Rethinking dormancy: Antibiotic persisters are metabolically active, non-growing cells

•Antibiotic bacterial persisters are a subpopulation known for their multidrug tolerance.•We challenge the longstanding belief that persisters are metabolically inactive.•Persisters actively produce RNA and adapt their transcriptome to enhance survival. Bacterial persisters are a subpopulation of multidrug-tolerant cells capable of surviving and resuming activity after exposure to bactericidal antibiotic concentrations, contributing to relapsing infections and the development of antibiotic resistance. In this study, we challenge the conventional view that persisters are metabolically dormant by providing compelling evidence that an isogenic population of Escherichia coli remains metabolically active in persistence. Using transcriptomic analysis, we examined E. coli persisters at multiple time points following exposure to bactericidal concentrations of ampicillin (Amp). Some genes were consistently upregulated in Amp treated persisters compared to the untreated controls, a change that can only occur in metabolically active cells capable of increasing RNA levels. Some of the identified genes have been previously linked to persister cells, while others have not been associated with them before. If persister cells were metabolically dormant, gene expression changes over time would be minimal during Amp treatment. However, network analysis revealed major shifts in gene network activity at various time points of antibiotic exposure. These findings reveal that persisters are metabolically active, non-dividing cells, thereby challenging the traditional view that they are dormant. [Display omitted]