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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., Author summary The protection provided by host commensal microbiota against pathogens is a long-known phenomenon fostering the notion that introducing beneficial bacteria could reduce or prevent infections. However, the identification of such protective microorganisms is hampered by the poor reproducibility and relevance of current in vivo models. We developed procedures to raise germ-free rainbow trout larvae to study the determinants of microbiota-associated resistance to infection. We showed that the fish pathogen Flavobacterium columnare rapidly kills infected germ-free but not conventional larvae. We then re-colonized germ-free larvae with bacteria originating from cultured fish microbiota and identified two bacterial species providing full protection against infection. Our approach constitutes a rational and potentially high throughput in vivo strategy to study host-pathogen interactions and resistance to infection in fish. The identification of probiotic bacteria protecting rainbow trout and potentially other fish against F. columnare could also contribute to improve aquaculture sustainability and health.

Additional file 1. Figure 1. Iris-plot representing the 20 most abundant genera. Each bar represents a sample, and bar colors represented the genera relative abundance.

Additional file 1: Supplementary Figure S1. Relative abundance (%) of the overall most prevalent bacteria at genus (or the next lowest taxonomic level) of the gut of rainbow trout fed different diets, food and raising water samples. Supplementary Table S1. Feed formulations for rainbow trout (g/100 g feed) of commercial-like feed (T0), full terrestrial-vegetal feed (Tv) and experimental feeds (F1 and F2). Supplementary Table S2. Summary of the sequencing pre-processing. The number and percentage of reads that passed the quality filter, denoising, chimera checking, and the paired-end merged reads are shown. Supplementary Table S3. PERMANOVA test comparison of bacterial composition under experimental diets. The distance matrix was based on the Bray-Curtis dissimilarity matrix or Weighted-Unifrac distance. Q-values under 0.05 were considered significant. Supplementary Table S4. Crude protein, lipid, fiber, chitin, starch and cinder percentage of each feed formulations for rainbow trout (% of dry matter) of commercial-like feed (T0), full terrestrial-vegetal feed (Tv) and experimental feeds (F1 and F2).

Additional file 2. Table 1. Posterior estimates of h2 and m2 for the 21 health-related traits.

Macroalgae contribute substantially to primary production in coastal ecosystems. Their biomass, mainly consisting of polysaccharides, is cycled into the environment by marine heterotrophic bacteria using largely uncharacterized mechanisms. Here we describe the complete catabolic pathway for carrageenans, major cell wall polysaccharides of red macroalgae, in the marine heterotrophic bacterium Zobellia galactanivorans. Carrageenan catabolism relies on a multifaceted carrageenan-induced regulon, including a non-canonical polysaccharide utilization locus (PUL) and genes distal to the PUL, including a susCD-like pair. The carrageenan utilization system is well conserved in marine Bacteroidetes but modified in other phyla of marine heterotrophic bacteria. The core system is completed by additional functions that might be assumed by non-orthologous genes in different species. This complex genetic structure may be the result of multiple evolutionary events including gene duplications and horizontal gene transfers. These results allow for an extension on the definition of bacterial PUL-mediated polysaccharide digestion., Carrageenans, major cell wall polysaccharides of red macroalgae, are metabolised by marine heterotrophic bacteria through unclear mechanisms. Here, the authors identify an unusual polysaccharide-utilization locus encoding carrageenan catabolism in a marine bacterium, and characterise the complete pathway.

International audience; Tenacibaculum maritimum is a devastating bacterial pathogen of wild and farmed marine fish with a broad host range and a worldwide distribution. We report here the complete genome sequence of the T. maritimum type strain NCIMB 2154 T. The genome consists of a 3,435,971-base pair circular chromosome with 2,866 predicted protein-coding genes. Genes encoding the biosynthesis of exopolysaccharides, the type IX secretion system, iron uptake systems, adhesins, hemolysins, proteases, and glycoside hydrolases were identified. They are likely involved in the virulence process including immune escape, invasion, colonization, destruction of host tissues, and nutrient scavenging. Among the predicted virulence factors, type IX secretion-mediated and cell-surface exposed proteins were identified including an atypical sialidase, a sphingomyelinase and a chondroitin AC lyase which activities were demonstrated in vitro.

Macroalgae contribute substantially to primary production in coastal ecosystems. Their biomass, mainly consisting of polysaccharides, is cycled into the environment by marine heterotrophic bacteria using largely uncharacterized mechanisms. Here we describe the complete catabolic pathway for carrageenans, major cell wall polysaccharides of red macroalgae, in the marine heterotrophic bacterium Zobellia galactanivorans. Carrageenan catabolism relies on a multifaceted carrageenan-induced regulon, including a non-canonical polysaccharide utilization locus (PUL) and genes distal to the PUL, including a susCD-like pair. The carrageenan utilization system is well conserved in marine Bacteroidetes but modified in other phyla of marine heterotrophic bacteria. The core system is completed by additional functions that might be assumed by non-orthologous genes in different species. This complex genetic structure may be the result of multiple evolutionary events including gene duplications and horizontal gene transfers. These results allow for an extension on the definition of bacterial PUL-mediated polysaccharide digestion.

This work was supported by grant AGL2006-07562 from the Spanish Ministry of Science and Education.

Poster presentado 10th Symposium on Aquatic Microbial Ecology (SAME10) september 2-7 2007, Faro After induction of the viable but nonculturable (VBNC) state in Escherichia coli populations, we have analysed abiotic and biotic factors suggested to promote the resuscitation process. The response to the stressing conditions implied the formation of three subpopulations, culturble VBNC and non viable. In most adverse situations studied, VBNC subpopulation did not represent the dominant fraction. Moreover, this fraction did not remain stable, decreasing along the time. This fact seems to indicate that in most of cases, the VBNC is not a successful phenotype. Our working resuscitation protocol is based on combination of methods of dilution and inhibition of remaining culturable cells in order to unequivocally distinguish between regrowth and resuscitation. Reversion of abiotic factors inducing nonculturability as well as prevention of additional oxidative stress did not provoke resuscitation of VBNC cells. Participation of biotic factors was studied by addition of cell free supernatants from cultures (E. coli STCC 416, Pseudomonas fluorescens CHA0 or Enterococcus faecalis pMW158GFP) with different growth phase. In cases, regardlees of the origin of cell free supernatants, the resuscitation was unsuccessful. These results indicate that the E. coli STCC 416 is not able to resuscitate from the VBNC state. Previously we have stated that VBNC cells release into the surrounding médium molecules which could aid the survival of persisting culturable cells. Combining the E. coli survival and resuscitation processes, the formation of a VBNC subpopulation could be considered as an intentional process, designed for the benefit of the population as a whole Research project UPV 9/UPV00093.310-14510/2002 from the Basque Country University

Gnotobiotic animal models reconventionalized under controlled laboratory conditions with multi-species bacterial communities are commonly used to study host-microbiota interactions under presumably more reproducible conditions than conventional animals. The usefulness of these models is however limited by inter-animal variability in bacterial colonization and our general lack of understanding of the inter-individual fluctuation and spatio-temporal dynamics of microbiota assemblies at the micron to millimeter scale. Here, we show underreported variability in gnotobiotic models by analyzing differences in gut colonization efficiency, bacterial composition, and host intestinal mucus production between conventional and gnotobiotic zebrafish larvae re-conventionalized with a mix of 9 bacteria isolated from conventional microbiota. Despite similar bacterial community composition, we observed high variability in the spatial distribution of bacteria along the intestinal tract in the reconventionalized model. We also observed that, whereas bacteria abundance and intestinal mucus per fish were not correlated, reconventionalized fish had lower intestinal mucus compared to conventional animals, indicating that the stimulation of mucus production depends on the microbiota composition. Our findings, therefore, suggest that variable colonization phenotypes affect host physiology and impact the reproducibility of experimental outcomes in studies that use gnotobiotic animals. This work provides insights into the heterogeneity of gnotobiotic models and the need to accurately assess re-conventionalization for reproducibility in host-microbiota studies., Competing Interests: Conflict of Interest The authors declare no conflict of interest.