Enhanced efficiency of virulent and temperate phage combination mediated through bacterial membrane vesicles

Abstract

Since multidrug-resistant Vibrio parahaemolyticus (VP), a deadly pathogenic bacterium responsible for acute hepatopancreatic necrosis disease (AHPND), which has increasingly emerged, bacteriophages have been focused on as an alternative therapy against drug-resistant bacterial infections. Here, we identifiedvarious distinct vibriophages, as evidenced by host range assays and restriction fragment length polymorphism analysis. Since we aimed to enhance the efficiencyof our previously developed vibriophage cocktail, Eric and Ariel (EA), which has been demonstrated to be effectiveagainst VPAHPND, each selected phage was combined with the EA cocktail to evaluate the efficiencyof formulated cocktails against VPAHPND isolates. The result revealed that supplementation of the EA cocktail with the phage PhiPS02 yielded outstanding outcomes, particularly against the VPAHPND strain KT1001. The enhanced efficacyof this formulated cocktail was further validated in vivo, where it rescued infected shrimp through a significantreduction of bacterial toxins, leading to approximately 75% survival. PhiPS02 is a novel temperate vibriophage and harbors a genome of 34,737 base pairs encoding genes involved in the lysogenic life cycle. Under physiological stresses that induced prophage activation, PhiPS02-lysogenized bacteria exhibited membrane blebbing and produced small membrane vesicles (MVs) with distinct biomolecular constituents. Interestingly, the combination of lysogen-derived MVs with the EA phage cocktail substantially enhanced bacterial suppression, compared to that of MVs from wild-type bacteria, suggesting a synergistic interaction between lysogen-derived MVs and phages. This study highlights the complex interplay among phages and their bacterial hosts, mediated through lysogen-derived MVs, and provides a novel strategy for the application of temperate phages in the management of VPAHPND in aquaculture.