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Background: Malnutrition in the first 1000 days can lead to lifelong developmental and growth deficits. Indigenous Mayans in Guatemala have some of the highest rates of malnutrition and stunting in the world. While research suggests an association of gut microbiota composition with early child development (ECD) and growth outcomes in healthy human infants, higher risk populations and the role of early localized gastrointestinal (GI) inflammation have been less studied. The main objective of this research was to determine if diet diversity, fecal microbiome composition, and GI inflammation were associated with infant development scores in a sub-population of Guatemalan infants enrolled in The Saqmolo’ Project. The central hypothesis was more optimal ECD and growth scores would be associated with higher diet diversity, a more mature GI microbiome composition, and lower levels of GI inflammation.
Methods: Endline data were used from the Saqmolo’ Project Microbiome Sub-Study of 11-16-month-old indigenous Mayan Guatemalan infants. The Saqmolo’ Project was a 6-month randomized controlled trial with infants receiving one egg a day compared to standard care controls. Fecal microbiome composition was assessed with 16S rRNA sequencing and QIIME2. Caregiver Reported Early Child Development Instruments (CREDI) long form measured ECD. Anthropometric Z-scores and WHO criteria for stunting were used for growth outcomes. The World Health Organization (WHO) Infant and Young Child Feeding indicators questionnaire measured minimum diet diversity and most frequently consumed milk type with additional questionnaires. Fecal microbiome co-abundance groups were created using guild-based clustering. Aim 1 explored the association of egg intake versus multiple micronutrients powders, diet diversity, and most frequently consumed milk type with fecal microbiome composition and fecal calprotectin (ng/ml). MaAsLin2, DESeq2, and LEfSe were used to assess the relationships between dietary factors with microbiome composition. Fecal calprotectin and dietary associations were evaluated with linear regression. Aim 2 examined the association of fecal microbiome composition with child development metrics using MaAsLin2. Aim 3 explored the association of fecal calprotectin with fecal microbiome composition using MaAsLin2, and ECD and growth metrics using linear regression.
Results: In Aim 1, among 200 infants (mean age 12.7 months, 49% female), microbiome composition differences between egg and control groups were more pronounced in non-breastfed than breastfed infants. LEfSe analysis identified 23 ASVs with higher and four ASVs with lower abundance in non-breastfed infants. Neither egg intake nor minimum diet diversity (MDD) was associated with specific ASVs or co-abundance groups. Fecal calprotectin levels were not significantly associated with treatment (β = -57.85, 95% CI: -119.08 to 98.52, P = 0.281) or MDD (β = -10.28, 95% CI: -187.01 to 71.31, P = 0.337), but were lower in non-breastfed infants (β = -174.70, 95% CI: -314.16 to -35.23, P = 0.014). In Aim 2, CREDI overall (co-abundance group 18: MaAsLin2 coefficient = 1.22, Q = 0.216) and language domain (co-abundance group 18: MaAsLin2 coefficient = 1.95, Q = 0.0226) were positively associated with co-abundance groups in non-breastmilk-fed infants. In Aim 3, fecal calprotectin was associated with higher relative abundance of co-abundance groups 8 (MaAsLin2 coefficient = 0.578, q = 0.105) and 28 (MaAsLin2 coefficient = 0.588, q = 0.245), and lower relative abundance of co-abundance group 1 (MaAsLin2 coefficient = -0.392, q = 0.136). Associations between fecal calprotectin and CREDI scores, Z-scores, or stunting in linear regression models were not significant (all P > 0.05).
Conclusion: The findings underscore the influence of breastfeeding on gut microbiome stability, with non-breastfed infants exhibiting greater microbial shifts in response to egg intervention. The positive association of the co-abundance group with CREDI language scores indicates these microbes could contribute to neurodevelopmental benefits. Localized GI inflammation does not appear to have a significant impact on ECD or growth outcomes, though it was linked to gut microbiome composition.