Mechanism of quinolone resistance in Staphylococcus aureus.
The resistance mechanisms to fluoroquinolones in Staphylococcus aureus were clarified by analyzing mutations in the genes encoding target enzymes, and examining the expression of the efflux pump, and determining the inhibitory activities of fluoroquinolones against the altered enzymes. Mutations in the grlA and gyrA genes of 344 clinical strains of S. aureus isolated in 1994 in Japan were identified by combinations of methods - single-strand conformation polymorphism analysis, restriction fragment length analysis, and direct sequencing - to identify possible relationships with fluoroquinolone resistance. Five types of single-point mutations and four types of double mutations were observed in the grlA gene in 204 strains (59.3%). Four types of single-point mutations and four types of double mutations were found in the gyrA gene in 188 strains (54.7%). Among these mutations, the grlA mutation of TCC --> TTC or TAC (Ser-80 --> Phe or Tyr) and the gyrA mutation of TCA --> TTA (Ser-84 --> Leu) were the principal ones, being detected in 137 (39.8%) and 121 (35.2%) isolates, respectively. A total of 15 types of mutation combinations within both genes were related to ciprofloxacin resistance (MIC greater than or equal 3.13 microg/ml) and were present in 193 mutants (56.1%). Strains containing mutations in both genes were highly resistant to ciprofloxacin (MIC50 =50 microg/ml). Those strains with the Ser-80 --> Phe or Tyr alteration in grlA, but wild type in gyrA showed a lower level of ciprofloxacin resistance (MIC50 less than or equal 12.5 microg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlA in the presence or absence of concomitant mutations at codons 73, 84, or 88 in gyrA (MIC less than or equal 6.25 microg/ml). Sitafloxacin (DU-6859a) showed good activity in 186 of 193 isolates (96.4%), with an MIC of less than or equal 6.25 microg/ml. The contribution of membrane-associated multidrug efflux protein (NorA) expression to fluoroquinolone resistance was clarified by the checker-board titration method for determining the MIC of norfloxacin alone and in combination with carbonyl cyanide m-chlorophenylhydrazone. Among 344 clinical isolates, 139 strains (40.4%), in which the MIC of norfloxacin varied from 1.56 to >800 microg/ml, overexpressed the NorA protein. GrlA and GrlB proteins of topoisomerase IV, and GyrA and GyrB proteins of DNA gyrase encoded by genes with or without mutations were purified separately. The inhibitory activities of fluoroquinolones against the topoisomerase IV which contained a single amino acid change (Ser --> Phe at codon 80, Glu --> Lys at codon 84 of grlA, and Asp --> Asn at codon 432 of grlB) were from 5 to 95 times weaker than the inhibitory activities against the non-altered enzyme. These results suggest that the mutations in the corresponding genes may confer quinolone resistance; the active efflux pump, NorA, was considered to be the third quinolone-resistance mechanism. The numerous and complicated mutations seen may explain the rapid and widespread development of quinolone resistance described in S. aureus. Sitafloxacin showed good antibacterial activity against ciprofloxacin- or levofloxacin-resistant mutants because of its high inhibitory activity against both topoisomerase IV and DNA gyrase.
Tanaka
M
M
New Product Research Laboratories I, Daiichi Pharmaceutical Co. Ltd., 16-13 Kitakasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan. tanakpmj@daiichipharm.co.jp
Wang
T
T
Onodera
Y
Y
Uchida
Y
Y
Sato
K
K
D027101:DNA Topoisomerase IV / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D024841:Fluoroquinolones / Q000494:pharmacology*; D009154:Mutation; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics
D024841:Fluoroquinolones; D027101:DNA Topoisomerase IV