Stochastic changes over time and not founder effects drive cage effects in microbial community assembly in a mouse model

Abstract

Maternal transmission and cage effects are powerful confounding factors in microbiome studies. To assess the consequences of cage microenvironment on the mouse gut microbiome, two groups of germ-free (GF) wild-type (WT) mice, one gavaged with a microbiota harvested from adult WT mice and another allowed to acquire the microbiome from the cage microenvironment, were monitored using Illumina 16S rRNA sequencing over a period of 8 weeks. Our results revealed that cage effects in WT mice moved from GF to specific pathogen free (SPF) conditions take several weeks to develop and are not eliminated by the initial gavage treatment. Initial gavage influenced, but did not eliminate a successional pattern in which Proteobacteria became less abundant over time. An analysis in which 16S rRNA sequences are mapped to the closest sequenced whole genome suggests that the functional potential of microbial genomes changes significantly over time shifting from an emphasis on pathogenesis and motility early in community assembly to metabolic processes at later time points. Functionally, mice allowed to naturally acquire a microbial community from their cage, but not mice gavaged with a common biome, exhibit a cage effect in Dextran Sulfate Sodium-induced inflammation. Our results argue that while there are long-term effects of the founding community, these effects are mitigated by cage microenvironment and successional community assembly over time, which must both be explicitly considered in the interpretation of microbiome mouse experiments.

Figure 1

Microbial community assembly over time after removal from GF conditions. Pie charts display the relative abundance of phyla at each time point. The donor microbiota is shown in the upper right hand corner. n=12 at all time points for both the acquired and gavage groups.

Figure 2

Richness as a function of time after removal from GF conditions for the acquired and gavage groups. The richness value for the donor biota was similar to week 1 values with a richness of 118.8. Values shown are the median of each cage.

Figure 3

(a) Bray–Curtis dissimilarity PCoA at the OTU level showing microbial community shifting over time. Gav: Gavage treatment; Acq: Acquired treatment. (b) Independent PCoA clusters were performed for each time point and are colored by treatment (top panel) and cage (bottom panel).

Figure 4

Cage effects illustrated through the use of Bray–Curtis PCoA performed at the OTU level. Shown for the first 12 PCoA co-ordinates are the P-values from a one-way ANOVA with a fixed factor of cage evaluating the null hypothesis that cage had no effect on the distribution of the co-ordinate.

Figure 5

For the first 20 co-ordinates from a PCoA at the OTU level, P-values from the mixed linear model evaluating the null hypothesis that the fixed effects of time, treatment (gavage vs acquired) and treatment × time interactions had no effect on the co-ordinate shown on the x axis. Dotted line represents P=0.05 significance level.

Figure 6

Relative abundance of genera in (a) gavage and (c) acquired at the 8-week time point broken down by cages. Each bar represents an individual mouse's microbial community before treatment with DSS. Differences in inflammation scores between cages were not significant for the gavage mice (b) but were for the acquired mice (d) with both a parametric one-way ANOVA and a non-parametric Kruskal–Wallis (with the indicated P-values). Error bars represent the s.d. or dispersion from the mean in each set of samples.