Changes in Gut Microbiota at 1-60 Days in 92 Preterm Infants in a Neonatal Intensive Care Unit Using 16S rRNA Gene Sequencing

Abstract

BACKGROUND Neonatal gut diversity is influenced by birth conditions and probiotic/antibiotic use. The gut microbiota affects brain development, immunity, and risk of diseases. Preterm infants, especially in neonatal intensive care units (NICUs), have different gut flora from full-term infants, suggesting in utero microbial colonization. This study examined gut microbiota changes in 92 NICU preterm infants in China. MATERIAL AND METHODS We collected data on 92 preterm infants admitted to the NICU immediately after birth, and fecal samples were collected on days 1, 3, 7, 14, 21, 28, and 60. We analyzed changes in intestinal bacteria through 16S rRNA sequencing, predicted the change in gut microbiota function over time, and compared the effects of main feeding modality on the intestinal bacteria of preterm infants. RESULTS At the phylum level, the top 5 phyla in total accounted for 99.69% of the abundance, in decreasing order of abundance: Proteobacteria, Firmicutes, Actinobacteria, Tenericutes, and Bacteroidetes. At the genus level, the top 10 genera in terms of abundance accounted for a total of 90.90%, in decreasing order of abundance: Pseudomonas, Staphylococcus, Klebsiella, Escherichia-Shigella, unclassified Enterobacteriaceae, Staphylococcus, Clostridium-sensu-stricto-1, Streptococcus, Sphingomonas, and Ureaplasma. The abundance of Proteobacteria and Pseudomonas showed a decreasing trend at first, reached a minimum at day 14, and then an increasing trend, while the opposite trend was observed for Firmicutes. The metabolic function of the bacterial community changed greatly at different time points. The abundance of Proteobacteria at the phylum level and Streptococcus at the genus level in formula-fed infants were significantly higher than in breast-fed infants. CONCLUSIONS Between 1 and 60 days, the gut microbiome in preterm infants in the NICU changed with changes in feeding patterns, with the main gut bacteria being from the phyla, Proteobacteria, and Pseudomonas.

Figure 1

Amplicon sequence variation (ASV) petal diagram (R-Studio v7.2).

Figure 2

Box plots of alpha diversity indices at 7 time points. (A) Chao1 index. (B) Observed species index. (C) Shannon index. (D) Simpson index. *** P<0.001. (R-Studio v7.2 and GraphPad Prism8.0 software).

Figure 3

2D classification plot of samples for PCoA analysis between 7 time points. The horizontal and vertical axes represent the 2 selected principal axes, and the percentages represent the values of the principal axes explaining the differences in sample composition; the scales of the horizontal and vertical axes are relative distances with no real meaning. The circles represent 95% confidence intervals. The different colors or shapes of the points represent different groups of samples. The dots of different colors or shapes represent different groups of samples, and the closer the dots are to each other, the more similar the species composition of each sample is. (R-Studio v7.2).

Figure 4

Histogram of the distribution of dominant intestinal bacteria in preterm infants. (A) Distribution of the top 5 phyla at different time points. (B) Distribution of the top10 genera at different time points. (GraphPad Prism8.0 software).

Figure 5

Line graph of relative abundance of dominant intestinal bacteria in preterm infants over time. (A) Trend of the top 5 phyla over time. (B) Trend of the top10 genera over time. (C) Trend of Bifidobacterium over time. (GraphPad Prism8.0 software).

Figure 6

Predictive function analysis of the top 10 pathways ranked in relative abundance among KEGG pathways with differences at 7 time points. (GraphPad Prism8.0 software). * P<0.05, ** P<0.01, *** P<0.001.