An OmpC- and O-antigen-targeting phage cocktail effectively eliminates E. coli O157:H7 from food via cooperative interactions

Escherichia coli O157:H7 is a major foodborne pathogen associated with severe illnesses, including hemolytic uremic syndrome. Although phage cocktails are promising biocontrol agents for eliminating E. coli O157:H7 in food, the understanding of cooperative interactions between phages is still in its infancy. In this study, two newly isolated E. coli O157:H7 phages, Jhoon1 and Jhoon3, were characterized. Jhoon1 is a T4-like (79.92 % sequence identity) Mosigvirus recognizing OmpC, while Jhoon3 is a CBA120-like (97.48 % identity) Kuttervirus cleaving the O-antigen. The combined phage cocktail exhibited strong growth-inhibitory activity against E. coli O157:H7 in broth and significantly delayed the emergence of bacteriophage-insensitive mutants compared to single-phage treatment. For both Jhoon3-sensitive (ATCC 43890) and -resistant (OE50) strains, the phage cocktail showed comparable antibacterial efficacy 48 h post-infection, reducing bacterial counts by 8.89–9.15 log CFU/mL in milk and 4.15–6.24 log CFU/cm² on lettuce at 25 °C. In beef stored at 4 °C, a greater reduction was observed for OE50 (2.03 log CFU/cm²) compared to ATCC 43890 (0.99 log CFU/cm²), further demonstrating the effectiveness of the phage cocktail. To investigate this synergistic antimicrobial effect, the recombinant tail spike protein of Jhoon3 (JH3 TSP) was expressed. The JH3 TSP exhibited O-antigen depolymerase activity, facilitating Jhoon1 adsorption and inhibiting the growth of E. coli O157:H7 in a dose-dependent manner. These results demonstrate that a phage cocktail targeting both OmpC and O-antigen enhances antibacterial efficacy through receptor cleavage-mediated synergy, offering novel insights into the strategic design of phage cocktails for food safety applications.