Dichotomous development of the gut microbiome in preterm infants

Abstract

Background: Preterm infants are at risk of developing intestinal dysbiosis with an increased proportion of Gammaproteobacteria. In this study, we sought the clinical determinants of the relative abundance of feces-associated Gammaproteobacteria in very low birth weight (VLBW) infants. Fecal microbiome was characterized at ≤ 2 weeks and during the 3rd and 4th weeks after birth, by 16S rRNA amplicon sequencing. Maternal and infant clinical characteristics were extracted from electronic medical records. Data were analyzed by linear mixed modeling and linear regression.

Results: Clinical data and fecal microbiome profiles of 45 VLBW infants (gestational age 27.9 ± 2.2 weeks; birth weight 1126 ± 208 g) were studied. Three stool samples were analyzed for each infant at mean postnatal ages of 9.9 ± 3, 20.7 ± 4.1, and 29.4 ± 4.9 days. The average relative abundance of Gammaproteobacteria was 42.5% (0-90%) at ≤ 2 weeks, 69.7% (29.9-86.9%) in the 3rd, and 75.5% (54.5-86%) in the 4th week (p < 0.001). Hierarchical and K-means clustering identified two distinct subgroups: cluster 1 started with comparatively low abundance that increased with time, whereas cluster 2 began with a greater abundance at ≤ 2 weeks (p < 0.001) that decreased over time. Both groups resembled each other by the 3rd week. Single variants of Klebsiella and Staphylococcus described variance in community structure between clusters and were shared between all infants, suggesting a common, hospital-derived source. Fecal Gammaproteobacteria was positively associated with vaginal delivery and antenatal steroids.

Conclusions: We detected a dichotomy in gut microbiome assembly in preterm infants: some preterm infants started with low relative gammaproteobacterial abundance in stool that increased as a function of postnatal age, whereas others began with and maintained high abundance. Vaginal birth and antenatal steroids were identified as predictors of Gammaproteobacteria abundance in the early (≤ 2 weeks) and later (3rd and 4th weeks) stool samples, respectively. These findings are important in understanding the development of the gut microbiome in premature infants.

Keywords: Abbreviations; Dysbiosis; Gammaproteobacteria; NECNecrotizing enterocolitis; OTUOperational taxonomic unit; VLBWVery low birth weight; Very low birth weight infant.

Fig. 1

Relative abundance of major bacterial taxonomic units in stool over time. Line diagrams (means ± standard deviation) show the relative abundances of major bacterial taxonomic units in stool, by cluster. Stool samples were collected during the first 2 weeks, and then during the 3rd and the 4th weeks, respectively. Repeated measures analysis of variance; *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 2

Clustering of VLBW infants by the relative abundance of fecal Gammaproteobacteria. a Dendrogram shows the average linkage (between the two groups) derived by hierarchical clustering. b. Scatter-plot shows that the VLBW infants included in our study were grouped into two distinct clusters based on the relative abundance of Gammaproteobacteria (percentages) in stool samples obtained during the first two postnatal weeks

Fig. 3

Relative abundance of major bacterial taxonomic units in stool, by cluster. Line diagrams (means ± standard deviation) show the relative abundances of major bacterial taxonomic units in stool in clusters 1 and 2. Stool samples were collected during the first 2 weeks, and then during the 3rd and the 4th weeks, respectively. Repeated measures analysis of variance; *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 4

Volatility analysis of the two clusters: Histogram shows the distribution of unweighted UniFrac distances between successive time-points. A distance of 1 means maximally different communities, while a distance of 0 implies identical communities, so a curve shifted toward 0 means lower variability between successive time-points. The two clusters showed a significant difference in variability (p = 0.038)

Fig. 5

Heat map of the most abundant single nucleotide variants (SNVs): Heat map shows the relative abundance of the 18 most abundant SNVs at each sample. The bar at the top is color coded according to time-point. Blue = most abundant, yellow = least abundant (minimum abundance displayed = 0.165% mean abundance across samples)

Fig. 6

Balance tree analysis for major bacterial taxa. Bifurcating tree relating the DADA2 sequence variants to each other by the time-point for stool collection highlights specific sequence variants that appeared only in early or late stages. Linear regression by cluster membership confirmed increased Klebsiella in cluster 2. Cluster 1 showed a true increase in Staphylococcus sequence variant