Project Summary/Abstract Bloodstream infection (BSI) due to methicillin-resistant Staphylococcus aureus (MRSA) carries ~20% mortality [1, 2]. MSRA displays tolerance to antibiotic killing [11], has a propensity to cause persistent BSI (pBSI) [3], and the duration of pBSI predicts mortality [2, 12-14]. MRSA rarely acquires frank antibiotic resistance during pBSI [3], highlighting tolerance as an important cause of poor patient outcomes. Antibiotic tolerance is a complex trait, which is distinct from resistance, and there are significant barriers to its study in vivo that have hampered progress on understanding the most important mechanisms in clinical settings. In this proposal, we advance an innovative genetic screening approach to overcome these barriers. Episodes of MRSA-pBSI that occur in different patients can be viewed as biological replicates of a naturally occurring experiment in microbial evolution. As bacterial population sizes collapse due to selection from antibiotic and immune pressure, tolerant mutants will become enriched. Mutations that arise independently in the same genetic loci at a rate that exceed chance alone, are biologically meaningful. In preliminary studies, using this “genotype-first” approach, we found evidence for in-host evolution of two genetic pathways strongly linked to antibiotic tolerance. Our central hypothesis is that mutants that arise during the treatment of MRSA-pBSI contain genetic adaptations for antibiotic and immune tolerance. We propose to identify and characterize these pathways through the following specific aims: Aim 1. Determine which genes evolving during MRSA-pBSI are associated with antibiotic tolerance and energy imbalance. Tolerance mechanisms often involve perturbations in metabolism, causing a ‘low energy’ state that leads to slow turnover of antibiotic targets [4, 5]. Such perturbations could arise through a variety of redundant pathways that converge on energy dysregulation. Alternatively, in vivo conditions may stress specific nodes in the cell’s metabolic networks and some pathways may dominate the antibiotic tolerance landscape. We will utilize our genetic screening approach to identify antibiotic tolerant mutants and determine which genes evolving during MRSA-pBSI are associated with antibiotic tolerance and energy imbalance. Aim 2. Determine if TCA cycle defects evolve during MRSA-pBSI due to a host-pathogen-drug interaction. Antibiotic tolerance can be induced by harsh environments and a leading model is that host immune pressure in the form of phagocyte-derived reactive oxygen species induces S. aureus into a drug-tolerant state by reducing flux through the tricarboxylic acid (TCA) cycle [6]. In our preliminary data, we identified TCA cycle mutants that evolved during MRSA-pBSI. If these mutants evolved by outcompeting wild-type MRSA in phagosomes, they will display a fitness advantage in this setting. We will utilize these mutants to test this model directly, by performing experime...