Abstract
Intensive fish rearing in aquaculture is challenged by infectious diseases, and although vaccines have been successfully developed for mature fish, alternative disease control measures are needed for fish larvae and juveniles. Probiotics offer a promising alternative to antibiotics, with the potential to reduce the risk of antibiotic resistance. Probiotics are typically isolated and used as pure cultures; however, in natural environments, it is the concerted effort of the complex microbiome that keeps pathogens at bay. Here, we developed an in vitro assay to evaluate the anti-pathogen efficacy of mixed algal microbiomes from the live feed microalgae Tetraselmis suecica and Isochrysis galbana. The inhibition of a green fluorescent protein (GFP)-tagged Vibrio anguillarum, a key fish pathogen, by microbial communities was measured and quantified as reduction in fluorescence. The Isochrysis galbana microbiome was more inhibitory to V. anguillarum than the Tetraselmis suecica microbiome. During co-culture with the pathogen, the bacterial density of the Isochrysis microbiomes increased, while the diversity was reduced as determined by metataxonomic analyses. Bacteria isolated from the fully inhibitory microbiomes were members of Alteromonadaceae, Halomonadaceae, Rhodobacteraceae, Vibrionaceae, Flavobacteriaceae, and Erythrobacteraceae. Although some strains individually inhibited the pathogen, these were not the key members of the microbiome, and enhanced inhibition was observed when Sulfitobacter pontiacus D3 and Vreelandella alkaliphila D2 were co-cultured, even though neither was inhibitory as monocultures. Thus, this study demonstrates that microbial communities derived from natural algal microbiomes can have anti-pathogen effects, and that bacterial co-cultures may offer synergistic advantages over monocultures as probiotics, highlighting their promise for aquaculture health strategies.IMPORTANCEAquaculture is the fastest-growing food protein-producing sector, and sustainable disease control measures are required. Probiotics have gained interest as a promising solution for combating fish pathogens, and using mixtures of microorganisms rather than pure cultures may represent a more stable pathogen control. We developed an assay using green fluorescent protein (GFP) tagging of a fish pathogen, enabling the quantitative assessment of the anti-pathogen effects of complex microbiomes. We show that the efficiency of pathogen suppression can be increased with co-cultures compared to monocultures, thus emphasizing the potential in using mixtures of bacteria as probiotics.