Mechanisms driving the dissemination of multidrug resistance plasmids – plasMED

Multidrug resistance (MDR), in particular in bacteria, is an increasingly serious threat to global public health. In September 2016, world leaders signaled an unprecedented level of attention to curb the emergence and spread of MDR, and committed to taking a broad coordinated approach to address the root causes of MDR. This complex problem requires actions across all government sectors and society, such as finding new antimicrobial molecules, developing technologies for vaccines or diagnosis, promoting best practices in the use of antibiotics in humans and animals, as well as fostering research to improve our understanding of the mechanisms by which MDR factors propagate within bacterial populations. This latter approach is the heart of the present collaborative fundamental research project.
MDR often results from the acquisition of foreign resistance genes, mainly by conjugation, which is the major mechanism of horizontal gene transfer (HGT) between bacterial cells in both intra- and inter-species. During conjugation, DNA molecules (mainly plasmids) are transferred from a donor cell to a recipient cell. If the acquired DNA is maintained, the recipient gains new genetic traits and is consequently definitively converted into a new donor, which in turn contributes to the exponential spread of MDR. Until now, conjugation has mainly been characterized using traditional genetic or biochemistry techniques and subsequent maintenance strategies have not been appreciated as deserved. In addition, regarding the emergence of MDR bacteria, the development of “superspreaders”, mutant plasmids exhibiting improved transfer efficiencies, has been overlooked.
Here, we propose an innovative and ambitious in-depth study of bacterial conjugation at both molecular and cellular scales. We will develop modern approaches to analyze three different conjugative plasmids for better grasp of overarching principle: pESBL is associated with severe MDR Escherichia coli outbreak; R388 contributes significantly to the spread of MDR with very large host spectrum; the E. coli sex factor F, paradigm of diverse and widespread incF plasmids. The proposed experimental strategy is based on the strong complementarities of each partner’s expertise within our consortium, i.e., Yoshiharu YAMAICHI (CR-CNRS) at I2BC, CNRS Gif-sur-Yvette, Christian LESTERLIN (CR-INSERM) at MMSB, CNRS/Univ. Lyon, and Jean-Yves BOUET (DR2-CNRS) at LMGM, CNRS/Univ. Toulouse.

We defined two major axes to be carried out:
[1] Dissecting the molecular mechanisms driving dissemination of conjugative plasmids.
[2] Uncovering the spatiotemporal dynamics of conjugative plasmids at the cellular scale.

Our key preliminary results include innovation of microfluidic-based system to track conjugative DNA transfer events and the fate of donor and recipient cells. In addition to providing original concept to this program, isolation of “superspreader” mutations also represents a totally new tool for the study of conjugation.

By combining promising preliminary data with modern, multidisciplinary and complementary approaches, including molecular bacterial genetics, protein biochemistry and state-of-the-art cell biology, the plasMED project is expected to provide an integrated understanding of gene transfer among bacteria, at the molecular and cellular scales. This fundamental research work is essential among other actions urging to curb the emergence of MDR bacteria in natural and clinical environments.