Microbial Biomarkers of Intestinal Barrier Maturation in Preterm Infants

Abstract

Intestinal barrier immaturity, or "leaky gut," is the proximate cause of susceptibility to necrotizing enterocolitis in preterm neonates. However, the impact of intestinal microbiota development on intestinal mucosal barrier maturation has not been evaluated in this population. In this study, we investigated a longitudinally sampled cohort of 38 preterm infants < 33 weeks gestation monitored for intestinal permeability (IP) and fecal microbiota during the first 2 weeks of life. Rapid decrease in IP indicating intestinal barrier function maturation correlated with significant increase in community diversity. In particular, members of the Clostridiales and Bifidobacterium were highly transcriptionally active, and progressively increasing abundance in Clostridiales was significantly associated with decreased intestinal permeability. Further, neonatal factors previously identified to promote intestinal barrier maturation, including early exclusive breastmilk feeding and shorter duration antibiotic exposure, associate with the early colonization of the intestinal microbiota by members of the Clostridiales, which altogether are associated with improved intestinal barrier function in preterm infants.

Keywords: Bifidobacterium; Clostridiales; breastmilk feeding; intestinal microbiota; intestinal permeability; leaky gut; necrotizing enterocolitis; preterm infant.

FIGURE 1

Heatmap of the 50 most abundant intestinal bacterial taxa relative abundance in samples collected from 38 preterm infants enrolled in the study. The microbiota of 64 fecal samples were successfully characterized by high-throughput sequencing of the V3–V4 variable regions of 16S rRNA genes. The three sidebars indicate cluster, time, and intestinal permeability category, respectively. Ward linkage clustering was used to cluster samples based on their Jensen-Shannon distance calculated in vegan package in R (Oksanen et al., 2011). The samples with no IP assessment were included to generated the clusters. The low and high intestinal permeability category was defined by a La/Rh > 0.05 or < = 0.05 respectively (Saleem et al., 2017). Taxonomic profiling of corresponding metagenomes further resolved Klebsiella spp. to Klebsiella pneumoniae, Enterococcus spp. to Enterococcus faecalis, and Bifidobacterium spp. to Bifidobacterium breve.

FIGURE 2

Boxplots comparing levels of intestinal permeability and microbial community diversity at study days 1, 8, and 15 in a cohort of 38 preterm infants (<33 weeks gestational age). Intestinal permeability is measured by non-metabolized sugar probes lactulose (La) (marker of intestinal paracellular transport)/rhamnose (Rh) (marker of intestinal transcellular transport). Microbial community diversity was calculated by OTU (Operational Taxonomic Units) richness. Wilcoxon rank sum test and a false discovery rate of 5% was used in significance test. Median values and interquartile of the values were shown in box. (A) Intestinal permeability (p-value = 0.002) and community diversity at the three study time points (p-value = 0.02). (B) Community diversity (p-value < 0.001) in infants with low and high intestinal permeability defined by a La/Rh > 0.05 or ≤0.05 respectively (Saleem et al., 2017).

FIGURE 3

Comparison of relative abundance of bacterial groups in stool samples associated with high and low IP (La/Rh) measurements. (A) Cumulative abundance between phase II/III subjects (6–24 months of age) and phase I infants (within first 2 weeks of life) with high and low IP; (B) Cumulative abundance at study day 1, 8, and 15 for phase I infants with high and low IP. (C) Cumulative abundance at study day 1, 8, and 15 for phase I infants that had first day starting breastmilk feeding at day 1 or 2, or day 3 and later. (D) The relative abundance of Clostridiales of each sampling point and the number of samples at study day 1, 8, and 15 for phase I infants with high or low IP. Bars represent the relative abundance of Clostridiales in each sample. Dotted line and solid line represent mean, and median relative abundance, respectively. Clostridiales was identified to be significantly discriminative with respect to the IP class [p-value = 0.0002, logarithmic linear discriminant analysis (LDA) score is 4.996] using LDA effect size (LEfSe) analysis (Segata et al., 2011). The alpha threshold value for the pairwise non-parametric Kruskal–Wallis test was 0.05 and the threshold for the logarithmic LDA model score (Fisher, 1936) for discriminative features was 2.0. An all-against-all comparison in multi-class analysis was performed. The low and high IP category was defined by a La/Rh > 0.05 or ≤0.05 respectively.

FIGURE 4

Bacterial species transcriptional activity in preterm infant stools. Fecal samples are represented in columns and taxonomic composition quantified using MetaPhlAn (Segata et al., 2012) version 2 are shown in rows, both are organized by hierarchical clustering. Normalization using Witten-Bell smoothing was performed, and the relative expression of a gene in a sample was calculated by normalizing the smoothed value of the expression level in the metatranscriptome by the smoothed value of the corresponding gene abundance in the metagenome (Franzosa et al., 2014; Franzosa et al., 2015). Color scheme indicates an approximate measure of the species’ clade-specific transcriptional activity (Franzosa et al., 2014). The colored branches show the clustering of bacterial species that are consistently transcriptionally active (yellow) or consistently transcriptionally inactive (blue) across samples.