Detection of novel mutations in the gyrA gene of Staphylococcus aureus by nonradioisotopic single-strand conformation polymorphism analysis and direct DNA sequencing.
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.
Tokue
Y
Y
Division of Clinical Laboratory, National Cancer Center Hospital, Tokyo, Japan.
Sugano
K
K
Saito
D
D
Noda
T
T
Ohkura
H
H
Shimosato
Y
Y
Sekiya
T
T
D001483:Base Sequence; D002939:Ciprofloxacin / Q000494:pharmacology; D004269:DNA, Bacterial / Q000032:analysis*; D004277:DNA, Single-Stranded / Q000032:analysis*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015242:Ofloxacin / Q000494:pharmacology; D009841:Oligonucleotides / Q000138:chemical synthesis; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics*
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D002939:Ciprofloxacin; D015242:Ofloxacin