58 JUNE 2025 • WORLD AQUACULTURE • WWW.WAS.ORG FIGURE 3. Schematic representation of experimental setup for probiotic candidate screening against Flavobacterium psychrophilum. Created with Biorender.com In light of the development of detrimental antimicrobial resistance to Flavobacterium psychrophilum and considering the possible effects this could have both for environmental biodiversity and public health (Reverter et al. 2020), more sustainable and innovative prevention biocontrol methods, such as probiotics, are necessary to counteract this pathogen, circumventing the need for chemo-therapeutic interventions. Native Candidates: A Systemic Approach Probiotics were defined by a joint WHO/FAO working group in 2002 as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.” The scientific research focus on the use of probiotics targeting microbial communities in aquaculture, has been mostly set on the gut microbiota of reared species, particularly on the ways in which the intestinal microbiome of fish could be restored or modulated through colonization of the gut, presenting beneficial downstream effects for the health of the fish host. However, beyond the gastrointestinal tract, bacterial populations extensively inhabit the water, the tank surfaces, and fish mucosal surfaces, such as the skin and gills. In the latter, they can act as entrance barriers by exerting antagonistic effects against potential pathogens, with a broader role in maintaining microbial diversity and thus ecological integrity. They can also be associated with immunological modulations and enhancement of mucosal tissue defense mechanisms (Sylvain et al. 2020), while they are also performing important functions like nitrogen cycling in the gills. Keeping in mind this inherent interconnectivity of the aquaculture microbiota, we thus pursued a more systemic approach in our search for probiotic microbial strains against F. psychrophilum. To identify potential probiotic bacterial candidates, we conducted samplings across different levels of a rainbow trout RAS system in Denmark. Water samples, biofilter material, and tank swabs from hatcheries and fingerling units served as a reservoir of microbial diversity. Our goal was to find indigenous bacterial strains capable of inhibiting the growth of F. psychrophilum (Figure 1). This approach of looking for ‘native’ bacterial strains has been previously adopted in the research of probiotic strains against marine aquaculture pathogens like vibrios (Grotkjær et al. 2016). The hypothesis here is that the re-introduction of bacteria which are already present within the aquaculture environment would merely shift the microbiome balance towards a more ‘favorable’ state, instead of introducing a completely new element that could generate adverse effects in the system (Bentzon-Tilia et al. 2016). This strategy increases the likelihood of ecological compatibility and the potential for long-term establishment of the probiotic in a desired quantity, thus representing a more systemic perspective for microbiome management. Our Methodology Our first goal was to create a screening assay that would help us evaluate different bacterial strains for their inhibitory activity against the pathogen. Using a specific growth substrate suitable for the growth of the fastidious F. psychrophilum, we fully embedded the pathogen directly into the substrate. We further optimized the embedding by testing different pathogen concentrations against a positive control strain (Pseudomonas fluorescens AH2) known to inhibit F. psychrophilum. This allowed us to observe potential antagonistic effects directly on the plates through the formation of inhibition/ clearing zones: clear areas surrounding each bacterial culture spot of potential probiotic bacteria, like a halo, indicative of pathogen inhibition (Figure 2). Following sampling in the rainbow trout farm, serial dilutions of the samples were prepared and plated on differently enriched solid media, allowing for differential nutrient access to the microbes in our samples. Then, the most diluted plates exhibiting distinct colonies were selected for replica plating. This technique further streamlined the screening process, allowing for the spatial organization of a sample plate with distinct bacterial colonies to be directly copied/replicated onto another plate in which F. psychrophilum was fully seeded. In this way, we were able to see how each of these separate bacterial colonies interacted with the pathogen-embedded agar. Approximately 600 colonies were developed from this first batch of samples and screened in this manner, yielding three promising candidates with consistent inhibitory activity against F. psychrophilum. Applying DNA-based identification methods, we identified which probiotic bacterial candidates were present in our samples by pinpointing the genus to which they belonged. The genetic information also provided us with detailed insights into their complete genetic characteristics, allowing us to potentially identify genetic structures that could be responsible for their exhibited antimicrobial abilities (Figure 3).
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