Single-cell analysis reveals critical toxin/antitoxin ratio triggering persister resuscitation

Persisters represent a transient, antibiotic-tolerant subpopulation within isogenic bacterial populations, contributing to infection relapses. However, the mechanisms driving persister formation and resuscitation remain elusive. Here, we developed nano-flow cytometry (nFCM)-based methods for single-cell quantification of toxin (T) RelE and antitoxin (A) RelB levels, as well as for monitoring persister states through cell wall growth. We demonstrate that bacteria elevate the T/A ratio through two distinct TA expression modalities to withstand bacteriostatic antibiotic challenge, with T/A = 1.0 as a critical threshold. Intriguingly, single-cell resuscitation dynamics revealed that subinhibitory antibiotic exposure promotes entry into a deeper dormant state characterized by elevated T/A ratios, underscoring the importance of maximizing therapeutic antibiotic concentrations. Crucially, we uncovered a triphasic detoxification process during resuscitation where progressive toxin depletion drives T/A ratio reduction to a critical proliferation-permissive threshold. Proteomic profiling unveiled that persisters with high RelE production have increased transmembrane transporter levels linked to stress response and drug efflux. Our findings offer pivotal molecular insights underlying persister transitions and underscore the need for high-throughput, single-cell analysis of these heterogeneity phenotypes.