结核分枝杆菌吡嗪酰胺耐药相关分子特征分析  

Molecular characteristics of pyrazinamide resistance in Mycobacterium tuberculosis strains

作　　者：罗振华[1,2] 钱雪琴[3] 范齐文[1] 方欢英[1] 郭建[1] 吴文娟[1] 

机构地区：[1]同济大学附属东方医院医学检验科,上海200123 [2]贵州省人民医院中心实验室,贵阳550002 [3]复旦大学附属公共卫生临床中心医学检验科

出　　处：《中华微生物学和免疫学杂志》2015年第9期660-665,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（81201340）;浦东新区卫生系统学科带头人计划（PWRd2012-09）;浦东新区卫生系统重点专科建设（PWZz2013-03）

摘　　要：目的：探讨结核分枝杆菌（ Mycobacterium tuberculosis，MTB）吡嗪酰胺（ pyrazinamide， PZA）耐药表型与耐药相关基因pncA的关系，对未发现pncA突变的菌株进行panD、rpsA基因分析。方法采用MGIT960仪器法检测108株结核分枝杆菌临床分离株对一线抗结核药物（包括PZA）的敏感性，采用PCR DNA测序技术对菌株进行16S rDNA和pncA、panD、rpsA基因扩增及序列分析。结果78株耐多药结核分枝杆菌（MDR-TB）及利福平单耐菌株中PZA耐药47株（60％），30株对一线抗结核药物（乙胺丁醇、异烟肼、利福平、链霉素，简称EIRS）敏感的菌株中PZA耐药4株（13％），耐药结核分枝杆菌中PZA耐药率显著高于EIRS敏感株。51株PZA耐药MTB菌株中49株（96％） pncA基因发生了突变，57株PZA敏感株中4株（7％）发生突变， PZA耐药株pncA基因突变率显著高于PZA敏感株。 PZA耐药菌株的pncA基因改变以碱基置换为主，His57 Asp氨基酸改变较为常见。碱基突变区域集中在160～169、203～289、309～396和413～467。 pncA基因突变序列分析发现7个新的突变位点：T175 C、C188 A、68位碱基插入G、235位碱基插入AGC、339位碱基插入C、392位碱基插入CC、395位碱基缺失GT。 PZA敏感菌株中发现的4个pncA基因突变位点与耐药株突变位点均不相同。 PZA表型耐药但pncA基因及上游调控序列无突变的菌株NJ108存在panD G419A突变，但rpsA基因为野生型。结论耐药结核分枝杆菌中PZA耐药率较高。吡嗪酰胺耐药结核分枝杆菌中pncA基因突变较为常见，panD基因突变的出现亦值得关注。Objective To investigate the correlation between the mutation of pncA gene and the susceptibility to pyrazinamide （ PZA） in Mycobacterium tuberculosis complex （ MTBC） strains and to analyze the mutation of panD and rpsA genes in wild type isolates without pncA gene mutation.Methods The sus-ceptibilities of 108 MTBC strains to first-line drugs including PZA were detected by using the MGIT 960 TB system.PCR was performed to amplify the 16S rDNA and pncA, panD and rpsA genes.The PCR products were analyzed by DNA sequencing analysis .Results Among the 78 multidrug-resistant MTBC strains , 47 isolates （60%） were resistant to PZA.Four out of 30 （13%） strains that were sensitive to ethambutol , iso-niazid, rifampicin and streptomycin （EIRS） were resistant to PZA.The drug-resistant MTBC strains showed higher resistance rate to PZA than that of the EIRS sensitive strains .There were 49 （ 96%） PZA-resistant isolates and 4 （7%） PZA-sensitive isolates occurred pncA gene mutation.Most of the pncA gene mutations in the genomes of PZA-resistant strains were base substitution mutation , especially the His57Asp substitu-tion.The pncA gene mutations centralized in the regions of 160-169, 203-289, 309-396 and 413-467.Seven novel mutation sites of pncA gene were observed including T175C, C188A, G insertion at 68, AGC insertion at 235, C insertion at 339, CC insertion at 392 and GT deletion at 395.The mutation sites founded in the genomes of PZA-sensitive strains were different from those of the PZA-resistant strains .No mutation of the pncA gene and the upstream regulatory sequence was found in two PZA-resistant strains , NJ44 and NJ108 .＆amp;nbsp;The sequence analysis of panD and rpsA gene showed that the NJ 108 strain had panD gene mutation at G419A, but no mutation was detected in the NJ 44 strain.Conclusion The multidrug-resistant MTBC strains showed higher resistance rate to PZA .The pncA gene mutation was common in PZA-resistant MTB strains and the panD gene mutation was also worthy of attention .

关 键 词：结核分枝杆菌 吡嗪酰胺 耐药 分子特征 

分 类 号：R378.911[医药卫生—病原生物学]