Ability of Bicarbonate Supplementation to Sensitize Selected Methicillin-ResistantStaphylococcus aureus(MRSA) Strains to β-Lactam Antibiotics in anEx VivoSimulated Endocardial Vegetation Model

Abstract

Word Count: 3963 25 2 ABSTRACT 26 Supplementation of standard growth media (cation-adjusted Mueller-Hinton Broth 27[CAMHB]) with bicarbonate (NaHCO 3 ) significantly increases β-lactam susceptibility of selected 28 MRSA strains ("NaHCO 3 -responsive"). This "sensitization" phenomenon translated to enhanced 29 β-lactam efficacy in a rabbit model of endocarditis. The present study evaluated NaHCO 3 -30 mediated β-lactam MRSA sensitization using an ex vivo pharmacodynamic model, featuring 31 simulated endocardial vegetations (SEVs), to more closely mimic the host microenvironment. 32Four previously described MRSA strains were used: two each exhibiting in vitro "NaHCO 3 -33 responsive" or "NaHCO 3 -nonresponsive" phenotypes. Cefazolin (CFZ) and oxacillin (OXA) were 34 evaluated in CAMHB±NaHCO 3 . Intra-SEV MRSA killing was determined over 72 hr exposure. In 35 both NaHCO 3 -responsive strains, supplementation with 25 mM or 44 mM NaHCO 3 significantly 36 reduced β-lactam MICs to below the OXA susceptibility breakpoint (≤ 4 mg/L) resulting in 37 bactericidal activity (≥ 3 log kill) in the model for both OXA and CFZ. In contrast, neither in vitro-38 defined NaHCO 3 -nonresponsive MRSA strains showed significant sensitization in the SEV 39 model to either β-lactam. At both NaHCO 3 concentrations, the fractional time-above-MIC was 40 >50% for both CFZ and OXA in the NaHCO 3 -responsive MRSA. Also, in RPMI+10% LB media 41 (proposed as a more host-mimicking microenvironment and containing 25 mM NaHCO 3 ), both 42 CFZ and OXA exhibited enhanced bactericidal activity against each NaHCO 3 -responsive strain 43 in the SEV model. Neither CFZ nor OXA exposures selected for high-level β-lactam-resistant 44 mutants within SEVs. Thus, in this ex vivo model of endocarditis, in the presence of NaHCO 3 45 supplementation, both CFZ and OXA are highly active against MRSA strains that demonstrate 46 similar enhanced susceptibility in NaHCO 3 -supplemented media in vitro. 47 3 INTRODUCTION 48

Antibiotics and Innate Immunity: A Cooperative Effort Towards the Successful Treatment of Infections

“…Additional studies have shown that buffering with bicarbonate, the major anionic buffer in mammalian physiology, is a major contributor to the superior predictability of physiological media in vitro antibiotic susceptibility to in vivo efficacy [ 11 ]. For example, some strains of methicillin-resistant Staphylococcus aureus (MRSA) demonstrate a methicillin-susceptible phenotype in bicarbonate-buffered media, and such strains can be effectively treated in animal models of endocarditis with beta-lactam monotherapy [ 11 , 13 ]. Azithromycin efficacy against multiple gram-negative bacterial species (eg, Acinetobacter baumannii , Klebsiella pneumoniae ) depends on bicarbonate, and this activity translates to in vivo efficacy in murine infection models [ 14 , 15 ].…”

Scope and Predictive Genetic/Phenotypic Signatures of Bicarbonate (NaHCO 3 ) Responsiveness and β-Lactam Sensitization in Methicillin-Resistant Staphylococcus aureus

“…Recently, we identified a novel MRSA AST phenotype, termed "NaHCO3-responsiveness", in which MRSA strains are highly susceptible in vitro to CFZ and OXA in media supplemented with NaHCO3 (7). Prototype MRSA strains with this in vitro phenotype were also highly susceptible to these same β-lactams in an ex vivo simulated endocarditis vegetation model, as well as in a rabbit model of infective endocarditis (7,10). In contrast, in vitro NaHCO3 non-responsive isolates were also resistant to CFZ and OXA under these same ex vivo and in vivo conditions.…”