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Home > Thèses et HDR > Thèses en 2021

30/03/2021 - Concepcion SANCHEZ-CID

by Laurent Krähenbühl - published on , updated on

Agenda

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Concepcion Sanchez-Cid defends her PhD on March 30, 2021 at 9:00 AM.
Place : visio conference only.

Characterization of antibiotic resistance genes in microbiomes from different ecosystems.

Jury :

  • Michael GILLINGS - Full Professor, Macquarie University, Department of Biological Sciences (Australie) - Rapporteur
  • Elizabeth WELLINGTON - Full Professor, University of Warwick, School of Life Sciences (Royaume Uni) - Rapporteur
  • Timothy VOGEL - Professeur, HDR, Université Claude Bernard - Directeur de thèse
  • Sabine Favre-Bonté - Maître de conférences, HDR, Université Claude Bernard - Examinatrice
  • Graeme NICOL - Directeur de recherche, HDR, CNRS - Examinateur
  • Kornelia SMALLA - Full Professor, Julius Kühn Institut, Institute for Epidemiology and Pathogen Diagnostics (Allemagne) - Examinatrice
  • Laurence DELAURIERE - Docteur, Senior Application Specialist (Promega France) - Invitée

Abstract :
Since the discovery of antibiotics 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 and the emergence of multidrug resistant (MDR) clones worldwide, which may result in antibiotic therapy failure and the beginning of a post-antibiotic era. 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) reach the environment mainly through wastewater treatment plants, manure fertilization or release from antibiotic producing plants. 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. Firstly, a methodological study comparing five DNA extraction methods and four RNA extraction methods was carried out to evaluate the effect of DNA extraction and sequencing depth on taxonomic, functional and ARG richness discovery in soil. Whereas sequencing depth had a stronger impact than DNA extraction on taxonomic and functional richness discovery, a high variability in ARG richness discovery was observed between triplicates regardless of sequencing depth. Furthermore, some methods arguably measured a higher ARG richness than others. Based on this study, a semi-automated method was selected for DNA extraction from antibiotic polluted soils.
Secondly, two microcosm studies were carried out to evaluate the effects of antibiotic pollution on the microbiome and resistome of an agricultural soil from La Côte de Saint André (France) and of water from the Rhône river (France) 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. Whereas inhibitory concentrations up to 1 mg of gentamicin per gram of soil were highly adsorbed onto soil particles and did not have significant effects on the soil microbiome nor resistome after 8-days exposure, a sub-inhibitory concentration of 50 ng of gentamicin per ml of water impacted in the composition of bacterial total and active communities and the abundance and expression of gentamicin resistance genes during 2-days exposure. These findings support the concern that sub-inhibitory concentrations of antibiotics may select for resistance in the environment and therefore deserve more attention when assessing the risks associated to antibiotic environmental pollution. 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. Since anthropogenic activities induce changes in the snow resistome without having a strong impact on the core microbiome, the effects of this pollution are probably caused by an increased input of organic matter from anthropogenic waste rather than by human microbiome ARB and ARG. 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).

Keywords :
antibiotic resistance, environmental resistome, ARGs, anthropogenic pollution, resistance development, DNA extraction, gentamicin, sub-inhibitory concentrations, inhibitory concentrations, metagenomics, Illumina MiSeq sequencing, Oxford Nanopore sequencing, qPCR/RT-qPCR.