Abstract The goal of the project is to determine the nature of bacterial drug tolerance. Two different types of mechanisms allow bacteria to evade killing by antibiotics – resistance; and tolerance conferred by persister cells. Unlike resistance, our knowledge of tolerance is limited. Paradoxically, most pathogens that cause chronic infections recalcitrant to antimicrobial chemotherapy are not drug resistant. Tolerance has been linked to persisters, a small subpopulation of dormant cells that survive antibiotics. Many chronic infections are associated with biofilms, which protect persisters from the immune system. An understanding of the mechanism of persister drug tolerance will close a significant gap in knowledge and will contribute to the development of better approaches to treat chronic infections. The current paradigm, based primarily on the study of E. coli, holds that mechanisms of persister formation are not conserved among bacteria, and are governed by toxin-antitoxin modules (TA). However, we recently reported that in S. aureus, TAs play no role in persister formation. Rather, a stochastic decrease in ATP in rare cells produces dormant persisters. We then found that a decrease in ATP is linked to persister formation in E. coli as well. We also established that while some TAs play a role in persister formation under specific conditions in E. coli, this is not the main mechanism. In this project, we will determine the general mechanism by which persisters form in bacteria using E. coli, a representative Gram negative pathogen, and S. aureus, a Gram positive species,. Our preliminary data indicate that stochastic variation in expression of energy producing components - Krebs cycle and glycolytic enzymes - leads to low ATP and persisters. In this project, we will use direct reporters for protein expression and ATP to establish causality between energy producing components and persisters. Apart from conventional time-lapse microscopy, we will take advantage of the “mother machine”, a massively parallel microfluidics instrument that allows simultaneous analysis of millions of individual cells. Another important unanswered question is the link between persisters and the clinical manifestation of disease. While indirect evidence points to persisters, causality is yet to be established. In this project, we will design pathogen strains with diminished; and overexpressed production of persisters, and link their levels to antibiotic tolerance in biofilm models of murine chronic infection. This project will provide a new paradigm for the understanding of recalcitrance of chronic diseases, and new tools for the study of persisters. This is a multi-PI collaboration between Dr. Kim Lewis, a microbiologist who pioneered the studies of persisters in chronic infections, and Dr. Johan Paulsson, a biophysicist who pioneered massively parallel single-cell analysis.