Isolation and characterization of four new bacteriophages against Aeromonas salmonicida, the causative agent of furunculosis

Aeromonas (A.) salmonicida, a Gram-negative bacteria belonging to the Aeromonadaceae family, is a primary fish pathogen that causes furunculosis in salmonids, carp and perch, as well as septicemia in a variety of fish. This species is considered as one of the main bacterial pathogens responsible for important economic losses in aquaculture industry. Large amounts of antibiotics such as oxytetracycline, quinolones and sulfonamides are used to treat this infection, which highly contributes to the emergence of antibiotic-resistant strains. The application of bacteriophages (phages) in aquaculture seems to be a promising solution to control pathogenic bacteria in this field because these organisms are well adapted to aquatic environments. The aim of this work was to isolate and characterize new lytic phages against A. salmonicida. The enrichment method used to isolate phages consists in mixing a centrifuged and filtered water sample with a bacterial culture in exponential phase. When clarification of the medium was observed, the supernatant of this mixture was spread on the surface of LB agar and covered with a bacterial overlay in exponential phase. Phages present in distinct clear lysis plaques were then purified three times by subculturing. For this purpose, a sampling campaign of water from fish farming ponds and natural aquatic environments in the south of Belgium was carried out in early 2022. Out of 48 water samples, four new lytic phages were isolated. A preliminary host spectrum test showed that three of these four phages were active against other wild A. salmonicida strains while the fourth one showed a narrower host spectrum. These four phages were not active against any of the A. hydrophila strains tested. After having determined temperature and pH stabilities, adsorption times and kinetics of these four new phages, further studies are needed to analyse their genomes and to assess the in vivo safety and efficacy of these phages.