Unraveling the Interplay Between Self-Organization and Antimicrobial Resistance Evolution Across Spatial Scales - Title: Unraveling the Interplay Between Self-Organization and Antimicrobial Resistance Evolution Across Spatial Scales The rise of antimicrobial resistance (AMR) is a serious threat to global health. Most microbial populations affecting human health are spatially self-organized across many scales, ranging from small surface attached biofilms to populations distributed across the lumen of the gut. Although laboratory evolution in tight synergy with theory has emerged as a powerful experimental approach to study AMR evolution in well-mixed test tube environments, we know little about the impact of spatial self-organization on AMR evolution. The challenges are to (i) track AMR evolution at high (ideally cell) resolution in space and time (ii) across the variety of spatial scales and structures using (iii) a single bacterial species and (iv) the joint modeling of population genetics and self- organization. Finally, while in vitro studies are ideal settings for deriving general rules for a wide range of structures, an in vivo implementation is needed to test whether those rules have any significance to actual within host AMR evolution. The objective of the proposed research is to measure and model how AMR evolution depends on the self- organization of bacterial populations, ranging from micro-scale biofilms to gut-lumen distributed populations. To this end, the P.I.s propose tightly-controlled experiments to track AMR evolution in a model pathogen (V. cholerae) both in vitro (microfluidics) and in vivo (zebrafish) spanning cellular to community-level scales, as well as extrapolating simulations and theory. The proposed research leverages an intense dialog between population genetic theory and experiment to achieve a predictive understanding of self-organization in microbial populations in terms of the joint actions of individual cells. The P.I.s have three specific aims. First, they will advance a synthetic mutagenesis system to engineer V. cholerae strains that enable tracking evolutionary processes in space and time. Second, they use advanced time-lapse imaging to monitor the spatio-temporal establishment of AMR clones from de novo mutations, standing variation and extrinsic invasion across a multi-scale panel of microfluidic cavities and in zebrafish as a host model system. Third, they will develop mathematically models to explain the observed structure-dependent rapidity of AMR evolution and generalize the obtained insights to conditions not tested in experiments. The P.I.s develop microfluidic techniques to control the self-organization of bacterial populations, synthetic biology and imaging techniques to track complex evolutionary processes via fluorescent imaging and employ state-of-the-art assays to study the zebrafish gut microbiome. These methods along with our computer simulations will be of broad utility to the biophysics community for the goal of dissecting collective properties of microbial populations. 1
