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Aquatic environments are important dissemination routes of antibiotic resistance genes (ARGs) from and to pathogenic bacteria. Nevertheless, in these complex matrices, identifying and characterizing the driving microbial actors and ARG dissemination mechanisms they are involved in remain difficult. We here explored the distribution/compartmentalization of a panel of ARGs and mobile genetic elements (MGEs) in bacteria and bacteriophages collected in the water, suspended material and surface sediments from the Orne River ecosystem (France). By using a new bacteriophage DNA extraction method, we showed that, when packaging bacterial DNA, bacteriophages rather encapsidate both ARGs and MGEs than 16S rRNA genes, i.e. chromosomal fragments. We also show that the bacteria and bacteriophage capsid contents in ARGs/MGEs were similarly influenced by seasonality but that the distribution of ARGs/MGEs between the river physical compartments (water vs. suspended mater vs. sediment) is more impacted when these markers were carried by bacteria. These demonstrations will likely modify our understanding of the formation and fate of transducing viral particles in the environment. Consequently, they will also likely modify our estimations of the relative frequencies of the different horizontal gene transfer mechanisms in disseminating antibiotic resistance by reinforcing the roles played by environmental bacteriophages and transduction.

BackgroundIn the environment, microbial communities are constantly exposed to invasion by antimicrobial resistant bacteria (ARB) and their associated antimicrobial resistance genes (ARGs) that were enriched in the anthroposphere. A successful invader has to overcome the biotic resilience of the habitat, which is more difficult with increasing biodiversity. The capacity to exploit resources in a given habitat is enhanced when communities exhibit greater diversity, reducing opportunities for invaders, leading to a lower persistence. In the context of antimicrobial resistance (AMR) dissemination, exogenous ARB reaching a natural community may persist longer if the biodiversity of the autochthonous community is low, increasing the chance of ARGs to transfer to community members. Reciprocally, high microbial diversity could serve as a natural long-term barrier towards invasion by ARB and ARGs.ResultsTo test this hypothesis, a sampling campaign across seven European countries was carried out to obtain 172 environmental samples from sites with low anthropogenic impact. Samples were collected from contrasting environments: stationary structured forest soils, or dynamic river biofilms and sediments. Microbial diversity and relative abundance of 27 ARGs and 5 mobile genetic element marker genes were determined. In soils, higher diversity, evenness and richness were all significantly negatively correlated with the relative abundance of the majority (>85%) of ARGs. Furthermore, the number of detected ARGs per sample was inversely correlated with diversity. However, no such effects were found for the more dynamic, regularly mixed rivers. Conclusions: In conclusion, we demonstrate that diversity can serve as barrier towards AMR dissemination in the environment. This effect is mainly observed in stationary, structured environments, where long-term, diversity-based resilience against invasion can evolve. Such barrier effects can in the future be exploited to limit the environmental proliferation of AMR.

Environmental microbiomes are constantly exposed to invasion events through foreign, antibiotic resistant bacteria that were enriched in the anthropic sphere. However, the biotic and abiotic factors, as well as the natural barriers that determine the invasion success of these invader bacteria into the environmental microbiomes are poorly understood. A great example of such invasion events are river microbial communities constantly exposed to resistant bacteria originating from wastewater effluents. Here, we aim at gaining comprehensive insights into the key factors that determine their invasion success with a particular focus on the effects of environmental stressors, regularly co-released in wastewater effluents. Understanding invasion dynamics of resistant bacteria is crucial for limiting the environmental spread of antibiotic resistance. To achieve this, we grew natural microbial biofilms on glass slides in rivers for one month. The biofilms were then transferred to laboratory, recirculating flume systems and exposed to a single pulse of a model resistant invader bacterium (E. coli) either in presence or absence of stress induced by Cu2+. The invasion dynamics ofE. coliinto the biofilms were then monitored for 14 days. Despite an initially successful introduction ofE. coliinto the biofilms, independent of the imposed stress, over time the invader perished in absence of stress. However, under stress c the invading strain successfully established and proliferated in the biofilms. Noteworthy, the increased establishment success of the invader coincided with a loss in microbial community diversity under stress conditions, likely due to additional niche space becoming available for the invader.

Background Mobile genetic elements (MGEs) are widely involved in the dissemination of antibiotic resistance genes and some of them, such as the integrative and conjugative element SXT, are even induced by specific antibiotics at sub-lethal concentrations. Objectives This work explores collateral effects of a broad range of antibiotics on the mobility of the SXTMO10 element using a specifically designed high-throughput screening test. Methods Twenty-five promoters involved in the mobility of SXT and six artificial constitutive promoters were transcriptionally fused to luxCDABE bioluminescent genes and introduced into Escherichia coli strains with or without SXT to build whole-cell biosensors for a large-scale screening involving 48 antibiotics. A bioluminescent assay implementing a classical agar diffusion approach was coupled to an automated data processing pipeline developed to extract and analyse luminescence data from over 2000 antibiotic/biosensor combination profiles. Results In addition to quinolones previously reported as inducing the expression of SXT mobility genes, we found that specific antibiotics belonging to other classes, such as imipenem and azithromycin, also behave as inducers. The use of a control set of constitutive biosensors also revealed an unexpected intricate relationship between cell respiration and light production that allowed the identification of antibiotics interfering with the respiration process. Conclusions The effect of antibiotics goes beyond the interaction with their primary cell targets and may lead to adverse effects such as triggering the dissemination of resistance by MGEs, sometimes in unpredictable ways. Identifying such MGE-triggering antibiotics is of prime importance for better controlling collateral effects during therapy.

International audience; EpicPCR (Emulsion, Paired Isolation and Concatenation PCR) is a recent single-cell genomic method based on a fusion-PCR allowing us to link a functional sequence of interest to a 16S rRNA gene fragment and use the mass sequencing of the resulting amplicons for taxonomic assignment of the functional sequence-carrying bacteria. Although it is interesting because it presents the highest efficiency for assigning a bacterial host to a marker, epicPCR remains a complex multistage procedure with technical difficulties that may easily impair the approach depth and quality. Here, we described how to adapt epicPCR to new gene targets and environmental matrices while identifying the natural host range of SXT/R391 integrative and conjugative elements in water microbial communities from the Meurthe River (France). We notably show that adding a supplementary PCR step allowed us to increase the amplicon yield and thus the number of reads obtained after sequencing. A comparison of operational taxonomic unit (OTU) identification approaches when using biological and technical replicates demonstrated that, although OTUs can be validated when obtained from three out of three technical replicates, up to now, results obtained from two or three biological replicates give a similar and even a better confidence level in OTU identification, while allowing us to detect poorly represented SXT/R391 hosts in microbial communities.

International audience; The elaboration of efficient hydrogel-based materials with antimicrobial properties requires a refined control of defining their physicochemical features, which includes mechanical stiffness, so as to properly mediate their antibacterial activity. In this work, we design hydrogels consisting of polyelectrolyte multilayer films for the loading of T4 and φX174 bacteria-killing viruses, also called bacteriophages. We investigate the antiadhesion and bactericidal performances of this biomaterial against Escherichia coli, with a specific focus on the effects of chemical cross-linking of the hydrogel matrix, which, in turn, mediates the hydrogel stiffness. Depending on the latter and on phage replication features, it is found that the hydrogels loaded with the bacteria-killing viruses make both contact killing (targeted bacteria are those adhered at the hydrogel surface) and release killing (planktonic bacteria are the targets) possible with ca. 20–80% efficiency after only 4 h of incubation at 25 °C as compared to cases where hydrogels are free of viruses. We further demonstrate the lack of dependence of virus diffusion within the hydrogel and of the maximal viral storage capacity on the hydrogel mechanical properties. In addition to the evidenced bacteriolytic activity of the phages loaded in the hydrogels, the antimicrobial property of the phage-loaded materials is shown to be partly controlled by the chemistry of the hydrogel skeleton and, more specifically, by the mobility of the peripheral free polycationic components, known for their ability to weaken and permeabilize membranes of bacteria, the latter then becoming “easier” targets for the viruses

International audience; Metal oxide nanoparticles (NPs), and among them metal oxides Quantum Dots (QDs), exhibit a multifactorial toxicity combining metal leaching, oxidative stress and possibly direct deleterious interactions, the relative contribution of each varying according to the NP composition and surface chemistry. Their wide use in public and industrial domains requires a good understanding and even a good control of their toxicity. To address this question, we engineered ZnO QDs with different surface chemistries, expecting that they would exhibit different photo-induced reactivities and possibly different levels of interaction with biological materials. No photo-induced toxicity could be detected on whole bacterial cell toxicity assays, indicating that ROS-dependent damages, albeit real, are hidden behind a stronger source of toxicity, which was comforted by the fact that the different ZnO QDs displayed the same level of cell toxicity. However, using in vitro DNA damage assays based on quantitative PCR, significant photo-induced reactivity could be measured precisely, showing that different NPs exhibiting similar inhibitory effects on whole bacteria could differ dramatically in terms of ROS-generated damages on biomolecules. We propose that direct interactions between NPs and bacterial cell surfaces prime over any kind of intracellular damages to explain the ZnO QDs toxicity on whole bacterial cells.

The dissemination of antimicrobial resistance (AMR) is one of the biggest challenges faced by mankind in the public health domains. It is currently favored by a lack of confinement between waste disposal and food production in the environmental compartment. To date, much effort has been devoted into the elucidation and control of cell-associated propagation of AMR. However, substantial knowledge gaps remain on the contribution of cell-free DNA to promote horizontal transfers of resistance genes in wastewater and downstream environments. Cell free DNA, which covers free extracellular DNA (exDNA) as well as DNA encapsulated in vesicles or bacteriophages, can persist after disinfection and promote gene transfer in the absence of physical and temporal contact between a donor and recipient bacteria. The increasing water scarcity associated to climatic change requires developing innovative wastewater reuse practices and, concomitantly, a robust evaluation of AMR occurrence by implementing treatment technologies able to exert a stringent control on AMR propagation in downstream environments exposed to treated or non-treated wastewater. This necessarily implies understanding the fate of ARGs on various forms of cell-free DNA, especially during treatment processes that are permissive to their formation. We propose that comprehensive approaches, investigating both the occurrence of ARGs and their compartmentalization in different forms of cellular or cell-free associated DNA should be established for each treatment technology. This should then allow selecting and tuning technologies for their capacity to limit the propagation of ARGs in any of their forms.

Metal oxide nanoparticles (NPs), and among them metal oxides Quantum Dots (QDs), exhibit a multifactorial toxicity combining metal leaching, oxidative stress and possibly direct deleterious interactions, the relative contribution of each varying according to the NP composition and surface chemistry. Their wide use in public and industrial domains requires a good understanding and even a good control of their toxicity. To address this question, we engineered ZnO QDs with different surface chemistries, expecting that they would exhibit different photo-induced reactivities and possibly different levels of interaction with biological materials. No photo-induced toxicity could be detected on whole bacterial cell toxicity assays, indicating that ROS-dependent damages, albeit real, are hidden behind a stronger source of toxicity, which was comforted by the fact that the different ZnO QDs displayed the same level of cell toxicity. However, using in vitro DNA damage assays based on quantitative PCR, significant photo-induced reactivity could be measured precisely, showing that different NPs exhibiting similar inhibitory effects on whole bacteria could differ dramatically in terms of ROS-generated damages on biomolecules. We propose that direct interactions between NPs and bacterial cell surfaces prime over any kind of intracellular damages to explain the ZnO QDs toxicity on whole bacterial cells.

Background Some antibiotics induce the dissemination of their own resistance genes by interfering with the regulation of specific mobile genetic elements. In Tn916, subinhibitory concentrations of tetracycline activate the transfer of the element through an anti-attenuation mechanism that relies on the Tet(M) resistance protein, itself encoded by the element. Objectives This work explores the effects of a broad range of antibiotics on the transfer of Tn916 and for which the element does not provide any selective advantage. Methods A sensitive promoter-reporter fusion approach was developed to test the effects of full antibiotic concentration gradients on gene promoter expression. Sixty molecules, covering most classes of antibiotics, were screened for their ability to modulate the activity of promoter Porf12 controlling the transfer of Tn916. Induction of Tn916 transfer was further demonstrated in mating assays with Enterococcus faecalis donors pre-exposed to subinhibitory concentrations of modulating antibiotics. Results Several antibiotics, other than tetracyclines, were identified as interfering with Tn916 regulation. Macrolides, lincosamides and streptogramins appeared to activate the transfer of Tn916 at unprecedented levels, in a Tet(M)-independent way that implies a yet undescribed regulatory mechanism for controlling the mobility of the element. Conclusions These results demonstrate that some ribosome-targeting antibiotics can induce the transfer of a given mobile genetic element, here Tn916, although it does not provide any resistance determinant for most of the triggering drugs. This implies that specific antibiotic therapies can have dramatic impacts on the dissemination of unexpected and unlinked resistance genes, with the clear risk of reducing our therapeutic potential for later treatments.

International audience; Salmonellosis is one of the most common causes of foodborne bacterial human disease worldwide, and the emergence of multidrug-resistant (MDR) strains of Salmonella enterica serovar Typhimurium (S. typhimurium) was associated to the incidence of invasive salmonellosis. The objective of the present work was to investigate the effects of the TiO2 photocatalysis process in terms of both bacteria inactivation and the emergence of mutants, on S. typhimurium TA102 water suspensions. The TiO2 photocatalysis was compared with a conventional disinfection process such as UV-C radiation. In spite of the faster bacterial inactivation obtained in UV-C disinfection experiments (45, 15, and 10 min for total inactivation for initial cell density 109, 108, and 107 CFU mL-1, respectively), photocatalytic disinfection (60, 30, and 15 min) was more energy efficient because of a lower energy requirement (2-20 mWs cm-2) compared to the UV-C disinfection process (5-30 mWs cm-2). During the photocatalytic experiments, the mutation frequency increased up to 1648-fold compared to background level for a 108 CFU mL-1 initial bacterial density, and mutants were inactivated after 1-10-min treatment, depending on initial bacterial cell density. In UV-C disinfection experiments, the mutation frequency increased up to 2181-fold for a 108 CFU mL-1 initial bacterial cell density, and UV-C doses in the range of 0.5-4.8 mWs cm-2 were necessary to decrease mutation frequency. In conclusion, both disinfection processes were effective in the inactivation of S. typhimurium cells, and mutants released into the environment can be avoided if cells are effectively inactivated.

Genomic islands, flanked by attachment sites, devoid of conjugation and recombination modules and related to the integrative and conjugative element (ICE) ICESt3, were previously found in Streptococcus thermophilus. Here, we show that ICESt3 transfers to a recipient harbouring a similar engineered genomic island, CIMEL3catR3, and integrates by site-specific recombination into its attachment sites, leading to their accretion. The resulting composite island can excise, showing that ICESt3 mobilizes CIMEL3catR3 in cis. ICESt3, CIMEL3catR3 and the whole composite element can transfer from the strain harbouring the composite structure. The ICESt3 transfer to a recipient bearing CIMEL3catR3 can also lead to retromobilization, i.e. its capture by the donor. This is the first demonstration of specific conjugative mobilization of a genomic island in cis and the first report of ICE-mediated retromobilization. CIMEL3catR3 would be the prototype of a novel class of non-autonomous mobile elements (CIMEs: CIs mobilizable elements), which hijack the recombination and conjugation machinery of related ICEs to excise, transfer and integrate. Few genome analyses have shown that CIMEs could be widespread and have revealed internal repeats that could result from accretions in numerous genomic islands, suggesting that accretion and cis mobilization have a key role in evolution of genomic islands.

The integrative and conjugative elements (ICEs) excise by site-specific recombination between attL and attR flanking sites, self-transfer the resulting circular form and integrate into the genome of the recipient cell. Two putative ICEs, ICESt1 and ICESt3, are integrated in the same locus in 2 strains of Streptococcusthermophilus. ICESt1 is a composite element harbouring an internal recombination site, attL’. The recombination between attL’ and attR leads to the excision of a shorter putative ICE, ICESt2. ICESt1/ICESt2 and ICESt3 carry related regulation modules sharing the open reading frame arp1 that encodes a protein related to the cI repressor of the phage λ. The repressors belonging to this family autoproteolyse in the presence of damaged DNA. Treatments with mitomycin C induce an increase in the excision of ICESt1, ICESt2 and ICESt3. Furthermore, the arp1 deletion leads to a 1,000-fold increase in the excision of ICESt1 and ICESt2 and to a decrease in the excision induction by mitomycin C. Thus, all together, these results suggest that the autocleavage of the arp1 repressor is involved in derepression of the S. thermophilus putative ICE excision by mitomycin C.

International audience; Plasmid-based dissemination of antibiotic resistance genes in environmental microbial communities is a matter of concern for public health, but it remains difficult to study for methodological reasons. In this study, we used the broad host range plasmid pB10 to compare and to point out the main drawbacks of the three different approaches currently used to evaluate plasmid transfer in natural communities. Culture-based selection of transconjugants appeared to be compromised by high prevalence of antibiotic resistances among natural communities, unless high loads of initial pB10-donor inocula were used. Fluorescence-based detection of transconjugants reached a dead-end consequently to the narrow host range of bacteria expressing fluorescent proteins from a genetically modified pB10 plasmid, in addition to the relatively high background level of fluorescence exhibited by some environmental matrices. The molecular-based approach was the only one to provide a mean to detect rare plasmid transfer events following a low but realistic initial pB10-donor inoculation. Whatever the method, culture-based or molecular-based, the detection of successful transfer events in a given environmental matrix seemed to be linked to the initial stability of the donor inoculum. Depending on the matrix considered, eukaryotic predation plays a significant role in either limiting or promoting the plasmid transfer events.

International audience; Horizontal transfer of genomic islands (GEIs), that is, chromosomal regions encoding functions that can be advantageous for the host, plays a key role in bacterial evolution, but their mechanisms of transfer remained elusive for a long time. Recent data suggest that numerous GEIs belong to noncanonical classes of mobile genetic elements (MGEs) that can transfer by conjugation. Among them, the integrative and conjugative elements encode their own excision, conjugative transfer, and integration, whereas the integrative mobilizable elements are autonomous for excision and integration but require the conjugation machinery of helper elements to transfer. Others can self-transfer but require the recombination machinery of the recipient cell to integrate. All these MGEs evolve by acquisition, deletion, or exchange of modules, that is, groups of genes involved in the same function. Moreover, composite GEIs can result from the insertion of a MGE within another or from the site-specific integration of an incoming MGE into one of the recombination sites flanking a resident GEI (tandem accretion). Tandem accretion enables the cis-conjugative mobilization of highly degenerated and nonautonomous GEIs, the cis-mobilizable elements. All these mechanisms contribute to the plasticity and complex evolution of GEIs and explain the highly diverse tableau revealed by more and more genome comparisons.

International audience; The toxicity of quantum dots (QDs) has been commonly attributed to the release of metal ions from thecore as well as to the production of reactive oxygen species. However, the information related to the stability of the nanoparticles are relatively scarce although this parameter may strongly influence their toxicity. The stability of aminosilane-capped ZnO QDs, here used as model nanoparticles, was investigated by inductively coupled plasma-optical emission spectrometer (ICP-OES) and whole cell biosensors using a dialysis setup to separate the QDs from the leaked Zn2+ ions. The integrity of the ZnO QDs appeared strongly affected by their dilution in aqueous medium, whereas the nanoparticles were slightly stabilized by bacteria. Our results demonstrate some inadequacy between the implementation and use of whole cell biosensors, and the monitoring of metal release from QDs.

Genomic islands, flanked by attachment sites, devoid of conjugation and recombination modules and related to the integrative and conjugative element (ICE) ICESt3, were previously found in Streptococcus thermophilus. Here, we show that ICESt3 transfers to a recipient harbouring a similar engineered genomic island, CIMEL₃catR₃, and integrates by site-specific recombination into its attachment sites, leading to their accretion. The resulting composite island can excise, showing that ICESt3 mobilizes CIMEL₃catR₃, in cis. ICESt3, CIMEL₃catR₃, and the whole composite element can transfer from the strain harbouring the composite structure. The ICESt3 transfer to a recipient bearing CIMEL₃catR₃, can also lead to retromobilization, i.e. its capture by the donor. This is the first demonstration of specific conjugative mobilization of a genomic island in cis and the first report of ICE-mediated retromobilization. CIMEL₃catR₃, would be the prototype of a novel class of non-autonomous mobile elements (CIMEs: CIs mobilizable elements), which hijack the recombination and conjugation machinery of related ICEs to excise, transfer and integrate. Few genome analyses have shown that CIMEs could be widespread and have revealed internal repeats that could result from accretions in numerous genomic islands, suggesting that accretion and cis mobilization have a key role in evolution of genomic islands.

Integrative and conjugative elements (ICEs), also called conjugative transposons, are genomic islands that excise, self-transfer by conjugation, and integrate in the genome of the recipient bacterium. The current investigation shows the intraspecies conjugative transfer of the first described ICEs in Streptococcus thermophilus , ICE St1 and ICE St3 . Mitomycin C, a DNA-damaging agent, derepresses ICE St3 conjugative transfer almost 25-fold. The ICE St3 host range was determined using various members of the Firmicutes as recipients. Whereas numerous ICE St3 transconjugants of Streptococcus pyogenes and Enterococcus faecalis were recovered, only one transconjugant of Lactococcus lactis was obtained. The newly incoming ICEs, except the one from L. lactis , are site-specifically integrated into the 3′ end of the fda gene and are still able to excise in these transconjugants. Furthermore, ICE St3 was retransferred from E. faecalis to S. thermophilus . Recombinant plasmids carrying different parts of the ICE St1 recombination module were used to show that the integrase gene is required for the site-specific integration and excision of the ICEs, whereas the excisionase gene is required for the site-specific excision only.

The integrative and conjugative elements (ICEs) excise by site-specific recombination between attL and attR flanking sites, self-transfer the resulting circular form and integrate into the genome of the recipient cell. Two putative ICEs, ICESt1 and ICESt3, are integrated in the same locus in 2 strains of Streptococcusthermophilus. ICESt1 is a composite element harbouring an internal recombination site, attL'. The recombination between attL' and attR leads to the excision of a shorter putative ICE, ICESt2. ICESt1/ICESt2 and ICESt3 carry related regulation modules sharing the open reading frame arp1 that encodes a protein related to the cI repressor of the phage lambda. The repressors belonging to this family autoproteolyse in the presence of damaged DNA. Treatments with mitomycin C induce an increase in the excision of ICESt1, ICESt2 and ICESt3. Furthermore, the arp1 deletion leads to a 1,000-fold increase in the excision of ICESt1 and ICESt2 and to a decrease in the excision induction by mitomycin C. Thus, all together, these results suggest that the autocleavage of the arp1 repressor is involved in derepression of the S. thermophilus putative ICE excision by mitomycin C.

A DNA-damaging agent, mitomycin C, derepresses the site-specific excision of two integrative and potentially conjugative elements from Streptococcus thermophilus , ICE St1 and ICE St3 . The regulation pathway involves a repressor related to phage lambda cI repressor. It could also involve a putative regulator related to another type of phage repressors, the “cI-like” repressors.

International audience

Mobile genetic elements (MGEs) are genetic structures frequently associated to the dissemination of antibiotic resistance genes (ARGs). In this work, we used two of them as proxies, class 1 integrons and IncP-1 plasmids, to better understand (i) the possible fate of ARGs once released in a river ecosystem (Orne, France), as well as (ii) the effect of anthropogenic pressures on their persistence. From river water analyses, we could show that the two MGEs do not behave the same way. The entry of class 1 integrons in the river system appeared to be diffuse rather than punctual, while the abundance of IncP-1 plasmid is relatively stable along the river section studied (23 km) thus indicating a rather indigenous origin. Anthropic inputs such as wastewater treatment plant did not seem to affect the abundance of MGEs because a too high level of effluent dilution. Interestingly, MGE-bearing bacteria appeared to be enriched on suspended material, which is likely to serve as a vehicle to drive MGE-richer communities of bacteria toward the sediments. The analysis of two sediment cores clearly indicates that only the top layers displayed an elevated level of MGE-bearing bacteria. These abundances decrease in deeper layers where only localized zones display micro-reservoirs of elevated MGE abundances. For one sediment core at least, we could show that the relative abundance of MGE negatively correlates with pollutants such as lead or certain PAHs; Les éléments génétiques mobiles (EGM) sont des structures génétiques fréquemment associées à la dissémination de gènes de résistance aux antibiotiques (GRA). Dans ce travail, nous avons utilisé deux EGM comme « proxies », les intégrons de classe 1 et les plasmides IncP-1, afin de mieux comprendre (i) le devenir possible des GRA une fois relargués dans un écosystème fluvial (l’Orne, France), ainsi que (ii) l’effet des pressions anthropiques sur leur persistance. À partir d'analyses de l'eau des rivières, nous avons pu montrer que les deux EGM ne se comportaient pas de la même manière. L'entrée des intégrons de classe 1 dans le système fluvial semblait être diffuse plutôt que ponctuelle, tandis que l'abondance du plasmide IncP-1 est relativement stable le long de la section de la rivière étudiée (23 km), indiquant ainsi une origine plutôt indigène. Les intrants anthropiques tels que les stations d’épuration des eaux usées ne semblent pas affecter l’abondance des EGM en raison d’un niveau trop élevé de dilution des effluents. Par ailleurs, il est intéressant de noter que les bactéries porteuses d’EGM semblaient être enrichies sur les matières en suspension, susceptibles de servir de véhicule pour amener des communautés de bactéries plus riches en EGM vers les sédiments. L'analyse de deux carottes de sédiment indique clairement que seules les couches supérieures présentent un niveau élevé de bactéries porteuses d’EGM. Ces abondances diminuent dans les couches plus profondes où seules des zones ponctuelles présentent des microréservoirs avec des abondances d’EGM plus élevées. Pour une carotte sédimentaire au moins, nous avons pu montrer que l'abondance relative d’EGM corrèle négativement la présence de polluants tel que le plomb ou certains HAP

Mobile genetic elements (MGEs) are genetic structures frequently associated to the dissemination of antibiotic resistance genes (ARGs). In this work, we used two of them as proxies, class 1 integrons and IncP-1 plasmids, to better understand (i) the possible fate of ARGs once released in a river ecosystem (Orne, France), as well as (ii) the effect of anthropogenic pressures on their persistence. From river water analyses, we could show that the two MGEs do not behave the same way. The entry of class 1 integrons in the river system appeared to be diffuse rather than punctual, while the abundance of IncP-1 plasmid is relatively stable along the river section studied (23 km) thus indicating a rather indigenous origin. Anthropic inputs such as wastewater treatment plant did not seem to affect the abundance of MGEs because a too high level of effluent dilution. Interestingly, MGE-bearing bacteria appeared to be enriched on suspended material, which is likely to serve as a vehicle to drive MGE-richer communities of bacteria toward the sediments. The analysis of two sediment cores clearly indicates that only the top layers displayed an elevated level of MGE-bearing bacteria. These abundances decrease in deeper layers where only localized zones display micro-reservoirs of elevated MGE abundances. For one sediment core at least, we could show that the relative abundance of MGE negatively correlates with pollutants such as lead or certain PAHs; Les éléments génétiques mobiles (EGM) sont des structures génétiques fréquemment associées à la dissémination de gènes de résistance aux antibiotiques (GRA). Dans ce travail, nous avons utilisé deux EGM comme « proxies », les intégrons de classe 1 et les plasmides IncP-1, afin de mieux comprendre (i) le devenir possible des GRA une fois relargués dans un écosystème fluvial (l’Orne, France), ainsi que (ii) l’effet des pressions anthropiques sur leur persistance. À partir d'analyses de l'eau des rivières, nous avons pu montrer que les deux EGM ne se comportaient pas de la même manière. L'entrée des intégrons de classe 1 dans le système fluvial semblait être diffuse plutôt que ponctuelle, tandis que l'abondance du plasmide IncP-1 est relativement stable le long de la section de la rivière étudiée (23 km), indiquant ainsi une origine plutôt indigène. Les intrants anthropiques tels que les stations d’épuration des eaux usées ne semblent pas affecter l’abondance des EGM en raison d’un niveau trop élevé de dilution des effluents. Par ailleurs, il est intéressant de noter que les bactéries porteuses d’EGM semblaient être enrichies sur les matières en suspension, susceptibles de servir de véhicule pour amener des communautés de bactéries plus riches en EGM vers les sédiments. L'analyse de deux carottes de sédiment indique clairement que seules les couches supérieures présentent un niveau élevé de bactéries porteuses d’EGM. Ces abondances diminuent dans les couches plus profondes où seules des zones ponctuelles présentent des microréservoirs avec des abondances d’EGM plus élevées. Pour une carotte sédimentaire au moins, nous avons pu montrer que l'abondance relative d’EGM corrèle négativement la présence de polluants tel que le plomb ou certains HAP

Les dysbioses intestinales sont les conséquences d'un déséquilibre impactant la microflore colonisant le système intestinal de l'Homme. Différentes causes peuvent être responsables de l'apparition de ce désordre microbien, comme la prise de médicaments antibiotiques ou une alimentation non optimale. La flore intestinale exerce de nombreux rôles physiologiques pour la santé de l'hôte et le déséquilibre pouvant ressortir entre les différentes populations microbiennes est susceptible d'entrainer de nombreuses conditions pathologiques. De nos jours, la connaissance du microbiote est en pleine expansion, tout comme les différents moyens thérapeutiques préventifs et/ou curatifs, notamment les probiotiques, qui sont un domaine en plein essor. L'objectif de cette thèse est de mettre en lumière la nécessité de posséder un écosystème intestinal optimal afin de préserver et d'optimiser la santé générale de patients atteints de pathologies liées aux dysbioses. En premier lieu, ce travail traitera de la microflore en général, de la caractérisation des espèces présentes dans notre système intestinal, de leur composition et leur implantation tout le long du tube digestif. La deuxième partie de la thèse concerne les différents rôles physiologiques attribués aux bactéries et des interactions qui s'établissent entre les espèces bactériennes et l'organisme de l'hôte. Pour la troisième partie, les dysbioses et les pathologies associées au déséquilibre des populations bactériennes sont détaillées. La quatrième partie de mon travail, nous nous intéresserons aux moyens diagnostiques et à l'arsenal thérapeutique dont on dispose à l'heure d'aujourd'hui, à la fois sur le plan préventif et sur le plan curatif, sans oublier de parler du rôle majeur de l'alimentation et des aliments la composant.

Les antibiotiques utilisés à concentrations subinhibitrices provoquent sur les bactéries des effets diversifiés sur leur phénotype et leur génotype. À ces faibles doses, ces molécules agissent comme des messagers et engendrent des réactions favorables ou défavorables pour la survie et le développement des bactéries. Chaque famille d'antibiotique provoque des effets qui lui sont propres ou non, et qui peuvent être récurrents, intenses, parfois rares mais notables, ou alors très différents d'une molécule à l'autre. Les modifications phénotypiques observées concernent notamment le métabolisme, l'expression de gènes, la formation de biofilm ou encore la morphologie des bactéries. Les trois bactéries les plus étudiées sur cette thématique, Escherichia coli, Pseudomonas aeruginosa et Staphylococcus aureus, réagissent de manières différentes à une exposition à des concentrations subinhibitrices d'antibiotique. Cela permet de mettre en évidence des associations fortes antibiotiquebactérie se traduisant par des effets intenses et récurrents.