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. 2022 Mar 15;11(3):392.
doi: 10.3390/antibiotics11030392.

Evaluation of Bacteriophage-Antibiotic Combination Therapy for Biofilm-Embedded MDR Enterococcus faecium

Katherine Lev  1 , Ashlan J Kunz Coyne  1 , Razieh Kebriaei  1 , Taylor Morrisette  2 , Kyle Stamper  1 , Dana J Holger  1 , Gregory S Canfield  3   4 , Breck A Duerkop  3 , Cesar A Arias  5   6 , Michael J Rybak  1   7
Affiliations

Evaluation of Bacteriophage-Antibiotic Combination Therapy for Biofilm-Embedded MDR Enterococcus faecium

Katherine Lev et al. Antibiotics (Basel). .

Abstract

Multidrug-resistant (MDR) Enterococcus faecium is a challenging pathogen known to cause biofilm-mediated infections with limited effective therapeutic options. Lytic bacteriophages target, infect, and lyse specific bacterial cells and have anti-biofilm activity, making them a possible treatment option. Here, we examine two biofilm-producing clinical E. faecium strains, daptomycin (DAP)-resistant R497 and DAP-susceptible dose-dependent (SDD) HOU503, with initial susceptibility to E. faecium bacteriophage 113 (ATCC 19950-B1). An initial synergy screening was performed with modified checkerboard MIC assays developed by our laboratory to efficiently screen for antibiotic and phage synergy, including at very low phage multiplicity of infection (MOI). The data were compared by one-way ANOVA and Tukey (HSD) tests. In 24 h time kill analyses (TKA), combinations with phage-DAP-ampicillin (AMP), phage-DAP-ceftaroline (CPT), and phage-DAP-ertapenem (ERT) were synergistic and bactericidal compared to any single agent (ANOVA range of mean differences 3.34 to 3.84 log10 CFU/mL; p < 0.001). Furthermore, phage-DAP-AMP and phage-DAP-CPT prevented the emergence of DAP and phage resistance. With HOU503, the combination of phage-DAP-AMP showed the best killing effect, followed closely by phage-DAP-CPT; both showed bactericidal and synergistic effects compared to any single agent (ANOVA range of mean differences 3.99 to 4.08 log10 CFU/mL; p < 0.001).

Keywords: Enterococcus faecium; antimicrobial; bacteriophage; biofilm; frequency of resistance; nontraditional antibacterial; phage sensitivity; phage therapy; resistance management.

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Conflict of interest statement

K.L., A.J.K.C., R.K., T.M., K.C.S., D.J.H., G.S.C., C.A.A. and M.J.R. have nothing to declare. M.J.R. has received grant support from and has consulted or spoken on behalf of Allergan, Melinta, Merck, Paratek, Shionogi, Spero, and Tetraphase. C.A.A. has received grant support from Merck and MeMed Diagnostics, and is a co-founder of Entasis Therapeutics and shareholder in Ancilia Biosciences, C.

Figures

Figure 1
Figure 1
(A–D) Checkerboard analysis of E. faecium strain R497. Additivity, defined as an FIC index >0.5 but <4, is indicated by the red outline in (A). Synergy, defined as an FIC index ≤ 0.5, is indicated by the red outline in (B). Comparisons are versus growth control and depicted by the blue color gradient as percentage of growth.
Figure 2
Figure 2
(A–D) Checkerboard analysis of E. faecium strain HOU503. Additivity, defined as an FIC index > 0.5 but <4, is indicated by the red outline in Figure 1A,B. Comparisons are versus growth control and depicted by the blue color gradient as percentage of growth.
Figure 3
Figure 3
(A–F) Time-kill analysis of DAP-resistant and DAP-SDD E. faecium strains R497 and HOU503, respectively. GC, growth control. (A) R497 vs. DAP (0.5xMIC) + AMP (free peak concentration = 72) + phage (MOI = 1). (B) R497 vs. DAP (0.5xMIC) + CPT (free peak concentration = 13.2) + phage (MOI = 1) (C) R497 vs. DAP (0.5xMIC) + ERT (free peak concentration = 15.5) + phage (MOI = 1) (D) HOU503 vs. DAP (0.5xMIC) + AMP (free peak concentration = 72) + phage (MOI = 1) (E) HOU503 vs. DAP (0.5xMIC) + CPT (free peak concentration = 13.2) + phage (MOI = 1) (F) R497 vs. DAP (0.5xMIC) + ERT (free peak concentration = 15.5) + phage (MOI = 1). DAP, daptomycin; AMP, ampicillin; CPT, ceftaroline; ERT, ertapenem.
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