Impact of Early Feeding: Metagenomics Analysis of the Infant Gut Microbiome

Matthew D Di Guglielmo, Karl R Franke, Alan Robbins, Erin L Crowgey, Matthew D Di Guglielmo, Karl R Franke, Alan Robbins, Erin L Crowgey

Abstract

Background: Different feeding regimens in infancy alter the gastrointestinal (gut) microbial environment. The fecal microbiota in turn influences gastrointestinal homeostasis including metabolism, immune function, and extra-/intra-intestinal signaling. Advances in next generation sequencing (NGS) have enhanced our ability to study the gut microbiome of breast-fed (BF) and formula-fed (FF) infants with a data-driven hypothesis approach.

Methods: Next generation sequencing libraries were constructed from fecal samples of BF (n=24) and FF (n=10) infants and sequenced on an Illumina HiSeq 2500. Taxonomic classification of the NGS data was performed using the Sunbeam/Kraken pipeline and a functional analysis at the gene level was performed using publicly available algorithms, including BLAST, and custom scripts. Differentially represented genera, genes, and NCBI Clusters of Orthologous Genes (COG) were determined between cohorts using count data and R (statistical packages edgeR and DESeq2).

Results: Thirty-nine genera were found to be differentially represented between the BF and FF cohorts (FDR ≤ 0.01) including Parabacteroides, Enterococcus, Haemophilus, Gardnerella, and Staphylococcus. A Welch t-test of the Shannon diversity index for BF and FF samples approached significance (p=0.061). Bray-Curtis and Jaccard distance analyses demonstrated clustering and overlap in each analysis. Sixty COGs were significantly overrepresented and those most significantly represented in BF vs. FF samples showed dichotomy of categories representing gene functions. Over 1,700 genes were found to be differentially represented (abundance) between the BF and FF cohorts.

Conclusions: Fecal samples analyzed from BF and FF infants demonstrated differences in microbiota genera. The BF cohort includes greater presence of beneficial genus Bifidobacterium. Several genes were identified as present at different abundances between cohorts indicating differences in functional pathways such as cellular defense mechanisms and carbohydrate metabolism influenced by feeding. Confirmation of gene level NGS data via PCR and electrophoresis analysis revealed distinct differences in gene abundances associated with important biologic pathways.

Keywords: breast-feeding; gut microbiome; infants; metagenomics; next generation sequencing; whole genome.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Di Guglielmo, Franke, Robbins and Crowgey.

Figures

Figure 1
Figure 1
Differentially Represented Genera. Distribution of genera identified in the gut microbiome of breast-fed and formula-fed infants. (A) Boxplot of the topmost abundant genera in breast-fed infants (red boxes) and the topmost abundant genera in formula-fed infants (blue boxes). The red asterisks represent the genera that were statistically different between the breast-fed and formula-fed cohorts. The y-axis represents phylogenetic abundance (percentage), and each genus is represented on the x-axis. Asterisk (*) represents some of the 39 genera in total that were differentially represented with FDR ≤ 0.01. (B) Shannon diversity index comparing breast-fed and formula-fed infants.
Figure 2
Figure 2
Clusters of Orthologous Genes (COG) Category Analysis. Statistically significant and overrepresented COGs are plotted by category and number from the total 60 COGs listed in Table 2.
Figure 3
Figure 3
Gene Amplification. (A, B) Non-quantitative PCR was used to validate the results of the bioinformatic analysis for 11 genes. Four representative samples from each cohort of breast-fed and formula-fed infants were analyzed using the purified DNA extracts. Next generation sequencing reads are shown under each PCR panel, as well as corresponding cohort with a higher abundance of the Clusters of Orthologous Genes category. (A) 326538, toxin component of the Txe-Axe toxin-antitoxin module, Txe/YoeB family; 156409, alkyl hydroperoxide reductase subunit AhpF; 309214, glycogen synthase; 316412, c-di-AMP phosphodiesterase, consists of a GGDEF-like and DHH domains; 288014, S-formylglutathione hydrolase FrmB. (B) 266471, chemotaxis protein CheY-P-specific phosphatase CheC; 234035, conjugal transfer/entry exclusion protein; 114703, phage DNA packaging protein, Nu1 subunit of terminase; 145511, adenylate cyclase, class 3; 178095, protein involved in initiation of plasmid replication; 345373, sensor histidine kinase regulating citrate/malate metabolism.

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