SUMMARY The long-term objective of this project is to understand how vancomycin-resistant enterococci (VRE) adapt during bloodstream infection (BSI) to better tolerate antibiotic and host immune defenses. Enterococci have evolved over hundreds of millions of years to colonize the gastrointestinal (GI) tract of animals, and they are well adapted to reside there. VRE causing BSI, however, face substantially different selective pressures, such as antibiotics in high concentrations, nutrient restriction, and host immune defenses. At our center over the past five years, patients with VRE-BSIs had a 30-day mortality rate of 36%, which was higher than BSIs due to all other ESKAPE pathogens. Additionally, VRE-BSIs are often difficult to treat, and nearly one third of patients with VRE- BSI experience either prolonged bacteremia (≥5 days), or recurrent infection within one year. Here we propose to study the population-level evolutionary dynamics of VRE sampled from the GI tract and blood of patients with VRE-BSI, and to characterize bacterial adaptations that promote VRE-BSI. Our central hypothesis is that VRE isolated from BSIs possess genetic adaptations that enable them to survive in the blood environment. In Aim 1, we will use bacterial population-level whole genome sequencing to identify genetic adaptations associated with VRE-BSI. We propose to collect matched samples from VRE GI tract surveillance specimens and VRE-BSI from approximately 150 patients, and to sequence them deeply to assess the diversity of the VRE population at each body site. We will also compare VRE-BSI populations collected over time from patients that have persistent or recurrent VRE-BSI. We will utilize comparative genomics and selection-based analyses to identify bacterial loci that are candidate targets for selection. In Aim 2, we will quantify the effect of mutations in VRE transcription and translation genes on antibiotic resistance and tolerance. We have already identified candidate adaptive mutations in RNA polymerase subunits, ribosomal proteins, a ribosome methyltransferase, and several transcriptional regulators. We will investigate: 1) The connection between antibiotic exposure and the occurrence of these mutations in the GI tract and blood of VRE-BSI patients, 2) The effects of these mutations on VRE transcription and translation, and 3) The contribution of these mutations to resistance and/or tolerance of antibiotics used to treat VRE-BSI. In Aim 3, we will determine whether mutations in the capsular polysaccharide (cps) and enterococcal polysaccharide antigen (epa) biosynthetic loci augment VRE growth and survival during BSI. We will investigate the impact of mutations that alter these cell surface-associated polysaccharides on VRE survival in whole human blood, in the presence of human neutrophils, as well as in a mouse model of VRE infection. Overall, this study has the potential to transform our understanding of how antibiotic-resistant bacteria adapt during hum...