Association of the Infant Gut Microbiome with Temperament at Nine Months of Age: A Michigan Cohort Study

Abstract

Though studies in animals and humans link the gut microbiota to brain development and control of behavior, little research has examined this connection in healthy infants. This prospective study could determine associations between infant gut microbiota at 3 months, and infant temperament at 9 months, in a prospective pregnancy cohort (Michigan Archive for Research on Child Health; n = 159). Microbiota profiling with 16S rRNA gene sequencing was conducted on fecal samples obtained at 3 months of age. Based on the relative abundance of gut microbiotas, three groups were identified, and each group was characterized by different microbes. Infant temperament outcomes were reported by mothers using the Infant Behavior Questionnaire-Revised Very Short Form at a mean age of 9.4 months. Fully adjusted multivariate linear regression models showed that certain clusters were associated with higher negative emotionality scores, prominently among infants who had poor vitamin D intake. However, no associations were evident between gut microbiota clusters and temperament scales after FDR correction. After using three differential abundance tools, Firmicutes was associated with higher positive affect/surgency scores, whereas Clostridioides was associated with lower scores. An association between the gut microbiota and early infancy temperament was observed; thus, this study warrants replication, with a particular focus on vitamin D moderation.

Keywords: Bacteroides; diet; gut microbiota; infant; microbiome clustering; neurodevelopment; temperament; vitamin D.

Figure 1

Dirichlet multinomial mixture clustering identified three optimal clusters from 159 fecal samples. (a) The number of clusters (k = 3) was chosen by selecting the minimal Laplace approximation, as per the negative log model’s evidence. (b,c) Boxplot of the alpha diversity (Shannon and Chao 1) distributed between the three clusters. Group differences were tested using Wilcoxon signed-rank test, and p-values were adjusted for multiple testing using Bonferroni. The adjusted p-value < 0.05 was labeled as *, and the adjusted p-value ≥ 0.05 was labeled as NS. (d) Principal component analysis (PCoA) ordinations of variation, based on the Bray–Curtis distance matrix. R² and the p-value were calculated using the univariate PERMANOVA test. (e) Heatmap of relative abundance of the top 10 genera in the three clusters. Within the clusters, samples were ordered in accordance with the relative abundance of the genus, Bacteroides.

Figure 2

Relative abundance of the top four genera that contributed to the clusters; each cluster is shown. Group differences were tested using the Wilcoxon signed–rank test. p-values were adjusted for multiple testing using Bonferroni. The FDR adjusted p-value < 0.05 was labeled as *, and the adjusted p-value ≥ 0.05 was labeled as NS. (a) Boxplot of the relative abundance of Bacteroides in each cluster. (b) Boxplot of the relative abundance of Bifidobacterium in each cluster. (c) Boxplot of the relative abundance of Veillonella in each cluster. (d) Boxplot of the relative abundance of Escherichia-Shigella in each cluster.