The Achères wastewater treatment plant, located just downstream of Paris, discharges its effluents into the lower Seine River. The effluents contain large numbers of heterotrophic bacteria, organic matter, and ammonium and are a source of nitrifying bacteria. As a result, degradation of organic matter by heterotrophic bacteria and subsequent oxygen depletion occur immediately downstream of the effluent outlet, whereas nitrifying bacteria apparently need to build up a significant biomass before ammonium oxidation significantly depletes the oxygen. We quantified the potential total nitrifying activity and the potential activities of the ammonia-and nitrite-oxidizing communities along the Seine River. In the summer, the maximum nitrifying activity occurs in the upper freshwater estuary, ϳ200 km downstream of Achères. The quantities of nitrifying bacteria, based on amoA gene copy numbers, and of Nitrobacter organisms, based on 16S rRNA gene copy numbers, were correlated with the potential nitrifying activities. The species composition of ammonia-oxidizing bacteria was investigated at two sites: the Triel station just downstream from Achères (km 84) and the Seine freshwater estuary at the Duclair station (km 278). By means of PCR primers targeting the amoA gene, a gene library was created. Phylogenetic analysis revealed that the majority of the analyzed clones at both sites were affiliated with the genus Nitrosomonas. The Nitrosomonas oligotropha-and Nitrosomonas urea-related clones represented nearly 81% of the community of ammonia-oxidizing bacteria at Triel and 60% at Duclair. Two other ammonia-oxidizing clusters of the ␤ subclass of the Proteobacteria, i.e., Nitrosomonas europaea-and Nitrosospira-like bacteria, were found in smaller numbers. The major change in the ammonia-oxidizing community between the two stations along the Seine River-upper estuary continuum was the replacement of the N. oligotropha-and N. urea-related bacteria by the Nitrosospira-affiliated bacteria. Although the diversities of the ammonia oxidizers appear to be similar for the two sites, only half of the restriction patterns are common to both sites, which could be explained by the differences in ammonium concentrations, which are much lower in the upper estuary than in the river at the effluent outlet. These results imply a significant immigration and/or selection of the ammonia-oxidizing bacterial population along the continuum of the Seine River from Paris to the estuary.The Seine River downstream of Paris is greatly affected by the discharge of treated effluents from the Achères wastewater treatment plant (WWTP) (8.5 million inhabitant equivalent from Paris and its suburbs, subjected only to a secondary treatment). A typical feature of the ecological functioning of the lower Seine River is a high summer oxygen deficit immediately downstream of the Achères effluent outlet due to degradation of organic matter (18,19,48) and another depletion in the freshwater estuary due to nitrification of ammonium that is also contained in the effluent ...

b Escherichia coli, a commensal bacterium from the intestinal tracts of humans and vertebrate animals, has been used as one of two bacterial indicators of fecal contamination, along with intestinal enterococci, to monitor the microbiological quality of water. However, water environments are now recognized as a secondary habitat where some strains can survive. We investigated the survival of E. coli isolates collected from bodies of water in France exhibiting distinct profiles of contamination, defined according to the following criteria: vicinity of the point sources of contamination, land use, hydrology, and physicochemical characteristics of the receiving water. We selected 88 E. coli strains among a collection of 352 strains to carry out a microcosm experiment in filtered estuarine water for 14 days at 10°C. The relationship between the survival of E. coli strains and genotypic and phenotypic characteristics was analyzed. This work showed that distinct E. coli survival types, able to survive from between 7 and 14 days to less than 2 days, coexisted in the water. E. coli isolates that rapidly lost their culturability were more frequently isolated in water recently contaminated by fecal bacteria of human origin, and most were multiresistant to antibiotics and harbored several virulence factors. In contrast, persistent strains able to survive from 4 to 14 days were more often found in water with low levels of fecal bacteria, belonged mainly to the B1 phylogroup, often harbored only one virulence factor, kspE or ompT, and were able to grow at 7°C.

Over 6 years, Escherichia coli were isolated from water samples from seven Seine estuary stations, characterized by a densely populated watershed (654 isolates). Resistances of these E. coli to 16 antibiotics were determined and compared with the same resistances in E. coli isolated from a small stream (120 isolates) and from the treated effluent of the largest estuary wastewater treatment plant (123 isolates). Between 30.2% and 56.6% of the estuary isolates were resistant, whatever the station or time of sampling; of these, 60.5-80% were resistant to at least two and up to 12 antibiotics. In the three contrasting sites, resistances to tetracycline, amoxicillin and ticarcillin were the commonest. DNA was extracted from 279 estuary isolates (January 2006) and class 1, 2 and 3 integrons were detected by multiplex real-time PCR and confirmed by classic PCR. IntI1 and intI2 genes were found in 11% of isolates. No intI3 gene was detected. The variable regions of the class 1 and 2 integrons sequenced contained predominantly gene cassettes aadA and dfr. However, for slightly over half of the E. coli isolates exhibiting the class 1 integron, the variable region could not be amplified, because part of the 3' conserved sequence was missing.

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.

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.

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.

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.

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