Ciprofloxacin (CIP) is used to treat P. aeruginosa biofilm infections. We showed that the pathways of CIP-resistance development during exposure of biofilms and planktonic P. aeruginosa populations to sub-inhibitory levels of CIP depend on the mode of growth. In this study, we analyzed CIP-resistant isolates obtained from previous evolution experiments, and we report a variety of evolved phenotypic and genotypic changes that occurred in parallel with the evolution of CIP-resistance. Cross-resistance to beta-lactam antibiotics was associated to mutations in genes involved in cell-wall recycling (ftsZ, murG); and could also be explained by mutations in the TCA cycle (sdhA) genes and in genes involved in arginine catabolism. We found that CIP-exposed isolates that lacked mutations in quorum-sensing genes and acquired mutations in type IV pili genes maintained swarming motility. and lost twitching motility, respectively. Evolved CIP-resistant isolates showed high fitness-cost, yet persisted in the biofilm under control conditions, compared with ancestor isolates; however, they had the advantage when exposed to CIP. Their persistence in competition experiments could be explained by their prolonged lag-phase. Interestingly, the set of mutated genes that we identified in these in vitro-evolved CIP-resistant colonies, overlap with a large number of patho-adaptive genes previously reported in P. aeruginosa isolates from cystic fibrosis (CF) patients. This suggests that the antibiotic stress is shaping the bacterial evolution in vivo, and that our model can be used to predict the in vivo evolutionary trajectories.
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