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Potential probiotic yeasts isolated from the fish gut protect zebrafish (Danio rerio) from a Vibrio anguillarum challenge.

ABSTRACT: Due to the negative consequences associated with the use of antibiotics, researchers, and food producers have studied alternatives, such as probiotics, for the control of fish diseases. The probiotic properties of yeasts in aquaculture have been scarcely considered. The present study investigated the probiotic properties of local yeast strains for aquaculture application in the protection of bacterial diseases. Yeast strains (n = 15), previously isolated from the intestinal gut of healthy salmonids, yellowtail, and croaker, were evaluated for their protection of zebrafish larvae following a Vibrio anguillarum challenge. We developed an infection model on zebrafish larvae with V. anguillarum, observing rapid mortality (?50%) 5 days post-immersion challenge. Infection of Tg(Lyz:DsRed)(nz50) larvae with fluorescent-marked V. anguillarum showed the oro-intestinal as the natural route of infection concomitant with an inflammatory response of the larvae reflected by neutrophil migration outside the hematopoietic tissue. Thirteen of 15 strains increased the percentage of larvae survival after the V. anguillarum challenge, although no yeast showed in vitro anti-V. anguillarum activity. In a subset of yeasts, we explored yeast-larvae interactions using fluorescent yeast and evaluated larvae colonization by culture analysis. All fluorescent yeasts were located in the gastrointestinal tract until 5 days post-inoculation (dpi). Yeasts reached 10(3) CFU/larvae at 0 dpi, although the persistence until 5 dpi of the viable yeast in the gut was different among the strains. These results reveal that some yeasts isolated from the gut of fish could be potential probiotics, reducing the mortality associated to V. anguillarum challenge, and suggest that gut colonization could be involved in the protective effect. Future studies should elucidate other mechanisms involved in yeast protection and verify the beneficial effects of probiotic use in commercial fish species.

SUBMITTER: Caruffo M

PROVIDER: S-EPMC4596066 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Potential probiotic yeasts isolated from the fish gut protect zebrafish (Danio rerio) from a Vibrio anguillarum challenge.

Caruffo Mario M Navarrete Natalie N Salgado Oscar O Díaz Angélica A López Paulina P García Katherine K Feijóo Carmen G CG Navarrete Paola P

Frontiers in microbiology 20151007

Due to the negative consequences associated with the use of antibiotics, researchers, and food producers have studied alternatives, such as probiotics, for the control of fish diseases. The probiotic properties of yeasts in aquaculture have been scarcely considered. The present study investigated the probiotic properties of local yeast strains for aquaculture application in the protection of bacterial diseases. Yeast strains (n = 15), previously isolated from the intestinal gut of healthy salmon ...[more]

PMID: 26500633

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Project description:The host microbiome plays an essential role in health and disease. Microbiome modification by pathogens or probiotics has been poorly explored especially in the case of probiotic yeasts. Next-generation sequencing currently provides the best tools for their characterization. Debaryomyces hansenii 97 (D. hansenii 97) and Yarrowia lipolytica 242 (Y. lipolytica 242) are yeasts that protect wildtype zebrafish (Danio rerio) larvae against a Vibrio anguillarum (V. anguillarum) infection, increasing their survival rate. We investigate the effect of these microorganisms on the microbiome and neutrophil response (inflammation) in zebrafish larvae line Tg(Bacmpx:GFP) i114. We postulated that preinoculation of larvae with yeasts would attenuate the intestinal neutrophil response and prevent modification of the larval microbiome induced by the pathogen. Microbiome study was performed by sequencing the V3-V4 region of the 16S rRNA gene and prediction of metabolic pathways by Piphillin in conventionally raised larvae. Survival and the neutrophil response were both evaluated in conventional and germ-free conditions. V. anguillarum infection resulted in higher neutrophil number in the intestinal area compared to non-infected larvae in both conditions. In germ-free conditions, infected larvae pre-inoculated with yeasts showed fewer neutrophil numbers than infected larvae. In both conditions, only D. hansenii 97 increased the survival of infected larvae. Beta diversity of the microbiota was modified by V. anguillarum and both yeasts, compared to non-inoculated larvae. At 3 days post-infection, V. anguillarum modified the relative abundance of 10 genera, and pre-inoculation with D. hansenii 97 and Y. lipolytica 242 prevented the modification of 5 and 6 of these genera, respectively. Both yeasts prevent the increase of Ensifer and Vogesella identified as negative predictors for larval survival (accounting for 40 and 27 of the variance, respectively). In addition, yeast pre-inoculation prevents changes in some metabolic pathways altered by V. anguillarum's infection. These results suggest that both yeasts and V. anguillarum can shape the larval microbiota configuration in the early developmental stage of D. rerio. Moreover, modulation of key taxa or metabolic pathways of the larval microbiome by yeasts can be associated with the survival of infected larvae. This study contributes to the understanding of yeast-pathogen-microbiome interactions, although further studies are needed to elucidate the mechanisms involved.

Project description:We investigated mechanisms involved in the protection of zebrafish (Danio rerio) larvae by two probiotic candidate yeasts, Debaryomyces hansenii 97 (Dh97) and Yarrowia lypolitica 242 (Yl242), against a Vibrio anguillarum challenge. We determined the effect of different yeast concentrations (104-107 CFU/mL) to: (i) protect larvae from the challenge, (ii) reduce the in vivo pathogen concentration and (iii) modulate the innate immune response of the host. To evaluate the role of zebrafish microbiota in protection, the experiments were performed in conventionally raised and germ-free larvae. In vitro co-aggregation assays were performed to determine a direct yeast-pathogen interaction. Results showed that both yeasts significantly increased the survival rate of conventionally raised larvae challenged with V. anguillarum. The concentration of yeasts in larvae tended to increase with yeast inoculum, which was more pronounced for Dh97. Better protection was observed with Dh97 at a concentration of 106 CFU/mL compared to 104 CFU/mL. In germ-free conditions V. anguillarum reached higher concentrations in larvae and provoked significantly more mortality than in conventional conditions, revealing the protective role of the host microbiota. Interestingly, yeasts were equally (Dh97) or more effective (Yl242) in protecting germ-free than conventionally-raised larvae, showing that protection can be exerted only by yeasts and is not necessarily related to modulation of the host microbiota. Although none of the yeasts co-aggregated with V. anguillarum, they were able to reduce its proliferation in conventionally raised larvae, reduce initial pathogen concentration in germ-free larvae and prevent the upregulation of key components of the inflammatory/anti-inflammatory response (il1b, tnfa, c3, mpx, and il10, respectively). These results show that protection by yeasts of zebrafish larvae challenged with V. anguillarum relates to an in vivo anti-pathogen effect, the modulation of the innate immune system, and suggests that yeasts avoid the host-pathogen interaction through mechanisms independent of co-aggregation. This study shows, for the first time, the protective role of zebrafish microbiota against V. anguillarum infection, and reveals mechanisms involved in protection by two non-Saccharomyces yeasts against this pathogen.

Dataset Information

Potential probiotic yeasts isolated from the fish gut protect zebrafish (Danio rerio) from a Vibrio anguillarum challenge.

Publications

Potential probiotic yeasts isolated from the fish gut protect zebrafish (Danio rerio) from a Vibrio anguillarum challenge.

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