Ninety-two and 33 methicillin-resistant (MRSA) strains were isolated in Japan and China respectively. They were categorised as ofloxacin-susceptible (MIC < 12.5 mg/L), moderately (MIC 12.5-25 mg/L) or highly (MIC ≧ 50 mg/L) ofloxacin-resistant. 4-Quinolone concentrations required to inhibit purified DNA gyrase from the moderately and highly quinolone-resistant MRSA were at least 20 times higher than those required to inhibit the equivalent enzyme from quinolone-susceptible strains. Reconstitution assays demonstrated that the 4-quinolone-resistant MRSA had a mutation in subunit A of DNA gyrase. A portion of the gene from amino acids codons 40–115 was sequenced. Four moderately resistant and seven highly resistant MRSA contained a Ser → Leu substitution at amino acid 84; one moderately and one highly resistant MRSA and one moderately resistant methicillin-susceptible (MSSA) strain contained a Glu → Lys substitution at amino acid 88. Eight MRSA, including one quinolone-susceptible strain and one MSSA contained a silent mutation at amino acid 86. Uptake of ofloxacin in moderately resistant strains was almost the same in the presence or absence of carbonyl cyanide mchlorophenylhydrazone (CCCP), whereas in highly resistant strains, uptake increased when CCCP was added. Restriction fragment length analysis of the gene with the restriction endonuclease I showed a mutation of nucleotide position 1085 in all MRSA strains tested except for one highly quinolone-resistant strain. Thus the mechanisms of 4-quinolone-resistance in these MRSA isolates involved alterations in both DNA gyrase and antimicrobial uptake and efflux.


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