耐多药结核分枝杆菌pncA基因突变特征及与吡嗪酰胺耐药相关性研究

Characteristics of pncA gene in multidrug-resistant Mycobacterium tuberculosis isolates and its correlation with drug resistance to pyrazinamide

目的了解耐多药MTB的pncA基因突变特征及其与吡嗪酰胺（PZA）耐药之间的关系。方法收集2007-2009年深圳市耐多药MTB共127株，采用BACTECMGIT960系统检测其PZA耐药性，并用Wayne法检测吡嗪酰胺酶（PZase）活性；对pncA基因进行测序，分析其突变情况和分布特征，对PZA药物敏感性与PZase活性、pncA基因突变进行相关性分析。结果127株耐多药菌株中有62株对PZA耐药，耐药率为48．8％。耐PZA菌株中有55株PZase阴性。62株耐药株中有48株发生了pncA基因突变，突变率为77．4％，突变形式包括碱基置换（33株）、移码突变（12株）和整码突变（3株）。48株pncA突变的耐药菌株中，PZase阴性为45株。全部65株敏感株酶活性均为阳性，其中1株菌株发生了pncA基因突变（N11D）。pneA基因突变分析中发现了5个新的突变类型位点：H57P、P62Q、G108R、D110Y、G162V。耐多药菌株对PZA的敏感性与PZase活性（r=0．895，P〈0．05）、pncA基因突变（r=0．779，P〈0．05）存在相关。结论pncA基因突变类型多样，突变随机分布整个基因片段，发现了5个此前未曾报道过的新突变类型，所有突变主要分布于PZase活性位点和金属结合位点及其相邻区域上，且与PZA耐药存在较高相关性。

Objective To understand the characteristics of pncA gene in nmhidrug-resistant Mycobacterium tuberculosis isolates and its correlation with drug resistance to pyrazinamide. Methods A total of 127 clinical isolates of muhidrug-resistant mycobacterium tuberculosis were collected from Shenzhen from year 2007 to 2009. PZA susceptibility was determined by the BACTEC MGIT 960 PZA method. Pyrazinamidase （PZase） activity testing and pncA gene sequencing were performed in all the isolates. The type and frequency of mutations in pncA were determined. Correlation analysis among PZA susceptibility and PZase activity, pncA mutation was performed. Results Among the 127 isolates, 62 isolates （48. 8% ） were found resistance to PZA. Among the 62 PZA resistant isolates, 45 isolates which had various pncA mutations were negative for PZase. Mutation rate was 77.4% （48/62） in total PZA resistance isolates. Different types of 48 resistant isolates were identified in the pncA gene, including base substitution （ 33 isolates ）, frame shift mutation （ 12 isolates） and codon mutation （3 isolates）. No mutations except one isolate （NllD） existed in all PZA- susceptibility isolates which were positive for PZase. A total of 5 mutations which have not been described previously were found as follows： H57P, P62Q, GIO8R, D110Y and G162V. The correlation among the PZA susceptibility and the PZase activity （ r = 0. 895, P 〈 0. 05 ）, the pncA mutation （ r = 0. 779, P 〈 0. 05 ） were significant in 127 multidrug-resistant isolates. Conclusion A high diversity of pncA gene mutation was found among PZA resistant strains of MTB. This study revealed five new mutations of the pncA gene that were not previously described, which scattered in the hot-spot regions located in the metal coordination site and active site of the enzyme. Mutations had a high correlation with the PZA resistance.