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.

Shifts in sediment bacterial communities reflect changes in depositional environments in a fluviatile context.

Sediments are complex heterogeneous matrices allowing to some extent the recording of past environmental conditions by integrating sediment characteristics, contamination and the microbial community assembly. In aquatic environments, abiotic environmental filtering is considered the primary deterministic mechanism shaping microbial communities in sediments. However, the number and relative contributions of geochemical and physical factors associated with biotic parameters (reservoir of microorganisms) complicate our understanding of community assembly dynamics. In this study, the sampling of a sedimentary archive in a site alternately subjected to contrasting inputs from the Eure and the Seine Rivers allowed us to study the response of microbial communities to changes in depositional environment over time. The coupling of the quantification and sequencing of the gene encoding the 16S rRNA with analyses of grain size, organic matter and major and trace metal contents demonstrated that microbial communities reflected contrasting sedimentary inputs over time. Total organic carbon (TOC) was the main factor influencing microbial biomass, while the quantity and quality of organic matter (R400, RC/TOC), major elements (i.e. Al, Fe, Ti) and trace metals (i.e. Zn, Pb, Cu, Cr, Ni, As, Co, Ag, Sb) shaped the structure of the microbial community. Besides the effect of geochemical factors, a specific microbial signature was associated with the contrasting sedimentary sources, highlighting the importance of the microbial reservoir in the assembly of microbial communities. Indeed, the main genera identified in the facies influenced by the Eure River were affiliated with the phyla Desulfobacterota (Syntrophus, Syntrophorhabdus, Smithella, Desulfatiglans), Firmicutes (Clostridium_sensu_stricto_1), Proteobacteria (Crenothrix), Verrucomicrobiota (Luteolibacter), while the contributions of the Seine River were characterised by some halophilic genera Salirhabdus (Firmicutes), Haliangium (Myxococcota) and SCGC-AB-539-J10 (Chloroflexi). This study sheds light on the overall processes determining the assembly of microbial communities in sediments and the importance of associating geochemical factors with reservoirs of microorganisms inherited from sediment sources.

Decoupled responses of the copepod Eurytemora affinis transcriptome and its microbiota to dissolved copper exposure.

Chemical contamination is a common threat to biota thriving in estuarine and coastal ecosystems. Of particular importance is that trace metals tend to accumulate and exert deleterious effects on small invertebrates such as zooplankton, which are essential trophic links between phytoplankton and higher-level consumers in aquatic food webs. Beyond the direct effects of the contamination, we hypothesized that metal exposure could also affect the zooplankton microbiota, which in turn might further impair host fitness. To assess this assumption, copepods (Eurytemora affinis) were sampled in the oligo-mesohaline zone of the Seine estuary and exposed to dissolved copper (25 µg.L-1) over a 72-hour time period. The copepod response to copper treatment was assessed by determining transcriptomic changes in E. affinis and the alteration of its microbiota. Unexpectedly, very few genes were differentially expressed in the copper-treated copepods compared to the controls for both male and female samples, while a clear dichotomy between sex was highlighted with 80% of the genes showing sex-biased expression. In contrast, copper increased the taxonomic diversity of the microbiota and resulted in substantial compositional changes at both the phyla and genus levels. Phylogenetic reconstruction of the microbiota further suggested that copper mitigated the phylogenetic relatedness of taxa at the basal tree structure of the phylogeny, whereas it strengthened it at the terminal branches. Increased terminal phylogenetic clustering in the copper-treated copepods coincided with higher proportions of bacterial genera previously identified as copper resistant (e.g., Pseudomonas, Acinetobacter, Alkanindiges, Colwellia) and a higher relative abundance of the copAox gene encoding a periplasmic inducible multi-copper oxidase. The enrichment in micro-organisms likely to perform copper sequestration and/or enzymatic transformation processes, underlines the need to consider the microbial component during evaluation of the vulnerability of zooplankton to metallic stress.

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.

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.

Anti-persister activity of squalamine against Acinetobacter baumannii.

Squalamine is a natural polycationic aminosterol extracted from the shark Squalus acanthias. Squalamine displays remarkable efficacy against antimicrobial-resistant Gram-negative and Gram-positive bacteria. Its membranolytic activity and low cytotoxicity make squalamine one of the most promising agents to fight nosocomial pathogens such as Acinetobacter baumannii. In the context of chronic diseases and therapeutic failures associated with this pathogen, the presence of dormant cells, i.e. persisters and viable but non-culturable cells (VBNCs), highly tolerant to antimicrobial compounds is problematic. The aim of this study was to investigate the antibacterial activity of squalamine against this bacterial population of A. baumannii. Bacterial dormancy was induced by cold shock and nutrient starvation in the presence of high doses of either colistin, ciprofloxacin or squalamine. Persisters and VBNCs induced by these treatments were then challenged with 100 mg/L squalamine. The efficacy of each treatment was determined by evaluating culturability on agar medium, membrane integrity (LIVE/DEAD®BacLightTM staining) and respiratory activity (BacLightTM RedoxSensorTM CTC staining) of bacteria. A. baumannii ATCC 17978 generated persisters as well as VBNCs in the presence of high doses of ciprofloxacin but not colistin or squalamine. Squalamine at 100 mg/L (below its haemolytic concentration) was able to kill dormant cells. Squalamine did not induce persister cell or VBNC formation in A. baumannii ATCC 17978. Interestingly, squalamine was significantly active against this type of dormant population generated by ciprofloxacin, making it a very promising anti-persister agent.

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.

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.

Diversity of active microbial communities subjected to long-term exposure to chemical contaminants along a 40-year-old sediment core.

In estuarine ecosystems, metallic and organic contaminants are mainly associated with fine grain sediments which settle on mudflats. Over time, the layers of sediment accumulate and are then transformed by diagenetic processes mainly controlled by microbial activity, recording the history of the estuary's chemical contamination. In an environment of this specific type, we investigated the evolution of the chemical contamination and the structure of both total and active microbial communities, based on PhyloChip analysis of a 4.6-m core corresponding to a 40-year sedimentary record. While the archaeal abundance remained constant along the core, a decrease by one order of magnitude in the bacterial abundance was observed with depth. Both total and active microbial communities were dominated by Proteobacteria, Actinobacteria, and Firmicutes in all sediment samples. Among Proteobacteria, alpha-Proteobacteria dominated both total (from 37 to 60 %) and metabolically active (from 19.7 to 34.6 %) communities, including the Rhizobiales, Rhodobacter, Caulobacterales, and Sphingomonadales orders. Co-inertia analysis revealed a relationship between polycyclic aromatic hydrocarbons, zinc and some polychlorobiphenyls concentrations, and the structure of total and active microbial communities in the oldest and most contaminated sediments (from 1970 to 1975), suggesting that long-term exposure to chemicals shaped the structure of the microbial community.

Detection of Salmonella in environmental water and sediment by a nested-multiplex polymerase chain reaction assay.

From 1995 to 2002, 53 serovars of Salmonella were isolated in the Seine estuary (France). The 3 serovars most frequently found were S. enterica serovar Typhimurium, S. enterica serovar Infantis and S. enterica serovar Virchow. A nested multiplex PCR (nm-PCR) assay was developed to detect the presence of Salmonella in estuarine water and sediment samples. The target gene used was the phase 1 flagellin fliC chromosomal gene, present in all Salmonella serovars. A set of 4 primers was first used to amplify an 890-bp sequence of the fliC gene, and then a second set of 3 primers was used for the nested PCR. The nmPCR method has been successfully tested for 28 serovars, 13 of which are of epidemiological significance. The detection limit of the assay, without any pre-enrichment step, was estimated at 1 CFU in deionized water, and at 4-5 CFU in the reaction mixture when tested on estuarine water seeded with a Salmonella strain. When the nmPCR was used together with the classical culture method in environmental samples, it gave additional positive results for 11.3% of the sediment samples and 20% of the water samples despite a high background of other bacteria. Overall, the results demonstrated that this molecular approach informed us about the contamination by Salmonella of estuarine water and sediment samples. Positive amplifications suggested the presence of Salmonella DNA and could thus provide information about a recent (culturable) or past (non-culturable, released DNA) contamination of environmental samples by this pathogenic bacteria.

Exposure to vancomycin causes a shift in the microbial community structure without affecting nitrate reduction rates in river sediments.

Antibiotics and antibiotic resistance genes have shown to be omnipresent in the environment. In this study, we investigated the effect of vancomycin (VA) on denitrifying bacteria in river sediments of a Waste Water Treatment Plant, receiving both domestic and hospital waste. We exposed these sediments continuously in flow-through reactors to different VA concentrations under denitrifying conditions (nitrate addition and anoxia) in order to determine potential nitrate reduction rates and changes in sedimentary microbial community structures. The presence of VA had no effect on sedimentary nitrate reduction rates at environmental concentrations, whereas a change in bacterial (16S rDNA) and denitrifying (nosZ) community structures was observed (determined by polymerase chain reaction-denaturing gradient gel electrophoresis). The bacterial and denitrifying community structure within the sediment changed upon VA exposure indicating a selection of a non-susceptible VA population.

Distinct diversity of the czcA gene in two sedimentary horizons from a contaminated estuarine core.

In estuarine ecosystems, trace metals are mainly associated with fine grain sediments which settle on mudflats. Over time, the layers of sediments accumulate and are then transformed by diagenetic processes, recording the history of the estuary's chemical contamination. In such a specific environment, we investigated to what extent a chronic exposure to contaminants could affect metal-resistant sedimentary bacteria in subsurface sediments. The occurrence and diversity of cadmium resistance genes (cadA, czcA) was investigated in 5- and 33-year-old sediments from a highly contaminated estuary (Seine France). Primers were designed to detect a 252-bp fragment of the czcA gene, specifically targeting a transmembrane helice domain (TMH IV) involved in the proton substrate antiport of this efflux pump. Although the cadA gene was not detected, the highest diversity of the sequence of the czcA gene was observed in the 5-year-old sediment. According to the percentage of identity at the amino acid level, the closest CzcA relatives were identified among Proteobacteria (α, ß, γ, and δ), Verrucomicrobia, Nitrospirae, and Bacteroidetes. The most abundant sequences were affiliated with Stenotrophomonas. In contrast, in the 33-year-old sediment, CzcA sequences were mainly related to Rhodanobacter thiooxydans and Stenotrophomonas, suggesting a shaping of the metal-resistant microbial communities over time by both diagenetic processes and trace metal contamination.