Background/Aims: The lungs of the patients with cystic fibrosis become chronically infected with the bacterium Pseudomonas aeruginosa. Infection can be treated with antibiotics such as ciprofloxacin but ciprofloxacin resistance in P. aeruginosa is increasing, making this drug less effective. The mechanisms of ciprofloxacin resistance in P. aeruginosa are partially understood. The purpose of this study was to find mutations in the P. aeruginosa genome associated with ciprofloxacin resistance in order to understand how the bacteria resist ciprofloxacin and how treatment influences the evolution of ciprofloxacin resistance.
Methods: Nine individual ciprofloxacin resistant strains driven from P. aeruginosa strain PAO1 were evolved in the laboratory using an antibiotic gradient agar plate method. Minimal inhibitory concentration (MIC) testing and Whole genome sequencing (WGS) was carried out.
Results: MIC showed that the evolved strains had ~4000 higher ciprofloxacin resistance than the parental PAO1 strain. WGS showed that mutations in gyrA (gyraseA), PA3491(probable ferredoxin), nfxB (negative regulator of efflux), parC/parE (topoisomerase IV subunits) and pil (pilin) genes are strong contributors of ciprofloxacin resistance. Mutations in parC were associated with high level of resistance. WGS of strains with intermediate resistance showed that mutations are acquired in a specific order. Comparison of mutations in the lab-evolved P. aeruginosa with the sequences of strains from cystic fibrosis patients showed that alleles conferring resistance in laboratory evolved samples are also present in strains from patients. Conclusion: Overall, our findings provide new insights into how P. aeruginosa evolves resistance to a key anti-Pseudomonal antibiotic.