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. 1994 Mar;38(3):428–431. doi: 10.1128/aac.38.3.428

Detection of novel mutations in the gyrA gene of Staphylococcus aureus by nonradioisotopic single-strand conformation polymorphism analysis and direct DNA sequencing.

Y Tokue 1, K Sugano 1, D Saito 1, T Noda 1, H Ohkura 1, Y Shimosato 1, T Sekiya 1
PMCID: PMC284475  PMID: 7911287

Abstract

A total of 36 clinical isolates of Staphylococcus aureus (29 fluoroquinolone-resistant strains and 7 fluoroquinolone-susceptible strains) were studied for the presence of point mutations in the gyrA gene by nonradioisotopic single-strand conformation polymorphism (Non-RI SSCP) analysis with silver stain. Direct DNA sequencing analysis of the PCR-amplified DNA fragments confirmed the results obtained by Non-RI SSCP analysis and revealed that fluoroquinolone resistance is closely associated with six types of mutations in the gyrA gene, of which three types of mutations were newly identified: (i) Ser-84-->Leu and Glu-88-->Gly, (ii) Ser-84-->Leu and Glu-88-->Lys, and (iii) Glu-88-->Gly. Furthermore, the novel ATT-->ATC mutation at codon 86 (silent mutation) was seen in only one fluoroquinolone-susceptible strain. All seven mutational types were separated from the wild type in a single electrophoretic step within 3 h after PCR amplification. Thus, we conclude that this new technique is a rapid, simple, and useful screening method for the genotyping of gyrA mutations associated with fluoroquinolone resistance.

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Selected References

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