Microbial Source Tracking Tools Research Articles

Pepper mild mottle virus as an effective tool in microbial source tracking for deficient domestic on-site water treatment systems

Pollution from domestic on-site wastewater treatment systems (OWTS) is a significant contaminant pressure in many rural catchments. However, due to their design, and dispersed proliferation, it is difficult to assess their impact. Water testing methodologies employ bacterial culturing methods and chemical analysis which may lose resolution and/or specificity being confounded by diffuse agricultural sources within a rural environment. In this study, we successfully assessed the applicability of Pepper Mild Mottle Virus (PMMoV) as a human faecal source tracker for deficient on-site wastewater treatment systems. The transport of PMMoV was first studied in the effluent of a 30 cm deep soil column which was dosed for 510 days with primary influent from a conventional septic system. The removal of PMMoV through the 30 cm deep soil column was quantified with a 5-day seeding trial employing primary influent mixed with PMMoV sourced from Tabasco pepper product ®. The trial was then carried out at field scale with the seeding solution dosed into an operational percolation trench receiving septic tank effluent which had been instrumented for porewater sampling. Samples were taken at depths of 10 cm, 30 cm, and 50 cm across the length of the trench at distances of 1 m, 7.5 m, and 17.5 m from the inlet of the trench. PMMoV was detected on all days of the trial, with a peak concentration of 1 × 106 found at the rear of the trench on day 2 of the seeding trial. Finally, to assess the effectiveness of PPMoV as a microbial source tracking tool from a water receptor perspective, three rural catchments with high densities of OWTSs were sampled and analysed for hourly variations in biological parameters which included total coliforms, Escherichia coli, PMMoV, and chemical parameters total organic carbon, total nitrogen, and total carbon. PMMoV was detected in all river samples over a 24-hour period, thereby indicating its suitability as a tracer of human wastewater effluent in such environments with multiple diffuse sources.

Integrated assessment of groundwater contamination: A multi-marker approach for comprehensive water quality monitoring

Abstract This study investigated the presence of adenoviral markers (Library-Independent microbial source tracking tool) in the groundwater of Ludhiana, Punjab, India, a hitherto unexplored area. While deep aquifers post-chlorination were adenovirus free, shallow aquifers near farm wastewater pits exhibited human adenovirus and bovine adenovirus. Coliform-negative samples also harboured pathogens, highlighting the limitations of conventional indicators. Surface water displayed higher viral contamination, potentially impacting groundwater. The use of farm wastewater for irrigation and open pit disposal emerged as key contributors, advocating for sustainable wastewater management. Physiochemical and microbial analyses revealed variations across sites, emphasising regional and temporal variations. The weighted arithmetic water quality index ranged from good to very poor, with deep aquifers showing better quality than shallow ones. A novel approach incorporating graphical representations of adenovirus estimations alongside water quality index provided a more comprehensive understanding. Intriguingly, the study revealed the presence of coliforms irrespective of water quality grade, questioning its reliability as a sole indicator. Correlations between specific water quality grades and adenovirus types suggested targeted control measures. The lack of significant correlations between viral markers and conventional parameters in groundwater compared to surface water studies highlighted the unique dynamics of groundwater contamination.

Assembly process and source tracking of microbial communities in sediments of Dongting Lake

Sediment source tracing can accurately provide a theoretical basis for controlling soil erosion effectively, by identifying the most serious types of land use. Traditional sediment tracing methods are based on physical, chemical, biological, and composite fingerprinting, which have not included microbes. As high-throughput sequencing becomes more prevalent, microorganisms can provide more information than what we think. Thus, whether the microorganism can also be used as a special fingerprint factor for sediment source identification during soil erosion, we have tested it by using microbial source tracking tool FEAST to quantify the microbe contribution from five types of eroded land (including dryland, urban, paddy field, forest and grassland) to the depositional areas (Niubitan) in the Yuanjiang basin. The source microbial community in the erosive area was heterogeneous, and assembly process analysis further demonstrated that the source tracking results could reach higher accuracy. The results of FEAST showed that dryland (35.50%), urban (17.21%), paddy field (8.14%), and forest (1.07%) were the major contributors to Niubitan. Our results follow the general soil erosion rules and prove its validity. Taken together, a new perspective is provided by these results for tracing sediment sources in erosion-sedimentary systems.

Prevalence and Characterization of CRISPR Locus 2.1 Spacers in Escherichia coli Isolates Obtained from Feces of Animals and Humans.

The clustered regularly interspaced short palindromic repeat (CRISPR) has been studied as an immune system in prokaryotes for the survival of bacteriophages. The CRISPR system in prokaryotes records the invasion of bacteriophages or other genetic materials in CRISPR loci. Accordingly, CRISPR loci can reveal a history of infection records of bacteriophages and other genetic materials. Therefore, identification of the CRISPR array may help trace the events that bacteria have undergone. In this study, we characterized and identified the spacers of the CRISPR loci in Escherichia coli isolates obtained from the feces of animals and humans. Most CRISPR spacers were found to stem from phages. Although we did not find any patterns in CRISPR spacers according to sources, our results showed that phage-derived spacers mainly originated from the families Inoviridae, Myoviridae, Podoviridae, and Siphoviridae and the order Caudovirales, whereas plasmid-derived CRISPR spacers were mainly from the Enterobacteriaceae family. In addition, it is worth noting that the isolates from each animal and human source harbored source-specific spacers. Considering that some of these taxa are likely found in the gut of mammalian animals, CRISPR spacers identified in these E. coli isolates were likely derived from the bacteriophageome and microbiome in closed gut environments. Although the bacteriophageome database limits the characterization of CRISPR arrays, the present study showed that some spacers were specifically found in both animal and human sources. Thus, this finding may suggest the possible use of E. coli CRISPR spacers as a microbial source tracking tool. IMPORTANCE We characterized spacers of CRISPR locus 2.1 in E. coli isolates obtained from the feces of various sources. Phage-derived CRISPR spacers are mainly acquired from the order Caudovirales, and plasmid-derived CRISPR spacers are mostly from the Enterobacteriaceae family. This is thought to reflect the microbiome and phageome of the gut environment of the sources. Hence, spacers may help track the encounter of bacterial cells with bacterial cells, viruses, or other genetic materials. Interestingly, source-specific spacers are also observed. The identification of source-specific spacers is thought to help develop the methodology of microbial source tracking and understanding the interactions between viruses and bacteria. However, very few spacers have been uncovered to track where they originate. The accumulation of genome sequences can help identify the hosts of spacers and can be applied for microbial source tracking.

Developing a novel Bifidobacterium phage quantitative polymerase chain reaction-based assay for tracking untreated wastewater

Microbial source tracking (MST) tools provide insights on fecal pollution levels in aquatic environments using predominantly quantitative PCR (qPCR) assays that target host-associated molecular marker genes. Existing wastewater-associated marker genes have shown limited or significant cross-reactions with non-human fecal samples. In this study, we mined the current Gut Phage Database (GPD) and designed a novel untreated wastewater-specific Bifidobacterium phage qPCR assay (i.e., Bifi assay). The sensitivity and specificity of the Bifi marker genes were assessed by collectively analyzing untreated (n = 33) and treated (n = 15) wastewater and non-human fecal samples (i.e., Rabbit, mouse, cow, horse, pig, chicken, sheep, dog, deer, kangaroos; n = 113) in Shenzhen, Guangdong Province, China and Brisbane, Australia. Bifi assay revealed 100% host-specificity against non-human fecal samples collected from Shenzhen and Brisbane. Furthermore, this marker gene was also detected in all untreated and treated wastewater samples, whose concentrations ranged from 5.54 to 6.83 log10 GC/L. In Shenzhen, the concentrations of Bifi marker gene were approximately two orders of magnitude lower than Bacteroides (HF183/BacR287 assay) and CrAssphage (CPQ_56 assay). The concentration of Bifi marker gene in untreated wastewater from Brisbane was 1.35 log10 greater than those in Shenzhen. Our results suggest that Bifi marker gene has the potential to detect and quantify the levels of human fecal pollution in Shenzhen and Brisbane. If additional detection sensitivity is required for environmental studies, Bifi marker gene should be paired with either CrAssphage or HF183/BacR287 marker genes.

A Combined Digital PCR and Next Generation DNA-Sequencing Based Approach for Tracking Nearshore Pollutant Dynamics Along the Southwest United States/Mexico Border.

Ocean currents, multiple fecal bacteria input sources, and jurisdictional boundaries can complicate pollution source tracking and associated mitigation and management efforts within the nearshore coastal environment. In this study, multiple microbial source tracking tools were employed to characterize the impact and reach of an ocean wastewater treatment facility discharge in Mexico northward along the coast and across the Southwest United States- Mexico Border. Water samples were evaluated for fecal indicator bacteria (FIB), Enterococcus by culture-based methods, and human-associated genetic marker (HF183) and Enterococcus by droplet digital polymerase chain reaction (ddPCR). In addition, 16S rRNA gene sequence analysis was performed and the SourceTracker algorithm was used to characterize the bacterial community of the wastewater treatment plume and its contribution to beach waters. Sampling dates were chosen based on ocean conditions associated with northern currents. Evidence of a gradient in human fecal pollution that extended north from the wastewater discharge across the United States/Mexico border from the point source was observed using human-associated genetic markers and microbial community analysis. The spatial extent of fecal contamination observed was largely dependent on swell and ocean conditions. These findings demonstrate the utility of a combination of molecular tools for understanding and tracking specific pollutant sources in dynamic coastal water environments.

Evaluation of Low-Cost Phage-Based Microbial Source Tracking Tools for Elucidating Human Fecal Contamination Pathways in Kolkata, India.

Phages, such as those infecting Bacteroides spp., have been proven to be reliable indicators of human fecal contamination in microbial source tracking (MST) studies, and the efficacy of these MST markers found to vary geographically. This study reports the application and evaluation of candidate MST methods (phages infecting previously isolated B. fragilis strain GB-124, newly isolated Bacteroides strains (K10, K29, and K33) and recently isolated Kluyvera intermedia strain ASH-08), along with non-source specific somatic coliphages (SOMCPH infecting strain WG-5) and indicator bacteria (Escherichia coli) for identifying fecal contamination pathways in Kolkata, India. Source specificity of the phage-based methods was first tested using 60 known non-human fecal samples from common animals, before being evaluated with 56 known human samples (municipal sewage) collected during both the rainy and dry season. SOMCPH were present in 40-90% of samples from different animal species and in 100% of sewage samples. Phages infecting Bacteroides strain GB-124 were not detected from the majority (95%) of animal samples (except in three porcine samples) and were present in 93 and 71% of the sewage samples in the rainy and dry season (Mean = 1.42 and 1.83 log10PFU/100mL, respectively), though at lower levels than SOMCPH (Mean = 3.27 and 3.02 log10PFU/100mL, respectively). Phages infecting strain ASH-08 were detected in 89 and 96% of the sewage samples in the rainy and dry season, respectively, but were also present in all animal samples tested (except goats). Strains K10, K29, and K30 were not found to be useful MST markers due to low levels of phages and/or co-presence in non-human sources. GB-124 and SOMCPH were subsequently deployed within two low-income neighborhoods to determine the levels and origin of fecal contamination in 110 environmental samples. E. coli, SOMCPH, and phages of GB-124 were detected in 68, 42, and 28% of the samples, respectively. Analyses of 166 wastewater samples from shared community toilets and 21 samples from sewage pumping stations from the same districts showed that SOMCPH were present in 100% and GB-124 phages in 31% of shared toilet samples (Median = 5.59 and <1 log10 PFU/100 mL, respectively), and both SOMCPH and GB-124 phages were detected in 95% of pumping station samples (Median = 5.82 and 4.04 log10 PFU/100 mL, respectively). Our findings suggest that GB-124 and SOMCPH have utility as low-cost fecal indicator tools which can facilitate environmental surveillance of enteric organisms, elucidate human and non-human fecal exposure pathways, and inform interventions to mitigate exposure to fecal contamination in the residential environment of Kolkata, India.

Investigation of relationships between fecal contamination, cattle grazing, human recreation, and microbial source tracking markers in a mixed-land-use rangeland watershed

The United States National Forests are mixed-use lands that support human recreation and cattle grazing. Overuse by humans or cattle, however, can lead to the fecal contamination of local waterways. Until recently, the source of these contaminants was a subject of conjecture; however, microbial source tracking tools have become widely used and are proving to be a valid methodology to identify the contamination source. This study aims to analyze and model the quantity and sources of fecal contamination in the Mink Creek watershed in southeastern Idaho. The U.S. Forest Service Caribou-Targhee National Forest (USFS) manages this watershed. Previous research has indicated that some localities within the watershed exceed US EPA standards for coliform bacteria. In 2019, water samples were collected before livestock began grazing and throughout the spring, summer, and fall after livestock grazing had ended. Fourteen sites were sampled seven times during the field season, allowing the water to be analyzed for total coliforms and E. coli bacteria. Microbial source tracking techniques using Bacteroides bacteria, which are known to live in specific digestive tracks, were used to identify the source of E. coli at each sampling location. The analysis indicated that E. coli counts exceeded state regulatory limits 35% of the time. These exceedances were associated with DNA source tracking markers for humans (58.8%), cattle (5.9%), or both cattle and humans (5.9%). Unknown sources were responsible for the Bacteroides bacteria 29.4% of the time. A statistical model was developed to estimate E. coli using the datasets of microbial source tracking measures, the presence or absence of humans, cattle, the proximity of the sampling date to a holiday, and other seasonal factors. The resulting model showed good performance indices at all the 14 sites based on a K-fold cross-validation scheme (R2 = 0.83 and NSE = 0.69). The results demonstrated that E. coli exceedances have a close association with human recreation and unknown sources and negatively influenced by dissolved oxygen.

Fecal pollution: new trends and challenges in microbial source tracking using next-generation sequencing.

In this minireview, we expand upon traditional microbial source tracking (MST) methods by discussing two recently developed, next-generation-sequencing (NGS)-based MST approaches to identify sources of fecal pollution in recreational waters. One method defines operational taxonomic units (OTUs) that are specific to a fecal source, e.g., humans and animals or shared among multiple fecal sources to determine the magnitude and likely source association of fecal pollution. The other method uses SourceTracker, a program using a Bayesian algorithm, to determine which OTUs have contributed to an environmental community based on the composition of microbial communities in multiple fecal sources. Contemporary NGS-based MST tools offer a promising avenue to rapidly characterize fecal source contributions for water monitoring and remediation efforts at a broader and more efficient scale than previous molecular MST methods. However, both NGS methods require optimized sequence processing methodologies (e.g. quality filtering and clustering algorithms) and are influenced by primer selection for amplicon sequencing. Therefore, care must be taken when extrapolating data or combining datasets. Furthermore, traditional limitations of library-dependent MST methods, including differential decay of source material in environmental waters and spatiotemporal variation in source communities, remain to be fully understood. Nevertheless, increasing use of these methods, as well as expanding fecal taxon libraries representative of source communities, will help improve the accuracy of these methods and provide promising tools for future MST investigations.

Human-specific phages infecting Enterococcus host strain MW47: are they reliable microbial source tracking markers?

The aim of this study was to determine the morphological diversity and environmental survival of human-specific phages infecting Enterococcus faecium host strain MW47, to support their use as microbial source tracking (MST) markers. Twenty phages capable of infecting strain MW47 were propagated and their morphologies were determined using transmission electron microscopy (TEM), which revealed that a heterogeneous group of phages was able to infect strain MW47. Three distinct morphologies from two different families (Myoviridae and Siphoviridae) were observed. In situ inactivation experiments were subsequently conducted to determine their environmental persistence. The findings revealed a statistically significant link between morphology and the rate of inactivation, with phages belonging to the Myoviridae family demonstrating more rapid inactivation in comparison to those belonging to the Siphoviridae family. The results suggest that while Enterococcus MW47 phages appear to be a potentially valuable MST tools, significant variations in the persistence of the different phages mean that the approach should be used with caution, as this may adversely affect the reliability of the approach, especially when comparing MW47 phage levels or the presence across different matrices (e.g. levels in sediments or shellfish). This highlights the importance of elucidating the ecological characteristics of newly proposed MST markers before they are used in full-scale MST investigations.

Resolution of habitat-associated ecogenomic signatures in bacteriophage genomes and application to microbial source tracking

Just as the expansion in genome sequencing has revealed and permitted the exploitation of phylogenetic signals embedded in bacterial genomes, the application of metagenomics has begun to provide similar insights at the ecosystem level for microbial communities. However, little is known regarding this aspect of bacteriophage associated with microbial ecosystems, and if phage encode discernible habitat-associated signals diagnostic of underlying microbiomes. Here we demonstrate that individual phage can encode clear habitat-related 'ecogenomic signatures', based on relative representation of phage-encoded gene homologues in metagenomic data sets. Furthermore, we show the ecogenomic signature encoded by the gut-associated ɸB124-14 can be used to segregate metagenomes according to environmental origin, and distinguish 'contaminated' environmental metagenomes (subject to simulated in silico human faecal pollution) from uncontaminated data sets. This indicates phage-encoded ecological signals likely possess sufficient discriminatory power for use in biotechnological applications, such as development of microbial source tracking tools for monitoring water quality.

Application of a microbial source tracking based on bacterial and chemical markers in headwater and coastal catchments

This study identified sources of fecal contamination in three different French headwater and coastal catchments (the Justiçou, Pen an Traon, and La Fresnaye) using a combination of microbial source tracking tools. The tools included bacterial markers (three host-associated Bacteroidales) and chemical markers (six fecal stanols), which were monitored monthly over one or two years in addition to fecal indicator bacteria. 168 of the 240 freshwater and marine water samples had Escherichia coli (E. coli) or enterococci concentrations higher than “excellent” European water quality threshold. In the three catchments, the results suggested that the fecal contamination appeared to be primarily from an animal origin and particularly from a bovine origin in 52% (Rum2Bac) and 46% (Bstanol) of the samples and to a lesser extent from a porcine origin in 19% (Pig2Bac) and 21% (Pstanol) of the samples. Our results suggested a human fecal contamination in 56% (HF183) and 32% (Hstanol) of the samples. Rainfall also impacted the source identification of microbial contamination. In general, these findings could inform effective implementation of microbial source tracking strategies, specifically that the location of sampling points must include variability at the landscape scale.

Comparison of the Performance of Different Microbial Source Tracking Markers among European and North African Regions.

Microbial source tracking (MST) has been extensively used to detect the sources of fecal pollution in water. The inclusion of MST in water management strategies improves the ecological status of the ecosystem and human and animal health under interdisciplinary analysis in all aspects of health care for humans, animals, and the environment (One Health approach). In this study, the performance of MST markers targeting host-specific Bacteroidales (HF183 and Rum-2-Bac) and species (HMBif and CWBif) were evaluated in raw sewage collected from human, ruminant, swine, and poultry sources in Tunisia, Cyprus, Ireland, and Spain. In addition, the ratio between somatic coliphages and bacteriophages infecting GA17 (SOMCPH/GA17PH) was measured in Tunisia and Spain. The obtained results showed variability of the bacterial markers between the four countries, suggesting that their usefulness could be affected by several conditions (dietary habits, agricultural practices, and climatic conditions) that differ between countries. The Rum-2-Bac marker stood out as a valid MST tool, particularly in Ireland, whereas CWBif was the best option in Tunisia, Spain, and Cyprus. The human-specific HMBif marker showed good sensitivity and specificity in Tunisia, Spain, and Ireland, whereas HF183 showed a low specificity. However, HF183 was suggested as a good human marker in Ireland and Cyprus because of its higher concentration than HMBif. Regarding viral markers, the ratio of SOMCPH/GA17PH showed a clear discrimination between human and nonhuman samples. The combined use of molecular bacterial markers and the ratio of SOMCPH/GA17PH may improve the success of MST.

Norwegian study on microbial source tracking for water quality control and pollution removal in constructed wetland treating catchment run-off.

This study describes the first Norwegian microbial source tracking (MST) approach for water quality control and pollution removal from catchment run-off in a nature-based treatment system (NBTS) with a constructed wetland. The applied MST tools combined microbial analyses and molecular tests to detect and define the source(s) and dominant origin(s) of faecal water contamination. Faecal indicator bacteria Escherichia coli and host-specific Bacteroidales 16 s rRNA gene markers have been employed. The study revealed that the newly developed contribution profiling of faecal origin derived from the Bacteroidales DNA could quantitatively distinguish between human and non-human pollution origins. Further, the outcomes of the MST test have been compared with the results of both physicochemical analyses and tests of pharmaceutical and personal care products (PPCPs). A strong positive correlation was discovered between the human marker and PPCPs. Gabapentin was the most frequently detected compound and it showed the uppermost positive correlation with the human marker. The study demonstrated that the NBTS performs satisfactorily with the removal of E. coli but not PPCPs. Interestingly, the presence of PPCPs in the water samples was not correlated with high concentrations of E. coli. Neither has the latter an apparent correlation with the human marker.

Genetic diversity and antimicrobial resistance of Escherichia coli as microbial source tracking tools of Karaj River, Iran

The aim of this study was to analyze the enterobacterial repetitive intergenic consensus (ERIC)-types, phylo-groups and antimicrobial resistance (AMR) patterns of Escherichia coli and to investigate if these approaches are suitable for microbial source tracking (MST). E. coli strains were isolated from cattle faeces and Karaj River. For genetic diversity, AMR profile, and phylo-grouping, we applied ERIC-PCR, disk diffusion, and multiplex-PCR, respectively. Fifty isolates from each sample group were used in the study. ERIC fingerprinting produced ten different bands, demonstrating 64 unique and 36 repetitive profiles. Six isolates from the river showed the same ERIC pattern of the cattle, of which four expressed the same AMR profile. E. coli isolates from water were represented in A, B1, C, and D phylo-groups. Phylo-groups A, B1, and E were more prevalent in the cattle isolates and B2 was absent in both sources. Three of the water isolates with the same ERIC-type and AMR to cattle isolates showed the same phylo-groups. Genetic characteristics, AMR, and phylo-groups of the isolates from the river are diverse and complex. For accurate MST, complementary approaches should be applied together and a comprehensive library should be provided.

Integrated Multivariate Analysis with Nondetects for the Development of Human Sewage Source-Tracking Tools Using Bacteriophages of Enterococcus faecalis.

We developed sewage-specific microbial source tracking (MST) tools using enterococci bacteriophages and evaluated their performance with univariate and multivariate analyses involving data below detection limits. Newly isolated Enterococci faecalis bacterial strains AIM06 (DSM100702) and SR14 (DSM100701) demonstrated 100% specificity and 90% sensitivity to human sewage without detecting 68 animal manure pooled samples of cats, chickens, cows, dogs, ducks, pigs, and pigeons. AIM06 and SR14 bacteriophages were present in human sewage at 2-4 orders of magnitude. A principal component analysis confirmed the importance of both phages as main water quality parameters. The phages presented only in the polluted water, as classified by a cluster analysis, and at median concentrations of 1.71 × 102 and 4.27 × 102 PFU/100 mL, respectively, higher than nonhost specific RYC2056 phages and sewage-specific KS148 phages (p < 0.05). Interestingly, AIM06 and SR14 phages exhibited significant correlations with each other and with total coliforms, E. coli, enterococci, and biochemical oxygen demand (Kendall's tau = 0.348 to 0.605, p < 0.05), a result supporting their roles as water quality indicators. This research demonstrates the multiregional applicability of enterococci hosts in MST application and highlights the significance of multivariate analysis with nondetects in evaluating the performance of new MST host strains.

A tool box strategy using Bacteroides genetic markers to differentiate human from non-human sources of fecal contamination in natural water

Bacteroides genetic markers have been widely used to identify fecal pollution of water originating from human and animal sources. Many of the assays currently used for detecting human-specific Bacteroides produce false positive results. The focus of this study was to develop a microbial source tracking (MST) tool box strategy for differentiating Bacteroides from human and animal sources.Bacteroides 16S rRNA gene sequences from fish and selected animals were aligned against human fecal Bacteroides isolates to compare and characterize the variable regions within the 16S rRNA gene sequence. Conserved sequences between 4 variable regions were deleted and the truncated sequences were combined to develop a hyper-variable genomic segment (HVGS). The cladogram created from truncated sequences show a clear separation of Bacteroides from human feces and those from animal sources. The proposed strategy was field tested by collecting water samples from central Arizona source waters and three different recreational ponds. PCR using HF134 and HF183 primer sets was performed and sequences from positive reactions were aligned against human Bacteroides sequences to identify the source of contamination. Based on PCR results, the source of fecal contamination was presumptively identified as either human or from another source. For samples testing positive using the HF183 primer set (8/13), fecal contamination was presumed to be from human sources, but to confirm the results, PCR products were sequenced and aligned against the four variable regions and then incorporated within the truncated cladogram. As expected, the sequences from water samples with human fecal contamination grouped in a separate clade.A variability matrix, developed after exclusion of conserved sequences among the four regions, was utilized to establish discrete groupings for sequences within the truncated cladogram, generally differentiating Bacteroides isolates from varying host animals, but most importantly, separating Bacteroides from human feces from Bacteroides from other animals. The proposed strategy offers a new tool box method for MST and a step-wise methodology essential for identifying human sources of fecal pollution.

Relevance of F-Specific RNA Bacteriophages in Assessing Human Norovirus Risk in Shellfish and Environmental Waters.

Human noroviruses (HuNoVs) are the main cause of shellfish-borne gastroenteritis outbreaks. In the absence of routine technical approaches allowing infectious particles to be detected, this viral pathogen is currently targeted by genome research, leading to difficult interpretations. In this study, we investigated the potential of F-specific RNA bacteriophages (FRNAPH) as fecal and viral contamination indicators in shellfish and water from a local harvesting area. FRNAPH were also used as microbial source tracking tools. Constraints imposed by detection limits are illustrated here by the detection of infectious FRNAPH in several samples in the absence of FRNAPH genomes. The opposite situation was also observed, likely explained by the persistence of the genomes being greater than infectivity. Similar considerations may be applied to HuNoVs, suggesting that HuNoV genome targeting is of limited relevance in assessing infectious risks. While FRNAPH did not provide any benefits compared to Escherichia coli as fecal pollution indicators in water, novel observations were made in shellfish: contrary to E. coli, a seasonal trend of infectious FRNAPH concentrations was observed. These concentrations were higher than those found in water, confirming bioaccumulation in shellfish. This study also underlines a relationship between the presence of HuNoV genomes and those of human-specific FRNAPH subgroup II (FRNAPH-II) in shellfish collected throughout Europe. Further research should be undertaken to evaluate FRNAPH potential as an indicator of the presence of infectious HuNoVs. To this end, shellfish involved in HuNoV-caused gastroenteritis outbreaks should be analyzed for the presence of infectious FRNAPH-II. This work provides new data about the use of F-specific RNA phages (FRNAPH) as a tool for evaluating fecal or viral contamination, especially in shellfish. In our case study, FRNAPH did not provide any benefits compared to E. coli as fecal pollution indicators in water but were found to be very useful in shellfish. Their concentrations in shellfish were higher than those found in the surrounding water, confirming bioaccumulation. This study also underlines a relationship between the presence of human norovirus genomes (HuNoVs) and those of FRNAPH subgroup II (FRNAPH-II). Considering that the two virus types have similar behaviors and since FRNAPH infectivity can be investigated, the specific detection of infectious FRNAPH-II could be regarded as an indication of the presence of infectious HuNoVs. The contribution of infectious human FRNAPH targeting for assessing the viral risk associated with HuNoVs in shellfish should thus be investigated.

Human-, Ovine-, and Bovine-Specific Viral Source Tracking Tools to Discriminate Between the Major Fecal Sources in Agricultural Waters.

This study evaluated the sources of fecal contamination in different river catchments, using a combination of microbial source tracking tools, for human, ruminant, ovine and bovine livestock, in order to define appropriate water management strategies. Every source of waterway pollution was evaluated in river water samples from one urban river catchment and two important farming regions in New Zealand. Fecal pollution was initially measured by testing Escherichia coli and evaluating the presence of human- and ruminant-associated DNA markers of Bacteroidales (BiAdo, BacHum-UCD, BacH, and BacR) and human and ruminant fecal sterols/stanols ratios. Then specific fecal pollution sources were assessed with previously reported quantitative PCR assays targeting human-, bovine-, and ovine-specific viruses: human adenoviruses (HAdV), human JC polyomaviruses, bovine polyomaviruses (BPyV), and ovine polyomaviruses (OPyV). High level of ruminant fecal contamination was detected all over the farming areas, whereas no ruminant sources were identified in the urban river sampling sites. BacR was the most frequently observed ruminant marker and OPyV and BPyV allowed the identification of ovine and bovine fecal sources. The human fecal viral marker (HAdV) was the most frequently observed human marker, highly abundant in the urban sites, and also present in farming areas. This is the first study using simultaneously the ovine and the bovine viral markers to identify and quantify both bovine and ovine fecal pollution.

Microbial health risks associated with exposure to stormwater in a water plaza

Climate change scenarios predict an increase of intense rainfall events in summer in Western Europe. Current urban drainage systems cannot cope with such intense precipitation events. Cities are constructing stormwater storage facilities to prevent pluvial flooding. Combining storage with other functions, such as recreation, may lead to exposure to contaminants. This study assessed the microbial quality of rainwater collected in a water plaza and the health risks associated with recreational exposure. The water plaza collects street run-off, diverges first flush to the sewer system and stores the rest of the run-off in the plaza as open water. Campylobacter, Cryptosporidium and Legionella pneumophila were the pathogens investigated. Microbial source tracking tools were used to determine the origin (human, animal) of the intestinal pathogens. Cryptosporidium was not found in any sample. Campylobacter was found in all samples, with higher concentrations in samples containing human Bacteroides than in samples with zoonotic contamination (15 vs 3.7 gc (genomic copies)/100 mL). In both cases, the estimated disease risk associated with Campylobacter and recreational exposure was higher than the Dutch national incidence. This indicates that the health risk associated with recreational exposure to the water plaza is significant. L. pneumophila was found only in two out of ten pond samples. Legionnaire's disease risks were lower than the Dutch national incidence. Presence of human Bacteroides indicates possible cross-connections with the CSS that should be identified and removed.