ABSTRACT Antibiotic treatment failure is one of the greatest global public health challenges of our generation. Antimicrobial- resistant bacteria directly account for over 1.2 million deaths annually worldwide. Microorganisms that are refractory to a new antibiotic often emerge shortly after the drug is introduced in the clinic. Besides becoming heritably resistant to antibiotics, some bacteria in clonal cultures—coined persisters—can reversibly reprogram their phenotypes and become transiently tolerant to a given drug. This can lead to infection relapse after a course of antibiotics, rendering the treatment ineffective. Evidence further suggests that persisters have a higher likelihood of acquiring resistance-conferring mutations. As such, the development of anti-persistence/resistance therapeutic strategies can increase the success of antimicrobial therapy. In this proposal, we focus on antibiotic persistence and resistance development in Escherichia coli and Pseudomonas aeruginosa cells in slow/non-growing cultures, which are less responsive to antibiotics and more difficult to eradicate than their growing counterparts. We aim to discover strategies to potentiate the activity of existing and new topoisomerase inhibitors against these gram-negative pathogens. We recently found that metabolic stimulation and loss of efflux pump action during topoisomerase inhibitor treatment reduce persistence and resistance in non-growing E. coli. To build upon these findings, we will execute the following aims: Aim 1: Discover metabolites that are abundant at infection sites that can modulate persistence and resistance development of E. coli and P. aeruginosa toward first-in-class non-fluoroquinolone topoisomerase inhibitors that are in clinical trials. Aim 2: Investigate the impact of metabolic stimulation and loss of efflux pump action on bacterial metabolism, DNA integrity, and viability during topoisomerase inhibitor treatment. Aim 3: Deduce the effects of metabolic stimulation and loss of efflux pump action during topoisomerase inhibitor treatment on the coordination of molecular events that are important for persister repair and resuscitation after topoisomerase treatment terminates. We envision that the successful completion of this project will expand our knowledge of persister survival strategies and vulnerabilities. This will enable us to develop methods to enhance the activities of existing antibiotics and preserve the efficacy of new drugs in development before their introduction to the clinic.