抗结核药物及其耐药相关突变位点的研究进展

结核病是结核分枝杆菌复合物引起的传染性疾病,致死率、致残率高,在全球传染病中居第2位。近年来耐药结核病所占比例逐年升高,成为消灭结核病面临的巨大挑战之一。传统的耐药诊断方法基于培养,费时费力,所需技术要求高;而现有分子检测方法仅能检测少量抗结核药物的少数耐药基因。因此,更好地理解抗结核药物的耐药机制有助于全面耐药诊断。本文对临床中使用频率较高的11类一线和二线抗结核药物及其相应耐药相关基因、突变位点的研究进展进行总结,尤其是对环丝氨酸、利奈唑胺、氯法齐明等二线药物的近期研究做了系统描述,为全面耐药诊断、精准治疗指导、新药研发及耐药机制深入研究提供了前期工作基础。     

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...     

结核分枝杆菌耐药机制研究进展

结核分枝杆菌为结核病的病原体.最近几年,由于基因突变导致多耐药及广泛耐药结核菌株的出现,以及抗结核病药近几十年来没有换代,使之前基本得到控制的结核痛死灰复燃,成为世界上病死率最高的传染病.为了遏制其进一步的恶化,必须从根本上透彻了解其耐药分子机制.近年来,各国学者采用先进分子生物学技术对结核杆菌耐药机制进行深入研究,定位了结核分枝杆菌耐药基因的位置和基因突变位点,比如耐异烟肼、利福平、乙胺丁醇、链霉素、吡嗪酰胺、喹诺酮类、外排泵等菌株新的基因突变位点引起新的功能改变有新的发现,特别是gidB基因、外排泵基因等有突破性的发现,对研制新一代抗结核病药提供了理论支持及新的方向,但仍有很多耐药机制未阐明,为后续研究者提供些许查考,故笔者就近几年来从分子水平对耐药机制的研究进展做一概述.     

结核病耐药原因概述

产生耐药性为结核杆菌的重要生物学特性之一,与治疗成败关系极大.了解耐药结核病的发生原因将有助于对耐药结核病采取有效防治措施.目前对结核病耐药原因尚不十分清楚,本文从药物因素和非药物因素两个方面对结核病耐药性发生相关的主要因素进行综述.     

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。     

非结核性分枝杆菌耐药机制的研究进展

非结核性分枝杆菌由于其天然的高度耐药性耐导致其感染的难治性已引起医学界的广泛关注,目前对非结核性分枝杆菌耐药机制的研究已包括细胞壁的通透性,药物泵,药物灭活,靶位点基因突变及质粒等各方面,本文就这些方面的最新进展做一综述。以期对进一步的研究及治疗有所帮助。     

新型抗结核活性化合物H37Ra的耐药菌筛选及其菌落表型

ATB-152E和ATB-152J为本实验室前期研究获得的具有良好抗结核活性的两种结构类似的小分子化合物,本文就其作用靶标及耐药机制进行探索。采用含药平板涂板筛选以及平板划线培养法逐步提高化合物浓度,分别筛选出结核分枝杆菌ATB-152E和ATB-152J耐药菌株。选取有代表性的耐药菌株,用微孔法测定结核分枝杆菌的最小抑菌浓度,分别对它们的菌落形态、生物膜形成等表型进行观察并与野生株进行比较。通过不断提高化合物筛选浓度最终筛选到ATB-152E耐药菌株17株、ATB-152J耐药菌株15株,这两种化合物的耐药频率均为10^－7。生长表型结果显示,与野生株结核分枝杆菌相比,ATB-152E耐药菌菌落褶皱变多,ATB-152J耐药菌菌落形态更为扁平,褶皱变少。耐药菌的生物膜形成所需时间与野生株也存在差异,提示活性化合物耐药菌的突变可能导致细菌脂质代谢异常。ATB-152E和ATB-152J耐药菌的获得,为后续深入探索这两种具有良好抗结核活性化合物的作用机制奠定了基础。     

结核分枝杆菌对异烟肼耐药的分子机制

抗结核一线药物异烟肼是应用最广泛的抗结核药物之一,自1952年应用于临床以来,异烟肼就成了治疗结核和潜在感染的基础药物.有报道,我国异烟肼耐药已排在首位.结核分枝杆菌对异烟肼耐药的分子机制十分复杂,涉及katG、inhA、kasA、ndh、axyR等多种基因,本研究仅就此方面的研究作一综述.     

结核分枝杆菌的耐药基因及其相关分子机制

结核分枝杆菌原发性和继发性耐药是当前控制和治疗结核病面临的重要问题,随着分子遗传学的发展,已经阐明了结核分枝杆菌耐药的分子基础是染色体的突变,影响了药靶本身或激活了药物前体的细菌酶,造成MTB的耐药。本文主要就MTB对其常用药物的耐药机制展开讨论,以便正确认识MTB对不同药物的耐药机制,建立快速检测耐药结核分枝杆菌基因型的分子生物学方法。     

结核分枝杆菌的耐药基因及其相关分子机制

结核分枝杆菌原发性和继发性耐药是当前控帝】和治疗结核病面临的重要问题,随着分子遗传学的发展,已经阐明了结核分枝杆菌耐药的分子基础是染色体的突变,影响了药靶本身或激活了药物前体的细菌酶,造成MTB的耐药。本文主要就MTB对其常用药物的耐药机制展开讨论,以便正确认识MTB对不同药物的耐药机制,建立快速检测耐药结核分枝杆菌基因型的分子生物学方法。     

Recent updates on drug resistance in Mycobacterium tuberculosis

Tuberculosis (TB) along with acquired immune deficiency syndrome and malaria rank among the top three fatal infectious diseases which pose threat to global public health, especially in middle and low income countries. TB caused by Mycobacterium tuberculosis (Mtb) is an airborne infectious disease and one-third of the world's population gets infected with TB leading to nearly 1·6 million deaths annually. TB drugs are administered in different combinations of four first-line drugs (rifampicin, isoniazid, pyrazinamide and ethambutol) which form the core of treatment regimens in the initial treatment phase of 6–9 months. Several reasons account for the failure of TB therapy such as (i) late diagnosis, (ii) lack of timely and proper administration of effective drugs, (iii) lower availability of less toxic, inexpensive and effective drugs, (iv) long treatment duration, (v) nonadherence to drug regimen and (vi) evolution of drug-resistant TB strains. Drug-resistant TB poses a significant challenge to TB therapy and control programs. In the background of worldwide emergence of 558 000 new TB cases with resistance to rifampicin in the year 2017 and of them, 82% becoming multidrug-resistant TB (MDR-TB), it is essential to continuously update the knowledge on the mechanisms and molecular basis for evolution of Mtb drug resistance. This narrative and traditional review summarizes the progress on the anti-tubercular agents, their mode of action and drug resistance mechanisms in Mtb. The aim of this review is to provide recent updates on drug resistance mechanisms, newly developed/repurposed anti-TB agents in pipeline and international recommendations to manage MDR-TB. It is based on recent literature and WHO guidelines and aims to facilitate better understanding of drug resistance for effective TB therapy and clinical management.     

结核分枝杆菌潜伏感染诊断方法的新进展

每年有超过8百万人感染结核,其中绝大部分没有发展为活动性结核病,而是表现为潜伏性结核感染。大多数活动性结核病是潜伏感染的结核杆菌重新被激活所致,因此结核潜伏感染者成为结核患者的重要来源。及早诊断和治疗结核潜伏感染者是控制结核传播的最有效手段之一。我们较要综述了目前国内外结核潜伏感染的诊断方法及其新进展。     

Dynamics of fluoroquinolones induced resistance in DNA gyrase of Mycobacterium tuberculosis

DNA gyrase is a validated target of fluoroquinolones which are key components of multidrug resistance tuberculosis (TB) treatment. Most frequent occurring mutations associated with high level of resistance to fluoroquinolone in clinical isolates of TB patients are A90V, D94G, and A90V–D94G (double mutant [DM]), present in the larger subunit of DNA Gyrase. In order to explicate the molecular mechanism of drug resistance corresponding to these mutations, molecular dynamics (MD) and mechanics approach was applied. Structure-based molecular docking of complex comprised of DNA bound with Gyrase A (large subunit) and Gyrase C (small subunit) with moxifloxacin (MFX) revealed high binding affinity to wild type with considerably high Glide XP docking score of ?7.88 kcal/mol. MFX affinity decreases toward single mutants and was minimum toward the DM with a docking score of ?3.82 kcal/mol. Docking studies were also performed against 8-Methyl-moxifloxacin which exhibited higher binding affinity against wild and mutants DNA gyrase when compared to MFX. Molecular Mechanics/Generalized Born Surface Area method predicted the binding free energy of the wild, A90V, D94G, and DM complexes to be ?55.81, ?25.87, ?20.45, and ?12.29 kcal/mol, respectively. These complexes were further subjected to 30 ns long MD simulations to examine significant interactions and conformational flexibilities in terms of root mean square deviation, root mean square fluctuation, and strength of hydrogen bond formed. This comparative drug interaction analysis provides systematic insights into the mechanism behind drug resistance and also paves way toward identifying potent lead compounds that could combat drug resistance of DNA gyrase due to mutations.     

A population-based study of first and second-line drug-resistant tuberculosis in a high-burden area of the Mexico/United States border

The resistance of 139 Mycobacterium tuberculosis (MTB) isolates from the city of Monterrey, Northeast Mexico, to first and second-line anti-TB drugs was analysed. A total of 73 isolates were susceptible and 66 were resistant to anti-TB drugs. Monoresistance to streptomycin, isoniazid (INH) and ethambutol was observed in 29 cases. Resistance to INH was found in 52 cases and in 29 cases INH resistance was combined with resistance to two or three drugs. A total of 24 isolates were multidrug-resistant (MDR) resistant to at least INH and rifampicin and 11 MDR cases were resistant to five drugs. The proportion of MDR-TB among new TB cases in our target population was 0.72% (1/139 cases). The proportion of MDR-TB among previously treated cases was 25.18% (35/139 cases). The 13 polyresistant and 24 MDR isolates were assayed against the following seven second-line drugs: amikacin (AMK), kanamycin (KAN), capreomycin (CAP), clofazimine (CLF), ethionamide (ETH), ofloxacin (OFL) and cycloserine (CLS). Resistance to CLF, OFL or CLS was not observed. Resistance was detected to ETH (10.80%) and to AMK (2.70%), KAN (2.70%) and CAP (2.70%). One isolate of MDR with primary resistance was also resistant to three second-line drugs. Monterrey has a high prevalence of MDR-TB among previously treated cases and extensively drug-resistant-MTB strains may soon appear.     

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.     

Structural implications of lipoarabinomannan glycans from global clinical isolates in diagnosis of Mycobacterium tuberculosis infection

In Mycobacterium tuberculosis (Mtb), surface-exposed Lipoarabinomannan (LAM) is a key determinant of immunogenicity, yet its intrinsic heterogeneity confounds typical structure–function analysis. Recently, LAM gained a strong foothold as a validated marker for active tuberculosis (TB) infection and has shown great potential in new diagnostic efforts. However, no efforts have yet been made to model or evaluate the impact of mixed polyclonal Mtb infections (infection with multiple strains) on TB diagnostic procedures other than antibiotic susceptibility testing. Here, we selected three TB clinical isolates (HN878, EAI, and IO) and purified LAM from these strains to present an integrated analytical approach of one-dimensional and two-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy, as well as enzymatic digestion and site-specific mass spectrometry (MS) to probe LAM structure and behavior at multiple levels. Overall, we found that the glycan was similar in all LAM preparations, albeit with subtle variations. Succinates, lactates, hydroxybutyrate, acetate, and the hallmark of Mtb LAM-methylthioxylose (MTX), adorned the nonreducing terminal arabinan of these LAM species. Newly identified acetoxy/hydroxybutyrate was present only in LAM from EAI and IO Mtb strains. Notably, detailed LC/MS-MS unambiguously showed that all acyl modifications and the lactyl ether in LAM are at the 3-OH position of the 2-linked arabinofuranose adjacent to the terminal β-arabinofuranose. Finally, after sequential enzymatic deglycosylation of LAM, the residual glycan that has ∼50% of α−arabinofuranose -(1→5) linked did not bind to monoclonal antibody CS35. These data clearly indicate the importance of the arabinan termini arrangements for the antigenicity of LAM.     

肺外结核病微生物学诊断方法的研究和应用进展

肺外结核病指由结核分枝杆菌（Mycobacterium tuberculosis, MTB）感染所引起的发生在肺部以外器官和部位的结核病。近年来肺外结核的发病率逐渐升高,未能得到早期有效治疗的肺外结核病患者可能并发畸形、截瘫甚至死亡等严重后果。微生物学检测方法对从病原学角度诊断肺外结核病至关重要。基于此,总结了近年来肺外结核病细菌学检查方法、结核分枝杆菌的抗原检测与分子生物学检测等微生物学诊断方法的概况及应用进展,并对这些检测方法的优缺点及适用范围进行了分析、比较,以期为今后肺外结核病病原学诊断的研究提供相关信息。