Radial expansion facilitates the maintenance of double antibiotic resistances.

Most microbes live in spatially confined sub-populations. Under spatial structure, the efficacy of natural selection is often reduced (relative to homogeneous conditions), due to the increased importance of genetic drift and local competition. Additionally, under spatial structure, the fittest genotype may not always be the one with better access to the heterogeneous distribution of nutrients. The effect of radial expansion may be particularly relevant for the elimination of antibiotic resistance mutations, as their dynamics within bacterial populations are strongly dependent on their growth rate. Here, we use Escherichia coli to systematically compare the allele frequency of streptomycin, rifampicin and fluoroquinolone single and double resistance mutants after 24h of coexistence with a susceptible strain under radial expansion (local competition) and homogeneous (global competition) conditions. We show that there is a significant effect of structure on the maintenance of double resistances which is not observed for single resistances. Radial expansion also facilitates the persistence of double resistances when competing against their single counterparts. Importantly, we found that spatial structure reduces the rate of compensation of the double mutant RpsLK43TRpoBH526Y and that a strongly compensatory mutation in homogeneous conditions becomes deleterious under spatial structure. Overall, our results unravel the importance of spatial structure for facilitating the maintenance and accumulation of multiple resistances over time and for determining the identity of compensatory mutations.