Stool microbiome, pH and short/branched chain fatty acids in infants receiving extensively hydrolyzed formula, amino acid formula, or human milk through two months of age

Abstract

Background: Early infant feeding with intact or extensively hydrolyzed (EH) proteins or free amino acids (AA) may differentially affect intestinal microbiota composition and immune reactivity. This multicenter, double-blind, controlled, parallel-group, pilot study compared stool microbiota from Baseline (1-7 days of age) up to 60 days of age in healthy term infants who received mother's own milk (assigned to human milk [HM] reference group) (n = 25) or were randomized to receive one of two infant formulas: AA-based (AAF; n = 25) or EH cow's milk protein (EHF; n = 28). Stool samples were collected (Baseline, Day 30, Day 60) and 16S rRNA genes were sequenced. Alpha (Shannon, Simpson, Chao1) and beta diversity (Bray Curtis) were analyzed. Relative taxonomic enrichment and fold changes were analyzed (Wilcoxon, DESEq2). Short/branched chain fatty acids (S/BCFA) were quantified by gas chromatography. Mean S/BCFA and pH were analyzed (repeated measures ANOVA).

Results: At baseline, alpha diversity measures were similar among all groups; however, both study formula groups were significantly higher versus the HM group by Day 60. Significant group differences in beta diversity at Day 60 were also detected, and study formula groups were compositionally more similar compared to HM. The relative abundance of Bifidobacterium increased over time and was significantly enriched at Day 60 in the HM group. In contrast, a significant increase in members of Firmicutes for study formula groups were detected at Day 60 along with butyrate-producing species in the EHF group. Stool pH was significantly higher in the AAF group at Days 30 and 60. Butyrate increased significantly from Baseline to Day 60 in the EHF group and was significantly higher in study formula groups vs HM at Day 60. Propionate was also significantly higher for EHF and AAF at Day 30 and AAF at Day 60 vs HM. Total and individual BCFA were higher for AAF and EHF groups vs HM through Day 60.

Conclusions: Distinct patterns of early neonatal microbiome, pH, and microbial metabolites were demonstrated for infants receiving mother's own milk compared to AA-based or extensively hydrolyzed protein formula. Providing different sources of dietary protein early in life may influence gut microbiota and metabolites.

Trial registration: ClinicalTrials.gov Identifier: NCT02500563 . Registered July 28, 2015.

Keywords: Bifidobacterium; Infant formula; Infant microbiota; Short chain fatty acids.

Fig. 1

Timeline and participant flow diagram. a Timeline for the entire study duration. Fecal samples were collected based on specified days (infant’s age) as stated for each visit. Baseline samples were collected after meconium. b Flow chart describing subject exclusion and participation. Subjects who did not provide a sample or did not complete all 3 visits were excluded from the final analysis

Fig. 2

Alpha and beta diversity measurements of the infant microbiome across feeding groups. a Pairwise comparisons of alpha diversity indices (Shannon, Simpson, Chao1) were carried out between visits to examine specific comparisons of interest in each feeding group; amino acid (AAF; red), extensively hydrolyzed formula (EHF; green) and human milk (HM; blue). Boxplots show the median, first and third quartiles and the whiskers extend out to the 1.5 x IQR (Interquartile Range) of the upper and lower limit. * indicates significant difference between a pairwise comparison of group-visit combinations (FDR < 0.05). ‡ and † indicates that at baseline and day 60 respectively, the HM group was significantly different compared to the AA and EHF groups (FDR < 0.05). ○ represents outliers. b Comparison of community profiles at the ASV level between feeding groups at day 60. Principle coordinates analysis (PCoA) using Bray Curtis distance was carried out followed by PERMANOVA analysis (p = 0.001)

Fig. 3

Heat trees comparing the relative abundances of taxa between a, c, e baseline and day 30 and b, d, f baseline and day 60 in the a. b amino acid (AAF), c, d extensively hydrolysed formula (EHF) and e,f human milk (HM) groups. Taxa in orange are enriched at day 30 or day 60 while taxa in purple are enriched at baseline. Size of nodes correspond to the number of genera and color intensity corresponds to proportions. Taxonomic log2 fold change is calculated using median proportions. Only taxa that were significantly different between visits, as determined through Wilcoxon signed rank test (FDR < 0.05) are shown. Taxonomic tree in g acts as a reference and represents all genera present in the samples

Fig. 4

DESeq2 analysis of differentially abundant ASVs between baseline and day 60 for each feeding group; the a amino acid (AAF), b extensively hydrolysed formula (EHF) and c human milk (HM) groups. Shrinkage estimations of log2 fold change values for each ASV were computed and each point represents an ASV that was significantly different (FDR < 0.05). Each color represents a phylum; Actinobacteria (red), Bacteroidetes (grey), Firmicutes (yellow), Proteobacteria (blue) and Verrucomicrobia (green)

Fig. 5

Differential heat trees based on pairwise comparisons of taxonomic relative abundance between feeding groups at day 60; a human milk (HM; yellow) vs amino acid (AA; purple), b extensively hydrolyzed formula (EHF; yellow) vs amino acid (AAF; purple) and c human milk (HM; yellow) vs extensively hydrolyzed formula (EHF; purple). Taxonomic tree in d acts as a reference and represents all genera present in the day 60 samples. Taxonomic log2 fold change is calculated using median proportions. All colored taxa are significantly different between feeding groups (Wilcoxon rank sum test; FDR p < 0.05). Size of nodes correspond to the number of genera and color intensity corresponds to proportions

Fig. 6

pH and S/BCFA analysis of stool samples. For the different feeding groups; amino acid (AAF; red), extensively hydrolyzed formula (EHF; green) and human milk (HM; blue), a pH, b acetate, c butyrate, d propionate, e total SCFA and f total BCFA were measured at each visit. A repeated measure analysis of variance (ANOVA) using a Toeplitz covariance structure was used with Bonferroni’s post-hoc test. Each point represents the mean and error bars represent upper and lower limits at a 95% confidence interval. * represents significant difference between visits for AAF in a pH and d propionate and EHF in c butyrate (p < 0.05). Points that do not share the same letter(s) at a given visit are significantly different from each other (p < 0.05)