614. Metabolic Interventions for the Resensitization of Daptomycin-Resistant (DAP-R) Streptococcus mitis-oralis Strains to DAP In vitro and Ex Vivo in a Simulated Model of Experimental Endocarditis (IE)

Abstract

Background Streptococcus mitis-oralis is a leading cause of IE. Treatment of this pathogen is limited by frequent high-level β-lactam resistance and the propensity to develop high-level DAP-R during DAP exposure. The current study elucidated key metabolic perturbations associated with high-level DAP-R in prototype S. mitis-oralis strain 351, following in vitro selection of DAP-R by 10-day serial passage in sub-inhibitory DAP. Furthermore, to test translatability of such metabolic changes (see below), the synergistic activity of combinations of DAP plus a strategic metabolic inhibitor (i.e., fosfomycin) vs. DAP or fosfomycin alone was assessed, using DAP-R S. mitis-oralis strain 351-D10 (MIC >256 µg/mL) in vitro and in an ex vivo IE model.MethodsMICs. E test Growth Curve: Optical density (OD600) determined spectrophotometrically at 0–8 hours glyceraldehyde-3-phosphatedehydrogenase (GAPDH) activity. Kit from BioVision®. Metabolomics: one-dimensional 1H NMR-MS and two-dimensional 1H-13C HSQC in vitro time-kill assay: Using sub-MIC/MIC drug concentrations (initial inoculum ~1 × 105 CFU/mL) for 0, 2, 4, 6, and 8 hours. Ex vivo IE model: Simulated endocardial vegetations (SEVs) quantitatively cultured at 0, 4, 8, 24, 32, 48, and 72 hours with DAP or fosfomycin alone or in combination.ResultsNMR metabolomics analysis identified a number of metabolite differences in the 351 D10 DAP-R vs. 351 DAP-S strain (Figure 1). These data are consistent with a significant reduction in GAPDH activity (a glycolytic enzyme) in 351-D10 vs. 351 strain. Based on these metabolic changes, fosfomycin (a phosphoenolpyruvate analog) was chosen as a strategic metabolic inhibitor to attempt to “resensitize” our DAP-R S. mitis-oralis strain to DAP. The combination of DAP + fosfomycin demonstrated synergistic killing of the DAP-R strain vs. DAP or fosfomycin alone in the in vitro time-killing assays. Moreover, the DAP-R strain was synergistically cleared from SEVs by DAP + fosfomycin in the ex vivo IE model.ConclusionTaken together, these data indicate there are unique metabolome signatures associated with the DAP-R phenotype in S. mitis-oralis. In addition, these data provide support for further studying the use of strategic S. mitis-oralis metabolic inhibitors in additional strain-sets to resensitize DAP-R strains to DAP, using in vitro, ex vivo and in vivo models. Disclosures All authors: No reported disclosures.