Seasonal Dynamics of Abundance, Structure, and Diversity of Methanogens and Methanotrophs in Lake Sediments.

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.

Uneven genotypic diversity of Escherichia coli in fecal sources limits the performance of a library-dependent method of microbial source tracking on the southwestern French Atlantic coast.

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 (<35%) and significant rates of incorrect 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 (<3%). Since efforts made to constitute such a library are not affordable for routine analyses, the results question the relevance of developing such a method for identifying sources of fecal contamination on such a coastline.

A comparison of enrichment and direct-plating methods for isolation of Listeria monocytogenes from surface water.

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.

Electroactivity of phototrophic river biofilms and constitutive cultivable bacteria.

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.

Spatial distribution of sediment archaeal and bacterial communities relates to the source of organic matter and hypoxia - a biogeographical study on Lake Remoray (France).

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.

Diversity, Functions and Antibiotic Resistance of Sediment Microbial Communities From Lake Geneva Are Driven by the Spatial Distribution of Anthropogenic Contamination.

Lake sediments are natural receptors for a wide range of anthropogenic contaminants including organic matter and toxicants such as trace metals, polycyclic aromatic hydrocarbons, polychlorinated biphenyls that accumulate over time. This contamination can impact benthic communities, including microorganisms which play a crucial role in biogeochemical cycling and food-webs. The present survey aimed at exploring whether anthropogenic contamination, at a large lake scale, can influence the diversity, structure and functions of microbial communities associated to surface sediment, as well as their genetic potential for resistance to metals and antibiotics. Changes in the characteristics of these communities were assessed in surface sediments collected in Lake Geneva from eight sampling sites in October 2017 and May 2018. These sampling sites were characterized by a large concentration range of metal and organic compound contamination. Variation between the two sampling periods were very limited for all sampling sites and measured microbial parameters. In contrast, spatial variations were observed, with two sites being distinct from each other, and from the other six sites. Benthic communities from the most contaminated sampling site (Vidy Bay, near the city of Lausanne) were characterized by the lowest bacterial and archaeal diversity, a distinct community composition, the highest abundance of antibiotic resistance genes and functional (respiration, denitrification, methanogenesis, phosphatase, and beta-glucosidase) activity levels. The second sampling site which is highly influenced by inputs from the Rhône River, exhibited low levels of diversity, a distinct community composition, high abundance of antibiotic resistance genes and the highest bacterial abundance. Overall, our results suggest that local anthropogenic contamination, including organic matter and toxicants, is a major driver of the diversity and functioning of sediment-microbial communities in Lake Geneva. This highlights the need to consider benthic microbial communities and a suite of complementary ecotoxicological endpoints for more effective environmental risk assessments of contaminants in lake sediments.

Distribution and diversity of Escherichia coli populations in the South Nation River drainage basin, eastern Ontario, Canada.

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 x 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.

Optimization and validation of rep-PCR genotypic libraries for microbial source tracking of environmental Escherichia coli isolates.

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.

Changes in sediment microbial diversity following chronic copper-exposure induce community copper-tolerance without increasing sensitivity to arsenic.

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.

Towards simple tools to assess functional effects of contaminants on natural microbial and invertebrate sediment communities.

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.

Seasonal relationships among indicator bacteria, pathogenic bacteria, Cryptosporidium oocysts, Giardia cysts, and hydrological indices for surface waters within an agricultural landscape.

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.

Nutrient effects on the genetic and functional diversity of aquatic bacterial 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.

Environmental Concentrations of Copper, Alone or in Mixture With Arsenic, Can Impact River Sediment Microbial Community Structure and Functions.

In many aquatic ecosystems, sediments are an essential compartment, which supports high levels of specific and functional biodiversity thus contributing to ecological functioning. Sediments are exposed to inputs from ground or surface waters and from surrounding watershed that can lead to the accumulation of toxic and persistent contaminants potentially harmful for benthic sediment-living communities, including microbial assemblages. As benthic microbial communities play crucial roles in ecological processes such as organic matter recycling and biomass production, we performed a 21-day laboratory channel experiment to assess the structural and functional impact of metals on natural microbial communities chronically exposed to sediments spiked with copper (Cu) and/or arsenic (As) alone or mixed at environmentally relevant concentrations (40 mg kg-1 for each metal). Heterotrophic microbial community responses to metals were evaluated both in terms of genetic structure (using ARISA analysis) and functional potential (using exoenzymatic, metabolic and functional genes analyses). Exposure to Cu had rapid marked effects on the structure and most of the functions of the exposed communities. Exposure to As had almost undetectable effects, possibly due to both lack of As bioavailability or toxicity toward the exposed communities. However, when the two metals were combined, certain functional responses suggested a possible interaction between Cu and As toxicity on heterotrophic communities. We also observed temporal dynamics in the functional response of sediment communities to chronic Cu exposure, alone or in mixture, with some functions being resilient and others being impacted throughout the experiment or only after several weeks of exposure. Taken together, these findings reveal that metal contamination of sediment could impact both the genetic structure and the functional potential of chronically exposed microbial communities. Given their functional role in aquatic ecosystems, it poses an ecological risk as it may impact ecosystem functioning.

Looking at biological community level to improve ecotoxicological assessment of freshwater sediments: report on a first French-Swiss workshop.

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 Villié-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.

Analysis of bacterial diversity in river biofilms using 16S rDNA PCR-DGGE: methodological settings and fingerprints interpretation.

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.

Bio-electrochemical characterization of air-cathode microbial fuel cells with microporous polyethylene/silica membrane as separator.

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®) in an air-cathode microbial fuel cell with a reticulated vitreous carbon foam bioanode. Performances of the microporous polyethylene membrane (RhinoHide®) were compared with Nafion®-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® 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.

Warming-induced changes in denitrifier community structure modulate the ability of phototrophic river biofilms to denitrify.

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 21days) 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.

Rotating disk electrodes to assess river biofilm thickness and elasticity.

The present study examined the relevance of an electrochemical method based on a rotating disk electrode (RDE) to assess river biofilm thickness and elasticity. An in situ colonisation experiment in the River Garonne (France) in August 2009 sought to obtain natural river biofilms exhibiting differentiated architecture. A constricted pipe providing two contrasted flow conditions (about 0.1 and 0.45 m s(-1) in inflow and constricted sections respectively) and containing 24 RDE was immersed in the river for 21 days. Biofilm thickness and elasticity were quantified using an electrochemical assay on 7 and 21 days old RDE-grown biofilms (t(7) and t(21), respectively). Biofilm thickness was affected by colonisation length and flow conditions and ranged from 36 ± 15 µm (mean ± standard deviation, n = 6) in the fast flow section at t(7) to 340 ± 140 µm (n = 3) in the slow flow section at t(21). Comparing the electrochemical signal to stereomicroscopic estimates of biofilms thickness indicated that the method consistently allowed (i) to detect early biofilm colonisation in the river and (ii) to measure biofilm thickness of up to a few hundred µm. Biofilm elasticity, i.e. biofilm squeeze by hydrodynamic constraint, was significantly higher in the slow (1300 ± 480 µm rpm(1/2), n = 8) than in the fast flow sections (790 ± 350 µm rpm(1/2), n = 11). Diatom and bacterial density, and biofilm-covered RDE surface analyses (i) confirmed that microbial accrual resulted in biofilm formation on the RDE surface, and (ii) indicated that thickness and elasticity represent useful integrative parameters of biofilm architecture that could be measured on natural river assemblages using the proposed electrochemical method.

Characteristics and frequency of detection of fecal Listeria monocytogenes shed by livestock, wildlife, and humans.

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.

Distribution and characteristics of Listeria monocytogenes isolates from surface waters of the South Nation River watershed, Ontario, Canada.

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.