Microbial Source Tracking

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.► The principal source of faecal pollution in most instances was non-human. ► Wet weather E. coli and enterococci were 1.1–1.2 logs higher than dry weather. ► Faecal indicator organisms were closely associated with increased turbidity levels. ► Cluster Analysis, reduced the fourteen monitoring sites to six ‘sentinel’ sites. ► Rainfall, animal and anthropogenic interferences influenced faecal contamination.

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 non-host 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 multi-regional applicability of enterococci hosts in MST application and highlights the significance of multivariate analysis with non-detects in evaluating the performance of new MST host strains.

Use of viral pathogens and indicators to differentiate ... Rachel T. Noble, Steven M. Allen, Angelia D. Blackwood, Weiping Chu, ... Sunny C. Jiang, Greg L. Lovelace, Mark D. Sobsey, Jill R. Stewart and ... Rachel T. Noble (corresponding author) Angelia D. Blackwood UNC-Chapel Hill ...

Conventional methods for fecal source tracking typically use single biomarkers to systematically identify or exclude sources. High-throughput DNA sequence analysis can potentially identify all sources of microbial contaminants in a single test by measuring the total diversity of fecal microbial communities. In this study, we used phylogenetic microarray analysis to determine the comprehensive suite of bacteria that define major sources of fecal contamination in coastal California. Fecal wastes were collected from 42 different populations of humans, birds, cows, horses, elk, and pinnipeds. We characterized bacterial community composition using a DNA microarray that probes for 16S rRNA genes of 59 316 different bacterial taxa. Cluster analysis revealed strong differences in community composition among fecal wastes from human, birds, pinnipeds, and grazers. Actinobacteria, Bacilli, and many Gammaproteobac-teria taxa discriminated birds from mammalian sources. Diverse families within the Clostridia and Bacteroidetes taxa discriminated human wastes, grazers, and pinnipeds from each other. We found 1058 different bacterial taxa that were unique to either human, grazing mammal, or bird fecal wastes. These OTUs can serve as specific identifier taxa for these sources in environmental waters. Two field tests in marine waters demonstrate the capacity of phylogenetic microarray analysis to track multiple sources with one test.

We report the design and validation of new TaqMan((R)) assays for microbial source tracking based on the amplification of fecal 16S rRNA marker sequences from uncultured cells of the order Bacteroidales. The assays were developed for the detection and enumeration of non-point source input of fecal pollution to watersheds. The quantitative &quot;universal&quot;Bacteroidales assay BacUni-UCD detected all tested stool samples from human volunteers (18 out of 18), cat (7 out of 7), dog (8 out of 8), seagull (10/10), cow (8/8), horse (8/8), and wastewater effluent (14/14). The human assay BacHum-UCD discriminated fully between human and cow stool samples but did not detect all stool samples from human volunteers (12/18). In addition, there was 12.5% detection of dog stool (1/8), but no cross-reactivity with cat, horse, or seagull fecal samples. In contrast, all wastewater samples were positive for the BacHum-UCD marker, supporting its designation as 100% sensitive for mixed-human source ...

We assessed the occurrence and specificity of bacteriophages of Bacteroides fragilis in swine farms for their potential microbial source tracking application. A local B. fragilis host strain SP25 (DSM29413) was isolated from a swine pooled feces sample from a non-antibiotic farm. This strain presented high specificity to swine fecal materials as it did not detect bacteriophages in any samples from human sewage, sheep, goats, cattle, dogs, and cats. The reference B. fragilis strain RYC2056 could detect phages in swine samples, but also in most of human sewage and polluted urban canal samples. Phages of SP25 existed in certain swine farms in

Assays for the detection and typing of adenoviruses, enteroviruses and F+ specific coliphages were performed on samples created as part of a national microbial source tracking methods comparison study. The samples were created blind to the researchers, and were inoculated with a variety of types of fecal contamination source (human, sewage, dog, seagull and cow) and mixtures of sources. Viral tracer and pathogen assays demonstrated a general ability to discriminate human from non-human fecal contamination. For example, samples inoculated with sewage were correctly identified as containing human fecal contamination because they contained human adenovirus or human enterovirus. In samples containing fecal material from individual humans, human pathogen analysis yielded negative results probably because the stool samples were taken from healthy individuals. False positive rates for the virus-based methods (0-8%) were among the lowest observed during the methods comparison study. It is s...

Microbiological source tracking (MST) methods are increasingly being used to identify fecal contamination sources in surface waters, but these methods have been subjected to limited comparative testing. In this study, 22 researchers employing 12 different methods were provided sets of identically prepared blind water samples. Each sample contained one to three of five possible fecal sources (human, dog, cattle, seagull or sewage). Researchers were also provided with portions of the fecal material used to inoculate the blind water samples for use as library material. No MST method that was tested predicted the source material in the blind samples perfectly. Host-specific PCR performed best at differentiating between human and non-human sources, but primers are not yet available for differentiating between all of the non-human sources. Virus and F+ coliphage methods reliably identified sewage, but were unable to identify fecal contamination from individual humans. Library-based isolat...

Culture-independent fecal source tracking methods have many potential advantages over library-dependent, isolate-culture methods, but they have been subjected to limited testing. The purpose of this study was to compare culture-independent, library-independent methods of fecal source tracking. Five laboratories analysed identical sets of aqueous samples that contained one or more of the following sources: sewage, human feces, dog feces, cattle feces and gull feces. Two investigators used methods based on PCR amplification of Bacteroidetes marker genes and both successfully discriminated between samples that did or did not contain human fecal material. One of these investigators was also able to identify the remaining sources, except for gull, with a low rate of false positives. A method based on E. coli toxin genes successfully identified samples containing sewage and cattle feces, but missed some samples with human feces because of low marker prevalence in individual human fecal sa...