Alteration of the rat cecal microbiome during colonization with the helminth Hymenolepis diminuta

Abstract

The microbiome is now widely recognized as being important in health and disease, and makes up a substantial subset of the biome within the ecosystem of the vertebrate body. At the same time, multicellular, eukaryotic organisms such as helminths are being recognized as an important component of the biome that shaped the evolution of our genes. The absence of these macroscopic organisms during the early development and life of humans in Western culture probably leads to a wide range of human immunological diseases. However, the interaction between the microbiome and macroscopic components of the biome remains poorly characterized. In this study, the microbiome of the cecum in rats colonized for 2 generations with the small intestinal helminth Hymenolepis diminuta was evaluated. The introduction of this benign helminth, which is of considerable therapeutic interest, led to several changes in the cecal microbiome. Most of the changes were within the Firmicutes phylum, involved about 20% of the total bacteria, and generally entailed a shift from Bacilli to Clostridia species in the presence of the helminth. The results point toward ecological relationships between various components of the biome, with the observed shifts in the microbiome suggesting potential mechanisms by which this helminth might exert therapeutic effects.

Keywords: ELB, enzymatic lysis buffer; GIT, gastrointestinal tract; Hymenolepis; LEfSe, Linear discriminant analysis Effect Size; OTU, operational taxonomic unit; PCoA, principle coordinate analysis; QIIME, Quantitative Insights Into Microbial Ecology; clostridia; helminths; microbiome; rat.

Figure 1.

The experimental design. The top portion of the diagram shows the division of animals during the study, and the bottom portion shows the timeline for specific events. Animals colonized with helminths are shown in green throughout the diagram, and green arrows indicate the feeding of helminths to the animals. Forty-two days after colonization of F₀ rats (F0) with helminths or sham (saline), the animals were bred. At the time of weaning (age 21 days), 32 F₁ males (F1) were randomly selected from the offspring, and were colonized with and without helminths as shown. Finally, when the animals reached an average age of 62 days (range 56–74), 96 hours before termination of the experiment, the animals were again divided further and received immune stimulation with LPS or with sham (saline) as shown in the diagram.

Figure 2.

Alpha (top) and β (bottom) diversity measures in animals from Groups S colonized with (n = 8) and without (n = 7) helminths and Group L colonized with (n = 6) and without (n = 7) helminths. Weighted pairwise UniFrac distances were averaged within each group to calculate an average β diversity value (a proxy for inter-individual variation). Analysis by 2-way ANOVA revealed no statistically significant changes in the α diversity. Results from the Simpson diversity metric are shown, and are similar to diversity measures using Chao1, Shannon-Weaver, and Faith's Phylogenetic Diversity indices. Significant changes in the β diversity (UniFrac) were observed for helminth treatment (p = 0.0001) and for the interaction between helminths and LPS treatment (p = 0.047). The means and standard errors are shown. The bars show the result of the post-hoc t-tests (p < 0.0001***; p < 0.005**; p < 0.05*) and indicate that the predominant characteristic associated with the β diversity measure is a relatively large value for the β diversity of animals without helminths in group L.

Figure 3.

Microbiome composition in animals from Groups S colonized with (n = 8) and without (n = 7) helminths and Group L colonized with (n = 6) and without (n = 7) helminths. The composition is based on 16S libraries isolated from digesta taken from the ceca when the animals reached an average age of 62 days (range 56–74 days). Results are shown at the (A) phylum and (B) genus level.

Figure 4.

Bacterial lineages with significantly different representation in rats inoculated with or without helminths in (A) Group S or (B) Group L. The log linear discriminant analysis (LDA) effect size quantifies the degree to which each lineage contributes to the uniqueness of each sample class.

Figure 4.

(Continued)

Figure 5.

Cladograms of bacterial lineages with significantly different representation in rats with or without helminths in (A) Group S or (B) Group L. Lineages on the bacterial trees are color-coded to indicate whether the taxon does (red or green) or does not (yellow) significantly differ between sample classes.

Figure 6.

Bacterial lineages with significantly different representation in rats treated with LPS or without LPS (saline). The results from animals (A) without and (B) with helminths are shown. The log linear discriminant analysis (LDA) effect size quantifies the degree to which each lineage contributes to the uniqueness of each sample class.

Figure 7.

Principle Coordinate Analysis (PCoA) of weighted Unifrac distances between libraries isolated from rats with (red) or without (blue) helminths in (A) Group S or (B) Group L. Each data point represents a library from a single animal (i.e., a single bar in Figure 3). Calculations were performed using the UniFrac method; values are weighted to account for differences in lineage frequencies. The distance between points represents unique branches on a phylogenetic tree (i.e. evolutionary history not shared between libraries in Figure 3), as well as differences in the relative abundance of lineages. Closer points share more branch length and have similar frequencies, while points more distant from one another have more unique or disparate gut microbiomes.