The Combination of Phage Therapy and β-Lactam Antibiotics for the Effective Treatment of Enterococcus faecalis Infections

Abstract

A phage-antibiotic synergy could be an alternative in urinary tract infection (UTI) therapy, as it leads to the elimination of bacteria and to the reduction in variants resistant to phages and antibiotics. The aims of the in vitro study were to determine whether phages vB_Efa29212_2e and vB_Efa29212_3e interact synergistically with selected antibiotics in the treatment of E. faecalis infections, to optimize antibiotic concentrations and phage titers for the most effective combinations, and to assess their impact on the number of spontaneous resistant variants and on the phages' reproductive cycles. The modified double-layer disc diffusion method, checkboard, time-kill assays, one-step growth method and the double agar overlay plaque assay were implemented. Synergistic interactions were most often observed after the combined action of phages 2e or 3e and β-lactam antibiotics on E. faecalis strains. The beneficial effects depended on the bacterial strain, phage and antibiotic used. The lowest minimum inhibitory concentration (MIC₅₀) values of the antibiotics were recorded, after the application of low titers of phage 2e, and high titers of phage 3e. The combined use of the tested agents resulted in a significant reduction in the number of resistant variants and had an impact on the reproductive cycle of the tested phages, e.g., a 50% increase in burst size, and a 5 min reduction in the latency period of 2e were observed. The study confirmed beneficial interactions between phages and β-lactam antibiotics against E. faecalis growth.

Keywords: Enterococcus faecalis; phage–antibiotic synergy; urinary tract infections; β-lactam antibiotics.

Figure 1

Disk diffusion method in the phage and antibiotic action against E. faecalis strains. (a) The differences obtained in clear zone diameters (in mm) after the application of antibiotics alone and antibiotics with phages. Green marked fields indicate the possibility of synergy, red marked fields indicate the possibility of antagonism. (b) The sample visualization of the morphology of phage plaques in the antibiotic-active zone and in the antibiotic-free zone. The white arrows indicate a typical plaque of the phage strain; the black arrows indicate plaques of increased size. (c) Visualization of the occurrence of PAS effect (increased inhibition zones after phage implementation). AMP or AM—ampicillin, AMC—amoxicillin/clavulanic acid, VA—vancomycin, CIP—ciprofloxacin, NOR—norfloxacin, TEC—teicoplanin, IMP—imipenem.

Figure 2

Heatmaps of checkerboard assay (after 24 h incubation) represent the mean E. faecalis strain growth inhibition percentage of each treatment. Fields marked with a black frame indicate MIC₅₀ values. AMP—ampicillin, AMC—amoxicillin/clavulanic acid, IMP—imipenem.

Figure 3

Time–kill assay of phage and antibiotic used individually and in combination against E. faecalis strains over 24 h. Phages vB_Efa29212_2e and vB_Efa29212_3e (multiplicity of infection, MOI 0.1), AMP (1/2 MIC), and their combination were used. E. faecalis strains: C13 (a), C27 (b), and ATCC (c,d) were tested. Statistical analysis: two-way ANOVA followed by Tukey’s post hoc test to identify statistically significant differences was performed.

Figure 4

The number of spontaneous resistant variants of E. faecalis strains observed after treatment with phage and antibiotics. Phages vB_Efa29212_2e or vB_Efa29212_3e and antibiotics (AMP, AMC, IMP) were used individually and in combination. E. faecalis strains: C13, C27 and ATCC were tested. ANOVA II test, the Tukey post hoc test, was used for statistical analysis. Brackets indicate differences between mean values, with statistical significance defined as p ≤ 0.01.

Figure 5

One-step growth curves of the phages vB_Efa29212_2e and vB_Efa29212_3e on a culture of E. faecalis strains: ATCC (a,c) and C27 (b), MOI 0.1 in BHI and BHI supplemented with 1/2MIC of AMP and AMC. (d) Biological properties of the phages (2e and 3e) propagated with or without sublethal doses of AMP or AMC: burst size, latency period, calculated on the basis of the one-step growth curves and the values of the adsorption constant, k. The nonparametric Mann–Whitney U test was used to compare the mean values of k with those obtained in controlled conditions (without an antibiotic), * p ≤ 0.05.