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

Stress tolerant slow- or non-growing bacterial subpopulations represent a key factor for chronic, recurrent, antibiotic-tolerant infections and necessitate prolonged antibiotic therapy. Consequently, new tools that facilitate investigation of these small subpopulations are highly needed. To characterize slow-/non-growing populations of mycobacteria, we here utilized Vybrant DiD, a lipophilic, fluorescent organic probe that detected pathogenic and non-pathogenic species in a rapid, specific and non-invasive manner. It enabled accurate, direct quantification of mycobacterial replication rate in vitro and in murine macrophages at the population and single-cell level, providing insight into population heterogeneity. We determined the preexisting slow-/non-growing fraction of mycobacteria during exponential growth, which increased upon stress. Monitoring mycobacterial resuscitation after antibacterial treatment and after low-oxygen-induced dormancy revealed the stochastic and heterogeneous nature of the resuscitation process. 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.