Marie-Paule Caprais

Development of microbial and chemical MST tools to identify the origin of the faecal pollution in bathing and shellfish harvesting waters in France

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.

Salmonella DNA persistence in natural seawaters using PCR analysis

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° and 20°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°C. But in summer, the persistence was shorter: 10 d or even 2 d for a smaller inoculum (3 × 103Salmonella 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°C, and from 3 to 8 d at 10°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.

Use of Rotavirus Virus-Like Particles as Surrogates To Evaluate Virus Persistence in Shellfish

ABSTRACT 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°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 1010 and 109 particles per g of pancreatic tissue) and for 37 days for lower contamination levels (105 particles per g of pancreatic tissue). These data suggest that viral particles may persist in shellfish tissues for several weeks.

Relative Decay of Fecal Indicator Bacteria and Human-Associated Markers: A Microcosm Study Simulating Wastewater Input into Seawater and Freshwater

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 in Coastal Areas: An Engineering Approach

Introduction : The occurrence of pathogenic microorganisms in seawater or in shellfish could exist anytime sewage from human or animal origin would be discharged to the coast (Metcalf, 1982). According to the diseases occurring in the human population or in animals, pathogens might be present in recreational waters or in shellfish. Thus, the presence of human enteric viruses (norovirus, astrovirus, rotavirus, hepatitisAvirus (HAV)) and pathogenic bacteria (Salmonella, Listeria monocytogenes, Shiga-toxin-producing Escherichia coli (STEC), Vibrio cholerae, Vibrio parahaemolyticus, etc.) has been reported in coastal areas for a long time (Colwell, 1978; Metcalf, 1978; Melnick et al., 1979; Grimes, 1991; Bosch et al., 2001;Kong et al., 2002). These microorganisms have been implicated in gastrointestinal and respiratory illnesses and other infections (skin, eyes, etc.), (Griffin et al., 2003). Using risk-assessment models for viruses, maximum risks were estimated to be 1.3 infections per 100 swimmers (Colwell et al., 1996). To evaluate the risk due to the presence of these pathogens in the environment, certain criteria have to be determined. Among them, the infectious dosewould be of a greatest importance (Table 14.1). Even if the infectious dose vary with the strains, the age of the patient, or other parameters, some pathogens are highly dangerous for men even at low concentrations (HAV, E. coli O157:H7, V. cholerae), whereas others have to be ingested in high concentrations to be harmful (V. parahaemolyticus) or are highly infectious but not very dangerous (norovirus). Thus, for some pathogens, a low contamination in seafood, for example, is not acceptable, based on risk-assessment models (Colwell et al., 1996). Among bacteria, the Vibrio family plays an important role in infections, waterborne or seafood diseases, especially in countries surrounded by warm marine waters. Toxigenic V...

Relevance of Bacteroidales and F-specific RNA bacteriophages for efficient fecal contamination tracking at the level of a catchment in France

ABSTRACT 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 (Fishers exact test, P < 0.01). Rum2Bac and HF183 enabled 73% of water samples that had ≥2.7 log10 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.

Microbial and chemical markers: runoff transfer in animal manure-amended soils

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.

Identification of the origin of faecal contamination in estuarine oysters using Bacteroidales and F-specific RNA bacteriophage markers.

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.