There is increasing evidence from pre-clinical and human studies implicating the microbiota–gut–brain axis in behavior and sleep physiology. Infancy is a critical time period for brain development and is vulnerable to the harmful effects of gut dysbiosis. Thus, it is crucial to understand how gut microbial colonization during this period may influence behavior and sleep physiology in the later stages of life.We analyzed data from 194 mother-infant pairs from the Michigan Archive for Research on Child Health (MARCH) cohort Study. Clinical and demographic information was obtained from the birth certificate and interview during pregnancy and childhood. Fecal samples from infants at 3-9 months of age were sequenced at the V4 region of the 16S rRNA gene. In the first study, which examined whether feeding practices may affect early gut microbial colonization, we found that the gut microbiota of infants who were exclusively breastfed displayed a significantly lower Shannon diversity (p-adjust < 0.001) and a different gut microbiota composition than infants who were not breastfed (p-value = 0.001). Among the exclusively breastfed infants, recipients of supplemental vitamin D displayed a significantly lower Shannon diversity (p-adjust = 0.007) and different gut microbiota composition structure than non-supplemented, breastfed infants (p-value = 0.02). In addition, several individual taxa were identified to be associated with different feeding practices. In the second study, we examined whether gut microbiota in early infancy was associated with temperament in the nine-month-old infants. We identified that a microbial cluster characterized by a higher abundance of Bifidobacterium, Veillonella, and Escherichia-Shigella that was associated with lower emotionality scores (coefficient = -0.58, p-value = 0.02) compared to a cluster characterized by a higher abundance of Bacteroides. This association was especially prominent among infants who were not supplemented with vitamin D (coefficient = -1.01, p-value = 0.01), while no significant association was found among infants who were supplemented (coefficient = -0.43, p-value = 0.20). In the final aim, we assessed the association between gut microbiota in early infancy and the difficulty of initiating and maintaining sleep at age of two years. The gut microbiota of children who had difficulty maintaining sleep displayed significantly higher Shannon index (OR: 2.41, 95% CI= 1.23-4.93, p-adjust < 0.04) and Chao 1 index (OR: 1.01, 95% CI= 1.0-1.03, p-adjust < 0.008) after adjustment for covariates. We also observed that gut microbiota composition was significantly different between children with difficulty initiating (p-value= 0.043) and maintaining sleep (p-value= 0.004) by PERMANOVA based on the unweighted UniFrac distance metric. In conclusion, these results from analysis in a prospective cohort study suggest that early gut microbial colonization is shaped by breastfeeding status, vitamin D supplement, and maternal characteristics including gestational age, delivery mode and education level. Our findings suggested that the infant gut microbiome clusters may be associated with the temperament characteristic of negative emotionality in 9-month-old infants. We also demonstrated a significant association between infant gut microbiome composition and sleep problems in 2-year-old children. Thus, our results add to the evidence that early gut microbial colonization may be linked with brain outcomes with potential long-term effects.
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