Reduction of the fitness burden of quinolone resistance in Pseudomonas aeruginosa

• Objectives: Quinolone resistance in the opportunistic pathogen Pseudomonas aeruginosa is commonly caused by mutations that alter the target molecules DNAgyrase/topoisomerase IV, or cause activation of various efflux systems.We have analysed the effect of quinolone resistance caused by DNA gyrase/topoisomerase IV mutations on bacterial fitness. Methods: Norfloxacin-resistant mutants were isolated and by DNA sequencing the mutations conferring resistance were identified. Mutant fitnesswas determined by measuring growth rates in vitro. Mutants with reducedgrowth rates were serially passaged to obtain growth-compensatedmutants. The level of DNA supercoiling was determined by isolatingplasmid DNA from the susceptible, resistant and compensated mutants andcomparing the topoisomer distribution patterns by gel electrophoresis inthe presence of chloroquine. Results: Low-level resistance (4-48 mg/L) was caused by single mutations in gyrA or gyrB. Among these strains, three out of eight mutants showed lower fitness,whereas high-level resistant (>256 mg/L) mutants with doublemutations in gyrA and parC, parE, nfxB or unknown genes all showed areduced fitness. Slow-growing resistantmutants with a gyrA mutation had decreased DNA supercoiling. Afterserial passage in laboratory medium, mutant fitnesswas increased by compensatory mutation(s) that restored supercoiling tonormal levels. The compensatory mutation(s) was not located in any ofthe genes (gyrAB, topA, parCE, hupB, fis, hupN, himAD or PA5348) thatwere expected to affect supercoiling. Conclusions: Our results show that 'no cost' and compensatory mutations are common in quinolone-resistant P. aeruginosa.

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