sites to six representative ‘sentinel’ sites. The correlation coefficient between presumptive E. coli and phages of Bacteroides GB-124 was very small (r = 0.05) whilst that between turbidity and suspended solids was high (r = 0.62). Variations in climate, animal and anthropogenic interferences were all, either directly or indirectly, related to faecal contamination. The findings show the importance of meteorological conditions, such as storm events, on microbial water quality, and suggest that any future increases in the frequency of storm events (associated with climate change) are likely to result in a greater incidence of FIO/pathogen loads. This low-cost approach could help to predict spatio-temporal ‘hotspots’ of elevated waterborne disease risk. The work also represents an important step towards integrating novel MST tools into river catchment modelling.     

Evaluating the operational utility of a Bacteroidales quantitative PCR-based MST approach in determining the source of faecal indicator organisms at a UK bathing water

Microbial source tracking techniques are used in the UK to provide an evidence-base to guide major expenditure decisions and/or regulatory action relating to sewage disposal. Consequently, it is imperative that the techniques used robustly index faecal indicator organisms (FIOs) that are the regulatory parameters for bathing and shellfish harvesting areas. This study reports a ‘field-scale’ test of microbial source tracking (MST) based on the quantitative PCR analyses of Bacteroidales 16S rRNA genetic marker sequences. The project acquired data to test the operational utility of quantitative Bacteroidales MST data, comparing it with FIO concentrations in streams, effluents and bathing waters. Overall, the data did not exhibit a consistent pattern of significant correlations between Bacteroidales MST parameters and FIOs within the different sample matrices (i.e. rivers, bathing waters and/or effluents). Consequently, there was little evidence from this study that reported concentrations and/or percentages of human and/or ruminant faecal loadings (that are based on Bacteroidales MST gene copy numbers) offer a credible evidence-base describing FIO contributions to receiving water ‘non-compliance’. The study also showed (i) there was no significant attenuation of the Bacteroidales gene copy number ‘signal’ through the UV disinfection process; and (ii) single non-compliant samples submitted for Bacteroidales MST analysis, do not reliably characterise the balance of faecal loadings due to the high variability in the MST signal observed.At this stage in the development of the MST tool deployed, it would be imprudent to use the percentage human and/or ruminant contributions (i.e. as indicated by MST data acquired at a bathing water) as the sole or principal element in the evidence-base used to guide major expenditure decisions and/or regulatory action.     

Evaluation of two spike-and-recovery controls for assessment of extraction efficiency in microbial source tracking studies

Quantitative PCR (qPCR), applied to complex environmental samples such as water, wastewater, and feces, is susceptible to methodological and sample related biases. In this study, we evaluated two exogenous DNA spike-and-recovery controls as proxies for recovery efficiency of Bacteroidales 16S rDNA gene sequences (AllBac and qHF183) that are used for microbial source tracking (MST) in river water. Two controls—(1) the plant pathogen Pantoea stewartii, carrying the chromosomal target gene cpsD, and (2) Escherichia coli, carrying the plasmid-borne target gene DsRed2—were added to raw water samples immediately prior to concentration and DNA extraction for qPCR. When applied to samples processed in replicate, recovery of each control was positively correlated with the observed concentration of each MST marker. Adjustment of MST marker concentrations according to recovery efficiency reduced variability in replicate analyses when consistent processing and extraction methodologies were applied. Although the effects of this procedure on accuracy could not be tested due to uncertainties in control DNA concentrations, the observed reduction in variability should improve the strength of statistical comparisons. These findings suggest that either of the tested spike-and-recovery controls can be useful to measure efficiency of extraction and recovery in routine laboratory processing.     

Presence of Enterococcus faecalis in broiler litter and wild bird feces for bacterial source tracking

When Enterococcus faecalis is isolated from fresh feces, its host range appears to be limited to humans and birds. Although E. faecalis is found in human sewage, the extent to which the bacterium is found in broiler litter and in the feces of wild birds is unclear. These results have implications for bacterial source tracking. We determined if media designed for the isolation of fecal enterococci affected this host range, and if E. faecalis was routinely found in broiler litter and in the feces of wild birds. Of five different isolation media, none affected the isolation of E. faecalis. Enterococcus faecalis was routinely found in fresh broiler feces (522 of 1092 isolates; 48%), but rarely in broiler litter (12 of 1452 isolates; <2%). Therefore, broiler litter selects against this bacterium, and broiler litter is an unlikely environmental source of this bacterium. The presence of E. faecalis in eight wild bird species was highly variable. Unless the fecal loading rate from migratory or resident wild birds is high, water samples collected during baseflow conditions with high numbers of E. faecalis may indicate human fecal contamination.     

Adaptation and evaluation of the Canadian Council of Ministers of the Environment Water Quality Index (CCME WQI) for use as an effective tool to characterize drinking source water quality

Protecting drinking source water quality is a critical step in ensuring a safe supply of drinking water. Increasingly, drinking source water protection programs rely on the active participation of various stakeholders with differing degrees of water science knowledge. A drinking source water quality index presents a potential communication and analysis tool to facilitate cooperation between diverse interest groups as well as represent composite water quality. We tested the effectiveness of the Canadian Council of Ministers of the Environment Water Quality Index (CCME WQI) in capturing expert assessments of drinking water quality. In cooperation with a panel of drinking water quality experts we identified a core set of parameters to reflect common source water concerns. Drinking source water target values were drafted for use in the index corresponding to two basic treatment levels. Index scores calculated using the core parameter set and associated source water target values were strongly correlated with expert assessments of water quality. We recommend a modified index calculation procedure to accommodate parameters measured at different frequencies within any particular study period. The resulting drinking source water CCME WQI provides a valuable means of monitoring, communicating, and understanding surface source water quality.     

Evaluating short-term changes in recreational water quality during a hydrograph event using a combination of microbial tracers, environmental microbiology, microbial source tracking and hydrological techniques: A case study in Southwest Wales, UK

Quantitative assessment of multiple sources to short-term variations in recreational water quality, as indexed by faecal indicator organism (FIO) concentrations, is becoming increasingly important with adoption of modern water quality standards and catchment-based water quality management requirements (e.g. the EU Water Framework Directive, Article 11 ‘Programmes of Measures’ and the US Clean Water Act, ‘Total Maximum Daily Loads’). This paper describes a study combining microbial tracers, intensive FIO measurement, open channel hydrology and molecular microbial source tracking (MST) to enhance understanding of recreational water quality at Amroth in southwest Wales, UK. Microbial tracers were released from four stream inputs during a moderate hydrograph event. Tracers from two local streams impacted simultaneously with a period of maximum FIO concentrations at the near-shore compliance monitoring site. Connection between these inputs and this site were rapid (9-33 min). Water quality impairment from a more remote stream input followed, 12.85 h after tracer release, sustaining FIO concentrations above desired compliance levels. MST analysis showed dominance of ruminant Bacteroidales genetic markers, associated with agricultural pollution. This integration of tracers and MST offers additional information on the movement and individual sources causing water quality impairment.     

Specificity and sensitivity evaluation of novel and existing Bacteroidales and Bifidobacteria-specific PCR assays on feces and sewage samples and their application for microbial source tracking in Ireland

Three novel ruminant-specific PCR assays, an existing ruminant-specific PCR assay and five existing human-specific PCR assays, which target 16S rDNA from Bacteroidales or Bifidobacteria, were evaluated. The assays were tested on DNA extracted from ruminant (n = 74), human (n = 59) and non-ruminant animal (n = 44) sewage/fecal samples collected in Ireland. The three novel PCR assays compared favourably to the existing ruminant-specific assay, exhibiting sensitivities of 91-100% and specificities of 95-100% as compared to a sensitivity of 95% and specificity of 94%, for the existing ruminant-specific assay. Of the five human-specific PCR assays, the assay targeting the Bifidobacterium catenulatum group was the most promising, exhibiting a sensitivity of 100% (with human sewage samples) and a specificity of 87%. When tested on rural water samples that were naturally contaminated by ruminant feces, the three novel PCR assays tested positive with a much greater percentage (52-87%) of samples than the existing ruminant-specific assay (17%). These novel ruminant-specific assays show promise for microbial source tracking and merit further field testing and specificity evaluation.