Viable but Nonculturable Mtb

可行但不可培养的山地车

基本信息

批准号：
10057813
负责人：
Michael Stephen Glickman
金额：
$ 94.76万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10057813
关键词：
Address Agar Animal Model Antioxidants Bacterial Infections Biological Assay Chemicals Clinical Clinical Trials Collaborations Colony-Forming Units Assay Colony-forming units Complement Detection Drug Tolerance Drug resistance in tuberculosis Enzymes Essential Genes Exposure to Extinction (Psychology)Fumarates Funding Future Genes Genetic Genomic DNA Genomics HIV Seronegativity Human Human Resources Hypoxia In Vitro Knowledge Left Leprosy Liquid substance M. tuberculosis genome Metabolic Metabolic Pathway Methods Molecular Target Mus Mycobacterium tuberculosis NBL1 gene Oxidants Pathway interactions Patients Pharmaceutical Preparations Phenotype Physiological Population Predisposition Preparation Pyruvate Quantitative Reverse Transcriptase PCR Regulon Relapse Reporting Reproducibility Research Resuscitation Rifampin Specimen Sputum Starvation Stress Supervision Technology Testing Tissues Training Transcript Tuberculosis Variant Work axenic culture bactericide base catalase chemotherapy clinically significant deep sequencing drug candidate drug testing improved in vivo evaluation inhibitor/antagonist knock-down mouse model pre-clinical response standard care standard of care transcriptome treatment duration tuberculosis drugs tuberculosis treatment

项目摘要

Tuberculosis (TB) takes longer to cure than almost any other bacterial infection. Subpopulations of Mycobacterium tuberculosis (Mtb) become phenotypically drug-tolerant in association with host-imposed stresses that put Mtb into a slowly-replicating or non-replicating state (here called NR for simplicity). We hypothesize that NR Mtb represent a major population of the "persisters" that survive the first months of chemotherapy. One subset of NR Mtb is termed "viable but nonculturable" (VBNC) because they elude detection as colony forming units (CFU) on agar. The nonculturability of VBNC Mtb has left us with almost no knowledge of their essential genes, metabolic pathways or susceptibilities to specific drugs and drug candidates. We have now improved a limiting dilution assay for VBNC Mtb and used it to confirm that an average of 87% of the viable Mtb detected in sputum from 33% of the untreated TB patients studied were VBNC forms missed both by CFU assays and by liquid culture in the BACTEC MGIT assay. Further, we developed a reproducible way to generate VBNC Mtb in vitro by prolonged starvation in axenic culture. In close collaboration with Project 5 and the Clinical Core, we will further improve and apply the resuscitation assay to characterize VBNC Mtb by chemical genomics in vitro, by genetics in the mouse, and by their presence in sputum from patients before and after standard therapy. We will deliver: compounds that eradicate VBNC Mtb in vitro and identification of the targets of these compounds; a mouse model where the efficacy of such inhibitors can be tested in vivo; and an assay that can be used in future clinical trials to complement the standard early bactericidal activity test on sputum so as to identify drugs that impact VBNC Mtb in the human host.

结核病 (TB) 的治愈时间比几乎所有其他细菌感染都要长。的亚群结核分枝杆菌 (Mtb) 变得与宿主施加相关的表型耐药使 Mtb 进入缓慢复制或非复制状态的压力（为简单起见，这里称为 NR）。我们假设 NR Mtb 代表了在最初几个月中存活下来的“坚持者”的主要群体化疗。 NR Mtb 的一个子集被称为“可生存但不可培养”(VBNC)，因为它们逃避检测为琼脂上的菌落形成单位 (CFU)。 VBNC Mtb 的非文化性让我们几乎没有了解其重要基因、代谢途径或对特定药物和药物的敏感性候选人。我们现在改进了 VBNC Mtb 的有限稀释测定，并用它来确认在所研究的 33% 的未经治疗的结核病患者的痰液中检测到的平均 87% 的活结核分枝杆菌 CFU 测定和 BACTEC MGIT 测定中的液体培养均未发现 VBNC 形式。此外，我们开发了一种可重复的方法，通过在无菌培养物中长时间饥饿来体外产生 VBNC Mtb。在与Project 5和Clinical Core密切合作，我们将进一步改进和应用复苏通过体外化学基因组学、小鼠遗传学以及它们的特性来表征 VBNC Mtb 标准治疗前后患者痰液中的存在。我们将提供：化合物体外根除 VBNC Mtb 并鉴定这些化合物的靶标；鼠标模型，其中可以在体内测试此类抑制剂的功效；以及一种可用于未来临床试验的检测方法补充标准的痰液早期杀菌活性测试，以确定影响 VBNC 的药物人类宿主中的 Mtb。