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

Objectives The Gram-negative bacteria Aeromonas (A.) salmonicida is a primary fish pathogen that causes furunculosis in salmonids as well as septicemia in a variety of fish. As this disease is responsible for significant losses in aquaculture worldwide, the aims of this study were to isolate new bacteriophages (phages) targeting this bacteria, phenotypically and genomically characterize them and finally assess their potential for phage therapy in a preliminary in vivo model. Methods Different water sources were collected in Belgium and France to isolate A. salmonicida-specific phages. Isolated phages were characterized in vitro for their ability to infect other A. salmonicida strains and their stability at different temperatures and pH. Phage morphologies and genomes were investigated by electron microscopy and Illumina sequencing. The safety and efficacy of these phages were finally evaluated using Galleria (G.) mellonella larvae. Results In 2022, four new phages targeting A. salmonicida were isolated. Among the characterization results, genomic analysis showed that 3 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. All four presented a myovirus morphotype. Four-day efficacy experiments in G. mellonella larvae showed that three of these phages were responsible for a significant extension in the larval survival time at the 2 treatment doses tested (MOI 10 and 100). Conclusion 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.