Abstract
Many opportunistic pathogens live in surface-attached communities called biofilms that generate ecological structure and can increase stress tolerance. Theory suggests that bacterial populations evolving in biofilms may harbor greater genetic diversity and become resistant to antibiotics by different pathways than in well-mixed environments. We used experimental evolution and whole genome sequencing to test how the mode of growth influences dynamics and mechanisms of antibiotic resistance in Acinetobacter baumannii populations. Biofilm and planktonic populations were propagated under conditions lacking antibiotics, under constant sub-inhibitory concentrations of ciprofloxacin, or under steadily increasing concentrations of this drug. As predicted, both the evolutionary dynamics and the identities of selected mutations differed between treatments and lifestyle. Planktonic populations exposed to ciprofloxacin underwent sequential selective sweeps of single mutations including the primary drug targets, gyrA and parC. In contrast, biofilm-adapted populations diversified by multiple contending mutations in regulators of efflux pumps. Mutants isolated from both lifestyles exhibited a trade-off between fitness and resistance level, wherein biofilm-adapted clones were less resistant but more fit in the absence of drug. Further, biofilm-adapted populations evolved collateral sensitivity to cephalosporins whereas the planktonic populations displayed cross-resistance with several classes of antibiotics. This study demonstrates that growth in biofilms, arguably the predominant bacterial lifestyle, may substantially alter the routes, dynamics, and consequences of the evolution of antibiotic resistance and is therefore an important consideration when treating infections.