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.
Subscribe to Journal
Get full journal access for 1 year
only $41.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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).
Rolig AS, Parthasarathy R, Burns AR, Bohannan BJ, Guillemin K. Individual members of the microbiota disproportionately modulate host innate immune responses. Cell Host Microbe. 2015;18:613–20.
McFall-Ngai MJ. Unseen forces: the influence of bacteria on animal development. Dev Biol. 2002;242:1–14.
van der Waaij D, Berghuis-de Vries JM, Lekkerkerk-van der Wees JEC. Colonization resistance of the digestive tract and the spread of bacteria to the lymphatic organs in mice. J Hyg. 1972;70:335–42.
Cani PD, Delzenne NM. The role of the gut microbiota in energy metabolism and metabolic disease. Curr Pharm Des. 2009;15:1546–58.
Stecher B, Hardt WD. The role of microbiota in infectious disease. Trends Microbiol. 2008;16:107–14.
Stecher B, Hardt WD. Mechanisms controlling pathogen colonization of the gut. Curr Opin Microbiol. 2011;14:82–91.
Falcinelli S, Rodiles A, Unniappan S, Picchietti S, Gioacchini G, Merrifield DL, et al. Probiotic treatment reduces appetite and glucose level in the zebrafish model. Sci Rep. 2016;6:18061.
Olsan EE, Byndloss MX, Faber F, Rivera-Chavez F, Tsolis RM, Baumler AJ. Colonization resistance: The deconvolution of a complex trait. J Biol Chem. 2017;292:8577–81.
Littman DR, Pamer EG. Role of the commensal microbiota in normal and pathogenic host immune responses. Cell Host Microbe. 2011;10:311–23.
Heselmans M, Reid G, Akkermans LM, Savelkoul H, Timmerman H, Rombouts FM. Gut flora in health and disease: potential role of probiotics. Curr Issues Intest Microbiol. 2005;6:1–7.
Boirivant M, Strober W. The mechanism of action of probiotics. Curr Opin Gastroenterol. 2007;23:679–92.
Robinson CJ, Bohannan BJ, Young VB. From structure to function: the ecology of host-associated microbial communities. Microbiol Mol Biol Rev. 2010;74:453–76.
Vollaard EJ, Clasener HA. Colonization resistance. Antimicrob Agents Chemother. 1994;38:409–14.
Gill HS. Probiotics to enhance anti-infective defences in the gastrointestinal tract. Best Pr Res Clin Gastroenterol. 2003;17:755–73.
Rawls JF, Samuel BS, Gordon JI. Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota. Proc Natl Acad Sci USA. 2004;101:4596–601.
Milligan-Myhre K, Charette JR, Phennicie RT, Stephens WZ, Rawls JF, Guillemin K, et al. Study of host-microbe interactions in zebrafish. Methods cell Biol. 2011;105:87–116.
Burns AR, Guillemin K. The scales of the zebrafish: host-microbiota interactions from proteins to populations. Curr Opin Microbiol. 2017;38:137–41.
Douglas AE. Simple animal models for microbiome research. Nat Rev Microbiol. 2019;17:764–75.
Flores EM, Nguyen AT, Odem MA, Eisenhoffer GT, Krachler AM. The zebrafish as a model for gastrointestinal tract-microbe interactions. Cell Microbiol. 2020;22:e13152.
Melancon E, Gomez De La Torre Canny S, Sichel S, Kelly M, Wiles TJ, Rawls JF, et al. Best practices for germ-free derivation and gnotobiotic zebrafish husbandry. Methods cell Biol. 2017;138:61–100.
Cantas L, Sorby JR, Alestrom P, Sorum H. Culturable gut microbiota diversity in zebrafish. Zebrafish. 2012;9:26–37.
Rendueles O, Ferrieres L, Fretaud M, Begaud E, Herbomel P, Levraud JP, et al. A new zebrafish model of oro-intestinal pathogen colonization reveals a key role for adhesion in protection by probiotic bacteria. PLoS Pathog. 2012;8:e1002815.
Caruffo M, Navarrete NC, Salgado OA, Faundez NB, Gajardo MC, Feijoo CG, et al. Protective Yeasts Control V. anguillarum Pathogenicity and Modulate the Innate Immune Response of Challenged Zebrafish (Danio rerio) Larvae. Front Cell Infect Microbiol. 2016;6:127.
Perez-Ramos A, Mohedano ML, Pardo MA, Lopez P. Beta-glucan-producing pediococcus parvulus 2.6: test of probiotic and immunomodulatory properties in zebrafish models. Front Microbiol. 2018;9:1684.
Chu W, Zhou S, Zhu W, Zhuang X. Quorum quenching bacteria Bacillus sp. QSI-1 protect zebrafish (Danio rerio) from Aeromonas hydrophila infection. Sci Rep. 2014;4:5446.
Wang Y, Ren Z, Fu L, Su X. Two highly adhesive lactic acid bacteria strains are protective in zebrafish infected with Aeromonas hydrophila by evocation of gut mucosal immunity. J Appl Microbiol. 2016;120:441–51.
Qin C, Zhang Z, Wang Y, Li S, Ran C, Hu J, et al. EPSP of L. casei BL23 Protected against the Infection Caused by Aeromonas veronii via Enhancement of Immune Response in Zebrafish. Front Microbiol. 2017;8:2406.
Girija V, Malaikozhundan B, Vaseeharan B, Vijayakumar S, Gobi N, Del Valle Herrera M, et al. In vitro antagonistic activity and the protective effect of probiotic Bacillus licheniformis Dahb1 in zebrafish challenged with GFP tagged Vibrio parahaemolyticus Dahv2. Microb Pathogenesis. 2018;114:274–80.
Lin YS, Saputra F, Chen YC, Hu SY. Dietary administration of Bacillus amyloliquefaciens R8 reduces hepatic oxidative stress and enhances nutrient metabolism and immunity against Aeromonas hydrophila and Streptococcus agalactiae in zebrafish (Danio rerio). Fish Shellfish Immunol. 2019;86:410–9.
Declercq AM, Haesebrouck F, Van den Broeck W, Bossier P, Decostere A. Columnaris disease in fish: a review with emphasis on bacterium-host interactions. Vet Res. 2013;44:27.
Decostere A, Haesebrouck F, Devriese LA. Characterization of four Flavobacterium columnare (Flexibacter columnaris) strains isolated from tropical fish. Vet Microbiol. 1998;62:35–45.
Figueiredo HC, Klesius PH, Arias CR, Evans J, Shoemaker CA, Pereira DJ Jr, et al. Isolation and characterization of strains of Flavobacterium columnare from Brazil. J fish Dis. 2005;28:199–204.
Soto E, Mauel MJ, Karsi A, Lawrence ML. Genetic and virulence characterization of Flavobacterium columnare from channel catfish (Ictalurus punctatus). J Appl Microbiol. 2008;104:1302–10.
Suomalainen LR, Bandilla M, Valtonen ET. Immunostimulants in prevention of columnaris disease of rainbow trout, Oncorhynchus mykiss (Walbaum). J fish Dis. 2009;32:723–6.
Pacha RE, Ordal EJ Myxobacterial infections of salmonids. American Fisheries Society, Diseases of Fishes and Shellfishes 1970:12.
Amin NE, Abdallah IS, Faisal M, Easa Me-S, Alaway T, Alyan SA. Columnaris infection among cultured Nile tilapia Oreochromis niloticus. Antonie Van Leeuwenhoek. 1988;54:509–20.
Decostere A, Haesebrouck F, Charlier G, Ducatelle R. The association of Flavobacterium columnare strains of high and low virulence with gill tissue of black mollies (Poecilia sphenops). Vet Microbiol. 1999;67:287–98.
Bernardet J-F, Bowman JP. The genus flavobacterium. Prokaryotes. 2006;7:481–531.
Li N, Zhu Y, LaFrentz BR, Evenhuis JP, Hunnicutt DW, Conrad RA, et al. The type IX secretion system is required for virulence of the fish pathogen flavobacterium columnare. Appl Environ Microbiol. 2017;83:e01769–17.
Garcia JC, LaFrentz BR, Waldbieser GC, Wong FS, Chang SF. Characterization of atypical Flavobacterium columnare and identification of a new genomovar. J Fish Dis. 2018;41:1159–64.
van der Vaart M, van Soest JJ, Spaink HP, Meijer AH. Functional analysis of a zebrafish myd88 mutant identifies key transcriptional components of the innate immune system. Dis Models Mech. 2013;6:841–54.
Pham LN, Kanther M, Semova I, Rawls JF. Methods for generating and colonizing gnotobiotic zebrafish. Nat Protoc. 2008;3:1862–75.
Lemon KP, Armitage GC, Relman DA, Fischbach MA. Microbiota-targeted therapies: an ecological perspective. Sci Transl Med. 2012;4:137rv5.
Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinforma. 2014;30:2114–20.
Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc, Ser B,57, 289-300.1995;57:289–300.
Ha SM, Kim CK, Roh J, Byun JH, Yang SJ, Choi SB, et al. Application of the whole genome-based bacterial identification system, TrueBac ID, using clinical isolates that were not identified with three matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems. Ann Lab Med. 2019;39:530–6.
Volant S, Lechat P, Woringer P, Motreff L, Campagne P, Malabat C, et al. SHAMAN: a user-friendly website for metataxonomic analysis from raw reads to statistical analysis. BMC Bioinforma. 2020;21:345.
Rognes T, Flouri T, Nichols B, Quince C, Mahe F. VSEARCH: a versatile open source tool for metagenomics. PeerJ. 2016;4:e2584.
Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2012;41:D590–D6.
Yarza P, Yilmaz P, Pruesse E, Glockner FO, Ludwig W, Schleifer KH, et al. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol. 2014;12:635–45.
Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.
Olivares-Fuster O, Bullard SA, McElwain A, Llosa MJ, Arias CR. Adhesion dynamics of Flavobacterium columnare to channel catfish Ictalurus punctatus and zebrafish Danio rerio after immersion challenge. Dis Aquat Org. 2011;96:221–7.
Cheesman SE, Neal JT, Mittge E, Seredick BM, Guillemin K. Epithelial cell proliferation in the developing zebrafish intestine is regulated by the Wnt pathway and microbial signaling via Myd88. Proc Natl Acad Sci USA. 2011;108:4570–7.
Stephens WZ, Burns AR, Stagaman K, Wong S, Rawls JF, Guillemin K, et al. The composition of the zebrafish intestinal microbial community varies across development. ISME J. 2016;10:644–54.
Mena KD, Gerba CP. Risk assessment of pseudomonas aeruginosa in water. Rev Environ contamination Toxicol. 2009;201:71–115.
Goncalves Pessoa RB, de Oliveira WF, Marques DSC, Dos Santos Correia MT, de Carvalho E, Coelho L. The genus Aeromonas: a general approach. Microb pathogenesis. 2019;130:81–94.
Russell AB, Wexler AG, Harding BN, Whitney JC, Bohn AJ, Goo YA, et al. A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism. Cell Host Microbe. 2014;16:227–36.
Chatzidaki-Livanis M, Coyne MJ, Comstock LE. An antimicrobial protein of the gut symbiont Bacteroides fragilis with a MACPF domain of host immune proteins. Mol Microbiol. 2014;94:1361–74.
Roelofs KG, Coyne MJ, Gentyala RR, Chatzidaki-Livanis M, Comstock LE. Bacteroidales secreted antimicrobial proteins target surface molecules necessary for gut colonization and mediate competition in vivo. mBio. 2016;7:e01055–16.
Jacobson A, Lam L, Rajendram M, Tamburini F, Honeycutt J, Pham T, et al. A gut commensal-produced metabolite mediates colonization resistance to salmonella infection. Cell host microbe. 2018;24:296–307.e7.
Larsbrink J, McKee LS. Bacteroidetes bacteria in the soil: glycan acquisition, enzyme secretion, and gliding motility. Adv Appl Microbiol. 2020;110:63–98.
Kamada N, Seo SU, Chen GY, Nunez G. Role of the gut microbiota in immunity and inflammatory disease. Nat Rev Immunol. 2013;13:321–35.
Ganz J, Melancon E, Eisen JS. Zebrafish as a model for understanding enteric nervous system interactions in the developing intestinal tract. Methods cell Biol. 2016;134:139–64.
Bates JM, Mittge E, Kuhlman J, Baden KN, Cheesman SE, Guillemin K. Distinct signals from the microbiota promote different aspects of zebrafish gut differentiation. Dev Biol. 2006;297:374–86.
Yan Q, van der Gast CJ, Yu Y. Bacterial community assembly and turnover within the intestines of developing zebrafish. PLoS ONE. 2012;7:e30603.
Costello EK, Stagaman K, Dethlefsen L, Bohannan BJ, Relman DA. The application of ecological theory toward an understanding of the human microbiome. Science. 2012;336:1255–62.
Rogers GB, Hoffman LR, Carroll MP, Bruce KD. Interpreting infective microbiota: the importance of an ecological perspective. Trends Microbiol. 2013;21:271–6.
Burns AR, Stephens WZ, Stagaman K, Wong S, Rawls JF, Guillemin K, et al. Contribution of neutral processes to the assembly of gut microbial communities in the zebrafish over host development. ISME J. 2016;10:655–64.
Hibbing ME, Fuqua C, Parsek MR, Peterson SB. Bacterial competition: surviving and thriving in the microbial jungle. Nat Rev Microbiol. 2010;8:15–25.
Shafquat A, Joice R, Simmons SL, Huttenhower C. Functional and phylogenetic assembly of microbial communities in the human microbiome. Trends Microbiol. 2014;22:261–6.
Shade A, Peter H, Allison SD, Baho DL, Berga M, Burgmann H, et al. Fundamentals of microbial community resistance and resilience. Front Microbiol. 2012;3:417.
Willing BP, Russell SL, Finlay BB. Shifting the balance: antibiotic effects on host-microbiota mutualism. Nat Rev Microbiol. 2011;9:233–43.
Brugman S, Liu KY, Lindenbergh-Kortleve D, Samsom JN, Furuta GT, Renshaw SA, et al. Oxazolone-induced enterocolitis in zebrafish depends on the composition of the intestinal microbiota. Gastroenterology. 2009;137:1757–67.e1.
Salonen A, Nikkila J, Jalanka-Tuovinen J, Immonen O, Rajilic-Stojanovic M, Kekkonen RA, et al. Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis. J Microbiol Methods. 2010;81:127–34.
Kunin V, Engelbrektson A, Ochman H, Hugenholtz P. Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates. Environ Microbiol. 2010;12:118–23.
Shabgah AG, Navashenaq JG, Shabgah OG, Mohammadi H, Sahebkar A. Interleukin-22 in human inflammatory diseases and viral infections. Autoimmun Rev. 2017;16:1209–18.
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).
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.
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.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
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