Rapid and efficient labeling by a selective organic fluorophore probe highlights heterogeneity of mycobacterial populations and persister resuscitation Dataset Figure3

Abstract Many bacteria enhance their survival by generating slow-/non-growing persister subpopulations. Persisters are stress tolerant and can reestablish populations after a stress. In mycobacterial infections, such as tuberculosis, these subpopulations necessitate prolonged antibiotic therapy, as they cause chronic, recurrent, and antibiotic-tolerant infections. These subpopulations are generally very small in size, making their molecular and physiological studies extremely challenging. Consequently, new tools that facilitate their investigation are highly needed. To characterize slow-/non-growing mycobacterial populations, we present an approach that utilizes Vybrant DiD, a lipophilic, small fluorescent organic probe, for mycobacterial labeling. Using this approach, pathogenic and non-pathogenic mycobacteria species can be detected and visualized within 5-20 minutes in a specific, sensitive and non-invasive manner. It enables accurate and direct quantification of mycobacterial replication rate in vitro under different growth conditions as well as intracellularly in murine macrophages at the population and single-cell level, providing insight into mycobacterial population heterogeneity. We determined the slow-/non-growing fraction of mycobacteria during exponential growth, which increased upon a stress, and we assessed the regrowth of antibiotic-tolerant, mycobacterial cells after antibacterial treatment. Monitoring mycobacterial resuscitation after low-oxygen-induced dormancy revealed the stochastic and heterogeneous nature of the resuscitation process. While the growth rate of resuscitated bacteria did not differ from the growth rate of untreated cells, their resuscitation time was found dependent on the duration of the applied hypoxia stress. Based on its simplicity and convenience, we anticipate that this method will be widely utilized in basic research on bacterial persistence and may also be included in applied settings, e.g. high-throughput drug characterization. (256 words) This dataset contains the data for Figure 3.摘要 诸多细菌可通过生成缓慢生长/不生长的持留菌（persister）亚群来提升自身生存能力。持留菌具有胁迫耐受性，可在胁迫解除后重新构建种群。在分枝杆菌（Mycobacterium）感染（如结核病）中，这类亚群会引发慢性、复发性且耐受抗生素的感染，因此需要延长抗生素治疗周期。这类亚群的规模通常极小，使得对其开展分子与生理学研究极具挑战性。因此，亟需能够助力这类亚群研究的新型工具。 为了表征缓慢生长/不生长的分枝杆菌亚群，我们开发了一种利用亲脂性小型荧光有机探针Vybrant DiD进行分枝杆菌标记的方法。借助该方法，可在5至20分钟内以特异性、高灵敏度且非侵入性的方式，检测并可视化致病性与非致病性分枝杆菌物种。该方法可在体外不同生长条件下，以及在小鼠巨噬细胞内的群体与单细胞水平上，精准直接地定量分枝杆菌的复制速率，从而助力解析分枝杆菌种群的异质性。 我们测定了指数生长期分枝杆菌的缓慢生长/不生长亚群比例，该比例会在胁迫作用下升高；同时我们还评估了抗菌处理后耐受抗生素的分枝杆菌细胞的再生情况。通过监测低氧诱导休眠后分枝杆菌的复苏过程，我们揭示了复苏过程的随机性与异质性特征。尽管复苏后细菌的生长速率与未处理细胞并无差异，但它们的复苏时长却取决于所施加的低氧胁迫持续时间。 鉴于该方法简便易行，我们预计其将在细菌持留性的基础研究中得到广泛应用，同时也可应用于高通量药物表征等实际场景中。（全文共256词） 本数据集包含图3的相关实验数据。