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.