98株河南省结核分枝杆菌氧氟沙星耐药株gyrA基因突变的研究

Mutation of gyrA gene in 98 strains ofloxacin-resistant Mycobacteria tuberculosis clinical isolates in Henan province

目的分析河南省结核分枝杆菌氧氟沙星耐药株氟喹诺酮类耐药基因gyrA突变的特点。方法应用比例法药敏试验筛选氧氟沙星耐药株126株中随机入选98株氧氟沙星耐药株,并选取28株氧氟沙星敏感株、M.tb H37Rv及H37Ra为对照;根据gyrA基因氟喹诺酮类耐药决定区域(QRDR)设计引物,PCR扩增,DNA测序。结果 98株耐氧氟沙星结核分枝杆菌中,81株(84%)检测到gyrA基因突变,其中30株为单位点突变,51株(63%)携有双位点突变(其中出现频率最多的是Asp94Gly和gyr90Val)。51株双位点突变菌株中7株(14%)携有Ala74Ser突变。gyrA基因突变位点发生频度依次为:gy-rA94(84%)、gyr90(71.6%)、gyrA91(19.8%)、gyrA74(14.8%);gyrA基因突变类型与MIC之间未观察到密切关系存在。测序中加入常规的M.tb H37Rv及H37Ra作为参照菌株,排除了95位AGC:ACC自然多态性的影响。结论河南省结核分枝杆菌中,84%的耐氧氟沙星菌株有gyrA基因突变,且具有gyrA基因QRDR双位点突变率高特点。

The purpose was to analyze the characteristics of fluoroquinolone resistance gene gyrA mutation in M. tuber- culosis in Henan Province. A total of 126 M. tuberculosis clinical isolates were conducted to test drug susceptibility, 98 resist- ance strains were chosen, while 28 susceptible strains, H37Rv and H37Ra was selected as comparison. According to DNA se- quencing, the primers were designed and PCR amplification was done. All strains were performed gyrA quinolone resistance determining region （QRDR） DNA sequencing. Results showed that gyrA mutation were found in 81 isolates （84%）, 30 iso- lates were single mutation among them, and other 51 isolates （63%, 51/81） were double mutations in gyrA. The ofloxacin-re- sistant Mycobacterium tuberculosis in our province was high in double mutations in gyrA QRDR. Ala74Ser mutation, which previously had seldom been found in M. tuberculosis, was found. The gyrA mutation occurred in frequence gyrA94 （84%）, gyr90 （71.6%）, gyrA91 （19.8%）, and gyrA74 （14.8%） ; no close relationship was observed between gyrA mutation type and ofloxacin MIC. By introducing M. tuberculosis H37Rv and M. tuberculosis H37Ra reference strains, interference from co- don 95 AGC： ACC polymorphism was successfully avoided. This study indicates that 84% of the ofloxacin-resistant resistant M. tuberculosis strains have gyrA genetic mutations. The 84% ofloxacin-resistant isolates indicates the high rate of gyrA QRDR double point mutations.