Viable but Nonculturable Mtb

结核病比几乎任何其他细菌感染都需要更长的时间才能治愈。亚种群 结核分枝杆菌(Mtb)与宿主强加相关的表型耐药 使结核分枝杆菌进入缓慢复制或非复制状态的压力(为简单起见，此处称为NR)。我们 假设NR Mtb代表在#年头几个月幸存下来的主要“坚持者”群体 化疗。NR Mtb的一个子集被称为“活的但不可培养的”(VBNC)，因为它们躲避了 琼脂上菌落形成单位(CFU)的检测。VBNC Mtb的不可培养性几乎没有给我们留下 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的限制稀释试验，并用它来确认 在33%的未经治疗的结核病患者的痰中检测到平均87%的活结核分枝杆菌是 CFU检测和BACTEC MGIT检测中的液体培养均未检出VBNC。此外，我们 建立了一种在体外通过无菌培养长期饥饿产生VBNC Mtb的可重复性方法。在……里面 与Project 5和临床核心密切合作，进一步完善和应用复苏 用体外化学基因组学、小鼠遗传学和它们的方法鉴定VBNC Mtb的方法 在标准治疗前后患者的痰中存在。我们将交付：化合物 体外清除VBNC Mtb并鉴定这些化合物的靶点；小鼠模型 这种抑制剂的有效性可以在体内进行测试；以及一种可以在未来的临床试验中使用的测试方法 补充标准的痰早期杀菌活性试验，以确定影响VBNC的药物 人类宿主中的结核杆菌。

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.