Impact of demographics on human gut microbial diversity in a US Midwest population

Abstract

The clinical utility of microbiome biomarkers depends on the reliable and reproducible nature of comparative results. Underappreciation of the variation associated with common demographic, health, and behavioral factors may confound associations of interest and generate false positives. Here, we present the Midwestern Reference Panel (MWRP), a resource for comparative gut microbiome studies conducted in the Midwestern United States. We analyzed the relationships between demographic and health behavior-related factors and the microbiota in this cohort, and estimated their effect sizes. Most variables investigated were associated with the gut microbiota. Specifically, body mass index (BMI), race, sex, and alcohol use were significantly associated with microbial β-diversity (P < 0.05, unweighted UniFrac). BMI, race and alcohol use were also significantly associated with microbial α-diversity (P < 0.05, species richness). Tobacco use showed a trend toward association with the microbiota (P < 0.1, unweighted UniFrac). The effect sizes of the associations, as quantified by adjusted R(2) values based on unweighted UniFrac distances, were small (< 1% for all variables), indicating that these factors explain only a small percentage of overall microbiota variability. Nevertheless, the significant associations between these variables and the gut microbiota suggest that they could still be potential confounders in comparative studies and that controlling for these variables in study design, which is the main objective of the MWRP, is important for increasing reproducibility in comparative microbiome studies.

Keywords: Demographics; Effect size; Microbial diversity; Microbiome; Target population.

Figure 1. Gut microbiota profile of the MWRP.

(A) Distribution of OTU abundance. (B) Distribution of OTU prevalence. (C) Relative abundance of major bacteria at the level of the phylum. (D) Relative abundance of major bacteria at the level of the family.

Figure 2. Associations between demographic and health behavior-related factors and the overall gut microbiota structure.

(A) Increased BMI is associated with decreased species richness (i.e., the observed number of OTUs). (B) Increased species richness was observed in white subjects. The three horizontal lines of the box represent the first, second (median), and third quartiles, respectively, with the whisk extending to the 1.5 interquartile range (IQR). (C) Principal coordinate analysis (PCoA) plot showing a sex effect. (D) PCoA plot showing an alcohol effect. Samples are colored according to group membership, and plotted on axes corresponding the first two principal coordinates (PCs). The percentage of variability explained by each PC is indicated in the parentheses.

Figure 3. Percentage of variability explained by the demographic and health behavior-related factors.

The unweighted UniFrac distance was used to summarize the microbiota variability. The distance-based R² was adjusted to reduce inflation due to a small sample size.

Figure 4. Microbial signatures of demographic and health behavior-related factors: BMI, race, sex, and alcohol use.

Barplots show the mean relative abundance and standard error of bacterial taxa in each subject subgroup. Taxa were selected based on univariate association tests with an FDR of 10%. Here, we have discretized BMI into normal and obese groups based on the cutoff of 30 kg/m² for visualization purposes. However, we treated BMI as a continuous outcome in our association tests.