Challenging the Traditional Dormancy Assumption: Antibiotic Persisters are Metabolically Active, Non-Growing Cells

Bacterial persisters are a subpopulation of multidrug-tolerant cells capable of surviving and resuming activity after exposure to lethal antibiotic treatments, contributing to relapsing infections and the development of antibiotic resistance. 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. Our transcriptomic analysis, conducted at various time points following exposure to lethal concentrations of ampicillin (Amp), revealed a number of genes with differential expression over time. 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 these genes have been previously linked to persister cells, while others have not been associated with them before. If persister cells were metabolically dormant, we would expect minimal changes in the gene network across different time points of Amp treatment. However, network analysis revealed major shifts in gene network activity at various time points of antibiotic exposure. To explore gene activation in persisters, we developed a new promoter system in which an Amp resistance gene is activated specifically in persister cells. Following treatment with lethal Amp, the Amp resistance gene conferred resistance after ~8 h. This gene's activation requires the production of both mRNA and protein, processes that can only occur in metabolically active cells. These findings reveal that persisters are metabolically active, non-dividing cells, thereby challenging the notion that they are dormant. Overall design: E.coli cells were grown to mid-log phase (OD ~0.5) and then treated with ampicillin (0.1 mg/ml) for 24 hours. RNA samples were collected at four time points: before antibiotic treatment (0 h) and after antibiotic treatment at 3 h, 6 h, and 24 h, in biological triplicates, for RNA sequencing analysis.