结核分枝杆菌耐药基因检测及中草药作用的生物学变化
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摘要
结核分枝杆菌(Mycobacterium tuberculosis，MTB)是结核病的病原菌。全世界
每年约有200余万人死于结核病。近年，由于耐药结核菌的出现，特别是多重耐药
结核菌的出现以及人类免疫缺陷病毒／艾滋病的流行，使这一全球性的疾病卷土重
来，其感染率、发病率和死亡率均居高不下。目前临床常用的一线抗结核药物主要
为INH、RFP、SM、PZA和EMB，但均已出现耐药菌，使得药物治疗效果不断下
降，而且耐药菌比例仍在不断上升。耐药结核菌使得临床治疗工作变得十分困难，
更增加了结核病的危险性，引起世界各国对结核菌耐药问题的极度重视。本实验在
对结核菌耐药性基因检测的基础上，筛选对耐药结核菌的耐药性有抑制作用或可增
强一线抗结核药物作用效果的中草药，为耐药结核菌的防治提供实验依据。
本实验通过BACTEC-TB460检测系统和MTT微量板法从临床分离出38株结核
菌，并确定分离菌株的耐药谱，从中筛选到17株耐利福平结核菌，9株耐链霉素结
核菌。根据结核菌耐利福平基因rpoB和耐链霉素基因rpsL的核酸序列设计合成引
物，对耐药基因进行PCR扩增、克隆及核酸序列测定，分析其耐药基因突变，检测
到3种耐利福平基因rpoB的突变，2种耐链霉素基因rpsL的突变，从分子水平确定
耐药产生的原因。
根据药性选择部分中草药通过煎煮、浸渍、蒸馏等方法制备成中草药粗提物。
采用MTT微量板法检测所制备中草药粗提物的敏感性对耐利福平、耐链霉素的结核
菌的最小抑菌浓度(MIC)，结果大部分中草药粗提物高浓度可抑菌或杀菌，低浓度
无作用。将上述耐药菌在含药培养基上培养及传代，用MTT法检测其对RFP、SM
的耐药性，通过分析菌株耐药性的变化，确定中草药对菌株耐药性的影响，获得2
种中草药(黄连、百部)对细菌SM耐药性有抑制作用，同时还发现有5种中草药
可以不同程度增强链霉素的作用效果；但未发现中草药对耐RFP菌耐药性有抑制作
用。根据细菌耐药机制，对中草药抑制耐药性的作用机制进行分析，相对应地检测
了中草药作用作用前后上述耐药菌的菌体形态，菌体蛋白图谱和SM耐药基因rpsL
的耐药突变变化情况。结果表明，细菌菌体形态和菌体蛋白图谱无明显变化。SM耐
药基因rpsL序列发生突变，突变点有4处。
Mycobacterium tuberculosis (MTB) is the pathogenic bacterium of tuberculosis (TB). One-third of the global population is sure to be infected with bacteria of the Mycobacterium tuberculosis complex. More than 8 million new cases of tuberculosis occur annually leading to 2 million deaths. Nowadays, because of occurrence of drug resistance tuberculosis, especially multiple-drug resistance tuberculosis (MDR-TB) and outbreak in association with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS), tuberculosis have became more dangerous. Therapeutic efficacy of the anti-tuberculosis drugs commonly used in clinical, such as INH RFP SM PZA and EMB, is on descent. There have been ample warnings that MDR-TB will continue to emerge if countries do not strengthen their control of TB. The aim of this study was to detect drug-resistance genes and screen herbs that could inhibit drug resistance of MTB or enhance the anti-tuberculosis drugs' effect in order to prevent and cure MTB.
In this study, we obtained 38 MTB isolations from clinical by BACTEC-TB 460 system. Using of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and microtiter plate, we assayed the minimal inhibitory concentration (MIC) of rifampin and streptomycin to the isolations. There are 17 rifampin-resistance isolations and 9 streptomycin-resistance isolations. According to the drug-resistance gene sequences of rpoB and rpsL, we designed 2 pairs of primers for PCR. By PCR and DNA sequencing, we analyzed the rifampin- and streptomycin-resistance isolations. The results were including 2 mutations identified in the rpsL gene and 3 mutations in the rpoB gene.
we made some crude preparation of Chinese medicine herb selected on the basis of nature of a drug, we detected crude preparation susceptibility to the rifampin- and streptomycin-resistance isolations by MTT and microtiter plate, resulting in most of non-sensitivity. Analyzing the changes of MIC of those resistance strains after cultivation and transferring of culture, we found 2 herbs could decrease the streptomycin-resistance of isolations and 5 herbs could enhance the drug's effect. But none could take effect on rifampin-resistance isolations. Based on mechanisms of drug resistance of MTB, we analyzed the changes of the morphology, protein electrophoresis map and drug-resistance genes occurred in those isolations. The results showed that no changes occurred in the two formers and the later changed for 4 sites by contrasted with the primordial drug-resistant isolatons.
每年约有200余万人死于结核病。近年，由于耐药结核菌的出现，特别是多重耐药
结核菌的出现以及人类免疫缺陷病毒／艾滋病的流行，使这一全球性的疾病卷土重
来，其感染率、发病率和死亡率均居高不下。目前临床常用的一线抗结核药物主要
为INH、RFP、SM、PZA和EMB，但均已出现耐药菌，使得药物治疗效果不断下
降，而且耐药菌比例仍在不断上升。耐药结核菌使得临床治疗工作变得十分困难，
更增加了结核病的危险性，引起世界各国对结核菌耐药问题的极度重视。本实验在
对结核菌耐药性基因检测的基础上，筛选对耐药结核菌的耐药性有抑制作用或可增
强一线抗结核药物作用效果的中草药，为耐药结核菌的防治提供实验依据。
本实验通过BACTEC-TB460检测系统和MTT微量板法从临床分离出38株结核
菌，并确定分离菌株的耐药谱，从中筛选到17株耐利福平结核菌，9株耐链霉素结
核菌。根据结核菌耐利福平基因rpoB和耐链霉素基因rpsL的核酸序列设计合成引
物，对耐药基因进行PCR扩增、克隆及核酸序列测定，分析其耐药基因突变，检测
到3种耐利福平基因rpoB的突变，2种耐链霉素基因rpsL的突变，从分子水平确定
耐药产生的原因。
根据药性选择部分中草药通过煎煮、浸渍、蒸馏等方法制备成中草药粗提物。
采用MTT微量板法检测所制备中草药粗提物的敏感性对耐利福平、耐链霉素的结核
菌的最小抑菌浓度(MIC)，结果大部分中草药粗提物高浓度可抑菌或杀菌，低浓度
无作用。将上述耐药菌在含药培养基上培养及传代，用MTT法检测其对RFP、SM
的耐药性，通过分析菌株耐药性的变化，确定中草药对菌株耐药性的影响，获得2
种中草药(黄连、百部)对细菌SM耐药性有抑制作用，同时还发现有5种中草药
可以不同程度增强链霉素的作用效果；但未发现中草药对耐RFP菌耐药性有抑制作
用。根据细菌耐药机制，对中草药抑制耐药性的作用机制进行分析，相对应地检测
了中草药作用作用前后上述耐药菌的菌体形态，菌体蛋白图谱和SM耐药基因rpsL
的耐药突变变化情况。结果表明，细菌菌体形态和菌体蛋白图谱无明显变化。SM耐
药基因rpsL序列发生突变，突变点有4处。
Mycobacterium tuberculosis (MTB) is the pathogenic bacterium of tuberculosis (TB). One-third of the global population is sure to be infected with bacteria of the Mycobacterium tuberculosis complex. More than 8 million new cases of tuberculosis occur annually leading to 2 million deaths. Nowadays, because of occurrence of drug resistance tuberculosis, especially multiple-drug resistance tuberculosis (MDR-TB) and outbreak in association with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS), tuberculosis have became more dangerous. Therapeutic efficacy of the anti-tuberculosis drugs commonly used in clinical, such as INH RFP SM PZA and EMB, is on descent. There have been ample warnings that MDR-TB will continue to emerge if countries do not strengthen their control of TB. The aim of this study was to detect drug-resistance genes and screen herbs that could inhibit drug resistance of MTB or enhance the anti-tuberculosis drugs' effect in order to prevent and cure MTB.
In this study, we obtained 38 MTB isolations from clinical by BACTEC-TB 460 system. Using of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and microtiter plate, we assayed the minimal inhibitory concentration (MIC) of rifampin and streptomycin to the isolations. There are 17 rifampin-resistance isolations and 9 streptomycin-resistance isolations. According to the drug-resistance gene sequences of rpoB and rpsL, we designed 2 pairs of primers for PCR. By PCR and DNA sequencing, we analyzed the rifampin- and streptomycin-resistance isolations. The results were including 2 mutations identified in the rpsL gene and 3 mutations in the rpoB gene.
we made some crude preparation of Chinese medicine herb selected on the basis of nature of a drug, we detected crude preparation susceptibility to the rifampin- and streptomycin-resistance isolations by MTT and microtiter plate, resulting in most of non-sensitivity. Analyzing the changes of MIC of those resistance strains after cultivation and transferring of culture, we found 2 herbs could decrease the streptomycin-resistance of isolations and 5 herbs could enhance the drug's effect. But none could take effect on rifampin-resistance isolations. Based on mechanisms of drug resistance of MTB, we analyzed the changes of the morphology, protein electrophoresis map and drug-resistance genes occurred in those isolations. The results showed that no changes occurred in the two formers and the later changed for 4 sites by contrasted with the primordial drug-resistant isolatons.
引文
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[2] Lu PL, Lee YW, Peng CF, et al. The decline of high drug resistance rate of pulmonary Mycobacterium tuberculosis isolates from a southern Taiwan medical centre, 1996-2000. Int J Antimicrob Agents, 2003, 21(3): 239-243.
[3] D.Y.Kunimoto, M.S.Peppler, J.Talbot, et al. Anakysis of Mycobacterium avium Complex Isolates from Blood Sample of AIDS Patients by Pulsed-Field Gel Eleetrophoresis J Clin Microbiol, 2003, 41(1): 498-499.
[4] Ferdinand S, Sola C, Verdol B, et al. Molecular characterization and drug resistance patterns of strains of Mycobacterium tuberculosis isolated from patients in an AIDS counseling center in Port-au-Prince, Haiti: al-year study. J Clin Microbiol, 2003,41(2): 694-702.
[5] Samman Y, Krayem A, Haidar M, et al. Treatment outcome of tuberculosis among Saudi nationals: role of drug resistance and compliance. Clm Microbiol Infect, 2003,9(4): 289-94.
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