Examination of single and multiple mutations involved in resistance to quinolones in Staphylococcus aureus by a combination of PCR and denaturing high-performance liquid chromatography (DHPLC).

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Abstract
Detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that has been used to detect mutations in human DNA. We report on the first study to use this technique as a tool to detect mutations in genes encoding antibiotic resistance in bacteria. Three methicillin-sensitive and three methicillin-resistant clinical Staphylococcus aureus isolates, susceptible to ciprofloxacin (MIC ≤ 0.4 mg/L), were used to derive mutants resistant to ciprofloxacin, levofloxacin, sparfloxacin, trovafloxacin and moxifloxacin. Genomic DNA from each strain was subjected to PCR amplification of 225–500 bp regions spanning the quinolone resistance determining regions of the gyrA, gyrB, grlA and grlB genes. Following DNA sequencing of these amplicons and identification of resistance mutations, DHPLC was undertaken to correlate the distinctive chromatogram with DNA sequence. The mutations detected by DHPLC resulted in the following amino acid substitutions: Ser-84→Leu, Ser-112→Pro, Glu-88→Lys in GyrA, Glu-84→Val, Ser-80→Phe in GrlA, Pro-456→Ser in GyrB and Glu-422→Asp, Pro-451→Ser, Asp-432→Gly in GrlB. Mutations could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This system facilitates the detection of resistance alleles, providing a rapid (5 min per sample), economic (96 sample per run) and reliable technique for characterizing antibiotic resistance in bacteria.

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