Agar concentration affects the evolution of antibiotic resistance and population genomics in experimental populations Pseudomonas aeruginosa

Bacteria live in a diversity of spatially structured environments, which can impact their evolutionary dynamics via local interactions and environmental variation. Spatial structure is expected to slow the rate of adaptive evolution, increase the amount of clonal competition and so increase diversity of evolutionary trajectories explored as a population evolves. In the lab, agar is a common way in which we add spatial structure to bacterial growth environments. In this study we explored the effects of agar concertation on experimental populations of Pseudomonas aeruginosa evolved in the presence/ absence of a sub-lethal concentration of an antibiotic, ciprofloxacin. We varied agar across four different concentrations which modified viscosity and so the rate at which bacteria move through their environment, as well as potentially shifting the mode of motility. We saw that increasing agar concentration decreased the rate of adaptation, and that the presence of antibiotics, amplified this effect. The number and frequency of evolved mutations also varied with agar concentration and the direction of the effect changed in the presence/ absence of antibiotics – for example, the number of high frequency mutation increased with agar concentration when antibiotics were absent but decreased when antibiotics were present. We also saw an increase in the degree of parallel evolution in populations evolved in the presence of antibiotics and even more so in higher concentrations of agar. Thus, we show that agar concentration, and so spatial structure, can be an important driver of evolutionary dynamics with important impacts on antibiotic resistance evolution including the rate and predictability of adaptation.