Mining zebrafish microbiota reveals key community-level resistance against fish pathogen infection

Abstract

The long-known resistance to pathogens provided by host-associated microbiota fostered the notion that adding protective bacteria could prevent or attenuate infection. However, the identification of endogenous or exogenous bacteria conferring such protection is often hindered by the complexity of host microbial communities. Here, we used zebrafish and the fish pathogen Flavobacterium columnare as a model system to study the determinants of microbiota-associated colonization resistance. We compared infection susceptibility in germ-free, conventional and reconventionalized larvae and showed that a consortium of 10 culturable bacterial species are sufficient to protect zebrafish. Whereas survival to F. columnare infection does not rely on host innate immunity, we used antibiotic dysbiosis to alter zebrafish microbiota composition, leading to the identification of two different protection strategies. We first identified that the bacterium Chryseobacterium massiliae individually protects both larvae and adult zebrafish. We also showed that an assembly of 9 endogenous zebrafish species that do not otherwise protect individually confer a community-level resistance to infection. Our study therefore provides a rational approach to identify key endogenous protecting bacteria and promising candidates to engineer resilient microbial communities. It also shows how direct experimental analysis of colonization resistance in low-complexity in vivo models can reveal unsuspected ecological strategies at play in microbiota-based protection against pathogens.

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Fig. 1: Flavobacterium columnare kills germ-free but not conventional zebrafish.
Fig. 2: Analysis of protection against F. columnare infection after antibiotic dysbiosis.
Fig. 3: Protection against F. columnare in zebrafish reconventionalized with individual or mixed bacterial strains isolated from zebrafish.
Fig. 4: Zebrafish immune response to F. columnare infection.
Fig. 5: Intestine of F. columnare infected germ-free zebrafish displays severe disorganization compared to conventional and reconventionalized larvae.
Fig. 6: Pre-exposure to C. massiliae protects larval and adult zebrafish against F. columnare infection.

Data availability

The raw sequences generated for the study can be found in the NCBI Short Read Archive under BioProject No. PRJNA649696. Bacterial genome sequences obtained in the present study are available at the European Nucleotide Archive with the project number PRJEB36872, under accession numbers ERS4385993 (Aeromonas veronii 1); ERS4386000 (Aeromonas veronii 2); ERS4385996 (Aeromonas caviae); ERS4385998 (Chryseobacterium massiliae); ERS4385999 (Phyllobacterium myrsinacearum); ERS4406247 (Pseudomonas sediminis); ERS4385994 (Pseudomonas mossellii) ERS4386001 (Pseudomonas nitroreducens); ERS4385997 (Pseudomonas peli); ERS4385995 (Stenotrophomas maltophilia).

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Acknowledgements

We thank Mark McBride, Pierre Boudinot, and Rebecca Stevick for critical reading of the manuscript. We are grateful to the late Covadonga Arias for the gift of F. columnare ALG 00–530, to Mark McBride for F. columnare C#2 strain and to Jean-François Bernardet for all other F. columnare strains. Prof. Annemarie Meijer (Leiden University) kindly provided the myd88 mutant zebrafish line. We thank Chloé Baron for her help, Julien Burlaud-Gaillard and Rustem Uzbekov from the IBiSA Microscopy facility, Tours University, France and the following zebrafish facility teams for providing eggs: José Perez and Yohann Rolin (Institut Pasteur), Nadia Soussi-Yanicostas (INSERM Robert Debré), Sylvie Schneider-Manoury and Isabelle Anselme (UMR7622, University Paris 6) and Frédéric Sohm (AMAGEN Gif-sur-Yvette).

Funding

This work was supported by the Institut Pasteur, the French Government’s Investissement d’Avenir program: Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (grant no. ANR-10-LABX-62-IBEID to  J-MG.), the Fondation pour la Recherche Médicale (grant no. DEQ20180339185 to  J-MG). FS was the recipient of a post-doctoral Marie Curie fellowship from the EU-FP7 program, JBB was the recipient of a long-term post-doctoral fellowship from the Federation of European Biochemical Societies (FEBS) and by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 842629. DP-P was supported by an Institut Carnot MS Postdoctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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FAS, JBB, DP-P, J-PL, and J-MG designed the experiments. OR contributed to the initial experiments. VB and J-PL provided zebrafish material and advice. FAS, JBB, DP-P, BA, VB, and J-PL performed the experiments. SB, SH performed bacterial genome sequencing and analysis, AG, SV, FAS, and DP-P performed the bioinformatic and sequence analyses. FAS, JBB, DP-P, J-PL, and J-MG analysed the data and wrote the paper with significant contribution from OR and ED.

Corresponding author

Correspondence to Jean-Marc Ghigo.

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Conflict of interest

The authors of this manuscript have the following conflict of interest: a provisional patent application has been filed: “bacterial strains for use as probiotics, compositions thereof, deposited strains and method to identify probiotic bacterial strains” by J-MG, FAS, DP-P, and JBB The other authors declare no conflict of interest in relation to the submitted work.

Ethics

All animal experiments described in the present study were conducted at the Institut Pasteur (larvae) or at INRA Jouy-en-Josas (adults) according to European Union guidelines for handling of laboratory animals (http://ec.europa.eu/environment/chemicals/lab_animals/home_en.htm) and were approved by the relevant institutional Animal Health and Care Committees.

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Stressmann, F.A., Bernal-Bayard, J., Perez-Pascual, D. et al. Mining zebrafish microbiota reveals key community-level resistance against fish pathogen infection. ISME J (2020). https://doi.org/10.1038/s41396-020-00807-8

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