From Isolation to Application: Designing a Multi-Target Phage Cocktail for Bivalve Safety

Narrow host specificity and bacterial resistance often limit single-phage treatments. Phage cocktails address these challenges by expanding the host range, reducing resistance, and enhancing bacterial inactivation. This study aimed to develop an optimised phage cocktail targeting Escherichia coli, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Aeromonas hydrophila, key pathogens in bivalve consumption. Twelve phages were isolated, purified, and screened for bacterial inactivation using resazurin-based viability assays. Host range analysis showed that all phages infected at least one additional bacterial species, with four (phEc4, phSE1, phAh2, phAh4) targeting three of the four bacteria. Cocktail formulation aimed to maximise bacterial reduction while balancing host range expansion with factors such as the risks of resistance development and inter-phage competition. Among the tested combinations, the most effective cocktail consisted of E. coli phage phEc3, S. Typhimurium phage phST1, S. Enteritidis phage phSE1, and A. hydrophila phage phAh2. Future studies should evaluate the cocktail's efficacy in vitro and assess both safety and performance in vivo in bivalve depuration systems.