(636d) Persisters: A Metabolically-Stimulated State

Bacterial persisters are rare, phenotypic variants with the ability to tolerate high levels of antibiotics, and they are a main cause of relapse from biofilm infections. The tolerance of persisters under antibiotic treatment has been commonly ascribed to transient quiescent state and the resulting inactivity of essential cell functions. Given that metabolism mediates persister shutdown, maintenance and reawakening, which are all essential processes for their antibiotic tolerances, it represents a rich source of potential anti-persister therapeutics. However, direct measurement of metabolites and metabolic activities in persisters using conventional approaches is not currently possible due to the lack of high fidelity isolation techniques. To circumvent these technical limitations, we had previously developed a method using fluorescence-activated cell sorting (FACS) and a fluorescent metabolic dye to measure persister metabolic activity in an exponential phase culture prior to antibiotic treatment. When we used this technique to analyze stationary phase cultures, we observed that cells exhibiting low metabolic activity were less likely to be a persister than cells with high metabolic activity. These observations contrast with cells from exponential phase and the general assumption that persisters are metabolically dormant. By dissecting this phenomenon with genetic, biochemical, and flow cytometric techniques, we have discovered that persisters are formed during stationary phase due to accumulated damage from continued stationary phase metabolism. We found that inhibition of metabolism prior to entry into stationary phase is an effective way to reduce persister levels, suggesting that such perturbations could be used to reduce persistence in nutrient-depleted environments, such as a biofilm.