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目的 分析重庆地区分离的耐多药结核分枝杆菌(MDR-MTB)菌株pncA和rpsA基因突变特征及其与吡嗪酰胺(PZA)耐药的相关性.方法 收集2014年11月至2016年2月重庆市结核病防治所“MTB耐多药项目”中所属39个区(县)的133株MDR-MTB临床分离菌株.应用BACTEC MGIT 960系统作PZA药物敏感性试验(简称“药敏试验”),再通过PCR-DNA测序技术,分析菌株pncA和rpsA基因突变类型及其与PZA耐药的相关性.结果 133株MDR-MTB中PZA耐药83株,耐药率为62.4％.83株PZA耐药株中73株pncA基因突变,突变率为87.9％;50株PZA敏感株中,4株pncA基因突变,突变率为8.0％;PZA耐药株pncA基因突变率明显高于PZA敏感株(x2=81.82,P=0.000).PZA耐药株pncA基因改变以碱基置换为主,碱基位点G395A的改变最多见(9株).突变位点随机散布在整个pncA以基因,位于核苷酸-11～515位.碱基突变的区域集中在位点20～40、136～146、185～226、392～408.pncA基因序列分析发现18种新的突变位点:T2C、T37G、T94G、C206T、G319A、C437T、T488C、52插入GCC、130插入C、139插入CA、232插入C、243插入T、288插入A、393插入GGT、408插入CA、341缺失ACGCC、342缺失GCCAC、376缺失GATGAGGTC.PZA敏感株pncA基因改变均为碱基置换,且突变位点与耐药株突变位点均不相同.133株MDR-TB均无rpsA基因突变.结论 重庆地区MDR-MTB中PZA耐药率较高.pncA基因突变是重庆地区MDR-MTB中PZA耐药的主要机制之一.“,”Objective To investigate the mutation characteristics in pncA and rpsA genes and its correlation with pyrazinamide (PZA) resistance in multidrug-resistant Mycobacterium tuberculosis (MDR-MTB) strains isolated from Chongqing.Methods A total of 133 MDR-MTB clinical isolates were collected from Chongqing from November 2014 to February 2016.PZA susceptibility was determined by BACTEC MGIT 960 TB system.The pncA and rpsA genes were amplified by PCR and the products were analyzed by DNA sequencing in order to identify the mutations in pncA and rpsA genes and the correlations with PZA resistance.Results Among the 133 MDR-TB clinical strains,83 isolates were resistant to PZA,yielding a resistance rate of 62.4％.Sequence analysis revealed that 73 out of 83 (87.9％) MDR-MTB strains harbored a mutation located in the pncA gene.Four out of 50 (8.0％) PZA-sensitive isolates occurred pncA gene mutation.The PZA-resistant strains showed higher mutation rate than the PZA-sensitive strains (x2 =81.82,P=0.000).Most of the pncA gene mutations in the genomes of PZA-resistant strains were base substitution mutation,especially the G395A substitution (9 isolates).The identified mutations were dispersed along the pncA gene,distributing in the region from nucleotide-11 to nucleotide 515.The pncA gene mutations centralized in the regions of 20-40,136-146,185-226,and 392-408.Eighteen novel mutation sites of pncA gene were observed including T2C,T37G,T94G,C206T,G319A,C437T,T488C,GCC insertion at 52,C insertion at 130,CA insertion at 139,C insertion at 232,T insertion at 243,A insertion at 288,GGT insertion at 393,CA insertion at 408,ACGCC deletion at 341,GCCAC deletion at 342,and GATGAGGTC deletion at 376.The pncA gene mutations in the genomes of PZA-sensitive strains were only the substitution mutations,and the sites were different from those of PZA-resistant strains.While no genetic mutation associated with PZA resistance was found in the rpsA gene.Conclusion The MDR-MTB strains showed higher resistance rate to PZA in Chongqing.Mutation in pncA gene might be one of the major mechanisms of resistance to PZA in MDR-MTB isolates in Chongqing.