Microbiome overview of the maternal and neonatal samples
The diversity of the microbiota in a given habitat reflects the composition and relative abundance of the community. Approximately 460 bp of PCR products were generated by amplifying the V3-V4 region of the 16S rRNA gene to compare the bacterial diversity among samples. DNA sequencing after quality filtering yielded 8.29 million paired-end reads, which further merged into 7.21 million tags, with a minimum of 64253 tags per sample (average of 92448 ± 8822), and ultimately formed 27801 operational taxonomic units (OTUs).
Rarefaction curves evaluated the OTU richness and represented whether a reasonable sampling size (sequencing depth) was used. As shown in Figure S2, the almost horizontal asymptotic curves indicated that the sequencing depth was sufficient for our research. The values of observed species (Sobs), Pielou evenness, Shannon, and inverse Simpson indices were calculated to thoroughly assess alpha-diversity. The Sobs index indicated the actual detected OTUs, while the Pielou evenness index referred to the species equitability within each group. The Simpson and Shannon indices measured the degree of species asynchrony and stability, and higher values represented higher richness, evenness, or both . As shown in Figure 1A, the highest Sobs value could be observed in the transitional stool samples of naturally delivered newborns (TSN), and the OTUs differed substantially between the transitional stool samples of caesarean-delivered newborns (TSC) and the TSN, which demonstrated that CD surgery reduced the actual number of species of newborn gut microbes compared with vaginal delivery. In contrast, the TSC group presented the highest evenness according to the value of the Pielou evenness index (Figure 1B) and there was a significant difference between the TSC and TSN groups (Wilcoxon test, PPielou<0.01). The above analyses revealed that CD surgery led to an obvious alteration in bacterial richness and evenness. Good agreement was exhibited in the inverse Simpson index and Shannon index, which suggested that the gut microbial composition of CD newborns was less diverse (Wilcoxon test, PSimpson<0.01, PShannon<0.01), while a lesser degree of dominant bacteria and a higher distribution homogeneity was observed in CD newborns with swabbing vaginal fluid (CS) (Figure 1C, 1D). The aggregate analyses of alpha-diversity indicated that the neonatal gut microbiota was affected by both the mode of delivery and the swabbing treatment. Beyond that, pregnant women who underwent different labour modes showed no significant differences in intestinal microbiota (Wilcoxon test, PSobs=0.13, PPielou=0.39, PSimpson=0.27, PShannon=0.32), whereas a disparate vaginal fluid microbiome was exhibited according to indices of alpha-diversity (Wilcoxon test, PPielou=0.03, PSimpson=0.04, PShannon=0.03), which might result from the different physical conditions and medical administrations during pregnancy.
We further investigated beta-diversity according to the Bray-Curtis distance to compare the microbial community structures among samples, as this method provided a model to describe the overall pattern of community composition based on OTUs. Similar trends as alpha-diversity were obtained (Figure 2). Briefly, the significant differences between the CD and CS newborns (Wilcoxon test, P = 0.045), as well as the CD and vaginally delivered newborns (Wilcoxon test, P < 0.01), showed that the offspring microbial communities were shaped by maternal vaginal fluids. The gut microbiota of CS and vaginally delivered newborns seemed to show no substantial difference in terms of beta-diversity. From the above analyses, it was concluded that a similar progression towards a natural configuration was observed with swabbing exposure (Wilcoxon test, P = 0.5), and a vaginal microbiome–like signature that was absent in CD newborns was exhibited in CS newborns. In parallel, the microbiota of the maternal vaginal fluid (Wilcoxon test, P = 0.35) and the stool samples (Wilcoxon test, P = 0.68) were not significantly different among the groups.
The visualization of non-metric multidimensional scaling (NMDS) analysis based on the Bray-Curtis distance showed clear ordinations that were able to distinguish the gut microbes of the CD and CS newborns from those of the vaginally delivered newborns (Adonis test, R2=0.24, PERMANOVA P < 0.01) (Figure 3). Furthermore, the OTU clustering distribution of CS newborns was mainly limited within the elliptical field (95% CI) of vaginally delivered newborns, suggesting that the microbial composition of transitional stool samples of caesarean-delivered newborns swabbed with maternal vaginal fluid (TSS) tended to be similar to that of TSN samples. In addition, the NMDS plot also reflected that the maternal vaginal microbiota (Adonis test, R2=0.13, PERMANOVA P = 0.08), rather than the maternal intestinal microbiota (Adonis test, R2=0.23, PERMANOVA P < 0.01), was the major source of neonatal gut colonization among vaginally delivered babies. However, the gut microbiota of CD newborns was inherited neither from the vaginal microbes nor from the intestinal microbes of their mothers. Similar conclusions could also be derived from the distance heatmap in Figure S3. Combined with the above results, swabbing treatment increased the richness of caesarean-delivered newborns’ intestinal microbiota and made it more similar to that of the vaginally delivered newborns.
The variation in microbiota caused by delivery modes and treatment
The gut microbiome of vaginally delivered newborns was enriched for Firmicutes (40.58%), Proteobacteria (26.45%), and Bacteroidetes (19.67%) at the phylum level. However, the highly abundant phyla in CD newborns were Proteobacteria (58.77%), Bacteroidetes (12.68%), Firmicutes (11.51%), and Cyanobacteria (10.95%), while the intestinal microbiota of CS newborns mainly consisted of Firmicutes (30.38%), Bacteroidetes (30.13%), and Proteobacteria (24.52%), as shown in Figure 4A. The bacterial composition and richness of CS newborns tended to be similar to those of vaginally delivered newborns. At the genus level, the microbial community of CS newborns changed clearly and consisted primarily of Bacteroides (17.51%) and Escherichia-Shigella (10.10%) after treatment (Figure 4B). The circos plots confirmed the above results from another aspect and are displayed in Figure S4. In addition, we used ternary enrichment and indicating taxa to evaluate the specific effects of swabbing exposure. At the phylum level, Firmicutes (Tukey HSD test, P < 0.01; Welch’s t test, PTSS-TSC=0.02, PTSN-TSC<0.01, PTSN-TSS=0.36) and Proteobacteria (Tukey HSD test, P < 0.01; Welch’s t test, PTSS-TSC=0.01, PTSN-TSC=0.03, PTSN-TSS=0.98) were recovered by swabbing (Figure 5A, Figure 5C), while Lactobacillus (Tukey HSD test, P < 0.01; Welch’s t test, PTSS-TSC=0.20, PTSN-TSC=0.80, PTSN-TSS=0.79), Bacteroides (Tukey HSD test, P < 0.01; Welch’s t test, PTSS-TSC<0.01, PTSN-TSC=0.99, PTSN-TSS<0.01), and Escherichia-Shigella (Tukey HSD test, P < 0.01; Welch’s t test, PTSS-TSC=0.11, PTSN-TSC=0.07, PTSN-TSS=0.63) showed similar patterns of restoration (Figure 5B, Figure 5D). The richness of the genera Akkermansia (Welch’s t test, PTSS-TSC=0.01) and Alistipes (Welch’s t test, PTSS-TSC<0.01) showed elevation in the CS newborns compared to CD newborns but there was no significant difference when compared with the vaginally delivered newborns (Figure 5B). It is worth noting that only vaginal fluid swabbing induced more shared taxa between CS and CD newborns, as displayed in Figure S5, and it could be speculated that the swabbing treatment introduced a considerable amount of microbial colonization via ‘mother-to-offspring transmission’. Interestingly, such bacterial transmission was not obviously related to maternal faeces or anal transmission.
The Metabolic Active Effects of the Vaginal Transferred Microbiota
Metagenomic functioning predictions using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) software provided further insight into the pathway abundances for investigating the functional implication of the treatment-induced changes. Compared with vaginally delivered newborns, the percentages of genes related to ‘metabolism of terpenoids and polyketides’ (Welch’s t test, P = 0.02) and ‘glycan biosynthesis and metabolism’ (Welch’s t test, P = 0.048) decreased in the gut environment in CD newborns, as displayed in Figure 6A. The TSS group had a higher number of bacterial genes related to ‘glycan biosynthesis and metabolism’ and ‘biosynthesis of other secondary metabolites’, whereas the future risks of ‘neurodegenerative diseases’ and ‘digestive system diseases’ were statistically larger in vaginally delivered newborns, as shown in Figure 6B. As a result of exposure to vaginal fluids, the number of bacterial genes related to metabolism, e.g., carbohydrates (Welch’s t test, P = 0.022), amino acids (Welch’s t test, P = 0.039), terpenoids and polyketides (Welch’s t test, P = 0.001), lipids (Welch’s t test, P < 0.001), glycans (Welch’s t test, P < 0.001), and nucleotide metabolism (Welch’s t test, P = 0.032), increased significantly. Beyond this, the predicted functions of ‘DNA replication and repair’ (Welch’s t test, P = 0.018) and ‘biosynthesis of other secondary metabolites’ (Welch’s t test, P = 0.041) were also enhanced by the treatment (Figure 6C). In summary, swabbing treatment can compensate for bacterial functional alterations in CD newborns, which could have potential benefits in decreasing the risk of digestive, cardiovascular, and immune diseases.