WWW.WAS.ORG • WORLD AQUACULTURE • JUNE 2025 59 Three top candidates, in terms of potent inhibition of F. psychrophilum, were a strain of Janthinobacterium tructae, a strain of Pseudomonas yamanorum and an unknown Pseudomonas strain. The latter showed significant genetic divergence from known Pseudomonas species based on its whole-genome data, which suggests that it may represent a previously uncharacterized or unknown species within the Pseudomonas genus. According to the whole genome data, both these Pseudomonas strains were shown to belong to the taxonomic group Pseudomonas fluorescens, which is not surprising. Other fluorescent Pseudomonas strains isolated from the skin of brown and rainbow trout had previously exhibited antagonism against freshwater pathogens like Saprolegnia parasitica and F. psychrophilum also (Hoseinifar et al. 2024). Exploring Possible Mechanisms of Antimicrobial Action One of the most exciting aspects of our research is the potential to unravel the mechanisms behind the antimicrobial effects of the probiotic strains. To investigate the possibility that the Pseudomonas and Janthinobacterium strains produce inhibitory compounds against Flavobacterium, we delved into their genomes and ‘scanned’ them for genes residing in so-called biosynthetic pathways which could lead to the production of the so-called secondary metabolites. These are small molecules of particular interest in the microbial universe, equipping bacteria with a diverse toolbox of compounds that may not be so indispensable for basic bacterial functions such as growth or reproduction, but can be useful for their survival and adaptation, when necessary. Secondary metabolites can be actors or mediators of several functions such as establishing microbial defenses against pathogens, communicating with other microbes via chemical signals, helping the microbial cells adapt to environmental stressors, or helping them acquire resources from their environment, such as iron. Pronounced examples of secondary metabolites are pigments, protecting microbial cells from ultraviolet radiation exposure acting as antioxidants, or they can also be antibiotic compounds, some of which are well-known and have been widely harvested for years by humans towards clinical applications. One possible mechanism contributing to the antagonistic nature of fluorescent Pseudomonas strains against pathogens like F. psychrophilum could be the secretion of siderophores, molecules which bind iron. Generally, siderophores can act as iron scavengers in environments where iron concentrations are scarce, subsequently helping the microbial cells to use that harvested iron for growth. Between two competing microbes, one of which could be a pathogen, the one that will be able to bind iron more efficiently will thus make it unavailable to the pathogen, thereby ‘starving’ it and indirectly suppressing its growth. As a matter of fact, iron stands out as an important molecule influencing the virulence of F. psychrophilum. The growth of the pathogen is enhanced when iron is present in the environment, along with its potential for infection and pathogenicity in the fish host tissue (Vaibarova and Cizek 2024). Supporting this mechanism, a recent study showed that iron supplementation in aquaculture settings influenced the severity of infections in rainbow trout, with higher iron levels correlating with increased disease severity (Macchia et al. 2022), while an older study showed that another fluorescent strain, Pseudomonas M174, was inhibiting F. psychrophilum under iron-depleted conditions in vitro (KorkeaAho et al. 2012). By analyzing the genetic data through a bioinformatics approach, we found a diverse array of secondary metabolite classes, emphasizing the metabolic potential of our candidate strains. For example, some of the biosynthetic genes identified in both Pseudomonas strains were indeed responsible for the production of secondary metabolites highly similar to ironbinding siderophores, called pyoverdines. The Pseudomonas secondary metabolite repertoire also included the production of compounds similar to viscosinamides and pseudodesmins, which have been known for their antimicrobial and antifungal effects (Thrane et al. 2000). When looking into the secondary metabolite arsenal of Janthinobacterium, we found compounds similar to terpenes, a well-known class of organic compounds, with reported biocontrol/modulatory effect towards soil-borne pathogens in plants (Chou et al. 2023). Looking Forward Our future research efforts will focus on expanding the microbial sample pool to include more aquaculture systems and tissue samples from healthy rainbow trout individuals. It is also crucial to understand the effect of the application of the isolated probiotic strains on the survival of rainbow trout eggs and fry, as well as their effect on the microbiome composition and stability of the entire rearing system, through different probiotic delivery modes and doses in aquaria-scale challenge trials. Once we have established that the probiotic strains are not harmful to the fish and to the ecological integrity of the rearing system, we aim to identify and characterize the specific compounds responsible for the observed antimicrobial activities. Overall, our research aims to refine our understanding of how probiotics can be effectively screened, integrated and upscaled into aquaculture practices. Despite, or exactly because of, the challenging nature of the aquatic microbial environment, a suite of adaptive solutions is being steadily researched and developed, tailored to aquaculture settings while keeping animal welfare at the forefront and ensuring the prevention of biodiversity disruption of the surrounding environment. Our current and future knowledge from studying probiotics and their interactions within rainbow trout rearing systems, can also inform strategies for other species, creating a ripple effect of sustainability and innovation across the sector. Stay tuned. Acknowledgments This project was funded by European Union’s Horizon Europe research and innovation program, under grant agreement No. 101084204 - Cure4Aqua - https://cure4aqua-project.eu/ Notes Despoina Athena-Vasileiadi,*, Mikkel Bentzon-Tilia and Lone Gram, Section for Microbial and Chemical Ecology, Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800 Kgs Lyngby, Denmark. * corresponding author, desvath@dtu.dk (CONTINUED ON PAGE 60)
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