Table_3_Probiotic Yeasts and Vibrio anguillarum Infection Modify the Microbiome of Zebrafish Larvae.xlsx

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.

宿主微生物组（host microbiome）在健康与疾病进程中发挥着至关重要的作用。针对病原体或益生菌介导的微生物组修饰效应的研究仍较为匮乏，针对益生菌酵母菌的相关探索尤为不足。当前，下一代测序技术（next-generation sequencing）为微生物组表征提供了最优工具。汉逊德巴利酵母97（Debaryomyces hansenii 97，简称D. hansenii 97）与解脂耶氏酵母242（Yarrowia lipolytica 242，简称Y. lipolytica 242）是两种可保护野生型斑马鱼（Danio rerio）幼体抵御鳗弧菌（Vibrio anguillarum，简称V. anguillarum）感染、提升幼体存活率的酵母菌。本研究旨在探究这两种微生物对Tg(Bacmpx:GFP)i114品系斑马鱼幼体微生物组及中性粒细胞应答（炎症反应）的影响。我们提出假说：提前用酵母菌接种幼体，可减弱肠道中性粒细胞应答，并阻止病原体诱导的幼体微生物组失衡。本研究通过对16S rRNA基因的V3-V4区域进行测序，并借助Piphillin预测常规饲养幼体的代谢通路，以此开展微生物组分析。同时在常规饲养与无菌两种培养条件下，分别评估幼体的存活率与中性粒细胞应答情况。实验结果显示：无论是常规饲养还是无菌条件下，鳗弧菌感染组幼体肠道区域的中性粒细胞数量均显著高于未感染组；在无菌条件下，提前接种酵母菌的感染组幼体，其中性粒细胞数量低于未提前接种的感染组；在两种培养条件中，仅汉逊德巴利酵母97可提升感染组幼体的存活率。与未接种的幼体相比，鳗弧菌与两种酵母菌均会改变微生物群落的β多样性。感染后3天，鳗弧菌可使10个菌属的相对丰度发生改变，而提前接种汉逊德巴利酵母97和解脂耶氏酵母242，可分别阻止其中5个和6个菌属的丰度变化。两种酵母菌均可抑制剑菌属（Ensifer）和沃斯氏菌属（Vogesella）的增殖——这两个菌属被鉴定为幼体存活率的负向预测因子，分别解释了40%和27%的存活率变异。此外，酵母菌提前接种可阻止鳗弧菌感染所引发的部分代谢通路异常。上述结果表明，在斑马鱼幼体的早期发育阶段，两种酵母菌与鳗弧菌均可塑造其肠道微生物组的结构。此外，酵母菌对幼体微生物组关键类群或代谢通路的调控作用，可能与感染幼体的存活率提升相关。本研究有助于深化对酵母菌-病原体-微生物组三者互作关系的理解，不过仍需开展进一步研究以阐明其中涉及的具体机制。