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An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10-50 gene copies or plaques × 50 ml(-1)) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40-70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aims: The aim of this study was to identify the origin of faecal pollution impacting the Elorn estuary (Brittany, France) by applying microbial source tracking (MST) markers in both oysters and estuarine waters., Methods and Results: The MST markers used were as follows: (i) human-, ruminant- and pig-associated Bacteroidales markers by real-time PCR and (ii) human genogroup II and animal genogroup I of F-specific RNA bacteriophages (FRNAPH) by culture/genotyping and by direct real-time reverse-transcriptase PCR. The higher occurrence of the human genogroup II of F-specific RNA bacteriophages using a culture/genotyping method, and human-associated Bacteroidales marker by real-time PCR, allowed the identification of human faecal contamination as the predominant source of contamination in oysters (total of 18 oyster batches tested) and waters (total of 24 water samples tested). The importance of using the intravalvular liquids instead of digestive tissues, when applying host-associated Bacteroidales markers in oysters, was also revealed., Conclusions: This study has shown that the application of a MST toolbox of diverse bacterial and viral methods can provide multiple lines of evidence to identify the predominant source of faecal contamination in shellfish from an estuarine environment., Significance and Impact of the Study: Application of this MST toolbox is a useful approach to understand the origin of faecal contamination in shellfish harvesting areas in an estuarine setting., (© 2013 The Society for Applied Microbiology.)

The relevance of three host-associated Bacteroidales markers (HF183, Rum2Bac, and Pig2Bac) and four F-specific RNA bacteriophage genogroups (FRNAPH I to IV) as microbial source tracking markers was assessed at the level of a catchment (Daoulas, France). They were monitored together with fecal indicators (Escherichia coli and enterococci) and chemophysical parameters (rainfall, temperature, salinity, pH, and turbidity) by monthly sampling over 2 years (n = 240 water samples) and one specific sampling following an accidental pig manure spillage (n = 5 samples). During the 2-year regular monitoring, levels of E. coli, enterococci, total F-specific RNA bacteriophages, and the general Bacteroidales marker AllBac were strongly correlated with one another and with Rum2Bac (r = 0.37 to 0.50, P < 0.0001). Their correlations with HF183 and FRNAPH I and II were lower (r = 0.21 to 0.29, P < 0.001 to P < 0.0001), and HF183 and enterococci were associated rather than correlated (Fisher's exact test, P < 0.01). Rum2Bac and HF183 enabled 73% of water samples that had ≥ 2.7 log(10) most probably number (MPN) of E. coli/100 ml to be classified. FRNAPH I and II enabled 33% of samples at this contamination level to be classified. FRNAPH I and II complemented the water sample classification obtained with the two Bacteroidales markers by an additional 8%. Pig2Bac and FRNAPH III and IV were observed in a small number of samples (n = 0 to 4 of 245). The present study validates Rum2Bac and HF183 as relevant tools to trace fecal contamination originating from ruminant or human waste, respectively, at the level of a whole catchment.

Fecal contaminations of inland and coastal waters induce risks to human health and economic losses. To improve water management, specific markers have been developed to differentiate between sources of contamination. This study investigates the relative decay of fecal indicator bacteria (FIB, Escherichia coli and enterococci) and six human-associated markers (two bacterial markers: Bacteroidales HF183 (HF183) and Bifidobacterium adolescentis (BifAd); one viral marker: genogroup II F-specific RNA bacteriophages (FRNAPH II); three chemical markers: caffeine and two fecal stanol ratios) in freshwater and seawater microcosms seeded with human wastewater. These experiments were performed in darkness, at 20 °C and under aerobic conditions. The modeling of the decay curves allows us (i) to compare FIB and markers and (ii) to classify markers according to their persistence in seawater (FRNAPH II < HF183, stanol ratios < BifAd, caffeine) and in freshwater (HF183, stanol ratios < FRNAPH II < BifAd < caffeine). Although those results depend on the experimental conditions, this study represents a necessary step to develop and validate an interdisciplinary toolbox for the investigation of the sources of fecal contaminations.

Fecal contamination of water resources is evaluated by the enumeration of the fecal coliforms and Enterococci. However, the enumeration of these indicators does not allow us to differentiate between the sources of fecal contamination. Therefore, it is important to use alternative indicators of fecal contamination to identify livestock contamination in surface waters. The concentration of fecal indicators (, enteroccoci, and F-specific bacteriophages), microbiological markers (Rum-2-bac, Pig-2-bac, and ), and chemical fingerprints (sterols and stanols and other chemical compounds analyzed by 3D-fluorescence excitation-matrix spectroscopy) were determined in runoff waters generated by an artificial rainfall simulator. Three replicate plot experiments were conducted with swine slurry and cattle manure at agronomic nitrogen application rates. Low amounts of bacterial indicators (1.9-4.7%) are released in runoff water from swine-slurry-amended soils, whereas greater amounts (1.1-28.3%) of these indicators are released in runoff water from cattle-manure-amended soils. Microbial and chemical markers from animal manure were transferred to runoff water, allowing discrimination between swine and cattle fecal contamination in the environment via runoff after manure spreading. Host-specific bacterial and chemical markers were quantified for the first time in runoff waters samples after the experimental spreading of swine slurry or cattle manure., (American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America.)

The microbiological quality of coastal or river waters can be affected by faecal pollution from human or animal sources. An efficient MST (Microbial Source Tracking) toolbox consisting of several host-specific markers would therefore be valuable for identifying the origin of the faecal pollution in the environment and thus for effective resource management and remediation. In this multidisciplinary study, after having tested some MST markers on faecal samples, we compared a selection of 17 parameters corresponding to chemical (steroid ratios, caffeine, and synthetic compounds), bacterial (host-specific Bacteroidales, Lactobacillus amylovorus and Bifidobacterium adolescentis) and viral (genotypes I-IV of F-specific bacteriophages, FRNAPH) markers on environmental water samples (n = 33; wastewater, runoff and river waters) with variable Escherichia coli concentrations. Eleven microbial and chemical parameters were finally chosen for our MST toolbox, based on their specificity for particular pollution sources represented by our samples and their detection in river waters impacted by human or animal pollution; these were: the human-specific chemical compounds caffeine, TCEP (tri(2-chloroethyl)phosphate) and benzophenone; the ratios of sitostanol/coprostanol and coprostanol/(coprostanol+24-ethylcopstanol); real-time PCR (Polymerase Chain Reaction) human-specific (HF183 and B. adolescentis), pig-specific (Pig-2-Bac and L. amylovorus) and ruminant-specific (Rum-2-Bac) markers; and human FRNAPH genogroup II., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Faecal contamination sources were identified in coastal areas around the Guerande-Atlantique peninsula using two microbial source tracking (MST) methods: (i) Bacteroidales host-specific 16S rRNA gene markers measured by real-time PCR and (ii) F-specific bacteriophage (FRNAPH) genotyping. Both methods were used on 63 water samples from 7 water courses. HF183 marker and bacteriophage genogroup II (FRNAPH II) were detected in all water samples and in the majority of water samples, respectively, from La Torre stream (W5), Piriac (W2), R2000 (W3) and Mazy (W7) rain water drains, and also detected, less frequently, in Le Nau drain (W4), suggesting contamination by human faecal sources at these sites. These human markers were weakly detected in Pouliguen channel (W6). Furthermore, BacR and bacteriophage genogroup I (FRNAPH I) were also detected, but at lower concentration and frequency. So, site W6 seems to be contaminated by multiple sources, though mainly human. Finally, BacR was detected twice in Pont d'Armes channel (W1), whereas HF183 was not detected. FRNAPH I and II were detected in only 3 out of 12 water samples. Site W1 seems mainly contaminated by animal sources. As a result of our findings, actions were taken to remediate water and shellfish quality.

In order to identify the origin of the fecal contamination observed in French estuaries, two library-independent microbial source tracking (MST) methods were selected: (i) Bacteroidales host-specific 16S rRNA gene markers and (ii) F-specific RNA bacteriophage genotyping. The specificity of the Bacteroidales markers was evaluated on human and animal (bovine, pig, sheep, and bird) feces. Two human-specific markers (HF183 and HF134), one ruminant-specific marker (CF193'), and one pig-specific marker (PF163) showed a high level of specificity (>90%). However, the data suggest that the proposed ruminant-specific CF128 marker would be better described as an animal marker, as it was observed in all bovine and sheep feces and 96% of pig feces. F RNA bacteriophages were detected in only 21% of individual fecal samples tested, in 60% of pig slurries, but in all sewage samples. Most detected F RNA bacteriophages were from genotypes II and III in sewage samples and from genotypes I and IV in bovine, pig, and bird feces and from pig slurries. Both MST methods were applied to 28 water samples collected from three watersheds at different times. Classification of water samples as subject to human, animal, or mixed fecal contamination was more frequent when using Bacteroidales markers (82.1% of water samples) than by bacteriophage genotyping (50%). The ability to classify a water sample increased with increasing Escherichia coli or enterococcus concentration. For the samples that could be classified by bacteriophage genotyping, 78% agreed with the classification obtained from Bacteroidales markers.

This study aimed to investigate the impact of small tributaries on seawater and shellfish quality in coastal area subjected to brief episodes leading to fecal contamination. Escherichia coli and F-RNA-specific bacteriophages were selected as fecal indicators and astroviruses were chosen as being representative of pathogens in the human population during winter viral epidemics. A two-dimensional hydrodynamic model was built to simulate the current and dispersion in the model domain, which includes areas uncovered at low tide. The model also includes decay rates to simulate microorganism behavior and assess the influence of fecal input on shellfish quality. The originality lies in the fact that specific features of the study area were considered. Modeling results indicate limited particle movements and long flushing times at the back of the bay, where shellfish are farmed. Computational results showed that under normal conditions, i.e. 94% of the time, when rainfall was less than 10 mm per day, the sector shows acceptable water quality. These results are in agreement with shellfish concentration measured in the field. Under high flow conditions, high concentrations of fecal indicators and astrovirus were measured in the river and tributaries. The corresponding fluxes were over 50 times higher than under normal weather conditions. The location of the shellfish beds near the coast makes them vulnerable and fecal indicators and viruses were detected in shellfish after short rainfall events. Our modeling approach makes a contribution to shellfish management and consumer protection, by indicating the "risk period" as defined by EU regulations. Molecular development such as viral quantification in conjunction with model developments will help to prevent shellfish contamination and thus provide safer products to consumers and an effective tool for shellfish producers.

Rotavirus virus-like particles (VLPs) and MS2 bacteriophages were bioaccumulated in bivalve mollusks to evaluate viral persistence in shellfish during depuration and relaying under natural conditions. Using this nonpathogenic surrogate virus, we were able to demonstrate that about 1 log10 of VLPs was depurated after 1 week in warm seawater (22 degrees C). Phage MS2 was depurated more rapidly (about 2 log10 in 1 week) than were VLPs, as determined using a single-compartment model and linear regression analysis. After being relayed in the estuary under the influence of the tides, VLPs were detected in oysters for up to 82 days following seeding with high levels of VLPs (concentration range between 10(10) and 10(9) particles per g of pancreatic tissue) and for 37 days for lower contamination levels (10(5) particles per g of pancreatic tissue). These data suggest that viral particles may persist in shellfish tissues for several weeks.

Coastal areas are frequently contaminated by microorganisms of human origin, due to high population density and low seawater renewal. To evaluate the impact of wastewater input on shellfish quality, a study was conducted in Brittany (France) over a period of 20 months. A hydrodynamic model was used to simulate wastewater impact on microbial water quality. To validate the model, wastewater from the three main sewage treatment plants and shellfish from three sites were sampled monthly. Bacterial indicators (E. coli), F-RNA phages were searched for by culture and noroviruses by RT-PCR and hybridisation. These microorganisms were detected in the three effluents and clams, with no marked seasonal variation. The microbial concentrations in the two oyster beds, distant from the effluent outfall, were low, and only three of the samples were positive for norovirus. For simulation, the winter wastewater inputs of E. coli and phages were calculated and an estimation for norovirus flux was made from the epidemic situation in the population. The microbial behaviour was included in the model by a decay-rate factor. Results from the model calculations were found to be very similar to E. coli and phage concentrations observed in shellfish. For noroviruses, the model indicated that shellfish distant from the wastewater input were under the detection limit of the RT-PCR method. This study demonstrated the use of modelisation to interpret norovirus contamination in various areas.

The risks of false-positive responses were examined when using the polymerase chain reaction (PCR) method for the detection of Salmonella in the marine environment (water and shellfish). The degradation rates of DNA, both free and from dead Salmonella, were evaluated in natural seawaters maintained at 10 degrees and 20 degrees C, using PCR with Vir and invA primers. The DNA of dead Salmonella was detected up to 55 d in seawater collected in winter and stored at 10 degrees C. But in summer, the persistence was shorter: 10 d or even 2 d for a smaller inoculum (3 x 10(3) Salmonella ml-1). The role of the planktonic organisms present in spring and summer was pinpointed. For free DNA, the persistence times were shorter: from 2 to 4 d at 20 degrees C, and from 3 to 8 d at 10 degrees C showing that the nuclease activity of marine organisms is higher at warm temperatures. These data led us to recommend careful interpretations of direct PCR results, especially during cold periods and for samples collected close to terrestrial discharges of high concentrations of live, dead or lysed Salmonella. PCR is a rapid, specific and sensitive method, but should be applied with care to marine samples, in order to avoid false-positive responses.

An investigation of beta-galactosidase activity of Escherichia coli strain H10407, under different physiological and environmental conditions, e.g. induced and uninduced osmotic stress, light, etc., was undertaken. In this study E. coli was employed as a model for faecal coliforms in waste water. beta-Galactosidase activity was induced by isopropyl-beta-D-thiogalactoside (IPTG). Enzyme activity (U cell-1)/cell for sewage bacteria and for induced E. coli was similar, i.e. log U cell-1 = -8.5 whereas uninduced E. coli yielded log U cell-1 = -12.1. Initial enzyme activity was not dependent on phase of growth of the cell (exponential vs stationary phase) or whether marine or fresh water at the time of initial dilution. However, osmotic change resulted in a decrease in culturable cells, even though enzyme activity remained constant. A significant decrease in the number of culturable bacteria, followed by a decrease in beta-galactosidase activity, was observed after exposure of cells to visible light radiation. It is concluded that beta-galactosidase enzyme is retained in viable but non-culturable E. coli. Furthermore, beta-galactosidase appears to offer a useful and rapid (25 min) measure of the viability of faecal coliforms, and therefore, of the water quality of bathing and shellfishing areas.

Enzyme assays for 4-methylumbelliferyl-beta-D-galactopyranosidase and 4-methylumbelliferyl-beta-D-glucuronidase activities were used for rapid detection (25 min) of fecal water pollution and to determine the impact of sewage discharge in coastal waters. Two coastal areas were investigated: (i) an estuary characterized by a high degree of contamination downstream of a discharge from a sewage treatment plant and a low degree of water renewal and (ii) a fjord with a low degree of pollution and a high degree of water renewal. Statistical analysis showed that a global correlation curve could be used to estimate concentrations of culturable fecal coliform bacteria in the two coastal areas, although environmental factors important for cell physiology (e.g., salinity) varied at different sampling locations. The sensitivity limit for detection of 4-methylumbelliferyl-beta-D-glucuronidase activity corresponded to bacterial concentrations on the order of 10 to 100 CFU/100 ml. The 4-methylumbelliferyl-beta-D-galactopyranosidase assay was less sensitive because of a higher rate of substrate autohydrolysis. The detection limit corresponded to bacterial concentrations on the order of 100 to 1,000 fecal coliforms per 100 ml.