Multiomics analysis reveals the presence of a microbiome in the gut of fetal lambs

Abstract

Objective: Microbial exposure is critical to neonatal and infant development, growth and immunity. However, whether a microbiome is present in the fetal gut prior to birth remains debated. In this study, lambs delivered by aseptic hysterectomy at full term were used as an animal model to investigate the presence of a microbiome in the prenatal gut using a multiomics approach.

Design: Lambs were euthanised immediately after aseptic caesarean section and their cecal content and umbilical cord blood samples were aseptically acquired. Cecal content samples were assessed using metagenomic and metatranscriptomic sequencing to characterise any existing microbiome. Both sample types were analysed using metabolomics in order to detect microbial metabolites.

Results: We detected a low-diversity and low-biomass microbiome in the prenatal fetal gut, which was mainly composed of bacteria belonging to the phyla Proteobacteria, Actinobacteria and Firmicutes. Escherichia coli was the most abundant species in the prenatal fetal gut. We also detected multiple microbial metabolites including short chain fatty acids, deoxynojirimycin, mitomycin and tobramycin, further indicating the presence of metabolically active microbiota. Additionally, bacteriophage phiX174 and Orf virus, as well as antibiotic resistance genes, were detected in the fetal gut, suggesting that bacteriophage, viruses and bacteria carrying antibiotic resistance genes can be transmitted from the mother to the fetus during the gestation period.

Conclusions: This study provides strong evidence that the prenatal gut harbours a microbiome and that microbial colonisation of the fetal gut commences in utero.

Keywords: intestinal microbiology.

Figure 1

Effect of data decontamination on gut microbial metagenomic and metatranscriptomic gene counts. (A) Total gene count for microbial metagenome (MG) and metatranscriptome (MT) before and after data decontamination in samples of fetal cecal content. ‘Before’ indicates gene counts before data decontamination; ‘after’ indicates gene counts after data decontamination. (B) Average gene count in microbial MG and MT per cecal content sample before and after data decontamination. In (B), the boxes represent IQRs between the first and third quartiles, and the horizontal line inside the box indicates the median; whiskers represent the minima or maxima within 1.5× IQR from the first or third quartiles.

Figure 2

Microbiome composition of metagenome (MG) and metatranscriptome (MT) in fetal gut. (A–C) Represent phylum-level, genus-level and species-level bacterial composition of the fetal gut microbiome (relative abundance ≥0.1%), respectively. Microbiome composition shows average relative abundance of bacterial presence in all samples. n=6. (D) Ratios of average relative microorganism abundance in MT relative to the MG (MT/MG).

Figure 3

Copy numbers of total bacteria and selected microbes per gram cecal content. The boxes represent IQRs between the first and third quartiles, and the horizontal line inside the box indicates the median; whiskers represent the minima or maxima within 1.5× IQR from the first or third quartiles. Boxes with different letters above their whiskers are significantly different at p<0.05.

Figure 4

KEGG and eggNOG classifications of the fetal gut metagenome and metatranscriptome. (A, B) KEGG classifications for the fetal gut metagenome and metatranscriptome, respectively. (C, D) eggNOG functional classifications for the fetal gut metagenome and metatranscriptome, respectively. eggNOG, non-supervised orthologous groups; KEGG, Kyoto Encyclopaedia of Genes and Genomes.

Figure 5

Associations of cecal microbiome with cecal content metabolites. (A) Analysis of co-occurrence networks between cecal content metabolites and microbial species in the fetal gut metatranscriptome. Nodes represent metabolites and microbial species. An increase in the number of lines through a node increases the size of a node. Red edges indicate positive correlations between metabolites and microbial species; Spearman’s rank correlation coefficient >0.6, p<0.05. Blue edges indicate negative correlations between metabolites and microbial species; Spearman’s rank correlation coefficient < −0.6, p<0.05. (B) Heat map of Spearman’s rank correlation coefficients between cecal content metabolites and microbial species in the fetal gut metatranscriptome. Spearman’s rank correlation coefficient >0.6 or < −0.6, *p<0.05, **p<0.01. Red and blue colours denote positive and negative correlations, respectively. Colour intensity is proportional to Spearman’s rank correlation values.