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

平行显微适应性测定 (PMDF) 评估结核分枝杆菌的生长和活力

• 批准号: 9089849
• 负责人: DAVID R SHERMAN
• 金额: $ 23.63万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9089849
• 关键词: Antibiotics; Antitubercular Agents; Area; Bacteria; Benchmarking; Biological; Biology; Carbon; Cells; Chemicals; Cholesterol; Clinical; Colony-forming units; Data; Deposition; Development; Disease; Environment; Event; Goals; Growth; Health; Heating; Heterogeneity; Immune; Individual; Life; Masks; Metabolic; Methodology; Methods; Microfluidics; Microscopic; Microscopy; Minimum Inhibitory Concentration measurement; Modification; Molecular and Cellular Biology; Monitor; Mycobacterium smegmatis; Mycobacterium tuberculosis; Natural History; Nutrient; Pharmaceutical Preparations; Phase; Phase-Contrast Microscopy; Phenotype; Population; Preclinical Drug Evaluation; Predisposition; Process; Protocols documentation; Proxy; Reagent; Research; Series; Soil; Solid; Source; Staining method; Stains; Stress; Technology; Testing; Time; Validation; Work; bacterial resistance; bactericide; drug discovery; fitness; improved; killings; microorganism; novel; novel strategies; novel therapeutics; pathogen; pressure; programs; research study; response; small molecule; time use

 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.

 描述(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批培养生长和表型--这是一种不精确的过程，产量适中，掩盖了反应的异质性，在过去100年中基本没有变化。像所有细菌一样，结核分枝杆菌(MTB)在其整个自然历史过程中都受到动态环境压力的影响。面对多变的环境，细菌以不同的方式做出反应--这些反应的广度在批量培养中是无法捕捉的。在这里，我们建议开发一种新的和稳健的方法，平行显微确定适合度(PMDF)，以监测细菌的生长和活力，同时保留和记录种群内的表型异质性。我们使用延时相和荧光显微镜进行了初步实验，以监测土壤腐生细菌污垢分枝杆菌的生长，跟踪每个实验中数千个独立的细胞沉积事件的独立命运。我们现在的目标是将这个实验平台适应MTB，进行一系列概念验证实验。我们将表征不同地理位置的结核分枝杆菌临床分离株随着时间的推移对明确的、与疾病相关的环境扰动的反应的特征，并开发这种方法来改进对小分子抗结核药物的敏感性分析。