Undetected Drug resistance and Tolerance in lesions of recurrent TB

结核病（TB）在世界上造成近150万人死亡。尽管有强大的化疗药物， 细菌通常无法被标准诊断方法检测到，在肺部病变中持续存在，并继续使患者生病， 即使在世界卫生组织标准规定的微生物“治愈”声明之后。 该项目的目标是表征“（表）基因组”（基因组和表观基因组）的组成， 需要手术干预治疗结核病的患者中的结核分枝杆菌（Mtb） (TB)，直接在感染部位，并创建一个知识库，帮助解释化疗失败 和标准诊断这些失败是由于痰中没有检测到耐药菌株吗？是 肺部病变中是否存在耐受药物压力的持久细胞？药物是否没有到达体内的细菌 在足够的浓度下损伤？或者是痰中的持久性或耐药杆菌， 而不被标准诊断发现吗解决这些问题将有助于开发新的和更多的 敏感的诊断，为开发新的治疗方法提供信息，以更有效地穿透病变， 靶向存活的细菌，或靶向所有细菌亚群，并使合理的设计和 开发一种疫苗，更全面地了解这种病原体的基因组变异性， 人类感染的背景。 结核分枝杆菌是一种专性人类病原体，但治疗决定取决于表型检测和基因组 表征从痰中分离并在体外生长的细菌作为结核分枝杆菌群落的代表 导致肺部感染痰和肺细菌群落之间的相互作用已经被证实。 被假设为疾病被错误描述的原因。在这个项目中，我们调查了 此外，旨在确定痰液中是否有其他细菌， 为开发新方法提供信息，以提供更全面的诊断。在此过程中，我们还将 确定持续性或重复感染是否在治疗失败中起作用，以及频率如何。 为了达到这些目标，该项目将研究200例肺损伤中的细菌。 传统治疗失败的患者。我们将首次直接测序， 肺病变和痰中Mtb的基因组和甲基化组，并确定区分Mtb的基因组 以及各植物群群的表观遗传特征。我们会发现隐藏的抵抗， 持久性，并通过差异培养，鉴定细菌是否已被抗生素破坏 治疗以及它们是否都被标准诊断检测到。我们现有的项目已经确定了新的 快速多样化的机制，使细菌群落能够承受免疫和药物压力。 作为该项目的一部分，我们将调查这些机制在肺部的流行和性质。这将 为开发更有效和新的治疗方法和疫苗提供了新的途径。

Tuberculosis (TB) kills nearly 1.5 million people in the world. Despite powerful chemotherapeutics, surviving bacteria often go undetected by standard diagnostics, persist in lung lesions, and continue to make patients ill, even after declaration of microbiological “cure” as specified by criteria from the World Health Organization. This project’s objective is to characterize the “(epi)genomic” (genomic and epigenomic) composition of Mycobacterium tuberculosis (Mtb) in patients who require surgical intervention for treatment of tuberculosis (TB), directly at the site of infection, and to create a knowledgebase that helps explain failure of chemotherapy and standard diagnostics. Are these failures due to undetected resistant strains absent in the sputum? Were there persister cells in pulmonary lesions that tolerated the drug pressure? Did drugs not reach bacteria within the lesion in sufficient concentrations? Or were persistent or resistant bacilli in the sputum that went undetected by standard diagnostics? Answering these questions will enable the development of new and more sensitive diagnostics, inform development of novel therapeutics that to more effectively penetrate lesions and target the surviving bacteria, or target all bacterial subpopulations, and enable rational design and development of a vaccine with a more comprehensive understanding of genomic variability of this pathogen in the context of human infection. Mtb is an obligate human pathogen, yet treatment decisions hinge on phenotypic testing and genomic characterization of bacteria isolated from sputum and grown in vitro as a proxy for the Mtb communities driving infection in the lungs. Inconsistencies between the sputum and lung bacterial communities have been hypothesized as reasons behind the mischaracterization of the disease. In this project, we investigate this hypothesis and, additionally, aim to determine whether there are additional bacteria in the sputum that can inform development of novel methods to provide more comprehensive diagnostics. In this process, we will also determine whether persistence or superinfection played a role in treatment failure, and how frequently. To reach these goals, this project will study bacteria residing within pulmonary lesions excised from 200 patients who failed traditional treatment. For the first time, we will directly sequence, and de novo assemble genomes and methylomes of Mtb in pulmonary lesions and sputum, and determine the distinguishing genomic and epigenetic characteristics of each subpopulation in these flora. We will detect hidden resistance or persistence, and, through differential culturing, identify whether bacteria have been damaged by antibiotic treatment and whether they are all detected by standard diagnostics. Our existing project has identified novel rapid diversifying mechanisms that allows the bacterial community to withstand immune and drug pressures. As part of this project, we will investigate the prevalence and nature of these mechanisms in the lung. This will inform new paths to development of more effective and novel therapeutics and vaccines.