Molecular characterization of fluoroquinolone resistance in Mycobacterium tuberculosis: functional analysis of gyrA mutation at position 74.
A PCR-sequencing assay was evaluated for direct detection of mutations in the quinolone resistance-determining region (QRDR) of gyrase A (gyrA) gene in fluoroquinolone-resistant Mycobacterium tuberculosis in respiratory specimens. As determined by gyrA QRDR analysis, complete concordance of genotypic and phenotypic fluoroquinolone resistance was demonstrated. Our results indicate that the assay is a rapid and reliable method for the diagnosis of fluoroquinolone-resistant tuberculosis, facilitating timely clinical management and public health control. Using the assay, we detected a novel gyrA Ala74Ser mutation in M. tuberculosis directly from sputum specimens. The functional effect of the Ala74Ser mutant was verified through the study of the DNA supercoiling inhibitory activity of fluoroquinolones against the recombinant gyrase. The drug-mediated gyrase-DNA cleavage complex model suggests perturbation of the gyrA-gyrA dimer interface caused by the Ala74Ser mutation probably disturbs the putative quinolone binding pocket and leads to the reduction of the drug binding affinity. A number of gyrA mutations (Glu21Gln, Ser95Thr, and Gly668Asp) were also characterized to be natural polymorphisms not associated with fluoroquinolone resistance.
Lau
Ricky W T
RW
Department of Microbiology, Queen Mary Hospital, University of Hong Kong, Hong Kong, China.
Ho
Pak-Leung
PL
Kao
Richard Y T
RY
Yew
Wing-Wai
WW
Lau
Terrence C K
TC
Cheng
Vincent C C
VC
Yuen
Kwok-Yung
KY
Tsui
Stephen K W
SK
Chen
Xinchun
X
Yam
Wing-Cheong
WC
D000890:Anti-Infective Agents / Q000494:pharmacology*; D027081:DNA Gyrase / Q000737:chemistry / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D024841:Fluoroquinolones / Q000494:pharmacology*; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D014376:Tuberculosis / Q000175:diagnosis / Q000382:microbiology
D000890:Anti-Infective Agents; D024841:Fluoroquinolones; D027081:DNA Gyrase