Parallel Evolution to Elucidate the Contributions of PA0625 and parE to Ciprofloxacin Sensitivity in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous pathogen that causes a wide range of acute and chronic infections. Ciprofloxacin, one of the first-line fluoroquinolone class antibiotics, is commonly used for the treatment of P. aeruginosa infections. However, ciprofloxacin-resistant P. aeruginosa is increasingly reported worldwide, making treatment difficult. To determine resistance-related mutations, we conducted an experimental evolution using a previously identified ciprofloxacin-resistant P. aeruginosa clinical isolate, CRP42. The evolved mutants could tolerate a 512-fold higher concentration of ciprofloxacin than CRP42. Genomic DNA reference mapping was performed, which revealed mutations in genes known to be associated with ciprofloxacin resistance as well as in those not previously linked to ciprofloxacin resistance, including the ParE(R586W) substitution and PA0625 frameshift insertion. Simulation of the ParE(R586W) substitution and PA0625 frameshift insertion by gene editing in CRP42 and the model strain PAO1 demonstrated that while the PA0625 mutation does contribute to resistance, mutation in the ParE(R586W) does not contribute to resistance but rather affects tolerance against ciprofloxacin. These findings advance our understanding of ciprofloxacin resistance in P. aeruginosa.