Evolution of Cost-Free Resistance under Fluctuating Drug Selection in Pseudomonas aeruginosa

Antibiotic resistance evolves rapidly in response to drug selection, but it can also persist at appreciable levels even after the removal of the antibiotic. This suggests that many resistant strains can both be resistant and have high fitness in the absence of antibiotics. To explore the conditions under which high-fitness, resistant strains evolve and the genetic changes responsible, we used a combination of experimental evolution and whole-genome sequencing to track the acquisition of ciprofloxacin resistance in the opportunistic pathogen Pseudomonas aeruginosa under conditions of constant and fluctuating antibiotic delivery patterns. We found that high-fitness, resistant strains evolved readily under fluctuating but not constant antibiotic conditions and that their evolution was underlain by a trade-off between resistance and fitness. Whole-genome sequencing of evolved isolates revealed that resistance was gained through mutations in known resistance genes and that second-site mutations generally compensated for costs associated with resistance in the fluctuating treatment, leading to the evolution of cost-free resistance. Our results suggest that current therapies involving intermittent administration of antibiotics are contributing to the maintenance of antibiotic resistance at high levels in clinical settings. IMPORTANCE Antibiotic resistance is a global problem that greatly impacts human health. How resistance persists, even in the absence of antibiotic treatment, is thus a public health problem of utmost importance. In this study, we explored the antibiotic treatment conditions under which cost-free resistance arises, using experimental evolution of the bacterium Pseudomonas aeruginosa and the quinolone antibiotic ciprofloxacin. We found that intermittent antibiotic treatment led to the evolution of cost-free resistance and demonstrate that compensatory evolution is the mechanism responsible for cost-free resistance. Our results suggest that discontinuous administration of antibiotic may be contributing to the high levels of antibiotic resistance currently found worldwide.