Parallel Microscopic Determination of Fitness (PMDF) to Assess Growth and Viability of Mycobacterium tuberculosis

 描述（由申请人提供）：尽管最近在了解结核分枝杆菌（MTB）的分子和细胞生物学方面取得了许多进展，但几乎所有实验中的限速步骤仍然是分批培养生长和表型分析-这是一种不精确的中等通量过程，掩盖了反应的异质性，并且在过去100年中基本没有变化。像所有的细菌一样，结核分枝杆菌（MTB）在其整个自然历史中受到动态环境压力的影响。面对各种各样不断变化的环境，细菌以不同的方式做出反应-这些反应的广度无法在批量培养中获得。在这里，我们建议开发一种新的和强大的方法，并行显微镜测定的健身（PMDF），监测细菌的生长和活力，同时保留和记录人口内的表型异质性。我们已经进行了试点实验，使用时间推移阶段和荧光显微镜监测生长的土壤saplete耻垢分枝杆菌，在每个实验中数以千计的独立的细胞沉积事件的独立命运。我们现在的目标是通过一系列概念验证实验，使这个实验平台适应MTB。我们将描述不同地理位置的结核分枝杆菌临床分离株的复制特征，因为它们随着时间的推移对确定的疾病相关环境扰动作出反应，并开发这种方法来改善对小分子抗结核药物敏感性的分析。

 DESCRIPTION (provided by applicant): Despite many recent advances in understanding the molecular and cellular biology of Mycobacterium tuberculosis (MTB), the rate-limiting step in nearly all experiments remains batch-culture growth and phenotyping - an imprecise process of modest throughput that masks the heterogeneity of responses and is largely unchanged in the last 100 years. Like all bacteria, Mycobacterium tuberculosis (MTB) is subjected to dynamic environmental stresses throughout its natural history. Faced with varied and changing environments, bacteria respond in diverse ways - and the breadth of these responses cannot be captured in batch culture. Here we propose to develop a novel and robust method, Parallel Microscopic Determination of Fitness (PMDF), to monitor bacterial growth and viability while preserving and recording phenotypic heterogeneity within the population. We have performed pilot experiments using time lapse phase and fluorescent microscopy to monitor growth of the soil saprophyte Mycobacterium smegmatis, following the independent fates of thousands of separate cell deposition events in each experiment. Our goal now is to adapt this experimental platform to MTB, with a series of proof-of-concept experiments. We will characterize the replication of geographically diverse clinical isolates of MTB as they respond over time to defined, disease- relevant environmental perturbations, and develop this approach to improve analysis of susceptibility to small molecule antitubercular agents.