Summary The longterm goals of the project are to identify the full array of efflux pumps of Staphylococcus aureus 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 genetic analysis of regulatory elements and on bacterial fitness and response to antimicrobials in a subcutaneous abscess model, collaborating with other project groups to assess the efficacy of novel antimicrobial compounds in abscesses and the extent to which efflux pumps affect that efficacy. There are four specific aims: 1) analyze the global array of efflux pumps of S. aureus for their effects on susceptibility to established antimicrobials and novel compounds identified by P01 collaborators; 2) analyze the effects of physiologic pump overexpression in the abscess environment and on treatment response to antimicrobials with focus on the Tet38 pump and tetracycline treatment; 3) dissect the regulatory networks affecting resistance effux pump expression using the high‐efficiency multiplex libraries developed by the Walker lab; and 4) test novel compounds from P01 collaborators for efficacy in mammalian infection and biofilm models and assess the moonlighting model in the abscess model. The work will utilize genetic manipulation and allelic exchange in S. aureus, measurements of gene expression with RT‐PCR, and established murine models of infection (subcutaneous abscess, renal abscess, lethality) utilizing a genomically defined strains of methicillin‐resistant and other S. aureus. The overall goal of the program project is to take a well‐integrated, multi‐disciplinary approach to understanding antibiotic resistance development and transmission, and to integrate that effort with the search for compounds that compromise resistant pathogens, including methicillin‐resistant S. aureus (MRSA), 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 mammalian models of a common MRSA infection to test compound activity in vivo.