Development of multiple-antibiotic-resistant (Mar) mutants of Pseudomonas aeruginosa after serial exposure to fluoroquinolones

Laboratory-derived fluoroquinolone-resistant mutants were created by serially passaging wild-type Pseudo-monas aeruginosa on fluoroquinolone-containing agar to obtain high-level fluoroquinolone resistance (e.g., ciprofloxacin MIC of 1,024 mg/ml). With increases of 4- to 32-fold in MICs of fluoroquinolones, these organisms demonstrated (relative to wild-type) normal morphology, resistance to fluoroquinolones only, no change in fluoroquinolone uptake, and no change in lipopolysaccharide profiles or outer membrane protein profiles. Complementation with wild-type Escherichia coli gyrA restored fluoroquinolone susceptibility, sug-gesting that these were gyrA mutants. After 4- to 32-fold increases in fluoroquinolone MICs (with continued passage on fluoroquinolone-containing agar) isolates demonstrated altered morphology, a multiple-antibiotic-resistant (Mar) phenotype (including cross-resistance to beta-lactams, chloramphenicol, and tetracycline), reduced fluoroquinolone uptake and altered outer membrane proteins (reductions in the 25- and 38-kDa bands as well as several bands in the 43- to 66-kDa region). Complementation with wild-type E. coli gyrA partially reduced the level of fluoroquinolone resistance by approximately 8- to 32-fold, suggesting that these mutants displayed both gyrA and non-gyrA mutations. Fluoroquinolones such as ciprofloxacin, norfloxacin, and ofloxacin have potent broad-spectrum antibiotic activitie