Viable but Nonculturable Mtb

可行但不可培养的山地车

• 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
• 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

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.

肺结核（TB）比几乎任何其他细菌感染都需要更长的时间才能治愈。亚群 结核分枝杆菌（Mtb）成为表型耐药与宿主强加的 使Mtb进入缓慢复制或非复制状态的应力（为简单起见，这里称为NR）。我们 假设NR Mtb代表存活最初几个月“坚持者”的主要群体， 化疗NR Mtb的一个子集被称为“可存活但不可培养”（VBNC），因为它们逃避了 检测为琼脂上的菌落形成单位（CFU）。VBNC Mtb的不可培养性给我们留下了几乎没有 了解他们的基本基因、代谢途径或对特定药物和药物的敏感性 候选人我们现在已经改进了VBNC Mtb的有限稀释测定法，并使用它来证实 在33%的未经治疗的结核病患者的痰中检测到的活结核杆菌中， 在BACTEC MGIT测定中，CFU测定和液体培养均遗漏VBNC形式。我们还 开发了通过在无菌培养物中延长饥饿在体外产生VBNC Mtb的可重复的方法。在 与项目5和临床核心密切合作，我们将进一步改进和应用复苏 通过体外化学基因组学、通过小鼠遗传学以及通过其 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; 这些抑制剂的功效可以在体内测试;以及可以在未来的临床试验中使用的测定， 补充痰液的标准早期杀菌活性试验，以确定影响VBNC的药物 结核病在人类宿主中。