Synergistic antimicrobial efficacy of phage cocktails and essential oils against Escherichia coli

This study was designed to evaluate the combined antimicrobial activity of selected phage cocktail (MS2+T7 phages) and essential oils (cinnamon, clove, oregano, and thymol) against Escherichia coli ATCC 15597. To select most effective phages, the lytic abilities of individual phages (MS2, phiX174, and T7) and their phage combinations were assessed using the phage spot test and plaque assay at various multiplicity of infections (MOIs) ranging from 0.01 to 100. The inhibitory effectiveness of selected phage combinations (MS2+T7), treated with and without essential oils (cinnamon, clove, oregano, and thymol; 1/2 × MIC each), against E. coli hosts was evaluated at various MOIs of 0, 0.1, and 1. The antimicrobial effect of phage cocktail (MS2+T7) combined with cinnamon (1/2 × MIC) and thymol (1/2 × MIC) were evaluated using time-kill curve assay for 48 h at 37°C. The 48-h treated cells, including the control, cinnamon, thymol, phage cocktail, and cinnamon-phage cocktail, and thymol-phage cocktail, were further tested for relative fitness, and phage mutant frequency assays. The highest lytic activity of MS2 and T7 phages was observed against E. coli (>6 log reduction) at an MOI of 0.1. The phage combination (MS2+T7) was considerably reduced the number of E. coli by 7 logs (p < 0.05). The lytic activity of MS2+T7 was significantly enhanced in the presence of cinnamon, clove, oregano, and thymol (1/2 × MIC each) after 24-h incubation at 37°C. The combinations of cinnamon-phage cocktail and thymol-phage cocktail effectively reduced the numbers of E. coli by more than 5 log CFU/mL compared to the control after 48 h of incubation at 37°C. The lowest relative fitness (<0.5) and mutant frequency (<10 %) were observed for E. coli treated with the combinations. These findings suggest that combining phage cocktails with essential oils can serve as synergistic antimicrobial agents. Consequently, this study provides valuable insights for developing effective phage therapy strategies and ensuring the sustained effectiveness of phage treatments.