Metabolites augment oxidative stress to sensitize antibiotic-tolerant Staphylococcus aureus to fluoroquinolones

If left unchecked, infections involving antibiotic-refractory bacteria are expected to cause millions of deaths per year in the coming decades. Beyond genetically resistant bacteria, persisters, which are genetically susceptible cells that survive antibiotic doses that kill the rest of the clonal population, can potentially contribute to treatment failure and infection relapse. Stationary-phase bacterial cultures are enriched with persisters, and it has been shown that stimulating these populations with exogenous nutrients can reduce persistence to different classes of antibiotics, including topoisomerase-targeting fluoroquinolones (FQs). In this study, we show that adding glucose and amino acids to nutrient-starved Staphylococcus aureus cultures enhanced their sensitivity to FQs, including delafloxacin (Dela)-a drug that was recently approved for treating staphylococcal infections. We found that while the added nutrients increased nucleic acid synthesis, this increase was not required to sensitize S. aureus to FQs. We further demonstrate that addition of these nutrients increases membrane potential and the ability to generate harmful reactive oxygen species (ROS) during FQ treatment. Chelating iron, scavenging hydroxyl radicals, and limiting oxygenation during FQ treatment and during recovery following FQ treatment rescued nutrient-stimulated S. aureus. In all, our data suggest that while nutrient stimulation increases the activity of FQ targets in stationary-phase S. aureus, the resulting generation of ROS, presumably made possible through metabolic upregulation, is the primary driver of increased sensitivity to these drugs.IMPORTANCEStaphylococcus aureus causes many chronic and relapsing infections because of its ability to endure host immunity and antibiotic therapy. While several studies have focused on the nutrient requirements for the formation and maintenance of staphylococcal infections, the effects of the nutrient environment on bacterial responses to antibiotic treatment remain understudied. Here, we show that adding nutrients to starved S. aureus activates biosynthetic processes, including DNA synthesis, but it is the generation of harmful reactive oxidants that sensitizes S. aureus to DNA topoisomerase-targeting FQs. Our results suggest that the development of approaches aimed at perturbing metabolism and increasing oxidative stress can potentiate the bactericidal activity of FQs against antibiotic-tolerant S. aureus.