Phage-Antibiotic Cocktail Rescues Daptomycin and Phage Susceptibility against Daptomycin-Nonsusceptible Enterococcus faecium in a Simulated Endocardial Vegetation Ex Vivo Model

Abstract

Enterococcus faecium is a difficult-to-treat pathogen with emerging resistance to most clinically available antibiotics. Daptomycin (DAP) is the standard of care, but even high DAP doses (12 mg/kg body weight/day) failed to eradicate some vancomycin-resistant strains. Combination DAP-ceftaroline (CPT) may increase β-lactam affinity for target penicillin binding proteins (PBP); however, in a simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic (PK/PD) model, DAP-CPT did not achieve therapeutic efficacy against a DAP-nonsusceptible (DNS) vancomycin-resistant E. faecium (VRE) isolate. Phage-antibiotic combinations (PAC) have been proposed for resistant high-inoculum infections. We aimed to identify PAC with maximum bactericidal activity and prevention/reversal of phage and antibiotic resistance in an SEV PK/PD model against DNS isolate R497. Phage-antibiotic synergy (PAS) was evaluated with modified checkerboard MIC and 24-h time-kill analyses (TKA). Human-simulated antibiotic doses of DAP and CPT with phages NV-497 and NV-503-01 were then evaluated in 96-h SEV PK/PD models against R497. Synergistic and bactericidal activity was identified with the PAC of DAP-CPT combined with phage cocktail NV-497-NV-503-01, demonstrating a significant reduction in viability down to 3-log₁₀ CFU/g (-Δ, 5.77-log₁₀ CFU/g; P < 0.001). This combination also demonstrated isolate resensitization to DAP. Evaluation of phage resistance post-SEV demonstrated prevention of phage resistance for PACs containing DAP-CPT. Our results provide novel data highlighting bactericidal and synergistic activity of PAC against a DNS E. faecium isolate in a high-inoculum ex vivo SEV PK/PD model with subsequent DAP resensitization and prevention of phage resistance. IMPORTANCE Our study supports the additional benefit of standard-of-care antibiotics combined with a phage cocktail compared to antibiotic alone against a daptomycin-nonsusceptible (DNS) E. faecium isolate in a high-inoculum simulated endocardial vegetation ex vivo PK/PD model. E. faecium is a leading cause of hospital-acquired infections and is associated with significant morbidity and mortality. Daptomycin is considered the first-line therapy for vancomycin-resistant E. faecium (VRE), but the highest published doses have failed to eradicate some VRE isolates. The addition of a β-lactam to daptomycin may result in synergistic activity, but previous in vitro data demonstrate that daptomycin plus ceftaroline failed to eradicate a VRE isolate. Phage therapy as an adjunct to antibiotic therapy has been proposed as a salvage therapy for high-inoculum infections; however, pragmatic clinical comparison trials for endocarditis are lacking and difficult to design, reinforcing the timeliness of such analysis.

Keywords: Enterococcus; antibiotic resistance; bacteriophage; infective endocarditis.

FIG 1

(A to C) Modified checkerboard MIC assay of (A) daptomycin, (B) ceftaroline, and (C) daptomycin plus ceftaroline combined with single phages NV-497 (A1-C1) and NV-503-01 (A2-C2) or two-phage cocktail NV-497 plus NV-503-01 (A3-C3) against daptomycin-nonsusceptible E. faecium. Phage was added to each non-GC and non-MC well in panels C1 to C3 at a phage MOI of 0.1. Squares within the yellow outline (C1 and C3) demonstrated synergy with a fractional inhibitory concentration (FIC) of 0.5. Squares within the orange outline demonstrated additivity with an FIC of 1. Percent growth was compared to the control via spectrophotometric analysis. GC, growth control; MC, medium control; MOI, multiplicity of infection; DAP, daptomycin; CPT, ceftaroline.

FIG 2

Bacterial quantification in 24-h time-kill experiments of daptomycin (0.5× MIC), ceftaroline (peak total concentration, 21.3), or daptomycin plus ceftaroline combined with phages NV-497 and NV-503-01 (each at a theoretical multiplicity of infection [MOI] of 0.1) against daptomycin nonsusceptible E. faecium R497. The error bars indicate standard deviation. P values were determined using a one-way ANOVA and Tukey’s post hoc test. **, P < 0.05; ***, P < 0.001. GC, growth control; DAP, daptomycin; CPT, ceftaroline.

FIG 3

Efficacy of bacteriophage-antibiotic combinations in a simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic (PK/PD) model (bacterial quantification) over a 96-h period. The error bars indicate standard deviation. GC, growth control; DAP, daptomycin; CPT, ceftaroline.

FIG 4

Final resistance in E. faecium R497 sensitive to each phage. Numbers are the PFU per milliliter for each combination of phage and bacterial isolate. Green, orange, and red cells indicate apparent, faint, and no bacterial lawn clearing, respectively. The lack of observed defined plaques is indicated by a titer of zero. S, sensitive; R, resistant; DAP, daptomycin; CPT, ceftaroline.

FIG 5

Postmodel sensitivity of bacteriophage not used in the SEV ex vivo models for daptomycin-nonsusceptible E. faecium isolate R497. Phage sensitivity was classified as high, medium, or low based on PFU counts as follows: >10⁷ PFU/mL was defined as high (green), ≥10³ to ≤10⁷ PFU/mL was defined as median (orange), and <10³ was defined as low (yellow). Phage was classified as nonsusceptible (NS) if no PFU were identified (red). SEV, simulated endocardial vegetation.