Oxidative damage and delayed replication allow viable Mycobacterium tuberculosis to go undetected [TnSeq]

“Viable but non-culturable” (VBNC) states pose challenges for environmental and clinical microbiology, but their biological mechanisms remain obscure. Mycobacterium tuberculosis (Mtb), the leading cause of death from infection until COVID-19, affords a striking example. Mtb can enter into a “differentially detectable” (DD) state associated with phenotypic antimicrobial resistance in which Mtb cells are viable but undetectable as colony-forming units. We found that Mtb cells enter the DD state when they undergo sublethal oxidative stress that damages their DNA, proteins, and lipids, and in addition, their replication is delayed, allowing repair. Mycobacterium bovis and BCG fail to enter the DD state under similar conditions. These findings have implications for TB latency, detection, relapse, treatment monitoring, and development of regimens that overcome phenotypic antimicrobial resistance. Overall design: H37Rv Transposon-library Sequencing of outgrowth cultures after exposure to the following conditions: growth in nutrient rich media; two weeks of nutrient starvation in PBS with tyloxapol; two weeks of nutrient starvation in PBS with tyloxapol followed by 5 days of 1% DMSO exposure; two weeks of nutrient starvation in PBS with tyloxapol followed by 5 days of exposure to 100 uM rifampin. For each listed condition, the subsequent outgrowth of the transposon library was carried out on nutrient rich solid agarose media, 5 mL of nutrient rich liquid media, or 50 mL of nutrient rich liquid media. 2 experiments with 1 sample for each condition/outgrowth in each experiment.

活的但不可培养（Viable but non-culturable，VBNC）状态给环境与临床微生物学研究带来了诸多挑战，但其生物学机制仍不甚明晰。结核分枝杆菌（Mycobacterium tuberculosis，Mtb）便是典型范例——在新冠疫情前，它曾是感染性疾病致死的首要病因。结核分枝杆菌可进入一种与表型耐药性相关的“差异检出（differentially detectable，DD）”状态，此时菌体虽存活却无法通过菌落形成单位检测到。本研究发现，结核分枝杆菌在遭遇可损伤DNA、蛋白质与脂质的亚致死氧化应激时，会进入DD状态；与此同时，其复制进程会延迟，以留出损伤修复的时间。牛分枝杆菌与卡介苗（BCG）在类似条件下无法进入DD状态。上述研究结果对结核潜伏感染、病原体检测、疾病复发、治疗监测以及克服表型耐药性的治疗方案开发均具有重要参考价值。实验整体设计：本研究针对暴露于以下条件后的复苏培养物开展H37Rv转座子文库测序：1. 在营养丰富的培养基中培养；2. 在含泰洛沙泊的磷酸盐缓冲液（PBS）中进行两周营养饥饿处理；3. 先在含泰洛沙泊的PBS中进行两周营养饥饿处理，随后暴露于1%二甲基亚砜（DMSO）环境中5天；4. 先在含泰洛沙泊的PBS中进行两周营养饥饿处理，随后暴露于100微摩尔利福平环境中5天。针对上述每一种处理条件，后续的转座子文库复苏培养分别在三种体系中进行：营养丰富的固体琼脂培养基、5mL营养丰富的液体培养基，以及50mL营养丰富的液体培养基。每个实验中，每种处理条件对应一种复苏培养体系均设置1个生物学重复，共开展2组独立实验。