The correlation of the intestinal with pharyngeal microbiota in early neonates

Abstract

Introduction: The gut-lung axis has long been recognized as an important mechanism affecting intestinal and lung immunity. Still, few studies have examined the correlation between the intestinal and pharyngeal microbiota in early neonates, especially when feeding patterns are one of the main drivers of microbiota development.

Methods: To explore the composition and function of intestinal and pharyngeal microbiota and to analyze the effect of limited formula feeding on the initial microbiota colonization in early full-term neonates, we characterized the stool and oropharyngeal microbiota of 20 healthy full-term newborns sampled on days 0 and 5-7 after birth using 16S rRNA gene sequencing. Based on the sequencing results, a comparison was made of the compositions and functions of the intestinal and oropharyngeal microbiota for analysis.

Results and discussion: At the phylum level, Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes were the most abundant in both niches. At the genus level, the species of pioneer bacteria were rich in the intestine and oropharynx but low in abundance on day 0. On days 5-7, Bifidobacterium (25.40%) and Escherichia-Shigella (22.16%) were dominant in the intestine, while Streptococcus (38.40%) and Staphylococcus (23.13%) were dominant in the oropharynx. There were eight core bacteria genera in the intestine and oropharynx on days 5-7, which were Bifidobacterium, Escherichia-Shigella, Staphylococcus, Streptococcus, Bacteroides, Parabacteroides, Rothia, and Acinetobacter. As indicated by PICRUSt analysis, on days 5-7, the intestinal microbiota was more predictive than the oropharyngeal microbiota in transcription, metabolism, cell motility, cellular processes and signaling, and organismal system function in the KEGG pathway. Compared to exclusive breastfeeding, limited formula feeding (40-60%) had no significant effect on the neonatal intestinal and oropharyngeal microbiota composition during the initial colonization period. Our results suggest that the initial colonization of microbiota is closely related to the ecological niche environment in the intestine and oropharynx, with their core microbiota being closely correlated. We found that early limited formula feeding could not significantly affect the initial colonization of microbiota in the intestine and oropharynx.

Keywords: 16S rRNA sequencing; gut-lung axis; intestinal microbiota; neonate; oropharyngeal microbiota.

Figure 1

The flowchart of participant enrollment.

Figure 2

The Venn diagram of operational taxonomic units (OTU) abundance in each group. The sequencing reads shared by S1 and T1 was 2,928 (A), and shared by S2 and T2 was 810 (B) (S1: stool on day 0; T1: oropharyngeal swab on day 0; S2: stool on day 5–7; T2: oropharyngeal swab on day 5–7).

Figure 3

Comparison of the microbial diversity between the intestine (red) and oropharynx (blue). (A, B) The Chao1 and Shannon index was shown as estimators. On day 0 and day 5–7, no significant difference was observed in the Shannon index between the intestine and oropharynx (A). On day 0, the Chao1 index between the intestine and oropharynx was not statistically significant, while it was higher in the oropharynx than in the intestine, with a statistically significant difference on day 5-7 (B). The Mann-Whitney U test was used between every two groups. ns p > 0.05, **p < 0.01 (S: stool; T: oropharyngeal swab). (C, D) Principal component analysis (PCA) at the OTU level of each group. The horizontal and vertical axes indicate the two selected principal component axes and the percentages indicate the value of the degree of the differences in sample composition by the principal components; each sign represents an individual sample, and the points of different colors or shapes indicate the samples of different groupings. The closer the points of the two samples, the higher the degree of similarity in the species composition of the two samples. The distance between S2 and T2 (D) was further than S1 and T1 (C). The PERMANOVA test was used between each two groups.

Figure 4

Comparison of the microbial structure of the newborns in different ecological niches. At the phylum level, both on day 0 and day 5–7, the relative abundances of Proteobacteria, Firmicutes, and Actinobacteria in the intestine and oropharynx all showed statistically significant differences, while Bacteroidetes showed no statistically significant difference (A). At the genus level, only Staphylococcus and Pseudomonas showed statistical differences between the intestinal and oropharyngeal microbiota on day 0, but Streptococcus, Staphylococcus, Escherichia-Shigella, Pseudomonas, Acinetobacter, and Bradyrhizobium showed statistical differences on day 5–7 (all with p < 0.05) (B). The Mann-Whitney U test was used between every two groups. *, **, and *** represent p ≤ 0.05, ≤0.01, and ≤0.001, respectively. (C) The core microbiota in the intestine and oropharynx at the genus level on day 5-7. Approximately 8 core microbiotas were observed in the intestine, while 16 core microbiotas were observed in the oropharynx. There were 8 core microbiotas shared by both, such as Bifidobacterium, Escherichia-Shigella, Staphylococcus, Streptococcus, Bacteroides, Parabacteroides, Rothia, and Acinetobacter. (D) Distinct bacterial taxa for the microbiota in the intestine and oropharynx at different time points that were identified by the LEfSe analysis. Green shaded areas indicate microbe orders to better describe the oropharyngeal microbiome from both time points of the neonates; red shaded areas indicate microbe orders to better describe the fecal microbiome from both time points of the neonates; The prefixes “p,” “c,” “o,” “f,” and “g” represents the annotated level of phylum, class, order, family, and genus; the genera with a linear discriminant analysis (LDA) score >3 shown for the data sets. The higher the LDA score, the greater the influence of species abundance on the difference.

Figure 5

Different analyses of the metabolic function enrichment in S2 and T2. The left indicates the proportion of the relative abundances of different microbiota functions in the two groups; the middle indicates the proportion of differences in the relative abundance of microbiota functions within the 95% confidence interval; the far right shows p; p < 0.05 indicates a significant difference, marked in red.