吡嗪酰胺耐药结核分枝杆菌pncA及rpsA基因突变特征分析

Characterization of pncA and rpsA mutations in pyrazinamide-resistant M.tuberculosis

目的 了解吡嗪酰胺（PZA）耐药结核分枝杆菌中pncA与rpsA的全基因突变特征与作用.方法 采用MGIT 960系统测定2010年9月至2012年11月广州市胸科医院的161株结核分枝杆菌的PZA药敏结果,分析pncA与rpsA全基因序列χ^2检验分析PZA耐药株和敏感株之间这2个基因的突变差异.结果 52株PZA耐药株的pncA突变率为84.6％ （44/52）,109株PZA敏感株无pncA突变,耐药株pncA突变率显著高于敏感株（χ^2=126.92,P=0.00）.3株PZA耐药株发生rpsA突变,1株PZA敏感株发生rpsA突变,耐药株与敏感株的rpsA突变率无显著性差异（χ^2=3.42,P=0.06）.PZA耐药株中,44株发生了34种pncA突变类型,其中包括12种新发突变类型（位点27缺失L、91缺失E、111缺失E、122缺失Q、130～132缺失VVG、131缺失V、D8A、S18P、H57Y、F58S、E174G及M175R）和1株启动子-11位核苷酸A→G突变株.pncA突变位点随机散布于pncA全基因,但有9株突变集中发生于G132-T142区域内.3株无pncA突变的PZA耐药株在rpsA基因的3＇羧基末端发生单点突变,分别是R474W、R474L和E433D突变.1株PZA敏感株rpsA基因发生Q162R单点突变.结论 pncA基因突变是结核分枝杆菌PZA耐药的主要机制,测定pncA基因突变有助于快速诊断结核分枝杆菌PZA敏感性,新发突变揭示了区域性研究pncA突变特征的必要性.rpsA基因3＇末端有望作为PZA敏感性分子诊断法的第2个耐药相关区域.

Objective To investigate the characterizations and contributions of mutations in pncA and rpsA whole genes sequence in pyrazinamide （PZA） resistant Mycobacterium tuberculosis （MTB）.Methods All of the 161 clinical strains of MTB collected from Guangzhou Chest Hospital during September 2010 and November 2012 were subjected to determine the susceptibilities to PZA by the MGIT 960 PZA system and to obtain pncA and rpsA whole gene sequences by DNA sequencing.Then the significant difference of pncA and rpsA mutation between the PZA resistant and susceptible isolates were analyzed by chi square test.Results The mutation frequency of 52 PZA resistant isolates was 84.6% （44/52）,but the 109PZA susceptible isolates had no mutations,which showed highly significant difference of pncA mutation frequency between the PZA resistant and the susceptible isolates （χ^2 =126.92,P =0.00）.rpsA mutation was observed in 3 PZA resistant MTB isolates while only 1 PZA susceptible ones was found rpsA mutation,which exhibited no significant difference of rpsA mutation between the PZA resistant and the susceptible isolates （χ^2 =3.42,P =0.06）.Thirty four types of pncA mutations were found in 44 PZA resistant isolates of MTB,including 12 novel mutations （L deletion at 27,E deletion at 91,E deletion at 111,Q deletion at 122,VVG deletions at 130 to 132,V deletion at 131,D8A,S18P,H57Y,F58S,E174G and M175R substitutions） and 1 promoter mutation at-11 nucleotide position with A to G.The identified mutations were randomly dispersed on the pncA whole gene sequence,but 9 isolates were observed with pncA mutations centralized in the region of G132-T142.Three PZA-resistant isolates,without pncA mutation,were observed with rpsA mutations of R474W,R474L,and E433D at C-terminus,and 1 PZA-susceptible strain showed mutation of Q162R in rpsA.Conclusions In the study,mutations in pncA gene is the major mechanism of PZA resistance and direct sequencing the pncA gene by PCR should help to rapidly identify PZA-resistant clinical strains of MTB.Furthermore,novel mutations of pncA in the study indicate the regional investigations are necessary for learning about the characteristics of pncA mutations in PZA-resistant strains of MTB.However,3＇ terminal of rpsA gene sequence may be added as the second PZA resistant relevant region for molecular identification of PZA susceptibility.