The health and environmental risks associated with antibiotic use in aquaculture have promoted bacterial probiotics as an alternative approach to control fish infections in vulnerable larval and juvenile stages. However, evidence-based identification of probiotics is often hindered by the complexity of bacteria-host interactions and host variability in microbiologically uncontrolled conditions. While these difficulties can be partially resolved using gnotobiotic models harboring no or reduced microbiota, most host-microbe interaction studies are carried out in animal models with little relevance for fish farming. Here we studied host-microbiota-pathogen interactions in a germ-free and gnotobiotic model of rainbow trout (Oncorhynchus mykiss), one of the most widely cultured salmonids. We demonstrated that germ-free larvae raised in sterile conditions displayed no significant difference in growth after 35 days compared to conventionally-raised larvae, but were extremely sensitive to infection by Flavobacterium columnare, a common freshwater fish pathogen causing major economic losses worldwide. Furthermore, re-conventionalization with 11 culturable species from the conventional trout microbiota conferred resistance to F. columnare infection. Using mono-re-conventionalized germ-free trout, we identified that this protection is determined by a commensal Flavobacterium strain displaying antibacterial activity against F. columnare. Finally, we demonstrated that use of gnotobiotic trout is a suitable approach for the identification of both endogenous and exogenous probiotic bacterial strains protecting teleostean hosts against F. columnare. This study therefore establishes an ecologically-relevant gnotobiotic model for the study of host-pathogen interactions and colonization resistance in farmed fish.

Bacteroides thetaiotaomicron is a gut symbiont that inhabits the mucus layer and adheres to and metabolizes food particles, contributing to gut physiology and maturation. Although adhesion and biofilm formation could be key features for B. thetaiotaomicron stress resistance and gut colonization, little is known about the determinants of B. thetaiotaomicron biofilm formation. We previously showed that the B. thetaiotaomicron reference strain VPI-5482 is a poor in vitro biofilm former. Here, we demonstrated that bile, a gut-relevant environmental cue, triggers the formation of biofilm in many B. thetaiotaomicron isolates and common gut Bacteroidales species. We determined that bile-dependent biofilm formation involves the production of the DNase BT3563 or its homologs, degrading extracellular DNA (eDNA) in several B. thetaiotaomicron strains. Our study therefore shows that, although biofilm matrix eDNA provides a biofilm-promoting scaffold in many studied Firmicutes and Proteobacteria, BT3563-mediated eDNA degradation is required to form B. thetaiotaomicron biofilm in the presence of bile.

Bacteroides thetaiotaomicron is one of the most abundant gut symbiont species, whose contribution to host health through its ability to degrade dietary polysaccharides and mature the immune system is under intense scrutiny. In contrast, adhesion and biofilm formation, which are potentially involved in gut colonization and microbiota structure and stability, have hardly been investigated in this intestinal bacterium. To uncover B. thetaiotaomicron biofilm-related functions, we performed a transposon mutagenesis in the poorly biofilm-forming reference strain VPI-5482 and showed that capsule 4, one of the eight B. thetaiotaomicron capsules, hinders biofilm formation. We then showed that the production of capsules 1, 2, 3, 5, and 6 also inhibits biofilm formation and that decreased capsulation of the population correlated with increased biofilm formation, suggesting that capsules could be masking adhesive surface structures. In contrast, we showed that capsule 8 displayed intrinsic adhesive properties. Finally, we demonstrated that BT2934, the wzx homolog of the B. thetaiotaomicron glycosylation locus, competes with capsule production and impacts its adhesion capacity. This study therefore establishes B. thetaiotaomicron capsule regulation as a major determinant of B. thetaiotaomicron biofilm formation, providing new insights into how modulation of different B. thetaiotaomicron surface structures affects in vitro biofilm formation. IMPORTANCE The human gut harbors a complex bacterial community that plays important roles in host health and disease, including nutrient acquisition, maturation of the immune system, and resistance to infections. The capacity to adhere to surfaces and form communities called biofilms is believed to be important for niche colonization and maintenance of gut bacteria. However, little is known about the adhesion capacity of most gut bacteria. In this study, we investigated biofilm formation in Bacteroides thetaiotaomicron, one of the most abundant bacteria of the normal mammalian intestine. We identified that B. thetaiotaomicron capsules, a group of eight surface-exposed polysaccharidic layers mediating important interactions with the gut environment, are also major determinants of biofilm formation that mask or unmask adhesion factors. Studying how B. thetaiotaomicron regulates its adhesion properties will allow us to better understand the physiology and specific properties of this important gut symbiont within anaerobic biofilms.