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

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 identified various distinct vibriophages, as evidenced by host range assays and restriction fragment length polymorphism analysis. Since we aimed to enhance the efficiency of our previously developed vibriophage cocktail, Eric and Ariel (EA), which has been demonstrated to be effective against VP_AHPND, each selected phage was combined with the EA cocktail to evaluate the efficiency of formulated cocktails against VP_AHPND isolates. The result revealed that supplementation of the EA cocktail with the phage PhiPS02 yielded outstanding outcomes, particularly against the VP_AHPND strain KT1001. The enhanced efficacy of this formulated cocktail was further validated in vivo, where it rescued infected shrimp through a significant reduction 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 VP_AHPND in aquaculture.

Since the resurgence of phage therapy as a strategy to combat antibiotic-resistant bacterial infections, the use of combined virulent phages has shown promising therapeutic potential. However, naturally occurring phages are predominantly temperate, restricting their use as therapeutic agents due to concerns regarding their ability in lysogenic conversion. Here, we demonstrate that temperate phages can confer a therapeutic potential by inducing lysogenized bacteria to produce small membrane vesicles (MVs) that synergize with virulent phages in combination to suppress bacterial growth. Since MVs originate from the bacterial outer membrane, they not only retain phage receptors but also carry phage-derived biomolecules influenced by resident prophage. Potentially, they can mediate receptor transfer to phage-resistant strains via cell-MV fusion and improve phage infectivity through the combined action of phages and prophage-derived enzymes. Our findings provide insights into the role of lysogen-derived MVs and offer strategy into a novel strategy for harnessing temperate phages in therapeutic applications.