PROJECT SUMMARY / ABSTRACT Osteomyelitis is an invasive infection of bone most commonly caused by the bacterial pathogen Staphylococcus aureus. Successful treatment of osteomyelitis requires prolonged antibiotic treatment as well as invasive surgical procedures to remove infected and nonviable bone. Despite these aggressive measures, nearly 1 in 5 patients with osteomyelitis will fail treatment. This includes patients who receive antibiotics with proven in vitro activity against the causative pathogen. These observations position osteomyelitis as a paradigm for treatment-recalcitrant infection. The goal of this proposal is to define bacterial and host factors that contribute to antibiotic failure during osteomyelitis. We hypothesize that bacterial virulence factors and host responses that promote physical shielding of bacteria in vivo contribute to antibiotic tolerance. Upon successful completion of the proposed experiments, we expect to have defined targetable mechanisms underlying antibiotic failure during osteomyelitis, with the ultimate goal of maximizing treatment efficacy, minimizing complications, and creating new approaches to detect and combat antibiotic tolerance in humans. In preliminary studies using our established murine model of osteomyelitis, we demonstrate that S. aureus rapidly develops tolerance to antibiotic killing in vivo. Histologic analyses reveal a characteristic “pseudocapsule” surrounding bacterial communities in infected bone. Pseudocapsule formation is postulated to involve key virulence factors known as coagulases and agglutinins, which together endow S. aureus with its distinctive ability to coagulate blood. We hypothesize that these virulence factors contribute to antibiotic tolerance during osteomyelitis by physically shielding bacteria within host tissues. Aim 1 will rigorously test this hypothesis, while also identifying novel determinants of antibiotic tolerance using an unbiased, in vivo bacterial screen. We will track the relative localization of antibiotics and bacteria using innovative new S. aureus reporter strains coupled with fluorescent antibiotics. Aim 2 will test the complementary hypothesis that canonical host coagulation contributes to shielding of bacteria and antibiotic tolerance. This Aim will also employ our new workflow for imaging mass spectrometry (IMS) of bone, which will enable discovery-based profiling of host analytes that form antibiotic barriers in vivo. We will also leverage IMS as a modality for label-free tracking of antibiotics in infected tissues. Aim 3 will use paired bacterial isolates and serum samples from patients with osteomyelitis to test how growth in a physiologically relevant medium alters antibiotic killing, and to link in vitro antibiotic tolerance to key clinical parameters. Collectively, the experiments in this proposal will define mechanisms leading to antibiotic tolerance during osteomyelitis, paving the way for new interventions that improve antibiotic therapy, l...