Line Roager

Aquaculture is an essential food production sector for meeting the global demand for high-quality protein. However, the sector faces significant challenges from bacterial pathogens, particularly Vibrio anguillarum , which causes vibriosis in numerous commercially important fish species. Current disease management strategies rely heavily on antibiotics, leading to antimicrobial resistance and environmental concerns. Microalgal microbiomes represent promising alternatives for sustainable pathogen control, yet the molecular mechanisms underlying their inhibitory activity remain poorly understood. Here, we employed an integrated multi-omics approach to elucidate the mechanisms by which the microbiome of microalga Isochrysis galbana inhibits the highly virulent fish pathogen V. anguillarum strain 90-11-286. Using a GFP-based inhibition assay, we confirmed potent pathogen suppression by the algal microbiome, achieving complete inhibition at a 1:1000 ratio of pathogen to microbiome. Through 16S rRNA gene amplicon sequencing, metagenomics, metatranscriptomics, and metabolomics, we characterized community composition, genomic potential, gene expression patterns, and metabolite production during pathogen challenge. The inhibitory microbiome was dominated by Alteromonas macleodii and Vreelandella alkaliphila , with high-quality metagenome-assembled genomes revealing substantial secondary metabolite biosynthetic potential. Metatranscriptomic analysis revealed active expression of biosynthetic gene clusters encoding, for example, non-ribosomal peptide synthetases, particularly a siderophore gene cluster in V. alkaliphila . Metabolomic profiling confirmed the production of hydroxamate siderophores in the microbiome, including desferrioxamine analogues, proferrioxamine G1t, and tenacibactin D, which accumulated during pathogen inhibition, as well as 10 putative new compounds. Notably, siderophore production was constitutive rather than pathogen-induced, suggesting iron competition as the primary inhibitory mechanism. Our findings demonstrate that iron sequestration through siderophore production represents a key strategy for pathogen suppression in marine microbial communities. This work provides molecular evidence for microbiome-mediated disease control and establishes a foundation for developing rationally designed multi-strain probiotic consortia for sustainable aquaculture applications, offering an environmentally friendly alternative to antibiotic-based pathogen management strategies.

The interactions between microalgae and the bacteria living in the phycosphere are pivotal to the role they play in aquatic ecosystems. This study examines how two representatives of common phycosphere bacteria, Yoonia sp. TsM2_T14_4 (Rhodobacteraceae) and Maribacter sp. IgM3_T14_3 (Flavobacteriaceae), interact with three microalgal hosts: Isochrysis galbana , Tetraselmis suecica , and Conticribra weissflogii (formerly Thalassiosira weissflogii ) using dual transcriptomic analyses of both bacteria and microalgae. Bacterial transcriptomes differed significantly depending on microalgal host, with notable changes in carbohydrate metabolism among other COG categories. Yoonia sp. expressed genes involved in anoxygenic photosynthesis in co-culture with I. galbana , presumably due to its inability to utilize carbohydrates from this algal host, whereas Maribacter sp. expressed polysaccharide degradation genes in co-culture with C. weissflogii along with T9SS genes, which can be employed to secrete these hydrolytic enzymes. Specifically, a putative glucan endo-1,3-beta-D-glucosidase was highly expressed, an enzyme that can hydrolyze laminarin and curdlan. Maribacter sp. IgM3_T14_3 could utilize laminarin as a sole carbon source in laboratory settings, a polysaccharide commonly found in marine environments and produced by C. weissflogii . Surprisingly, microalgal transcriptomes remained largely unaltered in the presence of either of the bacteria compared to transcriptomes of axenic algal cultures. These findings highlight the adaptability of phycosphere bacteria to different microalgal hosts. Furthermore, it also indicates a commensalism between microalgae, Yoonia sp. and Maribacter sp., in which the bacteria adapt to and benefit from microalgal host exudates, whereas under the conditions employed here the microalgae are unaffected by the presence of these bacterial symbionts.

Two Gram-negative, motile, rod-shaped bacteria, designated as C3_T13_0^T and A36a-5a^T, were isolated from marine environments collected off the coast of Crete, Greece, and from a biofilm in Jyllinge Harbor in Roskilde Fjord, Denmark, respectively. 16S rRNA gene phylogeny revealed that the two strains belong to the genus Phaeobacter. The major cellular fatty acids (>5%) were C_18 : 1  ω7c for strain A36a-5a^T and C_18 : 1  ω7c, C_18 : 2  ω7,13 and C_16 : 0 for strain C3_T13_0^T. The major respiratory quinone was ubiquinone-10, and the predominant polar lipids were phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine for both strains. C3_T13_0^T and A36a-5a^T both produce the antimicrobial compound tropodithietic acid. The C3_T13_0^T strain grows at temperatures between 8 and 37 °C with an optimum of 25 °C, whereas A36a-5a^T grows between 5 and 37 °C with an optimum of 30 °C. The salinity range for growth was 2–9 % NaCl (w/v) for C3_T13_0^T with an optimum of 3–4% and 0–10% NaCl (w/v) for A36a-5a^T with an optimum of 2–6%. The genomes of the two strains shared an average nucleotide identity (ANI) of 84% and had lower than 87% ANI when compared with genomes of Phaeobacter spp. type strains. The genome of C3_T13_0^T was 4,432,895 bp with a G+C content of 55.96 mol%, and the genome of A36a-5a^T was 4,050,005 bp with a G+C content of 61.37 mol%. Based on the phylogenetic and phenotypic analyses presented here, the two isolates C3_T13_0^T (=DSM 118664^T=LMG 33738^T) and A36a-5a^T (=DSM 118663^T=LMG 33737^T) are considered to represent two novel species belonging to the genus Phaeobacter, for which the names Phaeobacter cretensis sp. nov. and Phaeobacter bryozoorum sp. nov. are proposed, respectively.

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. IMPORTANCE: Aquaculture 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.

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 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 whilst 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 Halomonas campaniensis D2 were co-cultured, even though neither were 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. IMPORTANCE: Aquaculture 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 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 emphasising the potential in using mixtures of bacteria as probiotics.

Microalgal microbiomes play vital roles in the growth and health of their host, however, their composition and functions remain only partially characterized, especially across microalgal phyla. In this study, a natural seawater microbiome was introduced to three distinct, axenic species of microalgae, the haptophyte Isochrysis galbana, the chlorophyte Tetraselmis suecica, and the diatom Conticribra weissflogii (previously Thalassiosira ), and its divergence and assembly under constant illumination was monitored over 49 days using 16S rRNA amplicon and metagenomic analyses. The microbiomes had a high degree of host specificity in terms of taxonomic composition and potential functions, including CAZymes profiles. Rhodobacteraceae and Flavobacteriaceae families were abundant across all microalgal hosts, but I. galbana microbiomes diverged further from T. suecica and C. weissflogii microbiomes. I. galbana microbiomes had a much higher relative abundance of Flavobacteriaceae, whereas the two other algal microbiomes had higher relative abundances of Rhodobacteraceae. This could be due to the bacterivorous mixotrophic nature of I. galbana affecting the carbohydrate composition available to the microbiomes, which was supported by the CAZymes profile of I. galbana microbiomes diverging further from those of T. suecica and C. weissflogii microbiomes. Finally, the presence of denitrification and other anaerobic pathways was found exclusively in the microbiomes of C. weissflogii, which we speculate could be a result of anoxic microenvironments forming in aggregates formed by this diatom during the experiment. These results underline the significant role of the microalgal host species on microbiome composition and functional profiles along with other factors, such as the trophic mode of the microalgal host. IMPORTANCE: As the main primary producers of the oceans, microalgae serve as cornerstones of the ecosystems they are part of. Additionally, they are increasingly used for biotechnological purposes such as the production of nutraceuticals, pigments, and antioxidants. Since the bacterial microbiomes of microalgae can affect their hosts in beneficial and detrimental ways, understanding these microbiomes is crucial to both the ecological and applied roles of microalgae. The present study advances the understanding of microalgal microbiome assembly, composition, and functionality across microalgal phyla, which may inform the modeling and engineering of microalgal microbiomes for biotechnological purposes.

The globally important production of fish and shellfish in aquaculture is challenged by disease outbreaks caused by pathogens such as Vibrio crassostreae . These outbreaks not only lead to substantial economic loss and environmental damage, but treatment with antibiotics can also lead to antibiotic resistance affecting human health. Here, we evaluated the potential of probiotic bacteria, specifically the newly identified strain Phaeobacter piscinae S26, to counteract these threats in a sustainable manner. Through a systematic assessment of the antagonistic effect of P. piscinae S26 against V. crassostreae DMC-1, particularly within the context of algal feed systems, the study demonstrates the effectiveness of P. piscinae S26 as probiotic and thereby provides a strategic pathway for addressing disease outbreaks in aquaculture. This finding has the potential of significantly contributing to the long-term stability of the industry, highlighting the potential of probiotics as an efficient and environmentally conscious approach to safeguarding aquaculture productivity against the adverse impact of pathogens.

Microalgal microbiomes play vital roles in the growth and health of their host, however, their composition and functions remain only partially characterized, especially across microalgal phyla. In this study, a natural seawater microbiome was introduced to three distinct, axenic species of microalgae, the haptophyte Isochrysis galbana, the chlorophyte Tetraselmis suecica, and the diatom Conticribra weissflogii (previously Thalassiosira ), and its divergence and assembly was monitored over 49 days using 16S rRNA amplicon and metagenomic analyses. The microbiomes had a high degree of host specificity in terms of taxonomic composition and potential functions, including CAZymes profiles. Rhodobacteraceae and Flavobacteriaceae families were abundant across all microalgal hosts, but I .galbana microbiomes diverged further from T. suecica and C. weissflogii microbiomes. I .galbana microbiomes had a much higher relative abundance of Flavobacteriaceae, whereas the two other algal microbiomes had higher relative abundances of Rhodobacteraceae. This could be due to the mixotrophic nature of I. galbana affecting the carbohydrate composition available to the microbiomes, which was supported by the CAZymes profile of I. galbana microbiomes diverging further from those of T. suecica and C. weissflogii microbiomes. Finally, the presence of denitrification and other anaerobic pathways was found exclusively in microbiomes of C. weissflogii potentially resulting from anoxic microenvironments in aggregates formed by this diatom during the experiment. These results underline the deterministic role of the microalgal host species on microbiome composition and functional profiles along with other factors, such as trophic mode of the microalgal host. Importance: As the main primary producers of the oceans, microalgae serve as cornerstones of the ecosystems they are part of. Additionally, they are increasingly used for biotechnological purposes such as the production of nutraceuticals, pigments, and antioxidants. Since the bacterial microbiomes of microalgae can affect their hosts in beneficial and detrimental ways, understanding these microbiomes is crucial to both ecological and applied roles of microalgae. The present study advances the understanding of microalgal microbiome assembly, composition, and functionality across microalgal phyla, which may inform modeling and engineering of microalgal microbiomes for biotechnological purposes.

Microalgae are very important as primary producers in the ocean, but also as forthcoming sustainable producers of biotechnologically interesting compounds. Accordingly, the bacterial microbiomes associated with microalgae are attracting increasing attention due to their effects on the growth and health of microalgae.

Bacterial populations communicate using quorum‐sensing (QS) molecules and switch on QS regulation to engage in coordinated behaviour such as biofilm formation or virulence. The marine fish pathogen Vibrio anguillarum harbours several QS systems, and our understanding of its QS regulation is still fragmentary. Here, we identify the VanT‐QS regulon and explore the diversity and trajectory of traits under QS regulation in Vibrio anguillarum through comparative transcriptomics of two wildtype strains and their corresponding mutants artificially locked in QS‐on (Δ vanO ) or QS‐off (Δ vanT ) states. Intriguingly, the two wildtype populations showed different QS responses to cell density changes and operated primarily in the QS‐on and QS‐off spectrum, respectively. Examining 27 V. anguillarum strains revealed that ~11% were QS‐negative, and GFP‐reporter measurements of nine QS‐positive strains revealed a highly strain‐specific nature of the QS responses. We showed that QS controls a plethora of genes involved in processes such as central metabolism, biofilm formation, competence, T6SS, and virulence properties in V. anguillarum , with large strain‐specific differences. Moreover, we demonstrated that the QS state is an important driver of virulence towards fish larvae in one of two V. anguillarum strains. We speculate that infections by mixed‐strain communities spanning diverse QS strategies optimize the infection efficiency of the pathogen.