耐药基因和药物外排泵在广泛耐药结核分枝杆菌中的作用初探

The effects of gene mutation related to drug resistance and drug efflux pump in extensively drug-resistant tuberculosis clinical isolates

目的 初步探讨2种主要的耐药机制在广泛耐药（XDR）MTB临床分离株中的作用.方法 提取XDR-MTB临床分离株基因组DNA,采用PCR法扩增耐药相关基因,测序后分析其突变位点.根据XDR-MTB分离株KZN605的15个特有单核苷酸突变位点设计引物扩增后测序比对.检测药物外排泵抑制剂利血平、羰基氰化氯苯腙和盐酸维拉帕米对XDR-MTB分离株最低抑菌浓度（MIC）值的影响.结果 10株XDR-MTB菌株在rpoB、katG和rpsL位点均检测到突变,9株分离株在gyrA位点检测到突变,2株在gyrB位点检测到突变,6株分离株在rrs位点检测到突变,未检测到tlyA位点突变;未检测到大部分KZN605株特异的单核苷酸突变位点;药物外排泵抑制剂利血平导致1株XDR-MTB分离株的氧氟沙星MIC值降至原MIC的1/16,其余MIC值均无明显变化.结论 耐药相关基因突变是XDR-MTB分离株耐药的主要机制,药物外排泵可能部分参与氟喹诺酮类药物的耐药机制.

Objective To explore the effects of 2 major drug-resistant mechanisms in clinically isolated strains of extensively drug-resistant tuberculosis （ XDR-MTB ） . Methods Genomic DNA of 10 XDR-MTB strains isolated from Shanghai Pulmonary Hospital were extracted The main gene mutations related to drug resistance and 15 SNPs unique to XDR-MTB clinical isolate KZN605 reported by the Broad Institute in USA were detected by sequencing. The changes of minimal inhibition concentration （ MIC） of XDR-MTB isolates were detected before and after the addition of efflux pump inhibitors verapamil, CCCP and reserpine in liquid cultures. Results The mutation of rpoB, katG and rpsL occurred in all XDR-MTB strains. The mutation of gyrA, gyrB and rrs occurred in 9 strains, 2 strains and 6 strains respectively. There was no mutation of tlyA in all the strains. Most of the SNPs in KZN 605 strains were not detected in the clinical strains. The clinical strains showed no significant changes of MICs, except 1 strain for which the MIC of ofloxacin decreased by 16 times after addition of the efflux pump inhibitors. Conclusions The gene mutations related to drug resistance are the key mechanism for the clinical XDR-MTB strains, while the efflux pumps partly play a role in the drug resistance to fluoroquinolones. The detailed mechanism of efflux pump mediated drug resistance to other anti-TB drugs needs further study.