Spatial heterogeneity and co-occurrence patterns of human mucosal-associated intestinal microbiota

Abstract

Human gut microbiota shows high inter-subject variations, but the actual spatial distribution and co-occurrence patterns of gut mucosa microbiota that occur within a healthy human instestinal tract remain poorly understood. In this study, we illustrated a model of this mucosa bacterial communities' biogeography, based on the largest data set so far, obtained via 454-pyrosequencing of bacterial 16S rDNAs associated with 77 matched biopsy tissue samples taken from terminal ileum, ileocecal valve, ascending colon, transverse colon, descending colon, sigmoid colon and rectum of 11 healthy adult subjects. Borrowing from macro-ecology, we used both Taylor's power law analysis and phylogeny-based beta-diversity metrics to uncover a highly heterogeneous distribution pattern of mucosa microbial inhabitants along the length of the intestinal tract. We then developed a spatial dispersion model with an R-squared value greater than 0.950 to map out the gut mucosa-associated flora's non-linear spatial distribution pattern for 51.60% of the 188 most abundant gut bacterial species. Furthermore, spatial co-occurring network analysis of mucosa microbial inhabitants together with occupancy (that is habitat generalists, specialists and opportunist) analyses implies that ecological relationships (both oppositional and symbiotic) between mucosa microbial inhabitants may be important contributors to the observed spatial heterogeneity of mucosa microbiota along the human intestine and may even potentially be associated with mutual cooperation within and functional stability of the gut ecosystem.

Figure 1

Spatial distribution of microbial composition along the length of intestinal tract. (a) bacteria phyla (mean±S.E.); (b) the three most abundant bacterial genera (mean±S.E.); (c) Graphic illustration of seven sites in the gut that appear healthy.

Figure 2

Heat-map showing spatial distributions of 188 bacterial OTUs (species) with at least 10⁻³ relative abundance per subject. Individual cells are color-coded according to Z-scores to show the normalized abundance of a particular OTU in one site relative to the mean abundance across all seven sites. The relative intensity of the colors indicates how many standard deviations the observed OTU abundance is above or below the mean. All 188 OTUs belong to seven bacterial phyla, mapped left of the heatmap from top to bottom, including Firmicutes, Actinobacteria, Deinococcus-Thermus, Proteobacteria, TM, Fusobacteria and Bacteroidetes. TI, terminal ileum; IV, ileocecal valve; AC, ascending colon; TC, transverse colon; DC, descending colon; SC, sigmoid colon; R, rectum. Note: Heat-map was generated using R.

Figure 3

Community relatedness of human intestinal mucosa-associated microbiota. PCoA analyses were used to compare differences between mucosa-associated bacterial communities found in subjects based on non-phylogenetic beta diversity metrics (binary jaccard distance) (a) and phylogenetic beta diversity metrics (unweighted UniFrac full tree) (b) with 100 Monte Carlo randomizations. Each point corresponds to a sample colored by subjects. C, refers to the cluster analysis of 1  000 bootstrap heuristic searches according to parsimonious criterion, which uses the normalized OTU frequency data set with five ordered character states (0, 1, 2, 3 and 4). Branches were colored by the color of the representative individual of the cluster. TI, terminal ileum; IV, ileocecal valve; AC, ascending colon; TC, transverse colon; DC, descending colon; SC, sigmoid colon; R, rectum.

Figure 4

Summary of ADD modeling results. Within the inner line cycle, all 188 species from 11 subjects were used to analyze the abundance-distance dispersion (ADD) relationships of gut microbiota. The number in parentheses indicates that the ADD relationships in 172 of the total 188 species (91.49%) fit the ADD model (a, species at least shared by six subjects; b, species shared by 2 to 5 subjects; c, species unique to subject). Within the middle dashed cycle, the number in parentheses represents how many species in all 188 species existed in each subject (from S400 to S410). Within the outer dashed cycle, the bold number indicates how many species fit the ADD model in each subject (relative percentage was shown in parentheses); X/Y/Z in parentheses: X represents the ADD model fitted species shared by all seven gut sites, Y represents those shared by two to six gut sites and Z represents those unique to specific gut sites.

Figure 5

Co-occurrence networks of mucosa-associated microbiota along the length of intestinal tract. (a) the network is constructed using 396 spatially co-occurring OTU pairs along the intestine, all with an absolute Pearson's correlation above 0.81 at a 0.05 FDR-corrected significance level. Left panel: Node colors from bright to dark represent degrees from low to high of each node linked by other nodes. The node labels are the OTU codes. The nodes with the most numerous linkers are defined as hub nodes (square): OTU191, OTU318, OTU1369 and OTU1458. Right panel: OTUs are colored by occupancy (that is, generalists, specialists, habitat OTUs between generalists and specialists, as well as opportunists). Habitat generalist OTUs (in red) are defined as locally abundant (at least two sequences) and appearing in all seven sampling sites in at least six subjects. Habitat specialists OTUs (in blue) are defined as locally abundant (at least two sequences) and appearing in any one of seven sites in at least six subjects. Habitat OTUs between generalists and specialists were colored in gold. Habitat opportunists OTUs (in grey) are defined as locally abundant (one sequence) or appearance in any one of seven sites in less than six subjects. IV, ileocecal valve; AC, ascending colon; TC, transverse colon; DC, descending colon; SC, sigmoid colon; R, rectum. (b) two highly connected microbial clusters (modules). OTUs are colored by occupancy (that is, generalists, specialists and habitat OTUs between generalists and specialists). Edges are colored by positive or negative correlations as described in the left panel of (a). Inter-module interaction was implicated by edges with double-headed arrows. (c) spatial abundance distribution of the 11 bacterial OTUs within two modules (Figure b) as well as quantitative assessments (b-values) of their spatial heterogeneities. b>1 indicates a high heterogeneity in the population spatial distribution that signifies departure from random distribution.