RESUMO
The genes encoding topoisomerases (gyrA and grlA) and the norA promoter of 100 fluoroquinolone-susceptible and -resistant Staphylococcus aureus clinical isolates obtained in two geographically distant hospitals were analysed. The relationship between mutations found and the susceptibility to newer quinolones was determined. Thirty-nine strains were grouped in seven clones by pulsed-field gel electrophoresis (PFGE). The remaining 61 strains were classified as unrelated strains. In three clones, all strains showed the same grlA-gyrA-norA mutation profiles. Strains in the rest of the groups showed different mutation profiles, even though PFGE indicated that they possessed genetically similar populations. One cluster showed a high level of diversity; five different mutation profiles were detected in the six isolates belonging to this pattern. Two isolates had a Glu84 to Lys mutation in grlA and another isolate had this mutation combined with a Ser84 to Leu mutation in gyrA. Combination of a Ser80 to Phe mutation in grlA and a Ser84 to Leu in gyrA was found in the two other isolates. One of these also had a thymine to a guanine transversion at a position 89 nucleotides upstream of the norA start codon in the norA promoter. These results show that fluoroquinolone resistance in clinical S. aureus strains does not necessarily result from the spread of resistant clones. Fluoroquinolone resistance may develop independently in strains belonging to the same PFGE pattern by accumulation of different mutations over a quinolone-susceptible ancestor wild type or single grlA mutant.
Assuntos
Anti-Infecciosos/farmacologia , Variação Genética/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , 4-Quinolonas , Proteínas de Bactérias/genética , Northern Blotting , DNA Bacteriano/análise , Eletroforese em Gel de Campo Pulsado , Humanos , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Mutação/genética , Regiões Promotoras Genéticas/genética , Infecções Estafilocócicas/microbiologiaRESUMO
The aim of this study was to determine the in vitro activity of five quinolones against clinical strains of methicillin-susceptible and -resistant Staphylococcus aureus clinical isolates characterized at the molecular level with respect to the presence of mutations in genes coding for resistance to quinolones (grlA, gyrA and gyrB). The relationship between the mutations found and the activities of these quinolones was also analyzed. Trovafloxacin was the most active against methicillin-susceptible S. aureus and showed good activity against methicillin-resistant S. aureus, with a MIC90 of 2 mg/l. The grlA-gyrA double mutation was the most frequent (55% of the strains). Single mutation in grlA was detected only in 5% of strains; 39% of strains showed a wild-type genotype. The grlA-gyrA double mutants presented a high level of resistance against the fluoroquinolones tested except for trovafloxacin, with the MIC ranging between 0.5 and 4 mg/l. Wild-type strains were susceptible to all the fluoroquinolones tested and the single grlA mutants had a low level of quinolone resistance but were still below the breakpoint for resistance. Trovafloxacin and sparfloxacin were less affected by this mutation.
Assuntos
Anti-Infecciosos/farmacologia , DNA Topoisomerases Tipo II/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , DNA Girase , DNA Topoisomerase IV , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/genética , Fluoroquinolonas , Testes de Sensibilidade Microbiana , Mutação , Polimorfismo Conformacional de Fita Simples , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Se ha estudiado la actividad in vitro de cinco fluoroquinolonas frente a cepas clínicas de Staphylococcus aureus, sensibles y resistentes a la meticilina, caracterizadas desde el punto de vista genético respecto a la existencia de mutaciones en los genes causantes de la resistencia a las quinolonas (grlA, gyrA y gyrB). También se comprobó el efecto de estas mutaciones en la actividad de las quinolonas. El trovafloxacino apareció como el más activo de los antimicrobianos estudiados frente a S. aureus sensibles a la meticilina (SASM), presentando también una buena actividad frente a los resistentes a ésta (SARM), con una CMI90 de 2 mg/l. El patrón más frecuente fue la doble mutación gyrA y grlA (55 por ciento de las cepas), siendo mucho menos frecuente la mutación únicamente en grlA (5 por ciento). El 39 por ciento de las cepas presentaron un genotipo silvestre. Las cepas con doble mutación presentaron altos grados de resistencia a todas las fluoroquinolonas probadas excepto a trovafloxacino, con un rango de CMI de 0,5-4 mg/l. Las cepas silvestres fueron sensibles a todas las quinolonas ensayadas; en las cinco cepas con mutación en grlA se observó un ligero aumento de las CMI para todos los antimicrobianos, aunque siempre por debajo del punto de corte que determina resistencia. Las quinolonas menos afectadas por esta mutación fueron trovafloxacino y esparfloxacino (AU)
Assuntos
Staphylococcus aureus , Polimorfismo Conformacional de Fita Simples , Mutação , Fluoroquinolonas , DNA Girase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , DNA Topoisomerase IV , Anti-Infecciosos , DNA Bacteriano , Testes de Sensibilidade MicrobianaRESUMO
Fluoroquinolone efflux was studied in 47 Staphylococcus aureus clinical strains with MICs of ciprofloxacin (CFX) of < or = 2 micrograms/ml. Forty-three strains were wild type for gyrA, gyrB, and grlA quinolone resistance-determining regions and for norA and its promoter region. Forty of these strains (MICs of CFX, 0.1 to 0.2 microgram/ml) did not show efflux of fluoroquinolones. Three strains (MICs of CFX, 1 to 2 micrograms/ml) showed efflux. These results suggest that efflux can appear in S. aureus clinical strains in the absence of mutations in norA and its promoter.
