The presence or absence of a methicillin resistance gene in 58 clinical isolates of Staphylococcus aureus was examined by the polymerase chain reaction (PCR) and Southern blot analyses. The results were analyzed in relation to those of the MIC assay of methicillin and oxacillin. PCR assay results were identical to those of Southern blot analysis of genomic DNA digested with HindIII (positive, 28 strains; negative, 30 strains). Among the 28 PCR-positive strains, 6 strains showed methicillin susceptibility by the conventional susceptibility test (MICs, less than or equal to 8 micrograms/ml). Culturing of the six strains with ceftizoxime led to an increase in the phenotypic level of resistance to methicillin and oxacillin, indicating that these strains should be classified as methicillin-resistant S. aureus (MRSA). The PCR assay was found to be a sensitive and reliable procedure for the rapid diagnosis of MRSA infection, even in cases in which the conventional MIC assay failed to detect MRSA.
The distribution of fluoroquinolone resistance-associated point mutations in genes encoding the subunits of DNA gyrase and DNA topoisomerase i.v. was examined in 110 clinical isolates of Staphylococcus aureus. Point mutations were detected by polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis and mutations were further characterized by sequencing of PCR products. Mutations at Ser84 of GyrA were widely distributed among isolates exhibiting various degrees of fluoroquinolone resistance, and border zones between mutant and non-mutant strains based on drug susceptibility were generally distinct. Mutations at Ser80 of GrlA were also widely distributed, but border zones between mutant and non-mutant isolates were in this case less distinct and several GrlA mutants were highly susceptible to sparfloxacin and tosufloxacin. Only two gyrB mutants and one grlB mutant were observed among the isolates: all contained a previously unreported mutation. GyrA and grlA mutations thus appear to impart high levels of fluoroquinolone resistance in many S. aureus clinical isolates.
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