Bacteria from diverse habitats colonize and compete in the mouse gut

Abstract

To study how microbes establish themselves in a mammalian gut environment, we colonized germ-free mice with microbial communities from human, zebrafish, and termite guts, human skin and tongue, soil, and estuarine microbial mats. Bacteria from these foreign environments colonized and persisted in the mouse gut; their capacity to metabolize dietary and host carbohydrates and bile acids correlated with colonization success. Cohousing mice harboring these xenomicrobiota or a mouse cecal microbiota, along with germ-free "bystanders," revealed the success of particular bacterial taxa in invading guts with established communities and empty gut habitats. Unanticipated patterns of ecological succession were observed; for example, a soil-derived bacterium dominated even in the presence of bacteria from other gut communities (zebrafish and termite), and human-derived bacteria colonized germ-free bystander mice before mouse-derived organisms. This approach can be generalized to address a variety of mechanistic questions about succession, including succession in the context of microbiota-directed therapeutics.

Figure 1. Design of xenomicrobiota transplant experiments

(A,B) Summary of Stages 1–5. See text for details. Abbreviations: ‘m’, mouse cecal microbiota; ‘z’, zebrafish gut microbiota; ‘t’, termite hindgut microbiota; ‘s’, soil.

Figure 2. Comparison between input xenomicrobiota and gut-selected communities in gnotobiotic mice from Stages 1 and 2

(A) Pairwise unweighted UniFrac distances. Abbreviations: input, input community introduced into mice by gavage; output, fecal samples collected from mice harboring transplanted microbiota. (B) Analysis of the fecal output communities collected from mice in the Stage 1 transplant experiments shows that, despite the highly dissimilar input communities, the output communities cluster together, systematically excluding clades that fare poorly in the mouse gut. The large, gray phylogenetic tree in the upper left shows all of the 97%ID OTUs (collapsed into wedges at different taxonomical levels based on relative abundance) that are present in all samples collected from input and output communities. The numbers in parentheses next to each wedge indicate the percentage of the 97%ID OTUs collapsed into that wedge that were assigned to the specified taxonomy, i.e., 91% of the branches from the large clade at the top were assigned to the order Clostridiales, and 9% were assigned to other taxonomic groups. The smaller trees surrounding the network represent the input source (indicated by a triangle) and output fecal communities of mice (indicated by a square) at the end of Stage 1 (28 days after gavage). All smaller phylogenetic trees are formatted identically to the schematic tree; therefore, each branch corresponds to the taxon indicated in the schematic. Each wedge is colored if taxa from that clade were present in the corresponding community. The coloring for each tree is normalized to the relative abundance of OTUs for each source (i.e. darker colors represent taxa that were in higher relative abundance in a particular input or output community). Each phylogenetic tree is connected by a dotted line to the corresponding nodes within the network. In the network, the nodes represent genus-level OTUs and are connected by edges to either input communities (represented by triangles), output communities (represented by squares), or to both. The network is constructed to minimize the spring forces over all nodes and therefore to bring communities sharing more genera together. Each community’s nodes and edges in the network are uniquely colored to match their corresponding phylogenetic tree.

Figure 3. Correlations between fecal microbial community biomass and the functional properties of selected xenomicrobiota

(A) Fecal DNA concentrations (a proxy for microbial biomass) from mice harboring different microbiota, defined at the end of Stage 1. Mean values±SD are presented, with significant differences between bars denoted by different letters (P < 0.05; ANOVA; Tukey’s correction for multiple hypotheses). (B) Heatmap showing the normalized abundance (z-score) for CAZy-annotated glycoside hydrolases and polysaccharide lyases as determined by shotgun sequencing of Stage 1 output fecal microbiomes sampled 28 days after gavage. (C) Targeted and non-targeted GC-MS of the concentrations of SCFA and carbohydrates, respectively, in cecal contents obtained at sacrifice from mice harboring the indicated selected microbiota. Arrows at the end of each row indicate if the CAZyme or metabolite is significantly positively (green arrow) or negatively (red arrow) correlated with fecal DNA concentration (Pearson’s correlation, adjusted with Benjamini-Hochberg correction, P<0.05).

Figure 4. UPLC-MS of bile acids

(A) Analysis of cecal samples collected at the end of Stage 2 from mice harboring selected xenomicrobiota from zebrafish gut, termite hindgut or soil, or the cecal microbial community of conventionally-raised mice. *, P< 0.05 compared to CONV-D animals, as measured by two-way ANOVA with Holm-Šidák correction for multiple hypotheses. (B) Cecal and (C) ileal bile acids from samples collected at the end of Stage 5B from co-housed animals, and from control non-cohoused Stage 5A mice harboring a selected composite human fecal xenomicrobiota or a composite microbiota from conventionally-raised C57BL/6J and FVB/N mice (abbreviated CONV-D). Mean values±SEM are presented. *, P< 0.05 compared to CONV-D animals (two-way ANOVA with Holm-Šidák correction for multiple hypotheses). Also see Table S5C,D.

Figure 5. Analysis of ecological invasion in Stage 3 and 4 co-housing experiments

In Stage 3 experiments, the GF mouse was co-housed with three mice transferred from Stage 2; one with selected zebrafish gut xenomicrobiota, the other with a selected termite hindgut xenomicrobiota, and the third with a selected soil community. During the Stage 4 experiments, an ex-GF mouse from Stage 3 that had acquired a composite xenomicrobiota was co-housed with a CONV-D mouse. (A) The proportions of the different xenomicrobiota sources represented in the microbiota of the GF bystander over time defined using Microbial SourceTracker. Mean values±SD are presented. (B) Indicator species analysis identified bacterial 97%ID OTUs representative of the selected soil, termite hindgut, zebrafish hindgut, and mouse cecal microbiota at the end of Stage 2. The heatmap shows the mean relative abundances of these OTUs in the fecal microbiota of each group of mice at each sampling time in Stages 2–4.

Figure 6. Analysis of ecological invasion in Stage 5B co-housing experiments involving mice with selected composite human fecal microbiota, a composite mouse cecal microbiota, and GF bystanders

Microbial SourceTracker was used to estimate the proportions of human-derived and mouse-derived bacteria (mean values±SD) in (A) the GF bystander, (B) the mouse harboring a composite human fecal microbiota, and (C) the mouse harboring a composite mouse cecal community throughout the Stage 5B co-housing experiment. (D) The heatmap presents the mean percent relative abundances of mouse indicative and human indicative 97%ID OTUs in fecal samples collected from cagemates at the time points shown.