Analysis of gyrA mutation in Mycobacteria tuberculosis by denaturing HPLC

Objective To analyze the characteristic of fluoroquinolone resistance gene gyrA mutation in Mycobacterium tuberculosis and to evaluate the value of denaturing HPLC(DHPLC) method.Methods One hundred and nine Mycobacterium tuberculosis clinical isolates among which 87 were found ofloxacin-resistant while 22 susceptible according to routine method,received minimal inhibitory concentration (MIC) test,gyrA quinolone resistance determining region (QRDR) DNA sequencing and DHPLC analysis concurrently. Results Two new characteristics of ofloxacin-resistant Mycobacterium tuberculosis were found: one was the high rate of double mutations in gyrA QRDR (56.3% of 87 ofloxacin-resistant isolates were found carrying double mutations),the other was that among these double-mutated isolates,20.4% (10/49) harbored the Ala74Ser mutation,which previously was thought to exist only in other bacteria,and had never been found in Mycobacterium tuberculosis. No close relationship was observed between gyrA mutation type and ofloxacin MIC. By introducing M.tb H37Rv and M.tb Erdman two reference strains,interference from codon 95 AGC:ACC polymorphism was successfully avoided. Compared to DNA sequencing,the sensitivity and specificity of DHPLC method were all 100%.Conclusions Fluoroquinolone-resistance of Mycobacterium tuberculosis in our country is quite severe. The characteristic is the high rate of gyrA QRDR double point mutations. DHPLC method set up in this work is simple,rapid,stable,sensitive and may be valuable in large samples of drug-resistance survillence in large area. Key words: Mycobacterium tuberculosis; Mlcrobial sensitivity test; Gene mutation; High-performance liquid chromatography