The microbial communities resident in animal intestines are composed of multiple species that together play important roles in host development, health, and disease. Due to the complexity of these communities and the difficulty of characterizing them in situ, the determinants of microbial composition remain largely unknown. Further, it is unclear for many multispecies consortia whether their species-level makeup can be predicted based on an understanding of pairwise species interactions or whether higher-order interactions are needed to explain emergent compositions. To address this, we examine commensal intestinal microbes in larval zebrafish, initially raised germfree, to allow the introduction of controlled combinations of bacterial species. Using a dissection and plating assay, we demonstrate the construction of communities of one to five bacterial species and show that the outcomes from the two-species competitions fail to predict species abundances in more complex communities. With multiple species present, interbacterial interactions become weaker, suggesting that higher-order interactions in the vertebrate gut stabilize complex communities.
IMPORTANCE Understanding the rules governing the composition of the diverse microbial communities that reside in the vertebrate gut environment will enhance our ability to manipulate such communities for therapeutic ends. Synthetic microbial communities, assembled from specific combinations of microbial species in germfree animals, allow investigation of the fundamental question of whether multispecies community composition can be predicted solely based on the combined effects of interactions between pairs of species. If so, such predictability would enable the construction of communities with desired species from the bottom up. If not, the apparent higher-order interactions imply that emergent community-level characteristics are crucial. Our findings using up to five coexisting native bacterial species in larval zebrafish, a model vertebrate, provide experimental evidence for higher-order interactions and, moreover, show that these interactions promote the coexistence of microbial species in the gut.