Characterization of antibiotic resistance genes in microbiomes from different ecosystems

Au cours du XXème siècle, l'antibiothérapie a considérablement réduit la mortalité causée par les bactéries pathogènes. Cependant, au cours des dernières décennies, la communauté humaine est continuellement confrontée à de nouveaux cas cliniques de résistance aux antibiotiques, ce qui peut entraîner l’échec de l'antibiothérapie. Les antibiotiques sont utilisés dans le monde entier non seulement pour traiter les maladies causées par des agents pathogènes humains, mais aussi à des fins thérapeutiques et de stimulation de la croissance dans les fermes, l’aquaculture et l’agriculture. En raison des activités anthropiques, des concentrations résiduelles d'antibiotiques, des gènes de résistance aux antibiotiques et des bactéries résistantes aux antibiotiques atteignent l'environnement. Cela peut entraîner le développement et la sélection de résistance aux antibiotiques dans l’environnement et la dissémination de ces résistances aux microbiomes animaux et humains. Néanmoins, l’amplitude de ce phénomène reste inconnue. L’objectif de cette thèse était d'évaluer la réponse du microbiome et du résistome environnementaux à la pollution chimique par des antibiotiques, ainsi qu'à la pollution biologique causée par les activités humaines. Deux études utilisant des microcosmes ont été menées pour évaluer les effets de la pollution par des antibiotiques sur le microbiome et le résistome d'un sol agricole et de l'eau du Rhône en utilisant une combinaison des cultures et d’approches métagénomiques/qPCR. Les concentrations sous-inhibitrices et inhibitrices de gentamicine pour les bactéries du sol et de l'eau enrichies en milieu de culture ont été déterminées en évaluant la croissance bactérienne. Les microcosmes du sol ont été pollués avec une gamme de concentrations inhibitrices de gentamicine, tandis que les microcosmes de l'eau ont été pollués avec deux concentrations sous-inhibitrices et une concentration inhibitrice, afin d'établir des liens entre la dose de gentamicine et l'ampleur de la réponse dans le microbiome et le résistome environnementaux à différents temps d'exposition. Ces deux études illustrent comment les effets du même antibiotique sur différents environnements sont fortement dépendants des facteurs environnementaux et des propriétés physico-chimiques. Ces résultats supportent la crainte que les concentrations sous-inhibitrices d'antibiotiques puissent sélectionner des résistances dans l'environnement. De plus, cette recherche souligne les limites des termes «sous-inhibiteur» et «inhibiteur» dans des environnements complexes et l'importance d’effectuer des études en utilisant des microcosmes et des études de terrain afin d’évaluer les effets de la pollution par des antibiotiques sur le résistome environnemental. Pour terminer, une étude de terrain a été menée sur des échantillons de neige obtenus des Sudety Mountains (Pologne) soumis à différentes expositions aux activités humaines et à différentes quantités de végétation autour, afin d'évaluer l'impact de facteurs anthropiques et environnementaux sur le microbiome et le résistome de la neige en utilisant une approche métagénomique / qPCR. Cette recherche fournit des éléments qui supportent l'hypothèse selon laquelle des facteurs environnementaux et anthropiques induisent des changements dans le microbiome et le résistome de la neige en fournissant aux communautés bactériennes des niveaux plus élevés de carbone organique et d'autres nutriments. Cela favoriserait la croissance d'une communauté bactérienne plus abondante, qui à son tour augmenterait l'abondance des gènes de résistance aux antibiotiques qui pourraient ensuite être disséminée dans l'atmosphère ou à travers la fonte des neiges. Cette recherche surligne la nécessité de faire des études sur le développement de la résistance aux antibiotiques dans des environnements pollués par les activités humaines et la prise en compte des sources organiques de pollution en plus des polluants biologiques.During the 20th century, antibiotic therapy has drastically reduced the mortality caused by bacterial pathogens. However, during the last few decades, the human community is continuously facing new cases of antibiotic resistance in clinics, which may result in antibiotic therapy failure. Antibiotics and antibiotic resistance predate the discovery of antibiotics by thousands of years. However, the industrialization and extensive use of antibiotics in humans and animals has imposed a selective pressure without precedents on bacterial communities, accelerating the development of antibiotic resistance at a global scale. Antibiotics are used worldwide not only to treat diseases caused by human pathogens, but also with therapeutic and growth promotion purposes in farms, aquacultures and crops. As a consequence of anthropogenic activities, residual concentrations of antibiotics, antibiotic resistance genes (ARG) and antibiotic resistant bacteria (ARB) may reach the environment. This may result in the development and selection of antibiotic resistance in environmental settings and the dissemination of ARB and ARG from the environment to animal and human microbiomes. Nonetheless, the scope of this phenomenon remains unclear. The goal of this thesis was to evaluate the response of the environmental microbiome and resistome to chemical pollution with antibiotics, as well as to biological pollution caused by anthropogenic activities. Two microcosm studies were carried out to evaluate the effects of antibiotic pollution on the microbiome and resistome of an agricultural soil and of water from the Rhône river using a combination of culture-based and metagenomics/qPCR approaches. Gentamicin sub-inhibitory and inhibitory concentrations in soil and water bacteria enriched in culture media were determined by evaluating bacterial growth. Soil microcosms were polluted with a range of inhibitory concentrations of gentamicin, whereas water microcosms were polluted with two sub-inhibitory concentrations and an inhibitory concentration, in order to establish links between gentamicin dose and the magnitude of the response in the environmental microbiome and resistome at different exposure times. These two studies illustrate how the effects of the same antibiotic on different environments are strongly dependent of environmental factors and physicochemical properties. Our findings support the concern that sub-inhibitory concentrations of antibiotics may select for resistance in the environment. In addition, this research underlines the limitations of the terms “sub-inhibitory” and “inhibitory” in complex environments and the importance of microcosm and field studies for the evaluation of the effects of antibiotic pollution on the environmental resistome. Finally, a field study was carried out in snow samples from the Sudety Mountains (Poland) with a range of exposure to human activities and surrounding vegetation in order to evaluate the impact of both anthropogenic and environmental factors on the snow microbiome and resistome using a metagenomics and qPCR approach. This research supports the hypothesis that both environmental and anthropogenic factors impact snow ecology and induce changes in the snow microbiome and antibiotic resistome by providing bacterial communities with higher levels of organic carbon and other nutrients. This would support the growth of a more abundant bacterial community, which in turn increases the abundance of the antibiotic resistome and could stimulate competition and ARG proliferation in snow. This organic pollution could stimulate the development of antibiotic resistance in the snow microbiome that might be subsequently disseminated through the atmosphere or snow melting. The research carried out in this study highlights the need for survey of antibiotic resistance development in anthropogenic polluted sites and the consideration of organic sources of pollution in addition to biological pollutants (ARB and ARG)

Caractérisation de la diversité des gènes de résistance à des antibiotiques dans les microbiotes de différents écosystèmes environnementaux

During the 20th century, antibiotic therapy has drastically reduced the mortality caused by bacterial pathogens. However, during the last few decades, the human community is continuously facing new cases of antibiotic resistance in clinics, which may result in antibiotic therapy failure. Antibiotics and antibiotic resistance predate the discovery of antibiotics by thousands of years. However, the industrialization and extensive use of antibiotics in humans and animals has imposed a selective pressure without precedents on bacterial communities, accelerating the development of antibiotic resistance at a global scale. Antibiotics are used worldwide not only to treat diseases caused by human pathogens, but also with therapeutic and growth promotion purposes in farms, aquacultures and crops. As a consequence of anthropogenic activities, residual concentrations of antibiotics, antibiotic resistance genes (ARG) and antibiotic resistant bacteria (ARB) may reach the environment. This may result in the development and selection of antibiotic resistance in environmental settings and the dissemination of ARB and ARG from the environment to animal and human microbiomes. Nonetheless, the scope of this phenomenon remains unclear. The goal of this thesis was to evaluate the response of the environmental microbiome and resistome to chemical pollution with antibiotics, as well as to biological pollution caused by anthropogenic activities. Two microcosm studies were carried out to evaluate the effects of antibiotic pollution on the microbiome and resistome of an agricultural soil and of water from the Rhône river using a combination of culture-based and metagenomics/qPCR approaches. Gentamicin sub-inhibitory and inhibitory concentrations in soil and water bacteria enriched in culture media were determined by evaluating bacterial growth. Soil microcosms were polluted with a range of inhibitory concentrations of gentamicin, whereas water microcosms were polluted with two sub-inhibitory concentrations and an inhibitory concentration, in order to establish links between gentamicin dose and the magnitude of the response in the environmental microbiome and resistome at different exposure times. These two studies illustrate how the effects of the same antibiotic on different environments are strongly dependent of environmental factors and physicochemical properties. Our findings support the concern that sub-inhibitory concentrations of antibiotics may select for resistance in the environment. In addition, this research underlines the limitations of the terms “sub-inhibitory” and “inhibitory” in complex environments and the importance of microcosm and field studies for the evaluation of the effects of antibiotic pollution on the environmental resistome. Finally, a field study was carried out in snow samples from the Sudety Mountains (Poland) with a range of exposure to human activities and surrounding vegetation in order to evaluate the impact of both anthropogenic and environmental factors on the snow microbiome and resistome using a metagenomics and qPCR approach. This research supports the hypothesis that both environmental and anthropogenic factors impact snow ecology and induce changes in the snow microbiome and antibiotic resistome by providing bacterial communities with higher levels of organic carbon and other nutrients. This would support the growth of a more abundant bacterial community, which in turn increases the abundance of the antibiotic resistome and could stimulate competition and ARG proliferation in snow. This organic pollution could stimulate the development of antibiotic resistance in the snow microbiome that might be subsequently disseminated through the atmosphere or snow melting. The research carried out in this study highlights the need for survey of antibiotic resistance development in anthropogenic polluted sites and the consideration of organic sources of pollution in addition to biological pollutants (ARB and ARG).Au cours du XXème siècle, l'antibiothérapie a considérablement réduit la mortalité causée par les bactéries pathogènes. Cependant, au cours des dernières décennies, la communauté humaine est continuellement confrontée à de nouveaux cas cliniques de résistance aux antibiotiques, ce qui peut entraîner l’échec de l'antibiothérapie. Les antibiotiques sont utilisés dans le monde entier non seulement pour traiter les maladies causées par des agents pathogènes humains, mais aussi à des fins thérapeutiques et de stimulation de la croissance dans les fermes, l’aquaculture et l’agriculture. En raison des activités anthropiques, des concentrations résiduelles d'antibiotiques, des gènes de résistance aux antibiotiques et des bactéries résistantes aux antibiotiques atteignent l'environnement. Cela peut entraîner le développement et la sélection de résistance aux antibiotiques dans l’environnement et la dissémination de ces résistances aux microbiomes animaux et humains. Néanmoins, l’amplitude de ce phénomène reste inconnue. L’objectif de cette thèse était d'évaluer la réponse du microbiome et du résistome environnementaux à la pollution chimique par des antibiotiques, ainsi qu'à la pollution biologique causée par les activités humaines. Deux études utilisant des microcosmes ont été menées pour évaluer les effets de la pollution par des antibiotiques sur le microbiome et le résistome d'un sol agricole et de l'eau du Rhône en utilisant une combinaison des cultures et d’approches métagénomiques/qPCR. Les concentrations sous-inhibitrices et inhibitrices de gentamicine pour les bactéries du sol et de l'eau enrichies en milieu de culture ont été déterminées en évaluant la croissance bactérienne. Les microcosmes du sol ont été pollués avec une gamme de concentrations inhibitrices de gentamicine, tandis que les microcosmes de l'eau ont été pollués avec deux concentrations sous-inhibitrices et une concentration inhibitrice, afin d'établir des liens entre la dose de gentamicine et l'ampleur de la réponse dans le microbiome et le résistome environnementaux à différents temps d'exposition. Ces deux études illustrent comment les effets du même antibiotique sur différents environnements sont fortement dépendants des facteurs environnementaux et des propriétés physico-chimiques. Ces résultats supportent la crainte que les concentrations sous-inhibitrices d'antibiotiques puissent sélectionner des résistances dans l'environnement. De plus, cette recherche souligne les limites des termes «sous-inhibiteur» et «inhibiteur» dans des environnements complexes et l'importance d’effectuer des études en utilisant des microcosmes et des études de terrain afin d’évaluer les effets de la pollution par des antibiotiques sur le résistome environnemental. Pour terminer, une étude de terrain a été menée sur des échantillons de neige obtenus des Sudety Mountains (Pologne) soumis à différentes expositions aux activités humaines et à différentes quantités de végétation autour, afin d'évaluer l'impact de facteurs anthropiques et environnementaux sur le microbiome et le résistome de la neige en utilisant une approche métagénomique / qPCR. Cette recherche fournit des éléments qui supportent l'hypothèse selon laquelle des facteurs environnementaux et anthropiques induisent des changements dans le microbiome et le résistome de la neige en fournissant aux communautés bactériennes des niveaux plus élevés de carbone organique et d'autres nutriments. Cela favoriserait la croissance d'une communauté bactérienne plus abondante, qui à son tour augmenterait l'abondance des gènes de résistance aux antibiotiques qui pourraient ensuite être disséminée dans l'atmosphère ou à travers la fonte des neiges. Cette recherche surligne la nécessité de faire des études sur le développement de la résistance aux antibiotiques dans des environnements pollués par les activités humaines et la prise en compte des sources organiques de pollution en plus des polluants biologiques

Effect of environmental ARG expression under selective pressure on ARG transfer to bacteria

International audienceThe continuous increase of antibiotic-resistant bacteria (ARBs) in medical clinics is a major global health concern. Although the use of antibiotics for human and animal treatment seems to be related to the spread of ARBs, antibiotic resistance dispersion is a natural environmental process. Furthermore, several studies have documented the influence of anthropogenic activity on the environmental “resistome”. Novel antibiotic resistance genes (ARGs) may emerge in the non-clinical environment and spread to the human microbiome, from where the probability of their transfer to human pathogens increases. Thus, we are interested in the environmental factors that influence the rate and probability of ARGs transfer from non-human microbiome bacteria to human microbiome bacteria in non-clinical environments. Here we report on the influence of in situ transcription rates on the transfer of genes in soil. In order to determine whether the transcription of ARGs under different selective pressures increases their transfer rate to human microbiome bacteria, natural soils and soils incubated with antibiotics were inoculated with a labelled strain of E. coli. At different time points, these E. coli were isolated from soil microcosms by flow cytometry and a combination of metatranscriptomics and qPCR was used to evaluate the transfer of environmental ARG to E. coli as a function of their transcription rate in the donor bacteria. This approach provided a model for the study of environmental ARG transfer to human microbiome bacteria directly in soil and highlights the importance of gene expression monitoring in risk assessment studies

Sub-inhibitory gentamicin pollution induces gentamicin resistance gene integration in class 1 integrons in the environment

Abstract Antibiotics at sub-inhibitory concentrations are often found in the environment. Here they could impose selective pressure on bacteria, leading to the selection and dissemination of antibiotic resistance, despite being under the inhibitory threshold. The goal of this study was to evaluate the effects of sub-inhibitory concentrations of gentamicin on environmental class 1 integron cassettes in natural river microbial communities. Gentamicin at sub-inhibitory concentrations promoted the integration and selection of gentamicin resistance genes (GmRG) in class 1 integrons after only a one-day exposure. Therefore, sub-inhibitory concentrations of gentamicin induced integron rearrangements, increasing the mobilization potential of gentamicin resistance genes and potentially increasing their dissemination in the environment. This study demonstrates the effects of antibiotics at sub-inhibitory concentrations in the environment and supports concerns about antibiotics as emerging pollutants

Aminoglycosides analysis optimization using Ion pairing Liquid Chromatography coupled to tandem Mass Spectrometry and application on wastewater samples

International audienceAminoglycosides are mostly used as veterinary antibiotics. In France, their consumption accounts for about 10% of all prescribed animal medicine. Due to their high polarity nature (log Kow < -3), they require chromatographic separation by hydrophilic interaction liquid chromatography or ion-pairing chromatography. This study presents the development of an ion pairing liquid chromatography with alkanesulfonates coupled to tandem mass spectrometry for the analysis of 10 aminoglycosides (spectinomycin, streptomycin, dihydrostreptomycin, kanamycin, apramycin, gentamicin, neomycin and sisomicin) in wastewater samples. The novelty of this method lies in the addition of the ion paring salt directly and only into the sample vial and not in the mobile phase, lowering the amount of salt added and consequently reducing signal inhibition. The optimized method was validated and showed satisfactory resolution, performances suitable with the analysis of aminoglycosides in wastewater samples, with limits of quantifications less than 10 ng/mL for most of the compounds, low matrix effects, high accuracy (85%-115% recoveries) and reproducibility (2%-12%RSD). It was then applied successfully to raw and treated wastewater samples

Gentamicin Adsorption onto Soil Particles Prevents Overall Short-Term Effects on the Soil Microbiome and Resistome

Antibiotics used in agriculture may reach the environment and stimulate the development and dissemination of antibiotic resistance in the soil microbiome. However, the scope of this phenomenon and the link to soil properties needs to be elucidated. This study compared the short-term effects of a range of gentamicin concentrations on the microbiome and resistome of bacterial enrichments and microcosms of an agricultural soil using a metagenomic approach. Gentamicin impact on bacterial biomass was roughly estimated by the number of 16SrRNA gene copies. In addition, the soil microbiome and resistome response to gentamicin pollution was evaluated by 16SrRNA gene and metagenomic sequencing, respectively. Finally, gentamicin bioavailability in soil was determined. While gentamicin pollution at the scale of µg/g strongly influenced the bacterial communities in soil enrichments, concentrations up to 1 mg/g were strongly adsorbed onto soil particles and did not cause significant changes in the microbiome and resistome of soil microcosms. This study demonstrates the differences between the response of bacterial communities to antibiotic pollution in enriched media and in their environmental matrix, and exposes the limitations of culture-based studies in antibiotic-resistance surveillance. Furthermore, establishing links between the effects of antibiotic pollution and soil properties is needed

Gentamicin at sub-inhibitory concentrations selects for antibiotic resistance in the environment

International audienceAbstract Antibiotics released into the environment at low (sub-inhibitory) concentrations could select for antibiotic resistance that might disseminate to the human microbiome. In this case, low-level anthropogenic sources of antibiotics would have a significant impact on human health risk. In order to provide data necessary for the evaluation of this risk, we implemented river water microcosms at both sub-inhibitory and inhibitory concentrations of gentamicin as determined previously based on bacterial growth in enriched media. Using metagenomic sequencing and qPCR/RT-qPCR, we assessed the effects of gentamicin on water bacterial communities and their resistome. A change in the composition of total and active communities, as well as a gentamicin resistance gene selection identified via mobile genetic elements, was observed during a two-day exposure. We demonstrated the effects of sub-inhibitory concentrations of gentamicin on bacterial communities and their associated resistome in microcosms (simulating in situ conditions). In addition, we established relationships between antibiotic dose and the magnitude of the community response in the environment. The scope of resistance selection under sub-inhibitory concentrations of antibiotics and the mechanisms underlying this process might provide the basis for understanding resistance dispersion and associated risks in relatively low impacted ecosystems

Environmental resistome transcriptional response to gentamicin pollution at sub-inhibitory concentrations

National audienc

Sequencing Depth Has a Stronger Effect than DNA Extraction on Soil Bacterial Richness Discovery

Although Next-Generation Sequencing techniques have increased our access to the soil microbiome, each step of soil metagenomics presents inherent biases that prevent the accurate definition of the soil microbiome and its ecosystem function. In this study, we compared the effects of DNA extraction and sequencing depth on bacterial richness discovery from two soil samples. Four DNA extraction methods were used, and sequencing duplicates were generated for each DNA sample. The V3–V4 region of the 16S rRNA gene was sequenced to determine the taxonomical richness measured by each method at the amplicon sequence variant (ASV) level. Both the overall functional richness and antibiotic resistance gene (ARG) richness were evaluated by metagenomics sequencing. Despite variable DNA extraction methods, sequencing depth had a greater influence on bacterial richness discovery at both the taxonomical and functional levels. Sequencing duplicates from the same sample provided access to different portions of bacterial richness, and this was related to differences in the sequencing depth. Thus, the sequencing depth introduced biases in the comparison of DNA extraction methods. An optimisation of the soil metagenomics workflow is needed in order to sequence at a sufficient and equal depth. This would improve the accuracy of metagenomic comparisons and soil microbiome profiles

Environmental resistome transcriptional response to gentamicin pollution at sub-inhibitory concentrations

National audienc