Ability of Bicarbonate Supplementation To Sensitize Selected Methicillin-Resistant Staphylococcus aureus Strains to β-Lactam Antibiotics in an Ex Vivo Simulated Endocardial Vegetation Model

Abstract

Supplementation of standard growth media (cation-adjusted Mueller-Hinton Broth [CAMHB]) with bicarbonate (NaHCO3) increases β-lactam susceptibility of selected methicillin-resistant Staphylococcus aureus (MRSA) strains (“NaHCO3 responsive”). This “sensitization” phenomenon translated to enhanced β-lactam efficacy in a rabbit model of endocarditis. The present study evaluated NaHCO3-mediated β-lactam MRSA sensitization using an ex vivo pharmacodynamic model, featuring simulated endocardial vegetations (SEVs), to more closely mimic the host microenvironment. Four previously described MRSA strains were used: two each exhibiting in vitro NaHCO3-responsive or NaHCO3-nonresponsive phenotypes. Cefazolin (CFZ) and oxacillin (OXA) were evaluated in CAMHB with or without NaHCO3. Intra-SEV MRSA killing was determined over 72-h exposures. In both “responsive” strains, supplementation with 25 mM or 44 mM NaHCO3 significantly reduced β-lactam MICs to below the OXA susceptibility breakpoint (≤4 mg/liter) and resulted in bactericidal activity (≥3-log killing) in the model for both OXA and CFZ. In contrast, neither in vitro-defined nonresponsive MRSA strain showed significant sensitization in the SEV model to either β-lactam. At both NaHCO3 concentrations, the fractional time above MIC was >50% for both CFZ and OXA in the responsive MRSA strains. Also, in media containing RPMI plus 10% Luria-Bertani broth (proposed as a more host-mimicking microenvironment and containing 25 mM NaHCO3), both CFZ and OXA exhibited enhanced bactericidal activity against NaHCO3-responsive strains in the SEV model. Neither CFZ nor OXA exposures selected for emergence of high-level β-lactam-resistant mutants within SEVs. Thus, in this ex vivo model of endocarditis, in the presence of NaHCO3 supplementation, both CFZ and OXA are highly active against MRSA strains that demonstrate similar enhanced susceptibility in NaHCO3-supplemented media in vitro.

Impact of Bicarbonate on PBP2a Production, Maturation, and Functionality in Methicillin-Resistant Staphylococcus aureus

“…We recently discovered that the addition of NaHCO 3 , the body’s primary biological buffer, to CA-MHB rendered a relatively large proportion of MRSA strains susceptible to two standard β-lactams, oxacillin and cefazolin, by MIC testing; this phenotype has been termed NaHCO 3 responsiveness ( 11 , 12 ). The translational relevance of this NaHCO 3 -responsive phenotype was verified in a small strain set by successful β-lactam therapy in an ex vivo simulated endocarditis vegetation model as well as in a rabbit model of infective endocarditis ( 11 , 13 ).…”

Reliability of mecA in Predicting Phenotypic Susceptibilities of Coagulase-Negative Staphylococci and Staphylococcus aureus

“…Additionally, we no longer have the isolates and therefore are unable to test isolates for the presence of mecA in co-pathogens or inducible resistance/empty cassettes in CoNS. If it is possible to investigate this discrepancy in the future with existing isolates, efforts will be made to explore the influence of bicarbonate on resistance expression [ 17 , 18 ], as well as performing inducibility studies. Also, our center uses Biofire Film Array for detection of the mecA gene; other platforms may perform differently, and BCID2 (recently Food and Drug Administration–approved version of the Biofire test) may not report mecA for CONS.…”

A Combined Phenotypic-Genotypic Predictive Algorithm for In Vitro Detection of Bicarbonate: β-Lactam Sensitization among Methicillin-Resistant Staphylococcus aureus (MRSA)

“…Although 44 mM of NaHCO 3 is above standard human blood levels, it reflects tissue levels [ 46 ]. In addition, MIC determinations among MRSA using this latter NaHCO 3 level (vs. 25 mM) correlates better with ex vivo and in vivo infection model outcomes [ 23 , 27 ]. For MIC testing, isolates were grown overnight, then diluted into the specific AST testing media-of-interest, as previously described [ 23 ]).…”

“…This ex vivo model was performed as described previously [ 27 , 52 ], to attempt to recapitulate the in vitro designations of NaHCO 3 -responsive vs. nonresponsive phenotypes (as predicted using the three-metric algorithm of AMC susceptibility, plus mecA and spa genotyping). Three isolates fulfilling all three metrics were randomly selected and compared ex vivo to three isolates not fulfilling these three metrics.…”

“…Results of such studies suggest that it could be possible that “antibiotic resistance levels by MICs” determined in host-like media are better predictors of host infection outcomes in both murine and rabbit systemic infection models [ 8 , 9 , 23 , 24 , 25 ]. Our laboratory has recently published a series of investigations documenting the following in terms of MIC testing of MRSA in NaHCO 3 -supplemented MHB: (i) ~33% and ~75% frequency of in vitro susceptibility of MRSA to OXA and CFZ, respectively; (ii) better clearance of in vitro NaHCO 3 -responsive MRSA from experimental cardiac vegetations in both ex vivo and in vivo models; and (iii) definition of important mechanisms involved in this NaHCO 3 -responsive phenotype (e.g., penicillin-binding protein 2a (PBP2a) production, translocation and maturation) [ 23 , 26 , 27 , 28 ].…”