Isolation, in vitro characterization and efficacy assessment in Galleria mellonella larvae of four bacteriophages targeting Aeromonas salmonicida

The Gram-negative bacteria Aeromonas (A.) salmonicida 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 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. The aims of this work were to isolate new phages active against A. salmonicida, characterize them in vitro and assess their potential use for phage therapy in a preliminary in vivo model. Four new phages were isolated from water samples collected in fish farms and natural aquatic environments in southern Belgium. Transmission electron microscopy allowed to classify these four phages in the Caudoviricetes class which includes tailed-phages with dsDNA and an icosahedral capsid. Genomic analysis showed that three of these phages, named vB_AsaM_ULASA2 (170,823bp), vB_AsaM_ULASA3 (164,381bp) and vB_AsaM_ULASA4 (171,205bp), belong to the Straboviridae family while vB_AsaM_ULASA1 (47,813bp) stay in the unclassified part of the Caudoviricetes class. Four strains of A. salmonicida were tested for virulence on Galleria mellonella larvae and two of them, named ATCC7965 and Asa-CER1, were selected for further experiments. Inoculation doses were determined as 102 CFU/10 µl and 104 CFU/10 µl, respectively. The safety and efficacy of the four phages at MOI 10 and 100 in this model are currently assessed.