<i>In vitro</i> evaluation of phage cocktail efficiency against multiple aquaculture bacterial pathogens

AIMS: This study aimed to evaluate whether combining phages with different properties enhances antibacterial efficacy against individual strains and enables the simultaneous control of multiple bacterial species belonging to different genera. METHODS AND RESULTS: Our phage collection was screened for phages active against Aeromonas hydrophila, Escherichia coli, Salmonella enterica Typhimurium and Vibrio parahaemolyticus. Four phages were selected: AH-1, ECA2, phSE-5 and vB_VpS_LMAVpSH, originally isolated using A. hydrophila, S. Typhimurium, E. coli and V. parahaemolyticus as hosts, respectively. An initial phage cocktail (CK3) was prepared excluding phage SH, which was later included in a modified cocktail (CK4). Phage efficacy was evaluated in vitro (in nutrient-rich media and synthetic seawater) against bacterial strains individually and against mixed cultures of the four bacteria, for 12 h at 25°C. Both cocktails were associated with greater bacterial reduction compared with individual phage treatments, alongside reduced bacterial regrowth under the tested conditions. Although efficacy decreased in nutrient-limited conditions, phages still inhibited bacterial growth. CONCLUSIONS: The findings of this study provide experimental evidence of phage-bacteria interactions in multispecies systems and offer proof-of-concept evidence supporting further evaluation of phage cocktail approaches under aquaculture-relevant conditions.