Effects of repeated culture in sub-inhibitory concentrations of ciprofloxacin on resistance and genetic characteristics of an ocular Pseudomonas aeruginosa isolate

PURPOSE: To analyse the resistance and genetic changes of a sensitive ocular isolate of Pseudomonas aeruginosa to repeated exposure to subinhibitory concentrations of ciprofloxacin. METHODS: A ciprofloxacin-sensitive ocular isolate P. aeruginosa PA123 was serially passaged under gradually increasing sub-inhibitory concentrations of ciprofloxacin for 18 passages. The minimum inhibitory concentration (MIC) was determined daily using the broth microdilution method, and after 18 passages when resistance had plateaued, ciprofloxacin was withdrawn and passaging continued for a further 13 days. A control culture in the absence of ciprofloxacin was passaged sequentially for 31 days. Genomic DNA from selected passages was extracted and subjected to whole-genome sequencing. Sequencing reads were quality-trimmed using Trimmomatic 0.38 and then analysed with Snippy 4.6.0 to identify acquired mutations using parent PA123 (non-passaged) as a reference strain. RESULTS: The parent strain had an initial MIC of 1 µg/mL, this increased during growth in sub-MIC ciprofloxacin two-fold at passage 4 (2 µg/mL), then progressively increased to 64 µg/mL at passage 15, at which it remained for a further three passages. Upon removal of ciprofloxacin, there was no change of the MIC (64 µg/mL) during the subsequent 13 passages. In early passages (P1-P7), PA123 acquired a Leu240 frameshift mutation in mexS, while in intermediate and later passages, Thr83Ile, Ala51Val, Asp87Tyr, duplication of alanine at position 570 (Ala570dup) mutations occurred in gyrA gene. Isolates from control passages acquired only background mutations unrelated to ciprofloxacin resistance. CONCLUSION: Stepwise accumulation of mutations in mexS and gyrA under sub-inhibitory ciprofloxacin pressure might lead to high-level resistance in PA123, with MICs remaining elevated even after drug removal, demonstrating stable resistance and linking sub-MIC exposure to resistance evolution.