结核分枝杆菌对氧氟沙星的耐受程度与gyrA基因突变的相关性分析

Correlation analysis between gyrA gene mutations and the tolerance to ofloxacin in Mycobacterium tuberculosis strains

目的以氧氟沙星为例,探讨阈值的划分对实验室早期检测出耐氧氟沙星结核分枝杆菌的影响。结合DNA序列分析探讨gyrA基因点突变与结核分枝杆菌对氧氟沙星耐受程度的相关性。方法 (1)在含氧氟沙星改良罗氏培养基上检测101株结核分枝杆菌临床分离株对氧氟沙星的敏感性。(2)测序以检测耐氧氟沙星结核分枝杆菌gyrA基因点突变的特征。结果 (1)52株耐氧氟沙星5.0μg/ml的临床分离株中,17株能在含氧氟沙星10.0μg/ml的改良罗氏培养基上生长(约占32.69%),49株对氧氟沙星5.0μg/ml敏感的临床分离株中,有2株能在含氧氟沙星2.0μg/ml的改良罗氏培养基上生长(约占4.08%)。(2)基因扩增产物测序发现;耐氧氟沙星结核分枝杆菌gyrA基因点突变主要发生第90、91和94位点上,有10株在不同的gyrA基因位点上发生了碱基缺失或插入的变异,101株被检测的结核分枝杆菌临床分离株中,发生在第95位点的碱基突变率为100%。结论 (1)判定耐药结核分枝杆菌上、下限的阈值对耐药结核菌的早期检出具有重要的作用。(2)发生在耐氧氟沙星结核分枝杆菌gyrA基因上的点突变是多变的,但与结核分枝杆菌对氧氟沙星的耐受程度没有明显的相关性。

Objective Utilizing of ofloxacin as a target to investigate the effect of drug-resistant threshold on the early laboratory determination of drug-resistant Mycobacterium tuberculosis;combined with DNA sequence analysis,to probe the correlation between gyrA genetic mutations and the tolerance to ofloxacin in Mycobacterium tuberculosis strains.Methods（1） Utilizing L-J medium slant containing ofloxacin to detect the sensitivity to ofloxacin against 101 isolates by way of absolute concentration.（2） Sequencing to detect the ofloxacin-resistant Mycobacterium tuberculosis gyrA genetic mutation characteristics.Results（1） 17 of 52 ofloxacin-resistant 5.0,and 2 of 49 ofloxacin-sensitive strains can grow in 10.0 and 2.0 μg/ml of ofloxacin onto the ofloxacin containing Lowenstein-Jensen slants.Those were respectively contributed about 32.69% of 52 and about 4.08% of 49 of the tested isolates.（2） The sequencing results showed that genetic mutations of OFLX-resistant Mycobacterium tuberculosis clinical isolates occur mainly on codon 90,91 and 94.In addition,10 of 101 Mycobacterium tuberculosis isolates was found to occur different deletion or insertion on gyrA base chains.101 clinical isolates of Mycobacterium tuberculosis was totally genetic point mutation on codon 95,and the mutation rate was 100%,detected occuring Conclusions（1） DeterminingTB drug resistance on upper and lower threshold plays an important role in laboratory early detection of TB drug resistance.（2） gyrA genetic mutations occurring in the OFLX-resistant Mycobacterium tuberculosis were variable,and highly relevant to ofloxacin-resistance,but lack of significant correlation with the tolerance to ofloxacin in M.tuberculosis strains.