广泛耐药结核分枝杆菌耐药机制及其疾病诊断的研究进展

自20世纪90年代以来,全球结核病疫情回升,结核分枝杆菌耐药是其中的一个重要原因.广泛耐药结核病是指在耐多药结核病(即同时对异烟肼和利福平耐药的结核分枝杆菌引起的结核病)的基础上,还对氟喹诺酮类药物和至少3种二线静脉用抗结核药物(卷曲霉素、卡那霉素、阿米卡星)中的1种耐药的结核分枝杆菌引起的结核病.我国是结核病高流行国...     

导读与评议（2017年第6期）

精准医疗概念的提出开启了一个医学新时代,其实质包括精准诊断和精准治疗。张文宏课题组围绕结核病治疗中的精准医疗进行了阐述,涉及结核病的精准诊断,包括结核病的临床诊断及结核分枝杆菌的检测(分子检测及耐药检测技术等)、特殊人群的药理学参数与药物代谢相关的分子标记、针对病原体生命周期分子靶点的直接作用药物研发、通过正向调控或负向调控药物的使用实现宿主导向抗结核精准治疗。本期刊登了3篇关于结核病耐药的综述。鉴于耐药结核分枝杆菌的补偿性进化是其传播与流行的基础,高谦课题组从结核分枝杆菌的耐药分子机制、耐药突变的适应性代价与补偿性进化,以及补偿性进化如何影响耐药结核病传播等方面进行了综述。袁莉课题组就近年来结核分枝杆菌“毒素-抗毒素系统”(TAS)与生物膜的研究及抗结核药物对生物膜形成的影响进行综述......     

结核分枝杆菌蛋白抗原的研究进展

结核病当今世界人类致死的主要疾病之一,早期诊断发现病人、选择敏感的抗结核药物进行有效治疗是控制结核病的关键。而临床上对结核病患者检出率低,漏诊率和误诊率高,结果导致结核耐药的情况越来越严重。简便、快速、准确的免疫学检测方法在诊断结核病中起到了重要的作用。本文对用于免疫学检测的蛋白抗原作一综述。     

扩增子测序法筛选结核分枝杆菌的耐药突变

结核病是严重的公共健康问题之一,而耐药结核病的增加是控制结核病流行的难点之一。快速、准确的诊断是提高结核患者治愈率和降低死亡率的关键因素。本研究建立了基于二代测序技术的扩增子测序方法,对5种一线抗结核药物的17个耐药基因进行检测。在26个临床耐药结核菌株中共鉴定出65个突变,包括33个热点突变,9个稀有突变和23个新突变。对18个新发现的错义突变进行了蛋白质序列保守性和蛋白质局部结构的分析。结果表明,14个新的错义突变在9种分枝杆菌中显示出高度保守性,并且导致了该蛋白质局部结构的改变。根据本研究检测和分析结果,推测这些新发现的突变可能是潜在的耐药突变。在本研究中,构建了扩增子测序的检测方法,可同时检测10株临床结核菌株的17个耐药基因,是一种快速、准确并且全面的检测耐药结核分枝杆菌一线治疗药物耐药突变的方法,该方法不仅能检测热点突变和稀有突变,还能发现一些未报道过的新突变。该检测方法或可用于临床诊断和基础研究。     

非一线抗结核药物耐药机制及耐药性诊断研究进展

结核病(Tuberculosis, TB)至今仍是世界三大传染疾病之一。2014年,TB导致的死亡人数已经超过HIV。二线抗TB药物是临床治疗耐多药TB(Multidrug-resistant TB, MDR-TB)的主要药物,然而某些MDR-TB患者由于未及时诊断、治疗方案不合理、所处区域医疗条件差等原因,逐渐发展成为广泛耐药TB(Extensively drug-resistant TB, XDR-TB),使治疗更加困难,其死亡率甚至与肺癌接近。目前结核分枝杆菌(Mycobacterium tuberculosis)的耐药性机制研究已经转向非一线药物,如二线、三线和一些新研发的抗TB药物,揭示这些非一线药物的耐药机制对于耐药TB的治疗和新型抗TB药物的研发具有重要意义。本文对目前临床上使用的主要非一线药物的耐药机制研究进行了综述,并对目前常用的TB耐药性诊断方法的优缺点进行了归纳比较。     

结核分枝杆菌耐药性的分子生物学研究进展

结核病一直是世界性问题,我国其发病情况尤为严重,是亚洲的第二大结核病发病国家。结核病治疗方面常使用抗生素作为首选药物,随着抗菌药的滥用,结核杆菌对多种抗菌药产生耐药性,结核病耐药患者增多,治疗难度增加。因此,结核杆菌耐药分子机制的研究更加重要,新型抗结核药物研制更加迫切。结核分枝杆菌的基因突变是引起耐药的主要分子学依据,因此基于结核分枝杆菌耐药性相关基因的深入探索,对于预防结核病的传播及治疗皆具有深远影响。本文从分子生物学角度分析了近年来结核分枝杆菌耐药性产生的原因及相关研究进展。     

MTB新疆临床分离株MDR-TB和XDR-TB耐药情况初步调查

目的:分析新疆喀什地区结核分枝杆菌(MTB)临床分离株对4种一线和7种二线抗结核药物的耐药情况,初步探讨本地区耐多药结核病(MDR-TB)和广泛耐药结核病(XDR-TB)的流行情况。方法:收集2008.11.1~2009.10.31日期间新疆喀什地区结核病患者痰液标本,进行分离培养及菌种鉴定,并应用比例法对所分离得到的菌株进行耐药性检测。结果:102株分枝杆菌中,88株(86.3%)属于结核分枝杆菌复合群,7株(6.9%)属于牛型分枝杆菌,7株(6.9%)属于非结核分枝杆菌。对一种以上的抗结核药物具有耐药性的有20株,总耐药率为22.7%(20/88),耐多药率为6.8%(6/88),pre-XDR为33.3%(2/6),无XDR-TB病例。结论:新疆喀什地区结核病患者耐药率较高,在结核病治疗工作中应给予重视,尤其应加强对Pre-XDR的重视,以免发展成XDR-TB。     

结核分枝杆菌乙胺丁醇耐药机制的研究进展

耐多药结核病的出现和流行对结核病的防控造成了严重威胁。乙胺丁醇(Ethambutol, EMB)是一线抗结核药物,常与异烟肼、利福平等联合应用,还可用于耐药结核病的治疗。但近年来EMB耐药形势严峻,我国复治结核病患者中EMB耐药率已达17.2%,并呈上升趋势;耐多药结核病患者中,EMB耐药率约为51.3%~66.7%,情况不容乐观。明确EMB耐药的产生机制对于有效防控EMB耐药率的上升、充分发挥EMB的作用十分重要,因此本文对结核分枝杆菌EMB的耐药现状、EMB的作用机制及其耐药产生机制方面的研究进展进行了综述。     

耐药结核病及其防治

目前结核病呈现出高发病、高感染、高耐药、高死亡的特点。耐药结核病则是结核病防治工作的重点和难点。为有效控制其发展,本文分析了其产生和流行的医源性和社会性原因,并从7个方面阐述了耐药结核病的防治措施。即广泛普及结核病防治的知识;加强对流动人口的管理和监测;提高对耐药结核病的诊治水平;开展中西医结合和其他辅助治疗;预防社区和医院感染;完善结核病的耐药监测;积极开展抗结核新药、新手段和新方法的研究。     

蛋白质组学在结核分枝杆菌研究中的应用

蛋白质组学是在基因组学基础上发展起来的新兴学科, 其基本技术包括样品制备、蛋白质分离和蛋白质鉴定分析, 其中的核心技术是双向凝胶电泳技术(2-Dimensional Electrophoresis, 2-DE)和质谱技术(Mass Spectrometry, MS)。近年来, 蛋白质组学技术已应用于结核分枝杆菌的研究领域。应用蛋白质组学技术分离、鉴定、检测结核分枝杆菌致病株的全菌蛋白及分泌蛋白, 分析其蛋白组成, 可深入解析结核分枝杆菌的致病机理和耐药机制。通过对结核分枝杆菌致病株抗原的分析, 为研制预防结核病的新型疫苗拓展了空间。通过对结核分枝杆菌临床分离株的蛋白组成分析还发现了一些有意义的结核病早期诊断标志物。蛋白质组学技术还应用于寻找新的药物靶标, 在研制和筛选新的抗结核药物等方面展示了一些有价值的研究成果, 为更好地开展结核病的预防、早期诊断及治疗打下了基础。     

新型抗结核药物研发：微生物非常规培养与沉默基因激活策略

由结核分枝杆菌感染引起的结核病是人类重要传染病之一。临床上结核菌耐药性日趋严重,不断出现的耐多药及广泛耐药结核病患者,使现有的一线至五线药物不能满足结核病防控需求。微生物来源的天然产物是药物先导化合物的重要来源。环境中存在大量常规培养条件下未培养微生物,同时微生物基因组中也存在大量未被表达的"沉默代谢途径"。运用各种方法对未培养微生物进行再培养,同时激活微生物的沉默代谢途径,进而获得潜在的新型抗生素药物已成为目前研究热点。文中系统阐述了近年来获取天然化合物所采用的微生物非常规培养技术及沉默代谢途径激活策略,同时总结了利用这两种方法获得的新型抗结核天然产物,并展望了这些方法在抗结核药物进一步研发中的应用前景。     

2D-QSAR model development and analysis on variant groups of anti-tuberculosis drugs

A quantitative structure activity relationship study was performed on different groups of anti-tuberculosis drug compound for establishing quantitative relationship between biological activity and their physicochemical /structural properties. In recent years, a large number of herbal drugs are promoted in treatment of tuberculosis especially due to the emergence of MDR (multi drug resistance) and XDR (extensive drug resistance) tuberculosis. Multidrug-resistant TB (MDR-TB) is resistant to front-line drugs (isoniazid and rifampicin, the most powerful anti-TB drugs) and extensively drug-resistant TB (XDR-TB) is resistant to front-line and second-line drugs. The possibility of drug resistance TB increases when patient does not take prescribed drugs for defined time period. Natural products (secondary metabolites) isolated from the variety of sources including terrestrial and marine plants and animals, and microorganisms, have been recognized as having antituberculosis action and have recently been tested preclinically for their growth inhibitory activity towards Mycobacterium tuberculosis or related organisms. A quantitative structure activity relationship (QSAR) studies were performed to explore the antituberculosis compound from the derivatives of natural products . Theoretical results are in accord with the in vitro experimental data with reported growth inhibitory activity towards Mycobacterium tuberculosis or related organisms. Antitubercular activity was predicted through QSAR model, developed by forward feed multiple linear regression method with leave-one-out approach. Relationship correlating measure of QSAR model was 74% (R(2) = 0.74) and predictive accuracy was 72% (RCV(2) = 0.72). QSAR studies indicate that dipole energy and heat of formation correlate well with anti-tubercular activity. These results could offer useful references for understanding mechanisms and directing the molecular design of new lead compounds with improved anti-tubercular activity. The generated QSAR model revealed the importance of structural, thermodynamic and electro topological parameters. The quantitative structure activity relationship provides important structural insight in designing of potent antitubercular agent.     

结核病新抗生素靶标和抑制剂研发

随着结核病耐药现象越来越严重,迫切需要开发新型抗结核药物,而大部分的药物开发主要依赖于新型的抗生素靶标的发现。文中综述了2016年以来新出现的具有开发为抗结核药物潜力的新化合物,并重点阐述了这些新化合物所针对的抗生素靶标,将有助于针对这些靶标进一步开发新型抗结核药物。     

结核分枝杆菌耐氟喹诺酮类药物的分子机制研究进展

结核病是由结核分枝杆菌(Mycobacterium tuberculosis)通过空气传播引起人类感染的慢性传染病,耐药结核分枝杆菌的流行是目前结核病防治的世界难题。氟喹诺酮类药物是人工合成药物,应用于耐药结核的临床治疗中,在治疗中起着核心的作用。但近年来,氟喹诺酮类药物的抗性菌株不断出现,愈发增加了结核病治疗的困难与治疗失败风险。在临床中氟喹诺酮药物的靶点比较清楚,是结核分枝杆菌的DNA旋转酶。目前发现结核分枝杆菌耐氟喹诺酮类药物的机制主要包括药物靶点DNA旋转酶的关键氨基酸改变、药物外排泵系统、细菌细胞壁厚度的增加以及喹诺酮抗性蛋白MfpA介导的DNA旋转酶活性调控。其中在氟喹诺酮靶标DNA旋转酶功能活性改变的耐药机制方面,编码DNA旋转酶基因突变一直是研究的热点,但近年来发现DNA旋转酶的调控蛋白MfpA以及DNA旋转酶的修饰在细菌耐药性中起着重要的作用,相关机制还亟待发现。本文综述了当前结核分枝杆菌耐氟喹诺酮类药物的作用机制,旨在为研发精准诊断技术和药物发掘提供科学理论基础和参考。     

Comparison of Gene Expression Profiles Between Pansensitive and Multidrug-Resistant Strains of Mycobacterium tuberculosis

Mycobacterium tuberculosis has developed resistance to anti-tuberculosis first-line drugs. Multidrug-resistant strains complicate the control of tuberculosis and have converted it into a worldwide public health problem. Mutational studies of target genes have tried to envisage the resistance in clinical isolates; however, detection of these mutations in some cases is not sufficient to identify drug resistance, suggesting that other mechanisms are involved. Therefore, the identification of new markers of susceptibility or resistance to first-line drugs could contribute (1) to specifically diagnose the type of M. tuberculosis strain and prescribe an appropriate therapy, and (2) to elucidate the mechanisms of resistance in multidrug-resistant strains. In order to identify specific genes related to resistance in M. tuberculosis, we compared the gene expression profiles between the pansensitive H37Rv strain and a clinical CIBIN:UMF:15:99 multidrug-resistant isolate using microarray analysis. Quantitative real-time PCR confirmed that in the clinical multidrug-resistant isolate, the esxG, esxH, rpsA, esxI, and rpmI genes were upregulated, while the lipF, groES, and narG genes were downregulated. The modified genes could be involved in the mechanisms of resistance to first-line drugs in M. tuberculosis and could contribute to increased efficiency in molecular diagnosis approaches of infections with drug-resistant strains.     

Prediction of drug resistance in M. tuberculosis: molecular mechanisms,tools, and applications

Management of Tuberculosis is complicated by the emergence of drug resistant strains of Mycobacterium tuberculosis and this poses a threat to the success of Tuberculosis control programmes. Drug susceptibility testing by culture is time-consuming and technically difficult. It is known that resistance to drugs is due to a number of genomic mutations in specific genes of M. tuberculosis. These mutations in combination with molecular techniques can be used as markers for drug resistance, since drug susceptible isolates lack the corresponding gene mutations. This review focuses on molecular mechanisms, methods and applications as a possible new diagnostic tool for the early molecular detection of drug resistance in M. tuberculosis.     

Role of antimicrobial peptides in host defense against mycobacterial infections

Worldwide, tuberculosis remains the most important infectious disease causing morbidity and death. Currently, at least one-third of the world's population is infected with Mycobacterium tuberculosis. In addition, the World Health Organization estimates that about 8-10 million new tuberculosis cases occur annually worldwide and this incidence is currently increasing. Moreover, multidrug-resistant tuberculosis has been increasing in incidence in many areas during the past decade. These situations underscore the importance of the development of new therapeutic agents against mycobacterial infectious diseases. In this article, it is review current progress in the understanding of antimicrobial peptides as potential candidates to develop an alternative/adjunct therapeutic strategy against tuberculosis. This immunoadjunctive therapy might be evaluated in the context of possible drug resistance. This review also summarizes the knowledge about the functions of antimicrobial peptides in the pulmonary innate host defense system and their role in mycobacterial infection, and at the same time outlines recent advances in our understanding of the combined effect of antimicrobial peptides and anti-tuberculosis drugs against intracellular mycobacteria. A concerted effort should now focus on the clinical application of antimicrobial peptides for their practical use.