Phylogenetic and Metabolic Tracking of Gut Microbiota during Perinatal Development

Abstract

The colonization and development of gut microbiota immediately after birth is highly variable and depends on several factors, such as delivery mode and modality of feeding during the first months of life. A cohort of 31 mother and neonate pairs, including 25 at-term caesarean (CS) and 6 vaginally (V) delivered neonates (DNs), were included in this study and 121 meconium/faecal samples were collected at days 1 through 30 following birth. Operational taxonomic units (OTUs) were assessed in 69 stool samples by phylogenetic microarray HITChip and inter- and intra-individual distributions were established by inter-OTUs correlation matrices and OTUs co-occurrence or co-exclusion networks. 1H-NMR metabolites were determined in 70 stool samples, PCA analysis was performed on 55 CS DNs samples, and metabolome/OTUs co-correlations were assessed in 45 CS samples, providing an integrated map of the early microbiota OTUs-metabolome. A microbiota "core" of OTUs was identified that was independent of delivery mode and lactation stage, suggesting highly specialized communities that act as seminal colonizers of microbial networks. Correlations among OTUs, metabolites, and OTUs-metabolites revealed metabolic profiles associated with early microbial ecological dynamics, maturation of milk components, and host physiology.

Fig 1. Composition at phylum level of the gut microbiota profiles of 31 infants enrolled in the study for 30 days following birth.

At each time point, the gut microbiota was characterized by HITChip-based phylogeny.

Fig 2. Pie chart representing phyla taxa median values for 30 days following birth.

Statistically significant differences in relative abundance at each time point are reported below each graph (Kruskal-Wallis). Phyla correlation heat maps. Correlation levels (represented as colored squares) among phyla were calculated by Pearson’s test for all 6 time points. Only squares with a significance p<0.05 are shown. Blue squares represent a positive correlation and red squares represent a negative correlation. Panel A and Panel B report analyses for 1–3 days (phase “a”) and 7–30 days (phase “b”), respectively.

Fig 3. Pearson’s correlation heat maps for the 130 genus-like groups from the HITChip microarray.

Panels A, B, and C show the correlation levels (represented by colored squares) among groups, which were calculated by Pearson’s test for all 3 groups (CS-delivered 1–3 days, CS-delivered 7–30 days, and V1-3 delivered days). Only correlations with a significance p<0.05 are represented. The colored scale indicates the correlation values.

Fig 4. Graphical representation of OTUs co-occurrence networks.

Panel A shows the OTUs co-occurrence network for CS-delivered babies at 1–3 days following birth (see sheet B in S5 Table for details). Panel B shows OTUs co-occurrence network for CS-delivered babies at 7–30 days following birth (see sheet C in S5 Table for details). Panel C shows OTUs co-occurrence network for V-delivered babies at 1–3 days following birth (see sheet D in S5 Table for details). Red line indicates a positive correlation and a green line indicates a negative correlation. Pearson’s test was used to evaluate the correlation amongst OTUs (statistical significance was assessed with p<0.01).

Fig 5. Principal Components Analysis (PCA) of metabolic profiles from 55 CS-delivered newborns.

Samples were analysed over the 30 days following birth. Panel A: PC score plot. Panel B: loading plot. The first two components explained 42% of the total variance.

Fig 6. Correlation heat-map between OTUs and faecal metabolites.

Significant correlations (p<0.05) for Actinobacteria (Panel A), Bacteroidetes (Panel B), Proteobacteria (Panel C). 2OH3MB: 2-hydroxy-3-methylbutyrate; Isocapr: Isocaproate; Isoval: Isovalerate; Ile: Isoleucine; Leu: Leucine; EtOH: Ethanol; Fuc: Fucose; 3OH-Isoval: 3-hydroxyisovalerate; Lac: Lactate; Ala: Alanine; But: Butyrate; Ac: Acetate; N-Ac: N-Acetyl moiety; Glu: Glutamate; Suc: Succinate; DMA: Dimethylamine; Asp: Aspartate; TMA: Trimethylamine; Cr: Creatine; MA: Malonate; Cho: choline; MeOH: Methanol; Tyr: Tyrosine; Phe: Phenylalanine; For: Formate.

Fig 7. Correlation heat-map between OTUs and faecal metabolites.

Significant correlations (p<0.05) for Firmicutes (Panel A) and Verrucomicrobia (Panel B). 2OH3MB: 2-hydroxy-3-methylbutyrate; Isocapr: Isocaproate; Isoval: Isovalerate; Ile: Isoleucine; Leu: Leucine; EtOH: Ethanol; Fuc: Fucose; 3OH-Isoval: 3-hydroxyisovalerate; Lac: Lactate; Ala: Alanine; But: Butyrate; Ac: Acetate; N-Ac: N-Acetyl moiety; Glu: Glutamate; Suc: Succinate; DMA: Dimethylamine; Asp: Aspartate; TMA: Trimethylamine; Cr: Creatine; MA: Malonate; Cho: choline; MeOH: Methanol; Tyr: Tyrosine; Phe: Phenylalanine; For: Formate.

Fig 8. Model of microbial metabolism in perinatal babies under trophic pathways associated with transitional/mature milk.

The integration of genomic and metabolomic pipelines suggests a functional picture of microbial metabolism, primed by transitional/mature milk trophic pathways, during early development.