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International audience; Zooplankton can spend winter actively under the ice cover of lakes. However, dietary resources under lake ice are both quantitatively and qualitatively limited, and feeding might not be energetically rewarding for most zooplankton species. Many zooplankters are expected to fast throughout the winter, exhausting their previously accumulated fat storage. We hypothesised that only a fraction of the actively overwintering zooplankton contributes to an active food web under lake ice, leading to few trophic linkages within the planktonic community.2. Zooplankton habitats and feeding were investigated under the ice of Lake Onego. Zooplankton habitats and migrations were studied by coupling zooplankton sam-pling around the clock to measurements of particle movement using an acoustic Doppler current profiler. Secondly, fatty acid-specific stable isotope compositions were used to determine whether and which zooplankton fatty acids ultimately came from the assimilation of under-ice seston.3. The algal biomass was low under ice and mostly dominated by large diatoms. Copepods dominated the zooplankton community. Species present as late cope-podite and adult instars were confined to the deeper layers, while nauplii occupied the surface layer. Diel vertical migration by Cyclops was the most tangible ob-servation of persistent feeding under the ice. Previously accumulated fat storage represented most of zooplankton fatty acids, with few, yet detectable, fatty acids recently acquired by feeding under the ice.4. Although some zooplankton taxa maintained feeding activity under the ice of Lake Onego, the food source available beneath the ice was not sufficiently rewarding to leave an isotopic imprint upon the dominant fatty acids of bulk zooplankton. The seston fatty acids that were passed on to zooplankton from feeding under ice were not provided by diatoms, although they made up most of the phytoplank-ton biovolume. Instead, the zooplankton food web was supported by mixotrophic phytoplankton (i.e. cryptophytes and chrysophytes) that represented

Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2021.151783, Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring., The work is the result of the DNAqua-Net workshop held in Rome, on June 10–12, 2019, organised by IRSA-CNR and supported by European Cooperation in Science and Technology (COST) grant CA15219 (DNAqua-Net). It largely benefited from the discussions with DNAqua-Net members during this event as well as other DNAqua-Net conferences.This study was carried out within the UNIVPM VIRIDE Project (n. 2017EKFA98) funded by the Ministry of University and Research of Italy (MUR) and the Italian National Project on the use of environmental DNA for the identification of marine non-indigenous species funded by the Istituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA). JP, TC, and LAPG were supported by the Swiss National Science Foundation (grant 31003A_179125) as well as FL with an Early Postdoc.Mobility grant (P2GEP3_171829). MS-M was supported by the Ministry of Education, Youth and Sports of the Czech Republic, European Regional Development Fund-Project CZ.02.1.01/0.0/0.0/16_019/0000845 and LTC 20063. KP was supported by the European Regional Development Fund within National Programme for Addressing Socio-Economic Challenges through R&D (L190003VLVI RITA1/02-60-05) and base financed project P190250PKKH of Estonian University of Life Sciences, IM was supported by the Slovenian Research Agency (ARRS) programme P4-0085. SD was supported by the Portuguese Foundation for Science and Technology (FCT) (CEECIND/00667/2017). LC was supported by the PON AIM 1854833 – PON Ricerca e Innovazione 2014–2020 – Azione I.2 – D.D. n. 407, 27/02/2018 – Attraction and International Mobility.VK was supported by grants PUT1389 (ETAg) and Institute of Technology, University of Tartu basic funding grant.

International audience; Surface sediments can accumulate contaminants that affect microorganisms and invertebrates and disturb benthic ecological functions. However, effects of contaminants on ecological functions supported by sediment communities are understudied. Here, we tested the relevance of two simple tools to assess the ecotoxicological effects of metal contamination on natural sediment communities using particulate organic matter breakdown and decomposition as a functional descriptor. To this aim, we performed a 21-day laboratory microcosm experiment to assess the individual and combined effects of Cu and As (nominal concentration of 40 mg kg−1 dw each) using the bait-lamina method (cellulose, bran flakes, and active coal in PVC strips) as well as artificial tablets (cellulose, bran flakes and active coal embedded in an agar matrix). Sediment toxicity was also evaluated using the standardized ostracod toxicity test. Both the bait-lamina and artificial tablet methods showed low effects of As on organic matter breakdown and decomposition but strong effects of Cu on this important ecological function. Both also showed that the presence of Cu and As in mixture in the sediment induced total inhibition of organic matter breakdown and decomposition. The ostracod toxicity test also showed high toxicity of Cu-spiked and Cu-plus-As-spiked sediments and low toxicity of As-spiked sediments. Besides confirming that artificial organic matter substrates are relevant and useful for assessing the functional effects of contaminants on sediment micro-and macro-organism communities, these results suggest that the proposed methods offer promising perspectives for developing tools for use in assessing functional ecotoxicology in the sediment compartment.

Lake ecosystems contribute significantly to atmospheric methane and are likely to become even bigger methane emitters with the global spread of hypoxia/anoxia in freshwater ecosystems. Here we characterized the spatial heterogeneity of methane production potential, methane concentration, archaeal and bacterial and methanogen communities across Lake Remoray sediment during the summer period, when hypoxic conditions settle in the deepest part of the water column. It was hypothesized that methane concentration and production would be higher in the deeper part of lake, our results showed that some littoral areas exhibited similar or higher values than the deepest area. The full 16S rRNA gene sequencing dataset counted 41 methanogen OTUs in abundances that depended more on sampling-site location than on the water depth gradient. The methanogen co-occurrence network revealed the existence of 5 distinct sub communities, suggesting the presence of different methanogen niches across Lake Remoray. The variation in abundance of the two larger methanogen sub communities was significantly related to methanogenesis potential and sediment methane concentration across-lake. Taken together, our results highlight the need to consider the ecosystem at whole-lake scale when calculating methane budgets, and the need to couple spatial and temporal approaches in order to better estimate lake ecosystems contribution to atmospheric methane.

Bottom waters hypoxia spreads in many lakes worldwide causing severe consequences on whole lakes trophic network. Here, we aimed at understanding the origin of organic matter stored in the sediment compartment and the related diversity of sediment microbial communities in a lake with deoxygenated deep water layers. We used a geostatistical approach to map and compare both the variation of organic matter and microbial communities in sediment. Spatialisation of C/N ratio and δ13C signature of sediment organic matter suggested that Lake Remoray was characterized by an algal overproduction which could be related to an excess of nutrient due to the close lake-watershed connectivity. Three spatial patterns were observed for sediment microbial communities after the hypoxic event, each characterized by specific genetic structure, microbial diversity and composition. The relative abundance variation of dominant microbial groups across Lake Remoray such as Cyanobacteria, Gammaproteobacteria, Deltaproteobacteria and Chloroflexi provided us important information on the lake areas where hypoxia occurs. The presence of methanogenic species in the deeper part of the lake suggests important methane production during hypoxia period. Taken together, our results provide an extensive picture of microbial communities' distribution related to quantity and quality of organic matter in a seasonally hypoxic lake.

International audience; Understanding temporal and spatial microbial community abundance and diversity variations is necessary to assess the functional roles played by microbial actors in the environment. In this study, we investigated spatial variability and temporal dynamics of two functional microbial sediment communities, methanogenic Archaea and methanotrophic bacteria, in Lake Bourget, France. Microbial communities were studied from 3 sites sampled 4 times over a year, with one core sampled at each site and date, and 5 sediment layers per core were considered. Microbial abundance in the sediment were determined using flow cytometry. Methanogens and methanotrophs community structures, diversity, and abundance were assessed using T-RFLP, sequencing, and real-time PCR targeting mcrA and pmoA genes, respectively. Changes both in structure and abundance were detected mainly at the water-sediment interface in relation to the lake seasonal oxygenation dynamics. Methanogen diversity was dominated by Methanomicrobiales (mainly Methanoregula) members, followed by Methanosarcinales and Methanobacteriales. For methanotrophs, diversity was dominated by Methylobacter in the deeper area and by Methylococcus in the shallow area. Organic matter appeared to be the main environmental parameter controlling methanogens, while oxygen availability influenced both the structure and abundance of the methanotrophic community.

To develop a library-dependent method of tracking fecal sources of contamination of beaches on the Atlantic coast of southwestern France, a library of 6368 Escherichia coli isolates was constructed from samples of feces, from 40 known human or animal sources collected in the vicinity of Arcachon Bay in 2010, and in French Basque Country, Landes, and Béarn, between 2017 and 2018. Different schemes of source identification were tested: use of the complete or filtered reference library; characterization of the isolates by genotypic or proteomic profiling based on ERIC-PCR or MALDI-TOF mass spectrometry, respectively; isolate by isolate assignment using either classifiers based on the Pearson similarity or SVM (support vector machine). With the exception of one source identification scheme, which was discarded since it used self-assignment, all tested schemes resulted in low rates of correct classification (15%). The heterogeneous coverage of E. coli genotypic diversity between sources and the uneven distribution of E. coli genotypes in the library likely explain the difficulties encountered in identifying the sources of fecal contamination. Shannon diversity index of sources ranged from 0 for several wildlife species sampled once to 3.03 for sewage treatment plant effluents sampled on various occasions, showing discrepancies between sources. The uneven genotypic composition of the library was attested by the value of the Pielou index (0.54), the high proportion of nondiscriminatory genotypes (>91% of the isolates), and the very low proportion of discriminatory genotypes (

International audience; Since the early 20's antibiotics have been massively produced and consumed for the benefit of bothhuman and animal health. Nevertheless, antibiotics have also reached the aquatic environmentthrough diffuse sources (e.g. veterinary treatment, contaminated manure application...) and throughwastewater. Consequently, antibiotics concentrations between the ng/L and μg/L range are regularlydetected in surface water and those molecules have also been found in sediments and aquatic biota.The ubiquitous presence of antibiotics exerts a selective pressure on microbial communities leading tothe acquisition and dissemination of antibioresistance in the environment. While both antibiotics andantibioresistance have been found in different aquatic compartments, more investigation is requiredto better understand their distribution and to identify hot spots of accumulation.In this context, we investigated the repartition of antibiotics and antibioresistance in different aquaticcompartments on 4 stations belonging to regional observatories and presenting contrasting levels ofpharmaceuticals: 2 on the Arve river belonging to Sipibel observatory and 2 on Lake Geneva belongingto the Observatory on Lakes. On the Arve river, the 2 stations were located up- and down- stream thedischarge place of a wastewater treatment plant (WWTP) collecting both urban and hospitalwastewaters. On Lake Geneva, one station was located in a relatively pristine area while the secondwas close to the discharge of an urban WWTP. To better identify a potential temporal dynamic ofantibiotics and antibioresistances over seasons, 6 samplings were conducted during 1.5 year. On eachsampling campaign, the following parameters were determined: (i) antibiotics levels in water,sediments and periphyton; (ii) antibioresistance in periphyton and sediments using various techniques:detection of resistance genes, integrons quantification, detection of tolerance acquisition via a PICT(Pollution Induced Community Tolerance) approach; (iii) antibiotics biodegradation potential ofmicrobial community from sediments (by radiorespirometric measurement); (iv) diversity of bacteriaand diatoms in periphyton and sediments; (v) physico-chemical parameters and (vi) metalliccontamination in sediments.While microbial resistance to antibiotics is commonly assessed by quantifying resistance genes orisolating antibiotic resistant bacteria, antibiotic resistance can also be estimated by measuring theacquisition of antibiotics tolerance at community level, following a PICT approach. In our study,periphytic microbial communities from the Arve river were found to have a higher tolerance to thetested antibiotics (ciprofloxacin, ofloxacin, sulfamethazine and erythromycin) than communities fromLake Geneva, in agreement with the expected levels of contamination. In addition, in some cases, ahigher tolerance was also found at stations close to WWTP effluents than in upstream/protectedstations. For example, periphytic microbial communities collected downstream the WWTP on the Arveriver were generally found to have a higher tolerance to ciprofloxacin than the upstream communities.Comparing whole community tolerance to other classical indicators of antibioresistance and toantibiotics levels in the aquatic ecosystems allows us to better understand the interconnectionbetween pharmaceutical exposure, in situ tolerance and genetic potential for antibioresistance.

To develop a library-dependent method of tracking fecal sources of contamination of beaches on the Atlantic coast of southwestern France, a library of 6368

International audience; Sediment microbial communities were exposed for 21 days to an environmental concentration of copper to assess Cu-induced composition changes and resulting effects on microbial sensitivity to acute Cu and As toxicity. Chronic Cu exposure reduced the diversity of the bacterial and archaeal communities from Day 0 to Day 21. The pollution-induced community tolerance concept (PICT) predicts that loss of the most sensitive taxa and gain of more tolerant ones should increase the capacity of Cu-exposed communities to tolerate acute Cu toxicity. Although diversity loss and functional costs of adaptation could have increased their sensitivity to subsequent toxic stress, no increased sensitivity to As was observed. PICT responses varied according to heterotrophic activity, selected as the functional endpoint for toxicity testing, with different results for Cu and As. This suggests that induced tolerance to Cu and As was supported by different species with different metabolic capacities. Ecological risk assessment of contaminants would gain accuracy from further research on the relative contribution of tolerance acquisition and co-tolerance processes on the functional response of microbial communities

Global warming is expected to raise temperatures in freshwater lakes, which have been acknowledged to contribute up to 10% of the atmospheric methane concentrations. Increasing temperature enhances methane production and oxidation rates, but few studies have considered the balance between both processes at experimentally higher temperatures within lake sediments. The temperature dependence of methane concentrations, methane production rates, and methanogenic (mcrA) and methanotrophic (pmoA) community size was investigated in intact sediment cores incubated with aerobic hypolimnion water at 4, 8, and 12°C over 3 weeks. Sediment cores of 25 cm length were collected at two temperate lakes—Lake Stechlin (Germany; mesotrophic-oligotrophic, maximum depth 69.5 m) and Lake Geneva (France/Switzerland; mesotrophic, maximum depth 310 m). While methane production rates in Lake Stechlin sediments did not change with increasing temperatures, methane concentrations decreased significantly. In contrast, methane production rates increased in 20–25 cm in Lake Geneva sediments with increasing temperatures, but methane concentrations did not differ. Real-time PCR demonstrated the methanogenic and methanotrophic community size remained stable independently of the incubation temperature. Methane concentrations as well as community sizes were 1–2 orders of magnitude higher in Lake Stechlin than in Lake Geneva, while potential methane production rates after 24 h were similar in both lakes, with on average 2.5 and 1.9 nmol g−1 DW h−1, respectively. Our results suggest that at higher temperatures methane oxidation could balance, and even exceed, methane production. This suggests that anaerobic methane oxidation could be involved in the methane balance at a more important rate than previously anticipated.

The significance of methane production by lakes to the global production of greenhouse gas is well acknowledged while underlying processes sustaining the lacustrine methane budget remain largely unknown. We coupled biogeochemical data to functional and phylogenetic analyses to understand how sedimentary parameters characterize the methane cycle vertically and horizontally in the ice-covered bay of the second largest lake in Europe, Lake Onego, Russia. Our results support a heterogeneous winter methane cycle, with higher production and oxidation closest to riverine inputs. Close to the river mouth, the largest numbers of copies of methane-related functional genes pmoA and mcrA were associated with a specific functional community, and methane production potential exceeded oxidation, resulting in 6–10 times higher methane fluxes than in the rest of the bay. The elevated fluxes arise from the spatial differences in quantity and type (lacustrine versus riverine sources) of organic matter. More homogeneity is found toward the open lake, where the sediment is vertically structured into 3 zones: a shallow zone of methane oxidation; a transitional zone (5–10 cm) where anaerobic methane oxidation is dominant; and a methane production zone below. This vertical pattern is structured by the redox gradient and human-induced changes in sedimentary inputs to the bay. Retrieved 16S rRNA gene sequences from Candidatus Methanoperedens and Cand. Methylomirabilis suggest that anaerobic oxidation of methane occurs in these freshwater lake sediments. ISSN:2044-205X ISSN:2044-2041

The first French-Swiss workshop on ecotoxicology of freshwater sediment communities was co-organized by the French Research Institute of Science and Technology for Environment and Agriculture (Irstea) and the Swiss Centre for Applied Ecotoxicology (Ecotox Centre EAWAG-EPFL) in Villie-Morgon (Beaujolais Region, France) on April 27–28, 2017. The workshop brought together scientists working in different fields of expertise (ecotoxicologists, ecologists, environmental chemists…), environmental stakeholder groups and managers, as well as economic players (start-ups and consultancies) to better connect research needs of potential end-users with research outputs. The objectives of this workshop were (i) to establish the state of the art of research in the characterization of sediment contamination and in the evaluation of the effects on sediment-associated biological communities and ecosystem functioning and (ii) to give an overview of the French and Swiss regulations dealing with the assessment of contaminated sediments in freshwater ecosystems. The ultimate goal was to collectively identify research needs and knowledge gaps, as well as to highlight ways to improve the ecotoxicological assessment of sediments in freshwater environments by further considering the structure and functions of associated microbial and invertebrate communities.

Une approche de Microbial Source Tracking dite culture- et collection-dependante a ete developpee pendant 1 an pour faciliter l’identification des sources de pollution en cas d’episode de contamination fecale dans le bassin d’Arcachon (Gironde, France). Cette approche repose sur la comparaison d’une collection dite « environnementale » de genotypes obtenus dans des echantillons d’eau ou de sediment avec une collection dite « de reference » de genotypes provenant de matieres fecales d’origine connue (par exemple homme, animaux domestiques ou faune sauvage). La methode d’analyse consiste en l’isolement par culture sur milieu selectif d’indicateurs bacteriens de contamination fecale ( Escherichia coli et enterocoques fecaux) suivi d’un genotypage par Enterobacterial Repetitive Intragenic PCR (ERIC-PCR). Les caracteristiques genotypiques de chaque souche sont repertories dans une base de donnees.Pour E. coli , la base de donnees est constituee de 6 263 isolats obtenus a partir d’echantillons d’eau de mer ( n = 159), de sediments ( n = 119) et de feces d’animaux ou d’echantillons provenant de station d’epuration ( n = 169). La collection environnementale (3 565 isolats) contient 625 genotypes distinct dont 184, seulement, sont communs avec la collection de reference (2 698 isolats). Pour les enterocoques fecaux, elle est constituee de 3 941 isolats obtenus a partir d’echantillons d’eaux de mer ( n = 83), sediments ( n = 59) et feces d’animaux ou station d’epuration ( n = 128). La collection environnementale (2 013 isolats) represente 505 genotypes distincts dont seulement 137 sont communs avec la collection de reference (1 928 isolats). Seuls 29 % des 625 genotypes d’ E. coli et 27 % des 505 genotypes d’enterocoques fecaux etaient communs aux deux collections.L’analyse comparee des deux collections montre qu’une forte proportion de genotypes de la collection environnementale, 71 % pour E. coli et 73 % pour les enterocoques fecaux, n’ont ete detectes que dans l’eau ou les sediments. Cela peut suggerer l’existence d’une forte proportion de souches naturalisees.Des taux de classification correcte (ARCC) tres satisfaisants ont ete obtenus pour E. coli (65,2 %) et pour les enterocoques fecaux (ARCC = 88,7 %). De plus, nous avons pu confirmer l’origine aviaire d’une forte proportion d’isolats recueillis dans l’eau et les sediments d’un etang regulierement frequente par des canards colverts et des foulques macroules. Les valeurs d’ARCC et les resultants obtenus sur un site test valident la base de donnees constituee.

International audience; The aim of this work was to study the behavior over time of a separator made of a low-cost and non-selective microporous polyethylene membrane (RhinoHide (R)) in an air-cathode microbial fuel cell with a reticulated vitreous carbon foam bioanode. Performances of the microporous polyethylene membrane (RhinoHide (R)) were compared with Nafion (R)-117 as a cationic exchange membrane. A non-parametric test (Mann-Whitney) done on the different sets of coulombic or energy efficiency data showed no significant difference between the two types of tested membrane (p < 0.05). Volumetric power densities were ranging from 30 to 90 W.m(-3) of RVC foam for both membranes. Similar amounts of biomass were observed on both sides of the polyethylene membrane illustrating bacterial permeability of this type of separator. A monospecific denitrifying population on cathodic side of RhinoHide (R) membrane has been identified. Electrochemical impedance spectroscopy (EIS) was used at OCV conditions to characterize electrochemical behavior of MFCs by equivalent electrical circuit fitted on both Nyquist and Bode plots. Resistances and pseudo-capacitances from EIS analyses do not differ in such a way that the nature of the membrane could be considered as responsible.

Over a five year period (2004–08), 1171 surface water samples were collected from up to 24 sampling locations representing a wide range of stream orders, in a river basin in eastern Ontario, Canada. Water was analyzed for Cryptosporidium oocysts and Giardia cyst densities, the presence of Salmonella enterica subspecies enterica, Campylobacter spp., Listeria monocytogenes, and Escherichia coli O157:H7. The study objective was to explore associations among pathogen densities/occurrence and objectively defined land use, weather, hydrologic, and water quality variables using CART (Classification and Regression Tree) and binary logistical regression techniques. E. coli O157:H7 detections were infrequent, but detections were related to upstream livestock pasture density; 20% of the detections were located where cattle have access to the watercourses. The ratio of detections:non-detections for Campylobacter spp. was relatively higher (>1) when mean air temperatures were 6% below mean study period temperature values (relatively cooler periods). Cooler water temperatures, which can promote bacteria survival and represent times when land applications of manure typically occur (spring and fall), may have promoted increased frequency of Campylobacter spp. Fifty-nine percent of all Salmonella spp. detections occurred when river discharge on a branch of the river system of Shreve stream order = 9550 was >83 percentile. Hydrological events that promote off farm/off field/in stream transport must manifest themselves in order for detection of Salmonella spp. to occur in surface water in this region. Fifty seven percent of L. monocytogenes detections occurred in spring, relative to other seasons. It was speculated that a combination of winter livestock housing, silage feeding during winter, and spring application of manure that accrued during winter, contributed to elevated occurrences of this pathogen in spring. Cryptosporidium and Giardia oocyst and cyst densities were, overall, positively associated with surface water discharge, and negatively associated with air/water temperature during spring-summer-fall. Yet, some of the highest Cryptosporidium oocyst densities were associated with low discharge conditions on smaller order streams, suggesting wildlife as a contributing fecal source. Fifty six percent of all detections of ≥2 bacteria pathogens (including Campylobacter spp., Salmonella spp., and E. coli O157:H7) in water was associated with lower water temperatures (∼27 mm (62 percentile). During higher water temperatures (>∼14 °C), a higher amount of weekly rainfall was necessary to promote detection of ≥2 pathogens (primarily summer; weekly rainfall ∼>42 mm (>77 percentile); 15% of all ≥2 detections). Less rainfall may have been necessary to mobilize pathogens from adjacent land, and/or in stream sediments, during cooler water conditions; as these are times when manures are applied to fields in the area, and soil water contents and water table depths are relatively higher. Season, stream order, turbidity, mean daily temperature, surface water discharge, cropland coverage, and nearest upstream distance to a barn and pasture were variables that were relatively strong and recurrent with regard to discriminating pathogen presence and absence, and parasite densities in surface water in the region.

Escherichia coli is an indicator of fecal pollution used to mandate recreational and drinking water quality. Concentrations of culturable E. coli following contamination of surface water are determined by three factors: dilution; cell attachment to particulate material and settling or resuspension in the water column; and the net rate of change in viability. This study evaluated the variability in the latter parameter, and how predictive variation in death rate was of culturable population densities at the time of sampling. Water samples (N =232) with varying levels of E. coli contamination were collected from 46 discrete locations in four watersheds across Canada over a three-month period and enumerated for culturable E. coli by membrane filtration plate counting (T0EC ). Water samples were again enumerated following a laboratory 24 h holding period at 30°C in the dark, and the difference considered the death rate (ΔEC ). Relationships of T0EC and ΔEC with environmental and water chemistry factors were explored using step-wise multiple regression. The model predicting T0EC indicated that stream order, total rainfall seven days in advance of sampling day, total phosphorus, and ΔEC were the most significant contributors. The model predicting ΔEC indicated that turbidity and NH3 +NH4 were the most important contributors. A model suggests that the persistence factor is less important than dilution (i.e. stream order) in describing E. coli densities, followed by factors that influence the loading of E. coli into watersheds.

We investigated the prevalence and diversity of Escherichia coli strains isolated from surface waters from multiple watersheds within the South Nation River basin in eastern Ontario, Canada. The basin is composed of mixed but primarily agricultural land uses. From March 2004 to November 2007, a total of 2,004 surface water samples were collected from 24 sampling sites. E. coli densities ranged from undetectable to 1.64 × 10 5 CFU 100 ml −1 and were correlated with stream order and proximity to livestock production systems. The diversity of 21,307 E. coli isolates was characterized using repetitive extragenic palindromic PCR (rep-PCR), allowing for the identification of as many as 7,325 distinct genotypes, without capturing all of the diversity. The community was temporally and spatially dominated by a few dominant genotypes (clusters of more than 500 isolates) and several genotypes of intermediary abundance (clustering between 10 and 499 isolates). Simpson diversity indices, assessed on a normalized number of isolates per sample, ranged from 0.050 to 0.668. Simpson indices could be statistically discriminated on the basis of year and stream order, but land use, discharge, weather, and water physical-chemical properties were not statistically important discriminators. The detection of Campylobacter species was associated with statistically lower Simpson indices (greater diversity; P < 0.05). Waterborne E. coli isolates from genotypes of dominant and intermediary abundance were clustered with isolates obtained from fecal samples collected in the study area over the same period, and 90% of the isolates tested proved to share genotypes with fecal isolates. Overall, our data indicated that the densities and distribution of E. coli in these mixed-use watersheds were linked to stream order and livestock-based land uses. Waterborne E. coli populations that were distinct from fecal isolates were detected and, on this basis, were possibly naturalized E. coli strains.

Escherichia coli can be used to help identify sources of fecal contamination in the environment. Escherichia coli genotypic fecal libraries and pattern-matching algorithms were assessed for their effectiveness in correctly identifying sources. Fecal samples (n = 172) were collected from various sources from three agricultural landscapes in Canada. Escherichia coli isolates were fingerprinted using BOX- and enterobacterial repetitive intergenic consensus (ERIC) - polymerase chain reaction primers, revealing 769 and 1 057 distinct genotypes, respectively, for the 9 047 isolates collected in 2004 in Ontario. The average rate of correct classification (ARCC) was comparable for BOX- (48%) and ERIC-based (62%) libraries and between libraries with clones removed per sample (55%) and clones removed per unit (54%). ARCC increased with fewer classification units (from 44% to 65%). ARCC for k-nearest neighbour (64%) and maximum similarity (60%) algorithms were comparable, but maximum similarity had better sensitivity and specificity than k-nearest neighbour. Geographical and temporal shifts in community composition resulted in loss of accuracy. Several ERIC genotypes (n = 112) were common between sources and were removed from the library, improving ARCC (77%). The latter library proved to be more accurate, but its accuracy with respect to sourcing environmental isolates remains to be tested.

Intensive livestock production systems produce significant quantities of excreted material that must be managed to protect water, air, and crop quality. Many jurisdictions mandate how livestock wastes are managed to protect adjacent water quality from microbial and chemical contaminants that pose an environmental and human health challenge. Here, we consider innovative livestock waste treatment systems in the context of multi-barrier strategies for protecting water quality from agricultural contamination. Specifically, we consider some aspects of how enteric bacterial populations can evolve during manure storage, how their fate following land application of manure can vary according to manure composition, and finally the challenge of distinguishing enteric pathogens of agricultural provenance from those of other sources of fecal pollution at a policy-relevant watershed scale. The beneficial impacts of livestock waste treatment on risk to humans via exposure to manured land are illustrated using quantitative microbial risk assessment (QMRA) scenarios. Overall, innovative livestock treatment systems offer a crucially important strategy for making livestock wastes more benign before they are released into the broader environment.

The South Nation River basin in eastern Ontario, Canada is characterized by mixed agriculture. Over 1600 water samples were collected on a bi-weekly basis from up to 24 discrete sampling sites on river tributaries of varying stream order within the river basin between 2004 and 2006. Water samples were analyzed for: densities of indicator bacteria ( Escherichia coli , Clostridium perfringens , enterococci, total and fecal coliforms), the presence of pathogenic bacteria ( Listeria monocytogenes , E. coli O157:H7, Salmonella spp., Campylobacter spp.), and densities of parasite Giardia cysts and Cryptosporidium oocysts. Relationships between indicator bacteria, pathogens, and parasite oocysts/cysts were overall weak, seasonally dependent, site specific, but primarily positive. However, L. monocytogenes was inversely related with indicator bacteria densities. Campylobacter , Salmonella , Giardia cysts and Cryptosporidium oocysts were most frequently detected in the fall. E. coli O157:H7 was detected at a very low frequency. Exploratory decision tree analyses found overall that E. coli densities were the most utilitarian classifiers of parasite/pathogen presence and absence, followed closely by fecal coliforms, and to a lesser extent enterococci and total coliforms. Indicator bacteria densities that classified pathogen presence and absence groupings, were all below 100 CFU per 100 mL −1 . Microorganism relationships with rainfall indices and tributary discharge variables were globally weak to modest, and generally inconsistent among season, site and microorganism. But, overall rainfall and discharge were primarily positively associated with indicator bacteria densities and pathogen detection. Instances where a pathogen was detected in the absence of a detectable bacterial indicator were extremely infrequent; thus, the fecal indicators were conservative surrogates for a variety of pathogenic microorganisms in this agricultural setting. The results from this study indicate that no one indicator or simple hydrological index is entirely suitable for all environmental systems and pathogens/parasites, even within a common geographic setting. These results place more firmly into context that robust prediction and/or indicator utility will require a more firm understanding of microorganism distribution in the landscape, the nature of host sources, and transport/environmental fate affinities among pathogens and indicators.

International audience; In the natural environment, microbial community structure of river biofilm is controlled by biotic and abiotic factors. This study explored the capacity to manipulate the structure of microbial communities by modifying environmental conditions during the course of biofilm development. River epilithic biofilm was cultivated in situ on artificial substrates placed parallel to river water flow. Substrates were incubated for 3 and 5.5 weeks in river to allow natural biofilm development, at two sites with contrasting physico-chemical characteristics. The first site (Aurade' , Gers, France) was located in an agricultural watershed basin and the second site (Larroque, Haute-Garonne, France) was located in a forested watershed basin. After 3 weeks of biofilm development, a subset of substrates was collected from one site and transplanted to the second site where they remained for 2.5 further weeks. Epilithic bacterial community structure (at 3 weeks from each site and at 5.5 weeks from biofilms with and without transplantation) was assessed using PCR-DGGE of 16S rDNA fragment. Biofilm biomass was estimated using ash free dry mass (AFDM). After 3 weeks of development, biofilms from the two sites exhibited comparable AFDM values (average of 1.4¡0.2 g.mx2). A difference between the two sites was observed after 5.5 weeks of development: AFDM decreased for biofilms from the agricultural watershed basin (from 1.4 to 0.18 g.mx2) as a consequence of grazing pressure (Bithynia), and increased for biofilms from the forested agricultural watershed (from 1.4 to 2.6 g.mx2). Microbial community analyses revealed a differentiated community structure between biofilms from the different sites and exhibited a change of microbial community structure after 5.5 weeks of biofilm development. These observations confirm a process of ecological succession in microbial communities. Changing the incubation site during biofilm development modified the trajectory of these ecological successions, suggesting that site characteristics mainly conditioned the structure of these microbial communities.

Studies on the effects of biodiversity on ecosystem functioning have generally revealed a positive asymptotic relationship between biodiversity and single functions, suggesting species redundancy with respect to these functions. However, most research was performed on specific processes and did not consider ecosystem 'multifunctionality'. There is also little information on the relationship between genetic and functional diversity. To analyze this relationship, we performed a microcosm experiment on a complex lake assemblage of decomposers, in the presence of the green alga Scenedesmus obliquus, which acted as carbon source for decomposers. By manipulating nutrient enrichment and the N : P input ratio, we observed that the structures of particle-associated and free bacterial assemblages were highly predictable in response to stoichiometric constraints. For a given treatment, the taxonomic compositions of free and particle-associated bacterial communities appeared close to each other only when phosphorus was not depleted. A coinertia analysis revealed a clear coupling between the genetic diversity of the microbial community, assessed using PCR-denatured gradient gel electrophoresis, and its potential functional diversity, studied with Biolog Ecoplates. This suggests that an ecologically relevant fraction of bacterial communities is characterized by lower level of redundancy than frequently thought, highlighting the necessity of exploring further the role of biodiversity in multifunctionality within ecosystems.

International audience; The reconstruction of past pastoral activities based on microscopic methods (pollen and coprophilous fungal ascospores) does not accurately identify the domestic species involved. In contrast, source-specific DNA markers, commonly employed in water quality microbial source tracking (MST) studies, may represent a promising tool for retrospectively identifying species-specific fecal contamination in sediment deposition. In the present study, molecular methods were used to quantify Bacteroidales and identify ovine and bovine mitochondrial DNA extracted from sediment cores from two forest hollows comprising 2000 years of deposition. The DNA marker abundance was contrasted with the abundance of ascospores and plant-specific pollen throughout the sediment chronosequence. The distribution of DNA markers indicated an agro-pastoral practice transition from pasture/crop production to forested landscape from the second Iron Age/classical Antiquity to the end of the Roman period/modernity, in correlation with microscopic markers. During the second Iron Age/classical Antiquity, hollows were likely used to water herds, whereas during the late Antiquity, low Bacteroidales abundances and the sporadic detection of bovine and ovine DNA markers confirm the progressive afforestation observed using pollen data. For the end of the Roman period and modern times, reforested areas are characterized by the absence of ovine and bovine DNA markers while low Bacteroidales abundances suggest the presence of wild herbivores. The present study has established that in tandem with microscopic methods, sediment-archived fecal-specific bacterial and mitochondrial DNA are extremely useful for reconstructing agricultural practice over timeframes of millennia.

Summary Decennial changes in Planktothrix rubescens diversity and dynamics were reconstructed by applying molecular tools to analyse DNA and RNA extracted from lake sediments. The sediments studied were sampled from a deep peri-alpine lake that has experienced both dramatic shifts in trophic conditions and large-scale climatic changes. Palaeolimnological proxies were combined with statistical modelling to investigate the relative influence of phosphorus concentrations and temperature changes on the extent of Planktothrix blooms over the last century. Phylogenetic analysis revealed that the overall composition of the cyanobacterial community changed over the transition from oligotrophic to eutrophic conditions. When the relative abundance of Planktothrix decreased in the 1970s, concomitant with eutrophication, total cyanobacterial abundance remained high and more Anabaena and Microcystis sequences were detected. In spite of such drastic environmental changes, the lake provided a constant niche for one particular Planktothrix species, which was consistently present from the 1920s to the present day. Phosphorus concentration was found to be the dominant driver of the relative abundance of P. rubescens, with the highest abundances observed during mesotrophic conditions. The relative role of climate was nutrient-dependent, with warmer springs having a positive effect on P. rubescens abundance only during mesotrophic periods. Overall, this study confirms that analysis of genetic signatures preserved in sediment archives allows assessment of key palaeoenvironmental indicator species that have no diagnostic microscopic cellular features in the sediment record. In the case of cyanobacteria, palaeogenetics offer unique opportunities to anticipate how future climate change might affect the response of P. rubescens to phosphorus concentration.

Understanding spatial microbial community variation is necessary to assess diversity patterns. In this study, we investigated spatial heterogeneity and variability of functional and total microbial benthic community structures in Lake Bourget, France. Community structure variability was determined vertically by comparing three sediment layers per core, and horizontally at the intra-site level (between up to three cores per sites) and between three sites. Bacterial, archaeal, methanotrophs and methanogens community structures were assessed by genotyping the 16S rRNA–23S rRNA intergenic spacer, 16S rRNA, pmoA and mcrA genes, respectively. The consequence of pooling DNA extracts before genotyping was tested. After pooling, the OTU number decreased for all communities, but it had no effect on structure. At the core scale, bacterial community structure significantly differed between the sediment layers, but archaeal, methanogens and methanotrophs community structures only differed significantly between the superficial and deeper layers. Changes in environmental conditions affected microbial community structures between sites (sediment carbonates, total carbon contents, median particle sizes, and water O2 saturation), but not intra-site, as no significant changes were observed at the horizontal scale. These spatial scales should be considered to understand their importance for biogeochemical cycle when assessing benthic microbial community structure and diversity in lakes.

Temporal bacterial community changes in river biofilms were studied using 16S rRNA gene-based polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) followed by sequence analysis. Naturally occurring biofilms were sampled in 2001 during an undisturbed 7-month low-water period in the River Garonne (SW France). During the sampling period epilithic biomass exhibited a particular pattern: two 3-month periods of accumulation that resulted in two peaks in summer and fall, each at about 25 g ash-free dry mass per square meter. Bacterial community DGGE profiles differed between the summer and fall biomass peaks and shared only 30% common operational taxonomic units (OTUs), suggesting the influence of seasonal factors on these communities. During the second biomass accrual phase, bacterial richness and the appearance of new OTUs fitted a conceptual model of bacterial biofilm succession. During succession, five OTUs (corresponding to Dechloromonas sp., Nitrospira sp., and three different Spirosoma spp.) exhibited particular patterns and were present only during clearly defined successional stages, suggesting differences in life-history strategies for epilithic bacteria. Co-inertia analysis of DGGE banding patterns and physical-chemical data showed a significant relationship between community structure and environmental conditions suggesting that bacterial communities were mainly influenced by seasonal changes (temperature, light) and hydrodynamic stability. Within the periods of stability, analysis of environmental variables and community patterns showed the dominant influence of time and maturation on bacterial community structure. Thus, succession in these naturally occurring epilithic biofilm assemblages appears to occur through a combination of allogenic (seasonal) and autogenic changes.

PCR-DGGE analysis of a 16S rDNA fragment was used to determine spatial patterns of epilithic bacteria diversity over a 100 km river section. Epilithic biofilms were collected on natural substrata in a river reach (mid-slope of the river Garonne) where changes in epilithic bacterial activities and densities were expected due to the presence of a major urban centre (Toulouse city). Nitrogen and phosphorus forms increased 2- to 10-fold downstream of this urban centre. Close values of biomass (18 to 27 g m -2 in 2000 and 10 to 16 g m -2 in 2001), bacterial densities (3 to 11 × 10 12 cells m -2 for total bacteria) and activities (3.6 mg N m -2 h -1 for nitrification and 0.3 to 1.1 mg N m -2 h -1 for denitrification) were recorded at the different sampling sites. Conversely, in regard to community composition, up- and downstream samples were discriminated between according to their banding patterns: >60% similarity within clusters versus

Microbial denitrification is the main nitrogen removing process in freshwater ecosystems. The aim of this study was to show whether and how water warming (+ 2.5 °C) drives bacterial diversity and structuring and how bacterial diversity affects denitrification enzymatic activity in phototrophic river biofilms (PRB). We used water warming associated to the immediate thermal release of a nuclear power plant cooling circuit to produce natural PRB assemblages on glass slides while testing 2 temperatures (mean temperature of 17 °C versus 19.5 °C). PRB were sampled at 2 sampling times during PRB accretion (6 and 21 days) in both temperatures. Bacterial community composition was assessed using ARISA. Denitrifier community abundance and denitrification gene mRNA levels were estimated by q-PCR and qRT-PCR, respectively, of 5 genes encoding catalytic subunits of the denitrification key enzymes. Denitrification enzyme activity (DEA) was measured by the acetylene-block assay at 20 °C. A mean water warming of 2.5 °C was sufficient to produce contrasted total bacterial and denitrifier communities and, therefore, to affect DEA. Indirect temperature effect on DEA may have varied between sampling time, increasing by up to 10 the denitrification rate of 6-day-old PRB and decreasing by up to 5 the denitrification rate of 21-day-old PRB. The present results suggest that indirect effects of warming through changes in bacterial community composition, coupled to the strong direct effect of temperature on DEA already demonstrated in PRB, could modulate dissolved nitrogen removal by denitrification in rivers and streams.

EA MERS CT3 Enjeu 3; International audience; Listeria monocytogenes is a facultative intracellular pathogen distributed in the environment. Reference isolation methods include an enrichment step. We compared 2 protocols: one based on enrichment and the other on direct plating, with respect to the efficiency of isolation of L. monocytogenes from surface water samples. From March to May 2006, 126 surface water samples were collected biweekly from 24 discrete sampling sites located in the South Nation River basin (Ontario). Presumptive Listeria spp. were isolated using the 2 methods, and L. monocytogenes isolates were confirmed based on colony morphology and amplification of 3 pathogenicity genes: iap, inlA, and hlyA. Listeria monocytogenes was detected in 31 water samples using the enrichment approach and in 27 using the direct-plating approach, with only 13 samples positive using both approaches. No correlation was observed between the ability of the enrichment procedure to fail in the detection of the pathogenic bacterium and the abundance of several indicator bacteria. The proportion of samples carrying isolates from serovar groups 1/2a, 3a; 1/2b, 3b, 7; 4b, 4d, 4e; and Listeria spp. was significantly different between the 2 approaches. This data indicate that a direct-plating approach would be suitable for the detection of L. monocytogenes in water samples, but that enrichment and direct-plating protocols both have specific biases.; Listeria monocytogenes est un pathogène intracellulaire facultatif répandu dans l’environnement. La méthoded’isolement de référence inclut une étape d’enrichissement. Nous avons comparé 2 protocoles, basés sur un enrichissementou un étalement direct, pour l’efficacitéd’isolement de L. monocytogenes dans des échantillons d’eau de surface. De mars à mai 2006, 126 échantillons d’eau, provenant de 24 sites d’échantillonnage situés sur le bassin de la rivière South Nation (Ontario), ont été prélevés bimensuellement. Les colonies de Listeria spp. présumées ont été isolées par les 2 méthodes et les isolats confirmés sur la base de la morphologie des colonies et l’amplification de 3 gènes impliqués dans la pathogénicité de L. monocytogenes : iap, inlA et hlyA. Listeria monocytogenes a été détecté dans 31 échantillons par enrichissement et dans 27 échantillons par étalement direct, avec seulement 13 échantillons positifs par l’utilisation des 2 approches. Aucune corrélation n’a été observée entre la détection du pathogène et l’abondance de plusieurs bactéries indicatrices. La proportion d’échantillons contenant des isolats appartenant aux groupes sérologiques 1/2a, 3a; 1/2b, 3b, 7; 4b, 4d, 4e et Listeria spp. était significativement différente entre les 2 approches. Ceci indique que l’étalement direct serait adapté a` la détection de L. monocytogenes dans des échantillons d’eau, mais que chacune des approches présente ses propres biais.

Denitrification is an ecosystem service of nitrogen load regulation along the terrestrial-freshwater-marine continuum. The present study documents the short-term temperature sensitivity of denitrification enzyme activity in phototrophic river biofilms as a typical microbial assemblage of this continuum. Denitrification measurements were performed using the acetylene inhibition method at four incubation temperatures: 1.1, 12.1, 21.2 and 30.9°C. For this range of temperature, N(2)O production could be fitted to an exponential function of incubation temperature, yielding mean (±standard error) activation energy of 1.42 (±0.24) eV and Q(10) of 7.0 (±1.4). This first quantification of denitrification enzyme activity temperature dependence in phototrophic river biofilms compares with previous studies performed in soils and sediments. This demonstrates the high temperature dependence of denitrification as compared to other community-level metabolisms such as respiration or photosynthesis. This result suggests that global warming can unbalance natural community metabolisms in phototrophic river biofilms and affect their biogeochemical budget.

Electroactivity is a property of microorganisms assembled in biofilms that has been highlighted in a variety of environments. This characteristic was assessed for phototrophic river biofilms at the community scale and at the bacterial population scale. At the community scale, electroactivity was evaluated on stainless steel and copper alloy coupons used both as biofilm colonization supports and as working electrodes. At the population scale, the ability of environmental bacterial strains to catalyze oxygen reduction was assessed by cyclic voltammetry. Our data demonstrate that phototrophic river biofilm development on the electrodes, measured by dry mass and chlorophyll a content, resulted in significant increases of the recorded potentials, with potentials of up to +120 mV/saturated calomel electrode (SCE) on stainless steel electrodes and +60 mV/SCE on copper electrodes. Thirty-two bacterial strains isolated from natural phototrophic river biofilms were tested by cyclic voltammetry. Twenty-five were able to catalyze oxygen reduction, with shifts of potential ranging from 0.06 to 0.23 V, cathodic peak potentials ranging from −0.36 to −0.76 V/SCE, and peak amplitudes ranging from −9.5 to −19.4 μA. These isolates were diversified phylogenetically ( Actinobacteria , Firmicutes , Bacteroidetes , and Alpha -, Beta -, and Gammaproteobacteria ) and exhibited various phenotypic properties (Gram stain, oxidase, and catalase characteristics). These data suggest that phototrophic river biofilm communities and/or most of their constitutive bacterial populations present the ability to promote electronic exchange with a metallic electrode, supporting the following possibilities: (i) development of electrochemistry-based sensors allowing in situ phototrophic river biofilm detection and (ii) production of microbial fuel cell inocula under oligotrophic conditions.

Studies on the effects of biodiversity on ecosystem functioning have generally revealed a positive asymptotic relationship between biodiversity and single functions, suggesting species redundancy with respect to these functions. However, most research was performed on specific processes and did not consider ecosystem 'multifunctionality'. There is also little information on the relationship between genetic and functional diversity. To analyze this relationship, we performed a microcosm experiment on a complex lake assemblage of decomposers, in the presence of the green alga Scenedesmus obliquus, which acted as carbon source for decomposers. By manipulating nutrient enrichment and the N : P input ratio, we observed that the structures of particle-associated and free bacterial assemblages were highly predictable in response to stoichiometric constraints. For a given treatment, the taxonomic compositions of free and particle-associated bacterial communities appeared close to each other only when phosphorus was not depleted. A coinertia analysis revealed a clear coupling between the genetic diversity of the microbial community, assessed using PCR-denatured gradient gel electrophoresis, and its potential functional diversity, studied with Biolog Ecoplates. This suggests that an ecologically relevant fraction of bacterial communities is characterized by lower level of redundancy than frequently thought, highlighting the necessity of exploring further the role of biodiversity in multifunctionality within ecosystems.

International audience; This study assessed the functional significance of attached and free-living bacterial communities involved in the process of denitrification in a shallow aquifer of a riparian zone (Garonne River, SW France). Denitrification enzyme activity (DEA), bacterial density (BD) and bacterial community composition (BCC) were measured in two aquifer compartments: the groundwater and the sandy fraction of the sediment deposit. Samples were collected in wells located inside (IHD) and outside (OHD) identified hotspots of denitrification. Despite high BD values (up to 1.14 × 1012 cells m−3), DEA was not detected in the water compartment (< 0.32 mg N–N2O m−3 d−1). The sandy fraction showed detectable DEA (up to 1,389 mg N–N2O m−3 d−1) and, consistent with BD pattern, higher DEA values were measured in IHD zones than in OHD zones. The BCC assessed by 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) partly supported this result: attached and free-living communities were significantly different (< 30% similarity) but patterns of BCC did not cluster according to IHD and OHD zones. Targeting the denitrifying communities by means of a culture enrichment step prior to 16S rDNA PCR-DGGE showed that the free-living and sediment attached communities differed. Most sequences obtained from DGGE profiles of denitrifying communities were affiliated to Proteobacteria and showed low genetic distance with taxa that have already been detected in aquifers (e.g., Azoarcus sp., Acidovorax sp. and Pseudomonas spp.). This study confirms that in the aquifer the sediment-attached fraction exhibits different functions (DEA) from free-living communities and suggests that this functional difference is related to the communities’ structure.

International audience; Listeria monocytogenes is a facultative intracellular pathogen that can be carried asymptomatically in various animals and can be shed in feces. We investigated the prevalence and characteristics of L. monocytogenes isolated from livestock, wildlife, and human potential sources of contamination in 2 areas in Ontario, Canada. From February 2003 to November 2005, a total of 268 fecal samples were collected from different animals. Listeria monocytogenes was isolated using selective enrichment, isolation, and confirmation procedures, and 15 samples (6%) yielded to the isolation of 84 confirmed strains. Listeria monocytogenes was isolated from livestock (beef and dairy), wildlife (deer, moose, otter, and raccoon), and human (biosolids and septic) fecal sources. Thirty-two isolates were from serovar 1/2a, 34 from serovar 1/2b, 1 from serovar 3a, and 17 from serovar 4b. Listeria monocytogenes populations were resolved into 13 EcoRI ribotypes, and 18 ApaI and 18 AscI pulsotypes, with Simpson indexes of discrimination of 0.878 and 0.907, respectively. A majority (59%) of L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, which was supported by higher entry into Caco-2 cells (9.3%) than isolates producing truncated and secreted internalin A (1.3% of entry). Listeria monocytogenes fecal isolates were on average resistant to 6.4 ± 2.5 antibiotics out of 17 tested, and potentially virulent isolates exhibited an enhanced resistance to kanamycin, gentamicin, streptomycin, and rifampicin. Livestock, wildlife, and human L. monocytogenes fecal communities exhibited overlapping but distinct populations, and some genotypes and phenotypes were similar to those previously described for surface water isolates in the same area.; Listeria monocytogenes est un pathogène intracellulaire facultatif, porté de façon asymptomatique chez de nombreux animaux et excrété dans les fèces. La distribution et les caractéristiques de souches de L. monocytogenes isolées à partir de matières fécales d’origine animale (bétail et sauvage) et humaine ont été étudié dans deux régions de l’Ontario, Canada. De février 2003 à novembre 2005, 268 échantillons fécaux ont été collectés. Listeria monocytogenes a été isolé suivant une procédure d’enrichissement sélectif, d’isolation et de confirmation. Quinze échantillons positifs (6 %) ont permis d’isoler 84 souches confirmées. Listeria monocytogenes a été isolée à partir d’échantillons fécaux d’animaux d’élevage (boeuf et vache laitière), d’animaux sauvages (cerf, élan, loutre et raton laveur), et d’origine humaine (biosolides et fosse septique). Trente-deux des souches isolées sont du sérovar 1/2a, 34 du sérovar 1/2b, 1 du sérovar 3a, et 17 du sérovar 4b. Les souches de L. monocytogenes ont été différenciées en 13 ribotypes après digestion avec EcoRI, et en 18 pulsotypes après digestion avec ApaI et AscI, avec des valeurs de 0,878 et 0,907 pour l’indice de discrimination de Simpson, respectivement. Une majorité d’isolats (59 %) a démontré une virulence potentielle liée à la production d’une internaline A fonctionnelle, corrélée avec des pourcentages d’entrée plus importants dans des cellules Caco-2 (9,3 %) que pour les isolats produisant une internaline A tronquée (1,3 % d’entrée). En moyenne, les isolats fécaux de L. monocytogenes étaient résistants à 6,4 ± 2,5 antibiotiques sur les 17 testés. Les isolats potentiellement virulents présentaient une résistance accrue à la kanamycine, à la gentamicine, à la streptomycine et à la rifampicine. Les communautés fécales de L. monocytogenes d’origine animale (élevage et sauvage) et humaine présentent des populations communes bien que globalement distinctes, et certains génotypes et phénotypes étaient similaires à ceux précédemment décris pour des souches isolées d’eau de surface dans la même zone géographique.

Listeria monocytogenes is a facultative intracellular pathogen thought to be widely distributed in the environment. We investigated the prevalence and characteristics of L. monocytogenes isolates from surface waters derived from catchments within the South Nation River watershed (Ontario, Canada). This watershed is dominated by urban and rural development, livestock and crop production, and wildlife habitats. From June to November 2005, a total of 314 surface water samples were collected biweekly from 22 discrete sampling sites characterized by various upstream land uses. Presumptive Listeria spp. were isolated using a selective enrichment and isolation procedure, and 75 L. monocytogenes isolates were identified based on colony morphology, hemolytic activity, and amplification of three pathogenicity genes: iap, inlA , and hlyA . Thirty-two of 314 (10%) surface water samples were positive for the presence of L. monocytogenes , but detection ranged between 0 and 27% depending on the sampling date. Isolates belonging to serovar group 1/2a, 3a (50%) and group 4b, 4d, 4e (32%) were dominant. L. monocytogenes populations were resolved into 13 EcoRI ribotypes and 21 ApaI and 21 AscI pulsotypes. These had Simpson indexes of discrimination of up to 0.885. Lineage I-related isolates were dominant (61%) during the summer, whereas lineage II isolates were dominant (77%) in the fall. Isolates were, on average, resistant to 6.1 ± 2.1 antibiotics out of 17 tested. Half of the L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, and some isolates were found to be moderately to highly virulent by in vitro Caco-2 plaque formation assay (up to 28% of entry). There was a statistically significant link between the occurrence of L. monocytogenes and proximity to an upstream dairy farm and degree of cropped land. Our data indicate that L. monocytogenes is widespread in the studied catchments, where it could represent a public health issue related to agricultural land use.

Reliability of bacterial diversity assessment using polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA fragments was evaluated for a particular complex microbial assemblage: river epilithic biofilm. By comparing 3 routine protocols on replicates of one river biofilm sample, we found that common DNA extraction procedures gave comparable diversity (from 28.0 to 30.7 bands detected) and community composition (> 75% of homology) despite differences in the total amount of extracted DNA (from 0.9 to 4.2 microg). Therefore methodological improvements only concerned electrophoretic separation of DNA fragments (range of denaturing gradient from 35% to 70% and migration time=18h) and standardisation of DNA amounts used (PCR-template=50 ng, gel loading=700 ng). Using such a standardised methodology we found a good reproducibility of all steps of the procedure. When an Escherichia coli strain was introduced as a contaminant in a biofilm sample, we were able to recover ribotypes from the strain. As concerns fields sampling, a satisfactory repeatability of banding patterns from neighbouring pebbles (sampling point) allowed discriminating between the biofilm intrasite variability (various points from a cross-profile). These trials confirmed that PCR-DGGE is suitable to assess a reliable genetic fingerprint of epilithic biofilms in the river. Phylogenetic analysis of 40 partial sequences of 16S rDNA from DGGE gels of two sets of river biofilms samples proved evidences for the retrieval of DNA fragments related to phototroph Eukarya. However, in both cases plastidial 16S rDNA represented less than 25% of the analysed operational taxonomic units. Taking into account that Cyanobacteria, as members of the Bacteria, were also detected, sequence analysis of relevant bands from the pattern is required to target "bacteria", i.e. the functional group of prokaryotic microorganisms to which one commonly refers as a key component in sustaining the nutrient turnover.

International audience

Benthic microbial communities are actively involved in organic matter recycling and fact biogeochemical functioning of lake ecosystems. These communities comprise many phyla but their functional diversity is still incompletely known. This study is focused on the benthic microbial communities involved in the methane cycle in lacsutrine suystems. We aimed understanding the structural and abundance changes of functional genes related to the vertical distribution (redox gradient in sediment), the horizontal variability (coastal vs. pelagic benthic zone) and seasonal dynamics related to mixing of the water column (re-oxygenation of benthic interface). The composition of methanotrophic and methanogenic communities was characterized by sequencing analyses.For this study, sedimentary cores were sampled along a transect from coastal to pelagic zone, at different times during an annual cycle. In addition, each sediment core was analyzed in its verticality from the water-sediment interface to 20 cm depth. Microbial communities involved in the cycle of methane (methanogenesis and methanotrophy) were targeted by 2 functional genes (mcrA and pmoA). Furthermore, phylogenetic markers were used to characterize the total bacterial and archaeal communities. These communities are studied in terms of structure (genotyping), diversity (sequencing) and abundance (qPCR, DNA) of their functional genes.The results of the study showed that, on a spatial scale, a low heterogeneity was detected for a given sampling station in terms of structure of microbial communities (total and functional), however, a high variability was detected both at an horizontal scale along a transect (costal vs. pelagic zone), due to contrasted environmental conditions, and at a vertical scale (upper to deeper layers in the core) under the effect of redox conditions. The bacterial community being the most affected in the verticality, with structural changes among all strata studied. In the same study, a comparative analysis of the structure (for all of the communities), between pooled samples and individual samples, demonstrated that the analysis of individual samples provided a greater number of OTU for the majority of microbial communities.Moreover the study of the temporal dynamic of methanogen and methanotroph communities revealed changes in the structure and abundance, mainly at the water - sediment interface, according to the oxygenation levels that varied through time. The analysis of diversity showed a dominance of Methanomicrobiales (Methanoregula mainly) for methanogens, but Methanosarcinales (Methanosarcina) and Methanobacteriales (Methanobacterium) were also identified. The methanotrophs' community was dominated by Methylobacter on deeper stations and by Methylococcus in coastal station. Type II methanotrophs (Methylosinus and Methylocystis) were also identified.This work highlights the importance of taking into account both the spatial variability (horizontal and vertical) and the temporal variability of methanogen and methanotroph communities. Changes on their structures and abundances are significant parameters for understanding the processes involved in the methane cycle.; Les communautés microbiennes benthiques participent activement au recyclage de la matière organique et de fait au fonctionnement biogéochimique des écosystèmes lacustres. Ces communautés comportent de nombreux phyla mais leur diversité fonctionnelle est encore incomplètement connue. Ce travail vise à appréhender les modifications de structure et d'abondance des gènes fonctionnels en lien avec la distribution spatiale verticale (liée au gradient d'oxydoréduction), la variabilité spatiale horizontale (zone côtière vs pélagique) et la dynamique saisonnière liée au brassage de la colonne d'eau (ré-oxygénation des interfaces benthiques), de même que l'identification de la diversité des méthanotrophes et des méthanogènes.Pour cette étude, des carottes sédimentaires ont été prélevées sur un transect zone côtière – zone pélagique, à différentes dates au cours d'un cycle annuel. Chaque carotte sédimentaire a été analysée dans la verticalité entre l'interface eau-sédiment et 20 cm. Les communautés microbiennes participant au cycle du méthane ont été ciblées par 2 gènes de fonction et étudiées en termes de structure, de diversité et d'abondance; par ailleurs, des marqueurs phylogénétiques ont été utilisés pour caractériser les communautés bactériennes et archéennes totales.Les résultats de l'étude spatiale montrent que, si à l'échelle locale (station d'échantillonnage) une relative homogénéité des communautés microbiennes (totales et fonctionnelles) est observée, des variabilités fortes sont détectées d'une part à l'échelle des transects horizontaux en lien avec des changements de conditions environnementales et d'autre part dans la verticalité des sédiments sous l'effet des conditions d'oxydoréduction. La communauté bactérienne étant la plus affectée dans la verticalité, avec des changements de structure entre toutes les strates étudiées. Dans cette même étude, une analyse comparative de la structure des communautés (totales et fonctionelles) a démontré que l'analyse d'échantillons individuels permettait d'obtenir un plus grand nombre d'OTU que l'analyse des mêmes échantillons regroupés en pools.Les résultats de l'étude de la dynamique temporelle des communautés méthanogènes et méthanotrophes révèlent des changements de structure et abondance, principalement à l'interface eau - sédiment en lien avec la dynamique d'oxygénation du lac. Quant à l'analyse de la diversité, elle montre une dominance des Methanomicrobiales (Methanoregula principalement) pour les méthanogènes, mais les Methanosarcinales (Methanosarcina) et les Methanobacteriales (Methanobacterium) ont également été identifiés. Pour les méthanotrophes, la diversité est dominée par Methylobacter en zone profonde et par Methylococcus en zone côtière, les méthanotrophes de Type II (Methylosinus et Methylocystis) ont aussi été identifiés.L'ensemble de ces travaux souligne l'importance de prendre en compte, à la fois la variabilité spatiale (horizontale et verticale) et la variabilité temporelle, des communautés méthanogènes et méthanotrophes lors de l'étude de ces communautés. Les changements quant à leurs structures et leurs abondances sont des paramètres non négligeables pour comprendre les processus impliqués dans le cycle du méthane.