Summary The longterm goals of the project are to identify the full array of efflux pumps of Staphylococcus aureus and Enterococcus spp that contribute to multiple antimicrobial resistance and to elucidate the determinants of their expression, their role in microbial physiology and their effect on bacterial response to antimicrobials in infection. The work will focus on a structural and functional analysis of the Tet38 efflux pump that enables multidrug resistance, survival in abscesses, and invasion of epithelial cells, the potential contribution of multiple pumps to persister subpopulations in the presence of antimicrobials, expanding the understanding of efflux resistance in enterococci, and evaluating collaboratively novel novel antimicrobial compounds for effects of efflux, tissue tolerability, and in vivo efficacy. There are four specific aims: 1) assess the structural determinants of the S. aureus Tet38 efflux pump in resistance, physiology, and pathogenesis and determine the mechanism of its diverse effects on cellular physiology; 2) explore the conditions promoting and heterogeneity of S. aureus efflux pump expression in single-cell groups; 3) evaluate the role of effux pumps in resistance in enterococci; and 4) evaluate the effect of S. aureus efflux pumps on novel compounds from collaborating projects. The work will utilize genetic manipulation, allelic exchange, and expression vectors in S. aureus and E. faecalis, measurements of gene/protein expression with fluorescent reporters, and established murine models of infection (subcutaneous abscess, intestinal colonization) utilizing a genomically defined strains. The overall goal of the program project is to take a well‐integrated, multi‐disciplinary approach to understanding antibiotic resistance development and efflux within cellular physiology combined with the search for compounds that compromise resistant pathogens by inhibiting novel targets and pathways. This project will add to understanding of resistance mechanisms related to multidrug efflux pumps and provide strains for testing the effect of such pumps on novel compounds active against new targets and pathways. It will also utilize a mammalian model of infection to screen new compound tolerability and in vivo activity.