Center for Microbiology, VIB, 3001 Leuven, Belgium; Centre of Microbial and Plant Genetics, KU Leuven, 3001 Leuven, Belgium.
Department of Biology, KU Leuven, 3001 Leuven, Belgium.
STAR Protoc. 2022 Jun 17;3(3):101476. doi: 10.1016/j.xpro.2022.101476. eCollection 2022 Sep 16.
Given the low fraction of antibiotic-tolerant persisters and the transient nature of the persister phenotype, identifying molecular mechanisms underlying persister state exit, also called "awakening," is challenging. Here, we describe how persister awakening kinetics can be quantified at the single-cell level, enabling the identification of genes that are important for persister survival following antibiotic treatment. We report step-by-step sample preparation, dynamic recording, and data analysis. Although the setup is flexible, time-lapse microscopy requires a minimal number of persisters being present. For complete details on the use and execution of this protocol, please refer to Wilmaerts et al. (2022).
鉴于抗生素耐受持久菌的比例较低，以及持久菌表型的瞬时性，鉴定持久菌状态退出（也称为“唤醒”）的分子机制具有挑战性。在这里，我们描述了如何在单细胞水平上定量测定持久菌唤醒动力学，从而能够鉴定出在抗生素治疗后对持久菌存活很重要的基因。我们报告了分步的样本制备、动态记录和数据分析。尽管设置很灵活，但延时显微镜需要有最小数量的持久菌存在。如需了解本协议的使用和执行的完整详细信息，请参阅 Wilmaerts 等人（2022 年）。
