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

Introduction

The Gram-negative bacterium Aeromonas (A.) salmonicida is a primary fish pathogen that causes furunculosis in salmonids as well as septicemia in a variety of fish. In one hand because this disease is responsible for significant losses in salmonid production worldwide and in the other hand because of the frightening tendency of this bacteria to exhibit antimicrobial (multi-)resistances, phage therapy could represent a leading alternative to treat this infection in aquaculture. The aims of this study were to create a collection of A. salmonicida strains, isolate phages targeting these strains, phenotypically and genomically characterize these newly isolated phages and assess their potential for phage therapy in a preliminary in vivo model.

Materials and methods

An ATCC and three field strains of A. salmonicida isolated in Belgium were used for phage isolation. Different water sources were collected in Belgium and France to isolate A. salmonicida-specific phages: wastewater, fish farming water and natural aquatic environment water. Newly isolated phages were characterized in vitro for their ability to infect other A. salmonicida strains and their stability at different temperatures (25°C, 37°C, 45°C and 60°C) and units of pH (2, 4, 6, 8, 10, 12). Phage morphologies and genomes were investigated by electron microscopy and Illumina sequencing. The safety and efficacy of these phages were finally assessed at two treatment doses (MOI 10 and 100) during four days in a preliminary in vivo model using Galleria (G.) mellonella larvae.

Results and conclusion

Four new phages active against A. salmonicida, named vB_AsaM_ULASA1 (47,813bp), vB_AsaM_ULASA2 (170,823bp), vB_AsaM_ULASA3 (164,381bp) and vB_AsaM_ULASA4 (171,205bp), were isolated from water samples collected in fish farms and natural aquatic environments. Phages ULASA2, 3 and 4 were active against other A. salmonicida strains and showed a high resistance to temperatures and pH tested while the phage ULASA1 showed a more restricted host spectrum and less biochemical tolerance. Genomic analysis showed that phages ULASA2, 3 and 4 belong to the Straboviridae family but no family has already been attributed to the phage ULASA1. All four presented a myovirus morphotype. Four-day efficacy experiments in the preliminary in vivo model of G. mellonella larvae showed that phages ULASA1, 2 and 4 were responsible for a significant extension in the larval survival time at the two treatment doses tested (MOI 10 and 100) while the phage ULASA3 only showed a significant effect at MOI 100. In light of these results, these phages targeting A. salmonicida could represent potential new candidates for the development of anti-furunculosis phage treatments in aquaculture.