Rifampicin-induced Staphylococcus aureus persister formation is driven by CodY regulon and oxidative stress level

Staphylococcus aureus is a major human pathogen. Beyond resistance, antibiotic persistence and tolerance reduce treatment efficacy. Persisters are characterized by heterogenous sub-populations of non-growers within an otherwise susceptible replicative population. We leveraged the spectral properties of Timer and TimerFAST--two novel growth reporter fluorescent proteins in S. aureus--to identify non-growers and population heterogeneity using live-cell microscopy and flow cytometry. Single-cell phenotype analysis during rifampicin exposure revealed population heterogeneity, with replicating cells coexisting alongside non-growing but viable cells. The non-grower phenotype increased in the codY mutant upon rifampicin treatment. We uncovered that codY mutant exhibits higher ROS level than the wild-type. Treatment with both rifampicin and menadione--a ROS inducer--triggered a fully non-growing population independently of CodY. These findings highlight the major role of CodY regulon and oxidative stress in persister formation and pave the way to develop new therapeutic strategies.