Undetected Drug resistance and Tolerance in lesions of recurrent TB

世界上有近150万人死于结核病。尽管有强大的化疗药物，但幸存下来 细菌通常不会被标准的诊断方法发现，会持续存在于肺部病变中，并继续使患者患病， 即使在宣布了世界卫生组织标准所规定的微生物“治愈”之后。 该项目的目标是描述(Epi)基因组(基因组和表观基因组)的组成 结核分枝杆菌(Mtb)在需要外科手术治疗的结核病患者中的应用 (结核病)，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。这些失败是因为痰中没有检测到耐药菌株吗？是 肺部病变中有耐受药物压力的持久细胞吗？药物没有到达细菌体内吗 在足够的浓度下损伤？或者是痰中的顽固或耐药杆菌 未被标准诊断检测到？回答这些问题将使新的和更多的发展 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和 以存活细菌为目标，或以所有细菌亚群为目标，并使合理的设计和 一种更全面地了解该病原体基因组变异的疫苗的研制 人类感染的背景。 结核分枝杆菌是一种人类特有的病原体，但治疗决定取决于表型测试和基因组学 从痰中分离和体外培养的结核分枝杆菌菌群的特性 导致肺部感染。痰和肺部细菌群落之间的不一致一直是 被认为是错误描述这种疾病的原因。在这个项目中，我们对此进行了调查 假说，并旨在确定痰中是否有额外的细菌可以 通知新方法的发展，以提供更全面的诊断。在这个过程中，我们还将 确定持续性或双重感染是否在治疗失败中起了作用，以及发生的频率。 为了达到这些目标，这个项目将研究从200个肺部病变中切除的细菌。 传统治疗失败的患者。第一次，我们将直接排序，并重新组装 肺部病变和痰中结核分枝杆菌的基因组和甲基组，并确定区分基因组 以及这些植物区系中各亚群的表观遗传特征。我们将检测到隐藏的阻力或 持久性，并通过差异培养，确定细菌是否已被抗生素破坏 治疗以及它们是否都能通过标准诊断检测到。我们现有的项目已经确定了小说 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率和性质。这将是 为开发更有效和新的疗法和疫苗提供新的途径。

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.