Wild fish from a highly urbanized river (Orge, France) as vectors of culturable Enterobacterales resistant to antibiotics.

This study shows how wild fishes from urbanized rivers could be involved in the spread of antibiotic-resistant Enterobacterales. Antibiotic resistance profiles and molecular detection of clinical integron (IntI1) were carried out on 105 Enterobacterales isolated from 89 wildfish (skin or gut) belonging to 8 species. The proportion of isolates resistant to at least one antibiotic was independent of fish species and reached 28.3% within the Escherichia coli (E. coli) population and 84.7% in the non-E.coli Enterobacterales. Bacteria involved in nosocomial infections were isolated, such as E. coli, Klebsiella, and Enterobacter, as well as the environmental bacteria (Lelliottia, Butiauxella, and Kluyvera).

Urbanization Constrains Skin Bacterial Phylogenetic Diversity in Wild Fish Populations and Correlates with the Proliferation of Aeromonads.

Changes in the state of rivers resulting from the activity and expansion of urban areas are likely to affect aquatic populations by increasing stress and disease, with the microbiota playing a potentially important intermediary role. Unraveling the dynamics of microbial flora is therefore essential to better apprehend the impact of anthropogenic disturbances on the health of host populations and the ecological integrity of hydrosystems. In this context, the present study simultaneously examined changes in the microbial communities associated with mucosal skin and gut tissues of eight fish species along an urbanization gradient in the Orge River (France). 16S rRNA gene metabarcoding revealed that the structure and composition of the skin microbiota varied substantially along the disturbance gradient and to a lesser extent according to fish taxonomy. Sequences affiliated with the Gammaproteobacteria, in particular the genus Aeromonas, prevailed on fish caught in the most urbanized areas, whereas they were nearly absent upstream. This rise of opportunistic taxa was concomitant with a decline in phylogenetic diversity, suggesting more constraining environmental pressures. In comparison, fish gut microbiota varied much more moderately with the degree of urbanization, possibly because this niche might be less directly exposed to environmental stressors. Co-occurrence networks further identified pairs of associated bacterial taxa, co-existing more or less often than expected at random. Few correlations could be identified between skin and gut bacterial taxa, supporting the assumption that these two microbial niches are disconnected and do not suffer from the same vulnerability to anthropic pressures.

Toxicity of sediment-bound pollutants in the Seine estuary, France, using a Eurytemora affinis larval bioassay.

Coastal urbanisation exposes surrounding estuarine environments to urban-related contaminants such as polycyclic aromatic hydrocarbons (PAHs), polychlorobiphenyls (PCBs) and pesticide mixtures. Hydrophobic contaminants can adsorb on estuarine sediments. They can subsequently be released on a massive scale in the aquatic environment due to artificial or natural phenomena (e.g. dredging, tides), thereby threatening living organisms. The contamination of sediment is a significant ecological issue in the Seine estuary, France. However, few relevant methods have been developed to assess sediment toxicity and its ecological impacts in a cost-effective way. In this context, we aimed to assess the toxicity of natural sediments from the Seine estuary on the development of the calanoid copepod Eurytemora affinis using a previously developed larval bioassay. This assay involves direct exposure of nauplii to elutriates of sediments for six days. Sediments were collected along the Seine estuary from six polluted sites and one reference site. Pollutants in this estuary included PAHs, PCBs and OCPs (organochlorine pesticides). Nauplius survival was significantly more affected by exposure to all contaminated sediment elutriates, than by exposure to sediment from Yville-sur-Seine (the reference site), whereas nauplius growth was significantly reduced after exposure to contaminated sediment elutriates from four of the six contaminated sites. We identified two distinct site clusters, one including both the sand-rich and the least polluted sediments (Oissel, Quillebeuf-sur-Seine, Caudebec-en-Caux) and the other including both the clay- and silt-rich, and the most polluted sediments (La Bouille, Poses, Pont de Normandie). As expected, survival was significantly more impacted after exposure to elutriates from the second cluster than from the first. This work enables (i) assessment of the toxicity of natural sediments in the Seine estuary and (ii) validation of the larval bioassay previously developed using sorbed sediment with model molecules.

Modeling the impact of urban and hospital eco-exposomes on antibiotic-resistance dynamics in wastewaters.

The emergence and selection of antibiotic resistance is a major public health problem worldwide. The presence of antibiotic-resistant bacteria (ARBs) in natural and anthropogenic environments threatens the sustainability of efforts to reduce resistance in human and animal populations. Here, we use mathematical modeling of the selective effect of antibiotics and contaminants on the dynamics of bacterial resistance in water to analyze longitudinal spatio-temporal data collected in hospital and urban wastewater between 2012 and 2015. Samples were collected monthly during the study period at four different sites in Haute-Savoie, France: hospital and urban wastewater, before and after water treatment plants. Three different categories of exposure variables were collected simultaneously: 1) heavy metals, 2) antibiotics and 3) surfactants for a total of 13 drugs/molecules; in parallel to the normalized abundance of 88 individual genes and mobile genetic elements, mostly conferring resistance to antibiotics. A simple hypothesis-driven model describing weekly antibiotic resistance gene (ARG) dynamics was proposed to fit the available data, assuming that normalized gene abundance is proportional to antibiotic resistant bacteria (ARB) populations in water. The detected compounds were found to influence the dynamics of 17 genes found at multiple sites. While mercury and vancomycin were associated with increased ARG and affected the dynamics of 10 and 12 identified genes respectively, surfactants antagonistically affected the dynamics of three genes. The models proposed here make it possible to analyze the relationship between the persistence of resistance genes in the aquatic environment and specific compounds associated with human activities from longitudinal data. Our analysis of French data over 2012-2015 identified mercury and vancomycin as co-selectors for some ARGs.

Contrasted Antibiotics and Pesticides Occurrence in Fish Exposed In Situ to Urban Effluents: A 20-Day Caging Experiment.

Urban freshwater ecosystems receive a wide array of organic pollutants through wastewater-treatment plant (WWTP) discharges and agricultural runoff. Evaluating the fate and effects of antibiotics and pesticides can be a challenging task, especially the effects on freshwater vertebrates because of their abilities to metabolize and excrete these chemicals and because of their high mobility and escape behavior when exposed to stressful environmental conditions. In the present study, 37 wild gudgeons (Gobio gobio) were caged for a period of up to 20 days, upstream and downstream of a WWTP effluent discharge in the Orge River (a tributary of the Seine River, France). Levels of pesticides and antibiotics in fish muscles were monitored weekly and compared with environmental contamination (water and sediments). Our results highlighted a slight bioaccumulation of pesticides in the gudgeon muscles at the downstream site after 20 days of exposure. Concerning antibiotics, ofloxacin was the most detected compound in fish muscles (85% of occurrence) and ranged from undetectable to 8 ng g-1 dry weight. Antibiotic levels in fish muscle were not higher at the downstream site and did not increase with exposure duration, despite high levels in the water (up to 29 times greater than upstream). Potential ecotoxicological effects were also evaluated: Body condition did not differ between the caging location and exposure time. Three oxidative status markers in the fish livers showed significant shifts after 14 days of caging. Our results suggest a high clearance rate of antibiotics and, to a lesser extent, of pesticides in wild gudgeons, which could be explained by changes in xenobiotic metabolism with pollutant exposure. Environ Toxicol Chem 2024;43:701-711. © 2023 SETAC.

Changes in enterococcal populations and related antibiotic resistance along a medical center-wastewater treatment plant-river continuum.

To determine if hospital effluent input has an ecological impact on downstream aquatic environment, antibiotic resistance in Enterococcus spp. along a medical center-retirement home-wastewater treatment plant-river continuum in France was determined using a culture-based method. Data on antibiotic consumption among hospitalized and general populations and levels of water contamination by antibiotics were collected. All isolated enterococci were genotypically identified to the species level, tested for in vitro antibiotic susceptibility, and typed by multilocus sequence typing. The erm(B) and mef(A) (macrolide resistance) and tet(M) (tetracycline resistance) genes were detected by PCR. Along the continuum, from 89 to 98% of enterococci, according to the sampled site, were identified as Enterococcus faecium. All E. faecium isolates from hospital and retirement home effluents were multiply resistant to antibiotics, contained erm(B) and mef(A) genes, and belonged to hospital-adapted clonal complex 17 (CC17). Even though this species remained dominant in the downstream continuum, the relative proportion of CC17 isolates progressively decreased in favor of other subpopulations of E. faecium that were more diverse, less resistant to antibiotics, and devoid of the classical macrolide resistance genes and that belonged to various sequence types. Antibiotic concentrations in waters were far below the MICs for susceptible isolates. CC17 E. faecium was probably selected in the gastrointestinal tract of patients under the pressure of administered antibiotics and then excreted together with the resistance genes in waters to progressively decrease along the continuum.

Development of a larval bioassay using the calanoid copepod, Eurytemora affinis to assess the toxicity of sediment-bound pollutants.

Hydrophobic pollutants, in particular sediment-sorbed organic compounds, are widespread in the aquatic environment and could represent a threat to living organisms. Estuarine species, which live in turbulent ecosystems, are particularly exposed to this mode of contamination. For precise evaluation of the toxicity of hydrophobic contaminants desorbed from particles, a new larval assay using nauplii of the estuarine calanoid copepod Eurytemora affinis was developed. It consists of the direct exposure of copepods during naupliar development to elutriates of an unpolluted sediment spiked with different model contaminants. This bioassay measures the toxicity of the bioavailable fraction of particle-sorbed pollutants on the naupliar stage of copepods. Mortality and growth (non-invasive endpoints) in nauplii were analysed after six days of exposure. This approach was validated using six pollutants with different modes of action: benzo[a]pyrene (BaP), dimethylbenzo[a]anthracene (DMBA), phenanthrene (PHE), polychlorinated biphenyls (PCB 126, PCB 153) and 4-nonylphenol (4-NP). All these compounds induced a dose-dependent increase in toxic effects. Lethal effects only occurred at the highest tested concentrations: 58,541 and 6092 ng g(-1) dry weight sediment (dws), for PHE and DMBA, respectively. Sublethal effects (growth inhibition) were observed at lower concentrations for all tested compounds except PCB 153, from 8, 142, 297, 6092 and 8453 ng g(-1) dws for PCB 126, BaP, PHE, DMBA and 4-NP, respectively.

Evidence for a complex relationship between antibiotics and antibiotic-resistant Escherichia coli: from medical center patients to a receiving environment.

The aim of this study was to investigate the relationship between antibiotics and antibiotic-resistant fecal bacteria (E. coli) in water along a medical center-wastewater treatment plant-river continuum (4 km). A multiresidue chemical analysis methodology, using solid phase extraction coupled with liquid chromatography tandem mass spectrometry, was performed to detect whether low levels of contamination by 34 antibiotics were related to antibiotic resistance of E. coli and antibiotic use. The contamination of water by antibiotics and antibiotic-resistant E. coli decreased along the continuum. Although amoxicillin was predominantly prescribed, only ofloxacin (1 ng·L(-1)) and sulfamethoxazole (4 ng·L(-1)) persisted in the river. At the retirement home, in the medical center, even though no tetracycline and sulfamethoxazole were consumed, the highest occurrences of antibiotic resistance were in classes of quinolones (42.0%), sulfonamides (24.0%), tetracyclines (38.0%), and penicillins (38.0%), mainly due to the presence of multiple antibiotic-resistance genes on class 1 integrons. Along the continuum, the occurrence of E. coli resistant to antibiotics and those carrying class 1 integrons decreased in water samples (p-value <0.001). Interestingly, in the river, only persistent antibiotic compounds (ofloxacin, sulfamethoxazole) were found, but they did not correspond to the major resistances (tetracycline, amoxicillin) of E. coli.

Dysbiosis of fish gut microbiota is associated with helminths parasitism rather than exposure to PAHs at environmentally relevant concentrations.

Although parasite infection and pollution are common threats facing wild populations, the response of the gut microbiota to the joint impact of these stressors remains largely understudied. Here, we experimentally investigated the effects of exposure to Polycyclic Aromatic Hydrocarbons (PAHs) and infection by a common acanthocephalan intestinal parasite (Pomphorhynchus sp.) on the gut microbial flora of a freshwater fish, the European chub (Squalius cephalus). Naturally infected or uninfected individuals were exposed to PAHs at environmentally realistic concentrations over a five-week period. Characterization of the gut bacterial community through 16S rRNA gene amplicon sequencing revealed that parasitic infection was a more structuring factor of bacterial diversity and composition than PAH exposure. Specifically, chub infected by Pomphorhynchus sp. harbored significantly less evenly represented gut bacterial communities than the uninfected ones. In addition, substantial changes in sequence abundance were observed within the main bacterial phyla, including the Firmicutes, Fusobacteriota, Actinobacteriota, and Proteobacteria. Again, these compositional changes correlated with host infection with Pomphorhynchus sp., confirming its pivotal role in gut microbial assemblage. Overall, these results highlight the importance of defining the parasitic status of individuals when conducting microbial ecotoxicological analyses at the digestive tract level, as this should lead to better understanding of microbiota modulations and help to identify microbial markers specifically associated with chemicals.

Environmental contamination in a high-income country (France) by antibiotics, antibiotic-resistant bacteria, and antibiotic resistance genes: Status and possible causes.

Antimicrobial resistance (AMR) is a major global public health concern, shared by a large number of human and animal health actors. Within the framework of a One Health approach, actions should be implemented in the environmental realm, as well as the human and animal realms. The Government of France commissioned a report to provide policy and decision makers with an evidential basis for recommending or taking future actions to mitigate AMR in the environment. We first examined the mechanisms that underlie the emergence and persistence of antimicrobial resistance in the environment. This report drew up an inventory of the contamination of aquatic and terrestrial environments by AMR and antibiotics, anticipating that the findings will be representative of some other high-income countries. Effluents of wastewater treatment plants were identified as the major source of contamination on French territory, with spreading of organic waste products as a more diffuse and incidental contamination of aquatic environments. A limitation of this review is the heterogeneity of available data in space and time, as well as the lack of data for certain sources. Comparing the French Measured Environmental Concentrations (MECs) with predicted no effect concentrations (PNECs), fluoroquinolones and trimethoprim were identified as representing high and medium risk of favoring the selection of resistant bacteria in treated wastewater and in the most contaminated rivers. All other antibiotic molecules analyzed (erythromycin, clarithromycin, azithromycin, tetracycline) were at low risk of resistance selection in those environments. However, the heterogeneity of the data available impairs their full exploitation. Consequently, we listed indicators to survey AMR and antibiotics in the environment and recommended the harmonization of sampling strategies and endpoints for analyses. Finally, the objectives and methods used for the present work could comprise a useful example for how national authorities of countries sharing common socio-geographic characteristics with France could seek to better understand and define the environmental dimension of AMR in their particular settings.

Record of trace organic contaminants in a river sediment core: From historical wastewater management to historical use.

Some trace organic contaminants (TrOCs) can be considered as ubiquitous contaminants since the 1950s, and the study of their historical distribution within river sediments allows us to better understand the temporal variation of the chemical quality of sediments, and make assumptions about the most insightful forcings impacting these distributions. In this study, the occurrence of 41 TrOCs of various classes (i.e. pharmaceutical products and pesticides) was studied in a sedimentary core sampled in a disused dock along the Seine River, France. This core covers a 60 year-long period between 1944 and 2003, and 23 TrOCs were detected at least once. Their concentrations mainly ranged between 1 and 10 ng g-1 within the core, except for tetracycline that exhibited higher concentrations (~hundreds of ng·g-1). The dating of the core, based on previous studies, enabled the characterization of the changes since 1945, potentially impacted by (i) the sewer connectivity, (ii) the upgrading of wastewater treatment technologies, (iii) historical modifications in the use of each TrOC, and (iv) the sedimentary composition. In every case the deepest occurrence of each TrOC in the core matched its market authorization date, indicating the potential of TrOC to be used as chronomarkers. This study also reveals that the recent upgrading of wastewater treatment technologies within the watershed decreased the concentrations of each TrOC, despite an increase in TrOC diversity in the most recent years.

Influence of hydrological conditions on the Escherichia coli population structure in the water of a creek on a rural watershed.

BACKGROUND: Escherichia coli is a commensal bacterium of the gastro-intestinal tract of human and vertebrate animals, although the aquatic environment could be a secondary habitat. The aim of this study was to investigate the effect of hydrological conditions on the structure of the E. coli population in the water of a creek on a small rural watershed in France composed of pasture and with human occupation. RESULTS: It became apparent, after studying the distribution in the four main E. coli phylo-groups (A, B1, B2, D), the presence of the hly (hemolysin) gene and the antibiotic resistance pattern, that the E. coli population structure was modified not only by the hydrological conditions (dry versus wet periods, rainfall events), but also by how the watershed was used (presence or absence of cattle). Isolates of the B1 phylo-group devoid of hly and sensitive to antibiotics were particularly abundant during the dry period. During the wet period and the rainfall events, contamination from human sources was predominantly characterized by strains of the A phylo-group, whereas contamination by cattle mainly involved B1 phylo-group strains resistant to antibiotics and exhibiting hly. As E. coli B1 was the main phylo-group isolated in water, the diversity of 112 E. coli B1 isolates was further investigated by studying uidA alleles (beta-D-glucuronidase), the presence of hly, the O-type, and antibiotic resistance. Among the forty epidemiolgical types (ETs) identified, five E. coli B1 ETs were more abundant in slightly contaminated water. CONCLUSIONS: The structure of an E. coli population in water is not stable, but depends on the hydrological conditions and on current use of the land on the watershed. In our study it was the ratio of A to B1 phylo-groups that changed. However, a set of B1 phylo-group isolates seems to be persistent in water, strengthening the hypothesis that they may correspond to specifically adapted strains.

Comparative effects of mercury contamination and wastewater effluent input on Gram-negative merA gene abundance in mudflats of an anthropized estuary (Seine, France): a microcosm approach.

The macrotidal Seine estuary (France) is one of the most man-altered and mercury-contaminated European estuaries. Molecular quantification by competitive PCR has shown that the highest quantities of Gram-negative merA genes in intertidal freshwater mudflat sediments are located in recent sediment deposits independently of mercury concentrations, suggesting that particle-attached allochtonous mercury-resistant merA bacteria are deposited on mudflat surfaces. To investigate this hypothesis, a microcosm experiment was carried out to evaluate the respective contributions of (i) the input of allochtonous merA bacteria supplied by WWTP-treated effluents and (ii) merA gene abundance corresponding to a response of the sediment's autochthonous bacterial community to mercury contamination. Gram-negative merA gene quantification and T-RFLP analysis of both 16S rDNA and merA genes demonstrated that deposited allochtonous bacteria did not develop in estuarine sediments, whereas mercury contamination (10microg g(-1) wet sediment) selected an autochthonous mercury-resistant merA bacterial community. Thus, in mudflats of highly anthropized macrotidal estuaries, i.e. those subjected to intense hydrosedimentary processes and continuously contaminated by mercury and fecal bacteria, inputs of allochtonous merA bacteria are largely responsible for the high quantities of merA genes on the surface of mudflat sediments.

Faecal-indicator bacteria and sedimentary processes in estuarine mudflats (Seine, France).

Over a three-year period, quantification of faecal indicators and the molecular detection of Escherichia coli and Salmonella were monitored in sediments from three contrasting mudflats of the Seine estuary (France). The elevation of the mudflat surface was monitored concurrently using a high-resolution altimeter. During the period of the study, estuarine mudflats were areas of deposition for faecal-indicator bacteria and were mainly controlled by sedimentary processes. In the intertidal freshwater and subtidal mudflats, the highest abundances of faecal-indicator bacteria were counted during a depositional period. Maximum levels were observed in the freshwater mudflats during periods of high flow: thermotolerant coliforms: 3.9 x 10(4) cfu cm(-2), enterococci: 1.2 x 10(4) cfu cm(-2), Clostridium perfringens spores: 9.8 x 10(5) spores cm(-2). Loss of culturability of enteric bacteria in sediment microcosms demonstrated the remediatory capacity of the mudflats, even if they might be a secondary source of bacteria-forming spores to the water column through erosion and resuspension events.

Relationships between hydrosedimentary processes and occurrence of mercury-resistant bacteria (merA) in estuary mudflats (Seine, France).

The Seine estuary (France) is one of the world's macrotidal systems that is most contaminated with heavy metals. To study the mercury-resistant bacterial community in such an environment, we have developed a molecular tool, based on competitive PCR, enabling the quantification of Gram-negative merA gene abundance. The occurrence of the Gram-negative merA gene in relation with the topology (erosion/deposit periods) and the mercury contamination of three contrasted mudflats was investigated through a multidisciplinary approach and compared with a non-anthropized site (Authie, France). The higher abundance of the Gram-negative merA gene in the Seine estuary mudflats indicates a relationship between the degree of anthropization and the abundance of the merA gene in the mudflat sediments. In the Seine mudflats, the maxima of abundance are always located in fresh sediment deposits. Therefore, the abundance is closely related with the hydrosedimentary processes, which thus seem to be determining factors in the occurrence of the Gram-negative merA gene in the surface sediments of the Seine's mudflat.

Assessment of faecal contamination and the relationship between pathogens and faecal bacterial indicators in an estuarine environment (Seine, France).

The Seine estuary, one of the largest estuaries of the European northwest continental shelf, is subjected to numerous anthropogenic influences. Here we present an assessment of the microbial faecal contamination of the estuary water. The most vulnerable areas were defined on the basis of the fluxes of indicator organisms and the occurrence of Salmonella and Cryptosporidium sp. and Giardia sp. (oo)cysts. The microbial quality of the water changes from upstream to downstream: in the upstream area, contamination by faecal-indicator bacteria and Salmonella occurs during periods of high flow; in the urbanized area, mid-way between the uppermost areas of the estuary and its mouth, discharge from a wastewater treatment plant and a tributary degrade water quality; at the estuary mouth, the accumulation of microorganisms attached to particles in the maximum turbidity zone, particularly Clostridium perfringens spores and oocysts of Cryptosporidium, is accompanied by inputs of ThC and Escherichia coli from tributaries. In some areas, significant strong relations are observed between Salmonella, (oo)cysts of protozoan, and levels of faecal indicators.

Distinct Aeromonas Populations in Water Column and Associated with Copepods from Estuarine Environment (Seine, France).

Aeromonas spp. are ubiquitous bacteria primarily recovered from aquatic ecosystems. They are found in fresh water as well as estuarine and marine waters, and in association with numerous autochthonous aquatic organisms in these environments. However, aeromonads are also etiologic agents of fish diseases and are now recognized as emerging pathogens in humans. The estuary is therefore a key environment, harboring autochthonous aeromonads, and aeromonads originating from humans and animals, mainly released by treated WWTP effluent or watershed run-off via tributaries. The present study compares the abundance and the diversity of Aeromonas populations. Over 2 years of monitoring (eight campaigns from February 2013 to November 2015), the occurrence of Aeromonas was investigated within the water column (water and fluid mud) and in association with copepods. Moreover, the diversity of Aeromonas populations was ascertained by analyzing gyrB and radA sequences, and the antibiotic-resistance phenotypes were determined using the disk diffusion method. This study shows, for the first time, the presence of Aeromonas spp. in water (1.1 × 102 to 1.2 ± 0.3 × 103 CFU.100 mL-1), fluid mud (2.6 ± 2.6 × 102 to 9.8 ± 0.9 × 103 CFU.g-1) and in association with living copepods (1.9 ± 0.7 × 102 to >1.1 × 104 CFU.g-1) in the Seine estuary. Moreover, the diversity study, conducted on 36 strains isolated from the water column and 47 strains isolated from copepods, indicates distinct populations within these two compartments. Strains distributed in five clusters corresponding to A. bestiarum (n = 6; 5.45%), A. encheleia (n = 1; 0.91%), A. media (n = 22; 20.0%), A. rivipollensis (n = 34; 30.91%) and A. salmonicida (n = 47; 42.73%). A. salmonicida is the most abundant species associated with Eurytemora affinis (n = 35; 74.47%). In contrast, A. salmonicida accounts for only 30.56% (n = 11) of isolates in the water column. This study shows the coexistence of distinct populations of Aeromonas in the oligohaline area of an anthropized estuary. Moreover, A. media, a putative human pathogen, present in the water column and abundant in the WWTP samples, was not detected in association with living copepods.

Change in the Structure of Escherichia coli Population and the Pattern of Virulence Genes along a Rural Aquatic Continuum.

The aim of this study was to investigate the diversity of the Escherichia coli population, focusing on the occurrence of pathogenic E. coli, in surface water draining a rural catchment. Two sampling campaigns were carried out in similar hydrological conditions (wet period, low flow) along a river continuum, characterized by two opposite density gradients of animals (cattle and wild animals) and human populations. While the abundance of E. coli slightly increased along the river continuum, the abundance of both human and ruminant-associated Bacteroidales markers, as well as the number of E. coli multi-resistant to antibiotics, evidenced a fecal contamination originating from animals at upstream rural sites, and from humans at downstream urban sites. A strong spatial modification of the structure of the E. coli population was observed. At the upstream site close to a forest, a higher abundance of the B2 phylogroup and Escherichia clade strains were observed. At the pasture upstream site, a greater proportion of both E and B1 phylogroups was detected, therefore suggesting a fecal contamination of mainly bovine origin. Conversely, in downstream urban sites, A, D, and F phylogroups were more abundant. To assess the occurrence of intestinal pathogenic strains, virulence factors [afaD, stx1, stx2, eltB (LT), estA (ST), ipaH, bfpA, eae, aaiC and aatA] were screened among 651 E. coli isolates. Intestinal pathogenic strains STEC O174:H21 (stx2) and EHEC O26:H11 (eae, stx1) were isolated in water and sediments close to the pasture site. In contrast, in the downstream urban site aEPEC/EAEC and DAEC of human origin, as well as extra-intestinal pathogenic E. coli belonging to clonal group A of D phylogroup, were sampled. Even if the estimated input of STEC (Shiga toxin-producing E. coli) - released in water at the upstream pasture site - at the downstream site was low, we show that STEC could persist in sediment. These results show that, the run-off of small cattle farms contributed, as much as the wastewater effluent, in the dissemination of pathogenic E. coli in both water and sediments, even if the microbiological quality of the water was good or to average quality according to the French water index.

Delineation of Taxonomic Species within Complex of Species: Aeromonas media and Related Species as a Test Case.

Aeromonas media is an opportunistic pathogen for human and animals mainly found in aquatic habitats and which has been noted for significant genomic and phenotypic heterogeneities. We aimed to better understand the population structure and diversity of strains currently affiliated to A. media and the related species A. rivipollensis. Forty-one strains were included in a population study integrating, multilocus genetics, phylogenetics, comparative genomics, as well as phenotypics, lifestyle, and evolutionary features. Sixteen gene-based multilocus phylogeny delineated three clades. Clades corresponded to different genomic groups or genomospecies defined by phylogenomic metrics ANI (average nucleotide identity) and isDDH (in silico DNA-DNA hybridization) on 14 whole genome sequences. DL-lactate utilization, cefoxitin susceptibility, nucleotide signatures, ribosomal multi-operon diversity, and differences in relative effect of recombination and mutation (i.e., in evolution mode) distinguished the two species Aeromonas media and Aeromonas rivipollensis. The description of these two species was emended accordingly. The genome metrics and comparative genomics suggested that a third clade is a distinct genomospecies. Beside the species delineation, genetic and genomic data analysis provided a more comprehensive knowledge of the cladogenesis determinants at the root and inside A. media species complex among aeromonads. Particular lifestyles and phenotypes as well as major differences in evolution modes may represent putative factors associated with lineage emergence and speciation within the A. media complex. Finally, the integrative and populational approach presented in this study is considered broadly in order to conciliate the delineation of taxonomic species and the population structure in bacterial genera organized in species complexes.

Diversity of the dsrAB (dissimilatory sulfite reductase) gene sequences retrieved from two contrasting mudflats of the Seine estuary, France.

The diversity of sulfate-reducing microorganisms was investigated in two contrasting mudflats of the Seine estuary, by PCR amplification, cloning and sequencing of the genes coding for parts of the alpha and beta subunits of dissimilatory sulfite reductase (dsrAB). One site is located in the mixing-zone and shows marine characteristics, with high salinity and sulfate concentration, whereas the other site shows freshwater characteristics, with low salinity and sulfate concentration. Diversity and abundance of dsrAB genes differed between the two sites. In the mixing-zone sediments, most of the dsrAB sequences were affiliated to those of marine Gram-negative bacteria belonging to the order of Desulfobacterales, whereas in the freshwater sediments, a majority of dsrAB sequences was related to those of the Gram-positive bacteria belonging to the genus Desulfotomaculum. It is speculated that this is related to the salinity and the sulfate concentration in the two mudflats.