Reduction of the fitness burden of quinolone resistance in Pseudomonas aeruginosa.
OBJECTIVE
Quinolone resistance in the opportunistic pathogen Pseudomonas aeruginosa is commonly caused by mutations that alter the target molecules DNA gyrase/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 fitness was determined by measuring growth rates in vitro. Mutants with reduced growth rates were serially passaged to obtain growth-compensated mutants. The level of DNA supercoiling was determined by isolating plasmid DNA from the susceptible, resistant and compensated mutants and comparing the topoisomer distribution patterns by gel electrophoresis in the 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 double mutations in gyrA and parC, parE, nfxB or unknown genes all showed a reduced fitness. Slow-growing resistant mutants with a gyrA mutation had decreased DNA supercoiling. After serial passage in laboratory medium, mutant fitness was increased by compensatory mutation(s) that restored supercoiling to normal levels. The compensatory mutation(s) was not located in any of the genes (gyrAB, topA, parCE, hupB, fis, hupN, himAD or PA5348) that were expected to affect supercoiling.


CONCLUSIONS
Our results show that 'no cost' and compensatory mutations are common in quinolone-resistant P. aeruginosa.
Kugelberg
Elisabeth
E
Swedish Institute for Infectious Disease Control, Department of Bacteriology, S-171 82 Solna, Sweden.
Löfmark
Sonja
S
Wretlind
Bengt
B
Andersson
Dan I
DI
D000890:Anti-Infective Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D003470:Culture Media; D027081:DNA Gyrase / Q000235:genetics; D027101:DNA Topoisomerase IV / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D019143:Evolution, Molecular; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009643:Norfloxacin / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity; D015363:Quinolones / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics
D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D003470:Culture Media; D004268:DNA-Binding Proteins; C077901:NfxB protein, Pseudomonas aeruginosa; D015363:Quinolones; D014157:Transcription Factors; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase; D009643:Norfloxacin