Persisters are transiently nongrowing and antibiotic-tolerant phenotypic variants identified in major human pathogens, including intracellular Staphylococcus aureus. Due to their capacity to regrow once the environmental stress is relieved and to promote resistance, persisters possibly contribute to therapeutic failures. While persistence and its related quiescence have been mostly studied under starvation, little is known within host cell environments. Here, we examined how the level of reactive oxygen species (ROS) in different host cells affects dormancy depth of intracellular S. aureus. Using single-cell approaches, we found that host ROS induce variable dormant states in S. aureus persisters, displaying heterogeneous and increased lag times for resuscitation in liquid medium. Dormant persisters displayed decreased translation and energy metabolism, but remained infectious, exiting from dormancy and resuming growth when reinoculated in low-oxidative-stress cells. In high-oxidative-stress cells, ROS-induced ATP depletion was associated with the formation of visible dark foci similar to those induced by the protein aggregation inducer CCCP (carbonyl cyanide m-chlorophenylhydrazone) and with the recruitment of the DnaK-ClpB chaperone system involved in the clearance of protein aggregates. ATP depletion led to higher fractions of dormant persisters than ROS, due to a counterbalancing effect of ROS-induced translational repression, suggesting a pivotal role of translation in the dormant phenotype. Consistently, protein synthesis inhibition limited dormancy to levels similar to those observed in low-oxidative-stress cells. This study supports the hypothesis that intracellular S. aureus persisters can reach heterogeneous dormancy depths and highlights the link between ROS, ATP depletion, dark focus formation, and subsequent dormancy state. IMPORTANCE By their capacity to survive to antibiotic pressure and to regrow and give rise to a susceptible population once this pressure is relieved, intracellular persisters of S. aureus may contribute to explain therapeutic failures and recurrent infections. Here, we show that the level of dormancy and the subsequent capacity to resuscitate from this resting state are dependent on the level of oxidative stress in the host cells where bacteria survive. This observation nourishes the debate as whether the most appropriate strategy to cope with S. aureus intracellular infections would consist of trying to push persisters to a deep dormancy state from which wakening is improbable or, on the contrary, to prevent ROS-induced dormancy and force bacteria to maintain regular metabolism in order to restore their responsiveness to antibiotics. Importantly also, our data highlight the interest in single-cell analyses with conventional enumeration of CFU to quantify persisters and study host-pathogen interactions.
Keywords: Staphylococcus aureus; dormancy; intracellular infection; macrophages; oxidative stress; persisters.