Screening, selection, and identification of autoscthonous potential probiotic bacteria against the rainbow trout pathogen Flavobacterium psychrophilum

Introduction: Flavobacterium psychrophilum is a globally distributed pathogen affecting salmonid aquaculture and is responsible for the lethal Bacterial Cold Water Disease (BCWD) and Rainbow Trout Fry Syndrome (RBFS) in adult and fry/juvenile rainbow trout individuals, respectively. The prophylactic and therapeutic use of antimicrobials against this pathogen have resulted in the development of antimicrobial resistance (AMR), while there is no effective and commercially available vaccine for adults, to date. Therefore, the development of alternative sustainable biocontrol measures against this detrimental pathogen is required. The purpose of this study was to develop an assay for the primary identification of candidate probiotic strains against F. psychrophilum. These strains should be sourced from within the rainbow trout aquaculture environment, utilizing an element that already exists within the microbiome system, rather than introducing a new element, focusing on recirculating aquaculture units.

Methods: F. psychrophilum strain 950106-1/1 previously isolated from rainbow trout farms in Denmark was fully embedded in Tryptone Yeast Extract Salts agar (TYES-A) supplemented with glucose. Rainbow trout aquaculture tank biofilm and water samples were tenfold diluted and plated on TSA media. These plated samples were then replica plated on F. psychrophilum embedded plates. For this assay, Pseudomonas fluorescens AH2 was used as a positive inhibition control. Following the examination of the embedded plates for inhibition zones, the topologically corresponding colonies within the master plates were selected and purity-streaked to further confirm inhibitory activity through the same well diffusion assay, in triplicates. The colonies which produced clear inhibition zones were later selected for identification using MALDI-TOF via MALDI Biotyper. Whole Genome Sequencing of the inhibitory strains is underway, which, coupled with analyses of secondary metabolite potential (anti-SMASH), would reveal genomic structures pinpointing the molecules involved in the antagonistic activity of the strains.

Results: The MALDI-TOF results revealed 3 probiotic candidates following the screening of approximately 600 replica-plated colonies on the F. psychrophilum embedded agar. One of the candidates was identified to belong to the Pseudomonas genus, without a clear species identification. The other two candidates were identified as Pseudomonas gessardii and Janthinobacterium lividum. The latter has previously been identified as a member of the gut microbiome of salmonids, while Pseudomonas gessardii has also been retrieved from rainbow trout aquaculture units.

Conclusion: This assay paves the way for the continuation of exploration of autochthonous probiotic candidates against F. psychrophilum. The screening would follow the expansion of the microbial sample pool, including samples from more aquaculture units and healthy rainbow trout individuals.