ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) are a leading cause of invasive infections in both community-acquired and hospital-associated contexts. MRSA strains are intrinsically resistant by standard in vitro susceptibility testing to β-lactam antibiotics. In contrast, methicillin-susceptible S. aureus (MSSA) strains remain highly susceptible to many standard-of-care β-lactams (e.g. oxacillin; nafcillin; cefazolin). β-lactams are not recommended for treating MRSA infections: i) MRSA β-lactam MICs are above current CLSI “breakpoints”; ii) they bind relatively poorly to penicillin-binding protein (PBP) 2a (predominant PBP in MRSA strains responsible for cell wall synthesis and division); iii) β-lactam levels required to saturate PBP 2a exceed human serum levels achieved with standard clinical dose-regimens; and iv) treatment of experimental MRSA infections (e.g., endocarditis) with β-lactams are generally ineffective. Several labs recently showed that bicarbonate supplemention of standard MIC testing media can “sensitize” some (but not all) MRSA strains in vitro to β-lactams and host defense peptides (e.g., LL-37 from neutrophils; skin). Further, MRSA strains exhibiting a “bicarbonate- responsive” phenotype in vitro (i.e., β-lactam-resistant in standard media, but susceptible in bicarbonate-containing media) were effectively eradicated in murine bacteremia models with selected β-lactams. We amplified these observations using four prototype MRSA strains (LAC-USA-300; COL [USA 100] ; MW-2 [USA 400]; BMC1001 [USA 300] which demonstrated the following key outcomes: i) all strains were resistant in vitro in standard (MHB) to both oxacillin (OX) and cefazolin (CFZ); two strains exhibited a bicarbonate-responsive phenotype in bicarbonate- supplemented MHB, becoming highly susceptible to both β-lactams, while two did not; ii) two bicarbonate- responsive strains were heterotypic on population analyses, while the other two strains were homotypic (AUCs > 0.9); iii) both bicarbonate-responsive strains were effectively cleared from all target organs by both OX and CFZ in experimental endocarditis (IE), while two bicarbonate-nonresponsive strains were refractory to therapy; and iv) bicarbonate impacted both the mecA-pbp2a and sarA-sigB genetic pathways. The current proposal will investigate: i) the scope of the bicarbonate-responsive phenotype in vitro to β-lactams among a larger collection of clinical MRSA strains; ii) the overall large-scale translatability of such in vitro metrics to a relevant in vivo model of invasive MRSA infection (IE); and iii) the mechanism(s) underlying bicarbonate-responsiveness in MRSA. This proposal could lay the foundation for pivotal clinical trial(s) assessing predictability of modified in vitro testing of MRSA to β-lactams, utilizing bicarbonate supplementation of standard media. This research has the overarching potential to fundamentally transform current MRSA in vitro susceptibility testing methods for β-...