ABSTRACT Enterococcus faecium is a normal member of the healthy human microbiota; however, it has become a concerning opportunistic pathogen. E. faecium is resistant to many first-line antibiotics and most clinical isolates are also vancomycin-resistant (VREfm), leaving high-risk patients with few treatment options. As such, infections caused by VREfm are challenging to manage, and ~10% of patients with VREfm bloodstream infections will go on to develop recurrent disease. It is largely unknown how VREfm adapts within its host to evade eradication by standard antimicrobial therapies and cause recurrent infections, as few systematic genomic or phenotypic assessments have been performed in VREfm. The first two aims of this proposal plan to address this important knowledge gap by 1) comparative genomic analyses of VREfm bloodstream isolates collected from patients with recurrent infections to find genetic signatures associated with the potential to resist eradication and cause recurrence; and 2) determining the role of antibiotic tolerance in recurrent VREfm disease. Bacteriophage (phage) therapy has the potential to provide clinicians with an additional tool for treating challenging enterococcal infections, but transitioning phage therapy into clinical practice has been stymied by the limited generalizability of small pre-clinical studies and the lack of pharmacokinetic-guided dosing regimens. To address these limitations this proposal will 1) combine a broad-spectrum phage cocktail with VRE-targeting antibiotics against the cohort of recurrent VREfm isolates proposed above to assess for phage-antibiotic synergism and 2) develop a one-compartment pharmacokinetic model to determine optimal dosing strategies to inform future clinical use. This collection of VREfm isolates will provide a representative cross-section of strains likely to fail standard antibiotic therapies, and enable a thorough assessment of phage-antibiotic combinations as potential treatment options for complex VREfm infections. This K08 proposal is a five-year career development program for Madison Stellfox, MD, PhD that provides dedicated research training under the guidance of an expert mentoring team to enhance her existing laboratory and clinical background and provide additional instruction in the principles of comparative genomics, evolutionary biology, pharmacology, and phage therapy. Dr. Stellfox is currently an infectious diseases fellow and postdoctoral researcher in the laboratory of Dr. Van Tyne, and the resources and mentorship available at the University of Pittsburgh and in the Van Tyne laboratory provide an ideal educational environment. Through the successful execution of this training plan, Dr. Stellfox will gain the experience and guidance she needs to achieve her aspirations of becoming an independent physician-scientist with a research program focused on the in-host evolution of gram-positive bacteria during recurrent disease and the practical application of p...