Ciprofloxacin (CIP) is used to treat Pseudomonas aeruginosa biofilm infections. We showed that the pathways of CIP-resistance development during exposure of biofilms and planktonic P. aeruginosa populations to subinhibitory levels of CIP depend on the mode of growth. In the present 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 with 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 in planktonic competition experiments, yet persisted in the biofilm under control conditions, compared with ancestor isolates and had an advantage when exposed to CIP. Their persistence in biofilm competition experiments in spite of their fitness cost in planktonic growth 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 contributing to the bacterial evolution in vivo, and that adaptive laboratory evolution can be used to predict the in vivo evolutionary trajectories.

环丙沙星（CIP）用于治疗铜绿假单胞菌生物膜感染。我们表明，在生物膜和浮游铜绿假单胞菌种群暴露于CIP亚抑制水平期间，CIP耐药性发展的途径取决于生长方式。在本研究中，我们分析了从先前的进化实验中获得的耐CIP分离株，并且我们报告了与CIP抗性进化同时发生的多种进化表型和基因型变化。对β-内酰胺类抗生素的交叉耐药性与细胞壁回收利用相关基因的突变有关（ftsZ，murG）；也可以通过TCA周期的突变（sdhA）基因和涉及精氨酸分解代谢的基因。我们发现，CIP暴露的分离株在群体感应基因中没有突变，而在IV型菌毛基因中获得了突变，分别保持了群体运动和抽动运动。进化后的耐CIP分离株在浮游竞争实验中显示出较高的适应性成本，但与祖先分离株相比，在受控条件下仍可在生物膜中持续存在，并且在暴露于CIP时具有优势。尽管他们在浮游生物生长过程中付出了适当的代价，但他们仍在生物膜竞争实验中坚持不懈，这可以用他们的滞后期延长来解释。有趣的是，我们在这些体外进化的CIP抗性菌落中鉴定出的突变基因与先前在铜绿假单胞菌中报道的大量可适应病的基因重叠。囊性纤维化（CF）患者的分离株。这表明抗生素的应激有助于体内细菌的进化，而适应性实验室进化可用于预测体内的进化轨迹。