Infections caused by vancomycin-resistant Enterococcus faecium (VREfm) are plagued by limited treatment options and are associated with increased mortality compared to vancomycin-susceptible strains. Daptomycin (DAP), a lipopeptide antibiotic with activity contingent upon an optimized area under the concentration curve (AUC) over the minimum inhibitory concentration (MIC) of the VREfm strain, possesses potent activity against VREfm. However, resistance to DAP occurs clinically, and data demonstrate that doses above the currently approved 6 mg/kg/day are necessary to prevent DAP resistance emergence. Even elevated DAP doses may not be sufficient to prevent resistance, as VREfm with mutations in two different regulatory gene systems (liaFSR and yycFG) demonstrate the ability to resist treatment with DAP alone. These mutations are frequent among VREfm with DAP MICs in the higher levels of susceptible (3-4 g/ml). As such, novel therapeutic regimens involving combinations are necessary and warrant study. Beta-lactams are of interest in combination with DAP; as limited in vitro data have demonstrated the ability of beta-lactams to enhance DAP activity against VREfm. The overall objective of our study is to define the DAP dose exposure breakpoint (pharmacokinetic/pharmacodynamic [PK/PD] breakpoint) with DAP regimens alone against VREfm with known genetic changes giving them proclivity for DAP resistance and then evaluate the ability of beta-lactams to positively affect that breakpoint. These data will provide important information on the optimal DAP exposure (dosing regimens) in combination with beta-lactams to prevent DAP resistance and provide bactericidal activity. The long-term goal is to optimize VREfm infection patient outcomes and preserve DAP as a viable agent against these resistant pathogens while determining the optimal beta-lactam to use in combination for DAP resistance prevention when the DAP MIC is elevated. The central hypothesis is that beta-lactams will increase the DAP AUC0-24/MIC ratio by lowering the VREfm DAP MIC and thus provide improved resistance prevention and bactericidal activity. The rationale behind the proposed research is that data on the DAP dose relationship with VREfm and how it is affected by beta-lactam co-therapy will lead to optimal treatment regimens for patients with these insidious infections. The central hypothesis will be tested by pursuing three Specific Aims: 1) Determine if an AUC24h/MIC breakpoint is achievable at clinical dosages to overcome DAP resistance in VREfm with predisposition for DAP resistance in an in vitro PK/PD model, 2) Evaluate several key beta-lactams to optimize DAP AUC24h/MIC exposure and restore DAP activity against VREfm with predisposition for DAP resistance in an in vitro PK/PD model, and 3) test the in vitro derived DAP AUC24h/MIC exposures alone and in combination with a beta-lactam for VRE-faecium in a well-established animal endocarditis model to validate these parameters. Under the first two aims, a well-established in vitro model of simulated endocardial vegetations (SEVs) (previously validated against a rabbit endocarditis model) will be used to determine breakpoints (and corresponding DAP doses) using various clinical doses of DAP and beta-lactams. The proposed research is innovative because we will use both in vitro and in vivo PK/PD models to determine optimal beta-lactam plus DAP combination regimens against VREfm that have yet to be studied in-depth with complex modeling such as this. The research is significant because it is expected to provide knowledge integral to understanding optimal DAP dosing strategies and the best beta-lactam for synergy against VREfm with proclivity for DAP resistance. Once such knowledge is available, improved patient outcomes and the preservation of DAP as a viable therapeutic option in the treatment of VREfm infections will be the ultimate result.