Characterization of novel Vibrio phages as potential biocontrol agents against Vibrio alginolyticus and insights into its phage-resistant mutant

Pathogenic bacteria are an ongoing threat to intensive agriculture, including aquaculture. With the emergence of antimicrobial resistance (AMR), novel non-traditional antimicrobials are urgently needed to minimize the dependence on antibiotics. Bacteriophage (phage) therapy, which uses naturally occurring viruses to kill specific bacterium, is gaining interest and offers huge potential for targeted pathogen control in aquaculture. However, many challenges regarding stability and emergence of phage-resistance must be overcome. Here, we isolated and characterized eight Vibrio phages against an emerging aquaculture pathogen Vibrio alginolyticus and studied their bactericidal and antibiofilm potency. We then used next-generation sequencing to understand how the Vibrio species may gain phage resistance. Our results indicated that most of the isolated Vibrio phages (seven out of eight) shared < 40 % genomic similarity with phages isolated elsewhere, possibly suggesting novel strains. The phages were stable in different temperatures (4-40 °C), pHs (3-10) and salinities (0-50 ppt) up to 6 h without significant loss in viability. Although individual phages had variable bactericidal efficiency and bacteria rapidly developed phage-resistance, phage cocktail formulations were highly efficient and significantly suppressed bacterial growth up to 15 h, inhibited biofilm formation (p < 0.05) and eradicated established biofilms (p < 0.05). Sequencing confirmed absence of lysogeny modules, known toxins and AMR genes in seven of the phages. Further, tRNAs and a putative anti-CRISPR (Acr) protein was found in two of the most efficient phages. Though bacteria rapidly developed phage-resistance, we observed increased antibiotic sensitivity as a trade-off which possibly resulted from defective efflux pump. Our findings support potential applications of Vibrio phages in aquaculture systems for minimizing the burden of Vibriosis. However, further research is required to elucidate the role of efflux pump system in phage-resistance and antimicrobial resistance.