结核病复发及结核分枝杆菌耐药机制的研究

发布时间：2018-04-04 02:20

本文选题：结核病　切入点：复发　出处：《复旦大学》2006年博士论文

【摘要】： 结核病是一种古老的传染性疾病，在历史上曾经夺取数以万计的生命。近年来由于全球人口流动的加速，人类免疫缺陷病毒感染的流行，耐药菌株的出现和传播，以及其它一些经济和政策等方面的原因，使得结核病卷土重来，再次构成对人类健康的重大危害。我国的结核病控制工作尽管已经取得了很大的进步，但是形势依然很严峻。结核病的复发已成为我国结核病实际防治工作中面临的难题。本研究根据我国较高的结核病复发率，选择上海地区的复发病人作为研究对象，利用基因型分型技术，对复发的病因学进行了研究。同时，针对复发患者中较高的耐药率，在文献综述的基础上，对一线抗结核药物乙胺丁醇(ethambutol，EMB)特殊的耐药现象及其耐药分子机制进行了探索性研究。全文分二个部分：第一部分结核病复发的病因学研究为研究结核病复发的病因学，首先评估了一种新的结核分枝杆菌基因型分型法—分枝杆菌散在分布重复单位基因型分析法(MIRU)。随机抽取上海地区2000年至2002年分离保存的91株菌株，对其进行MIRU分型。发现91株菌株用该分型法可得到46种基因型。12个MIRU位点的多态性分析表明各位点之间有较大的区别，其中位点26显示了较高的多态性，位点16、31、40显示了中等程度的多态性。与另一种以PCR为基础的基因型分析技术Spoligotyping相比，MIRU显示了较好的分辨率。进一步以上海地区1999年至2004年复发肺结核患者为对象，，通过比较复发患者前后两次发病时结核分枝杆菌MIRU基因型的差异，来判断结核病复发的真正原因。在52例结核病复发患者中，32例患者两次发病时结核分枝杆菌的MIRU基因型发生了变化，提示62％的结核病患者的复发是由于外源性再感染而引起的。随着年龄的增加，由外源性再感染所致复发的可能性逐步降低，≤30岁、31～60岁、≥60岁复发患者中外源性再感染的比例分别为100％(4／4)、67％(12／18)、53％(16／30)。随着复发间隔时间的延长，结核病复发患者中由外源性再感染所致的可能性逐步增加，6个月内复发的患者中47％(7／15)是由外源性再感染引起的，而1年以后这个比例达到74％(17／23)。复发患者中较高的外源性再感染比例提示结核病近期传播现象较严重，结核病防治工作中应当加强传染源的早期发现，切断传播途径。外源性再感染在结核病患者中常见，也说明人体免疫系统缺乏有效的保护机制，不能保护机体免受结核分枝杆菌再次感染，这对结核病预防性疫苗的研究和开发提出了新的挑战。第二部分结核分枝杆菌EMB耐药的分子机制研究 EMB是一种目前普遍采用的一线抗结核药物。先前的研究发现乙胺丁醇耐药常常与其它耐药联系在一起，为了观察这种现象是否与不同地区的菌株流行相关，本研究对上海市10年来保存的菌株的药敏结果进行了分析。结果表明，单耐EMB的菌株数显著少于单耐其它常用抗结核药物(异烟肼、利福平和链霉素)的菌株数。在耐两种以上药物的菌株中，耐EMB的菌株数量与菌株耐药种类的数目成正相关，提示菌株耐EMB与耐其它抗结核药物存在相关性。为研究embB306位点突变与菌株耐药的关系，本研究分析了116株耐药菌株和54株全敏感菌株embB306位点的突变情况。结果发现，在所有耐EMB的27株菌株中，共检测到embB306位点突变17株，在不耐EMB但耐其它药物的89株菌株中也检测到突变17株，在全敏感菌株中没有检测到embB306位点的突变。经统计学检验，embB306突变不仅与EMB耐药之间存在相关性，也与耐其它常用抗结核药物存在相关性。同时也发现embB306突变所占的比例随着耐药种类的增加而增加。由于临床上EMB单独耐药菌株数目较少，因而检测embB306位点的突变对检测菌株EMB单独耐药的实际意义不大。但embB306位点突变与菌株耐多药之间存在一定的相关性，因而检测该位点的突变可以作为快速筛查临床耐多药菌株的候选分子标记。结核分枝杆菌产生耐药的机制与其它细菌略有不同，质粒、转座子以及整合子等以基因水平传播方式介导的耐药机制较少见，染色体上的缺失和突变被认为是其产生耐药的主要方式。为进一步深入理解分枝杆菌EMB耐药产生的分子机制，以耻垢分枝杆菌作为研究模型，采用转座子突变技术研究与EMB耐药相关的新基因。使用基于Himar1转座子的MycoMarT7转座子系统，构建了耻垢分枝杆菌转座子突变文库。Southern blot实验证实Himar1转座子以随机单拷贝插入的方式分布在染色体上。进一步对该文库进行EMB耐药筛选，分离获得了4个耐药突变株分别命名为ER2A、ER4A、ER5A和ER7A，经MIC测定其耐药能力比野生型提高了20倍以上。使用接头PCR和标记挽救方法鉴定了各突变株中转座子在基因组中的插入位点。生物信息学分析表明，ER2A突变菌株中转座子插入部位基因编码单加氧酶，该酶可能参与分枝杆菌中间产物的代谢；ER4A突变株插入部位的基因为lprE，编码一种细胞内的脂蛋白；ER5A突变株插入部位的基因为编码转运蛋白的arsA；ER7A突变株中插入部位编码的基因功能未知。这些新分离到基因耐乙胺丁醇的机制有待进一步深入研究。
[Abstract]:Tuberculosis is an infectious disease of old, in history has killed millions of people. In recent years due to the acceleration of global population, the prevalence of human immunodeficiency virus infection, the emergence and spread of resistant strains, and other economic and policy issues, the TB comeback again pose a significant harm to human health our work in tuberculosis control. Although great progress has been made, but the situation is still very serious. The recurrence of tuberculosis has become a problem for the actual work of prevention and control of tuberculosis in China in this study. According to our country's higher recurrence rate of tuberculosis in Shanghai area, the recurrence of patients as the research object, using gene typing technique the etiology of recurrence, were studied. At the same time, the drug resistance in patients with high recurrence rate, on the basis of literature review, on a The special drug resistance of ethambutol (EMB) and its molecular mechanism were studied. The full text is divided into two parts:
The etiological study of the first part of tuberculosis recurrence
As the etiology of recurrent tuberculosis, first evaluate a new Mycobacterium tuberculosis genotyping method - mycobacterial interspersed repetitive unit genotype analysis (MIRU) were randomly selected in Shanghai. From 2000 to 2002 from the collection of 91 strains were genotyped by MIRU. 91 strains with the classification method can obtain polymorphism analysis of 46 genotypes of.12 MIRU loci showed a larger difference between you, the site 26 showed high polymorphism loci, 16,31,40 showed moderate polymorphism. Compared with another PCR based genotyping technology Spoligotyping, MIRU show good resolution. Further in Shanghai from 1999 to 2004 the recurrence of pulmonary tuberculosis patients as the object, by comparing the recurrence of patients before and after two times at the onset of Mycobacterium tuberculosis MIRU gene type, to determine the node The real reason for the nuclear attack. In 52 cases of recurrent tuberculosis patients, 32 patients changed two times at the onset of Mycobacterium tuberculosis MIRU gene, showed that 62% of the patients with tuberculosis recurrence is due to exogenous re infection. With the increase of age, the possibility of exogenous re infection caused by recurrence gradually reduced, less than 30 years, 31~60 years, more than 60 years old patients with recurrent exogenous reinfection rates were 100% (4 / 4), 67% (12 / 18), 53% (16 / 30). With prolonging recurrence interval, the possibility of recurrence in patients with tuberculosis by exogenous re infection gradually increase the recurrence within 6 months were 47% (7 / 15) is caused by exogenous infection, and 1 years later, this proportion reached 74% (17 / 23). Exogenous re infection is higher in patients with recurrent ratio that recent transmission of tuberculosis is serious, tuberculosis In the prevention and treatment should be found early strengthen the source of infection, cut off the transmission. Exogenous re infection is common in patients with tuberculosis, also shows that the lack of effective protection mechanisms of the human immune system, can protect the body against Mycobacterium tuberculosis infection again, the prevention of TB vaccine research and development has brought new challenges.
Molecular mechanism of EMB resistance in the second part of Mycobacterium tuberculosis
EMB is a widely used first-line anti tuberculosis drugs. Previous studies found that ethambutol resistance and other resistant often together, in order to observe whether this phenomenon is related with the prevalent strains in different regions, the study of drug sensitive strains in Shanghai city for 10 years to save the results were analyzed. The results showed that the bacteria the number of single EMB resistance is significantly less than the single resistance to other antituberculosis drugs (isoniazid, rifampicin and streptomycin) of the strains. In more than two kinds of drug resistant strains of EMB resistant strains, the number and the number of drug-resistant strains of species are positively related, suggesting that EMB resistant strains and resistant to other anti tuberculosis drugs are associated. For the study of embB306 mutation with the strain resistance, this study analyzed the mutation of 116 strains of resistant strains and 54 strains of sensitive strain embB306 loci. The results showed that in all the 27 strains resistant to EMB, EmbB306 mutations were detected in 17 strains, not resistant to EMB but resistant to other drugs among the 89 strains have mutation was detected in 17 strains, no mutation detected embB306 loci in all strains. By statistical analysis, the correlation between embB306 mutation with EMB drug resistance, and resistance to other antituberculosis drugs there is a correlation. Also found that the proportion of embB306 mutations account for the increased with the increase of resistant species. Due to the clinical EMB alone resistant strains of the small number of the detection of embB306 mutations is of practical significance to detect drug resistance strain EMB alone. But the mutation of embB306 is associated with multi drug resistant strains, and detection of the mutation sites of the candidate molecular markers can be used for rapid screening of clinical multi drug resistant strains.
Mycobacterium tuberculosis drug resistance mechanism is slightly different, with other bacterial plasmid, transposon and integron resistance mechanism to gene level communication mediated by a rare, lack of chromosome and mutation is considered the main resistance. For further understanding the molecular mechanism of Mycobacterium tuberculosis drug resistance to EMB. In Mycobacterium smegmatis as the research model, transposon mutagenesis technique and the related research of EMB resistance gene by using Himar1. The new transposon based MycoMarT7 transposon system, constructs a transposon mutant library of.Southern blot confirmed that Himar1 transposon random single copy insertion mode on the chromosomes of Mycobacterium smegmatis further. The library was EMB resistance screening, obtained 4 resistant mutants were named as ER2A, ER4A, ER5A and ER7A, determined by MIC resistant ability than the wild Type increased more than 20 times. The use of PCR markers and joint rescue method of the mutants the transposon insertion sites in the genome were identified. Bioinformatics analysis showed that ER2A mutant strains of the transposon insertion site gene encoding monooxygenase, this enzyme may be involved in mycobacterial intermediate metabolism; ER4A insertion mutant part of the gene is lprE, encoding an intracellular lipid protein; ER5A mutant gene encoding the insertion site for transporter arsA; ER7A mutation gene encoding unknown function insertion site lines. These new isolated resistance gene B ETHAMBUTOLE mechanism needs further research.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R378;R52

【引证文献】