堪萨斯分枝杆菌临床分离株对异烟肼敏感性差异机制研究  

The mechanisms identification of the susceptibility discrepancy to isoniazid among clinical isolates of Mycobacterium kansasii

作　　者：任汝颜 薛毅 梁倩 黄海荣 Ren Ruyan;Xue Yi;Liang Qian;Huang Hairong(National Tuberculosis Clinical Laboratory,Beijing Chest Hospital,Capital Medical University,Beijing 101149,China)

机构地区：[1]首都医科大学附属北京胸科医院国家结核病临床实验室,北京101149

出　　处：《中国防痨杂志》2023年第8期728-733,共6页Chinese Journal of Antituberculosis

基　　金：北京市卫生健康委员会高层次公共卫生技术人才建设项目(G2003-2-002)。

摘　　要：目的:测定堪萨斯分枝杆菌临床分离株对异烟肼的敏感性,并探讨引发其差异的原因。方法:结核分枝杆菌标准株H37Rv(ATCC27294)、堪萨斯分枝杆菌标准株(ATCC12478),以及35株分离自非结核分枝杆菌感染患者的堪萨斯分枝杆菌临床分离株均来源于首都医科大学附属北京胸科医院。微孔板法测定35株堪萨斯分枝杆菌临床分离株加入外排泵抑制剂(氰化羰基-3-氯苯腙、二环己基碳二亚胺、维拉帕米和利血平)前后的异烟肼体外最低抑菌浓度(minimum inhibitory concentration,MIC)变化,并评估其关联性;测定已知与异烟肼耐药相关的基因序列,了解基因序列多态性与MIC值的相关性。过表达结核分枝杆菌和堪萨斯分枝杆菌过氧化氢-过氧化物酶(KatG),利用生物层干涉技术检测蛋白与异烟肼亲和力,利用酶联免疫法检测蛋白过氧化物-过氧化氢酶活性。结果:35株堪萨斯分枝杆菌临床分离株中,异烟肼对5.7%(2/35)、71.4%(25/35)、2.9%(1/35)和20.0%(7/35)的菌株的MIC值分别为0.5、1、4和>32μg/ml;分别加入适量氰化羰基-3-氯苯腙、二环己基碳二亚胺、维拉帕米和利血平后,分别有5株(14.3%)、2株(5.7%)、9株(25.7%)和0株菌株的异烟肼MIC降低了50%以上。测序结果显示,堪萨斯分枝杆菌临床分离株的katG、inhA、kasA、ahpC、oxyR基因序列均与其标准菌株一致。堪萨斯分枝杆菌KatG蛋白与异烟肼亲和力常数(KD=1.45×10^(-4)mol/L)略低于结核分枝杆菌(KD=7.44×10^(-4)mol/L),但结核分枝杆菌KatG蛋白的过氧化物酶活性(318.69 U/mg)高于堪萨斯分枝杆菌(87.62 U/mg)。结论:不同堪萨斯分枝杆菌临床分离株对异烟肼体外药物敏感性差异明显,但已知的异烟肼耐药相关机制均不是该差异的主要原因。结核分枝杆菌比堪萨斯分枝杆菌对异烟肼更敏感可能与其KatG蛋白过氧化物酶活性更高有关。Objective:To measure the susceptibility of clinical isolates of Mycobacterium kansasii(M.kansasii)to isoniazid(INH),and investigate the mechanism that causes the differences.Methods:The standard strain of Mycobacterium tuberculosis H37Rv(MTB,ATCC27294)and M.kansasii(ATCC12478),as well as 35 clinical strains of M.kansasii isolated from patients infected with nontuberculous mycobacteria,all of which come from Beijing Chest Hospital affiliated to Capital Medical University.The changes of minimum inhibitory concentration(MIC)of INH in vitro before and after the addition of effluvium pump inhibitors,including carbonyl cyanide 3-chlorophenzone,N,N′-dicyclohexylcarbodiimide,verapamil and reserpine,were measured by microplate method and their correlation was evaluated.Sequences of known genes associated with INH resistance were identified to understand the correlation between gene sequence polymorphisms and MIC values in clinical isolates.Catalase-peroxidase(KatG)of MTB and M.kansasii were overexpressed,the affinity of KatG proteins to INH was tested with the Biolayer Interferometry(BLI)technique,and the peroxide-peroxidase activity of KatG proteins was tested with enzyme-linked immunoassay.Results:5.7%(2/35),71.4%(25/35),2.9%(1/35)and 20.0%(7/35)of the 35 clinical strains of M.kansasii had MIC to INH at 0.5μg/ml,1μg/ml,4μg/ml and>32μg/ml,respectively.After adding appropriate amounts of carbonyl cyanide 3-chlorophenzone,N,N′-dicyclohexylcarbodiimide,verapamil and reserpine,5(14.3%),2(5.7%),9(25.7%)and 0 strains showed more than 50%reduction in MIC values.The katG,inhA,kasA,ahpC and oxyR gene sequences of all clinical isolates were consistent with reference strain of M.kansasii.The dissociation constant between INH and KatG proteins derived from M.kansasii(K D=1.45×10^(-4) mol/L)was slightly lower than that from MTB(K D=7.44×10^(-4) mol/L),but the specific peroxidase activity of KatG proteins from MTB(318.69 U/mg)was higher than that from M.kansasii(87.62 U/mg).Conclusion:Clinical isolates M.kansasii showed vari

关 键 词：分枝杆菌 堪萨斯 分枝杆菌 结核 异烟肼 抗药性 微生物 

分 类 号：R378[医药卫生—病原生物学]