Single-cell study of the emergence of antibiotic resistance and bacterial genome diversification – UniBac

We have used the universal platform developed, patented and published by Charles Baroud's laboratory, which enables us to track the growth dynamics of thousands of individual colonies from single cells, which are separated into microfluidic droplets at the nanoliter scale. In this way, descriptors of growth parameters (e.g. lag time, maximum growth rate, final colony size, ...) can be determined for thousands of individual colonies in parallel, providing a distribution of values for each condition.

We were able to screen 5 different ABs in this system. Analysis of these data enabled us to compare the response of individual cells to these antibiotics, which have different mechanisms of action, and to relate this response to the mechanism of action of the ABs. We were also able to compare the evolution of cell morphology under the action of different molecules, notably as a signature of AB stress. Thanks to a new artificial intelligence tool, we were able to identify changes in cell morphology in the images (around 200,000 images of individual drops), in order to link them to the mechanism of action of the ABs. At the same time, we have continued the molecular characterization of the different responses to AB stress, in E. coli and V. cholerae, and made several important discoveries about their impact on the persistence of bacteria during treatment

The project has already led to several publications. We have identified various factors, all linked to the translation machinery and the maintenance of protein homeostasis, which play an important role in the phenotype of tolerance and persistence to aminoglycosides (AG). We have also shown an interaction between quorum sensing and AGs, suggesting that targeting QS signaling could be a strategy for improving the efficacy of these ABs in V. cholerae. The validation of the platform, its detailed description and the first results on monitoring during treatment with ciprofloxacin have been submitted for publication.
This project formalized the collaboration between our two teams and led to a new collaboration on the role of membrane vesicles in ABs resistance.

7 publications and softwares

Antimicrobial resistance is a major threat worldwide that requires a strong investment in fundamental studies. Resistance, as well as transient tolerance (persistence) to antibiotics, involve a network of intracellular stress responses: e.g., the stringent response, the SOS response, and the RpoS-regulated general stress response. We and others have shown that these stress responses are induced by antibiotic (AB) doses below the minimum inhibitory concentration (sub-MIC) and that they can accelerate acquisition of heritable AB resistance through increased mutagenesis and horizontal gene transfer (HGT). Although low concentrations of antibiotics do not kill bacteria, they can have a major impact on bacterial populations. In particular, it was shown that AB concentrations as low as hundred-fold below the MIC can lead to mutations and the selection of AB resistant cells.
Most of the studies describing how bacteria acquire resistance or become persisters are based on experiments dealing with populations of cells. Such measurements yield average quantities for the whole population but they cannot provide a distribution of responses, nor can they follow the temporal evolution of individuals within the population. By contrast, there is mounting evidence that cells within a given population can display widely heterogeneous responses to an AB stress.
This project aims at describing precisely individual cell fate during stress responses to low doses of antibiotics, and understanding the emergence of antibiotic resistance on the level of a single cell. We propose to address the profile of induction of four stress responses at the single-cell level: SOS, stringent response, RpoS general stress response and oxidative stress response, in response to three ABs from different families (fluoroquinolones, aminoglycosides, ß-lactams). To this end, we will use a microfluidic platform to culture bacteria, while submitting them to controlled AB stresses to assess heterogeneity and growth on chip. We will develop the theoretical description of bacterial growth dynamics taking into account the AB stress through mathematical modelling relating large-scale heterogeneity to the variability on the scale of individual cells. We will then isolate and extract cells that show phenotypic diversity. The large statistics will allow us to get access to rare events. The extracted cells will be subjected to analysis (NGS, dPCR) in order to detect horizontal gene transfers, mutations or changes of protein expression that can explain the behavior of these cells. This will first require the development of technological tools to genotype the small number of bacterial cells that can be recovered from the microchannel. The second step will be to explore different conditions that lead to the emergence of antibiotic resistance in order to gain insight into the underlying mechanisms and devise strategies to counter them.
The impact of this project will be threefold: (i) Concerning the fundamental biological knowledge it will bring, (ii) the technological and quantitative developments that accompany it, and (iii) in understanding the emergence of resistance mechanisms and their implications for the development of new therapeutic strategies.