Oxygenation and intestinal perfusion and its association with perturbations of the early life gut microbiota composition of children with congenital heart disease

Abstract

Background: Early life gut microbiota is known to shape the immune system and has a crucial role in immune homeostasis. Only little is known about composition and dynamics of the intestinal microbiota in infants with congenital heart disease (CHD) and potential influencing factors.

Methods: We evaluated the intestinal microbial composition of neonates with CHD (n = 13) compared to healthy controls (HC, n = 30). Fecal samples were analyzed by shotgun metagenomics. Different approaches of statistical modeling were applied to assess the impact of influencing factors on variation in species composition. Unsupervised hierarchical clustering of the microbial composition of neonates with CHD was used to detect associations of distinct clusters with intestinal tissue oxygenation and perfusion parameters, obtained by the "oxygen to see" (O2C) method.

Results: Overall, neonates with CHD showed an intestinal core microbiota dominated by the genera Enterococcus (27%) and Staphylococcus (20%). Furthermore, a lower abundance of the genera Bacteroides (8% vs. 14%), Parabacteroides (1% vs. 3%), Bifidobacterium (4% vs. 12%), and Escherichia (8% vs. 23%) was observed in CHD compared to HCs. CHD patients that were born by vaginal delivery showed a lower fraction of the genera Bacteroides (15% vs. 21%) and Bifidobacterium (7% vs. 22%) compared to HCs and in those born by cesarean section, these genera were not found at all. In infants with CHD, we found a significant impact of oxygen saturation (SpO2) on relative abundances of the intestinal core microbiota by multivariate analysis of variance (F[8,2] = 24.9, p = 0.04). Statistical modeling suggested a large proportional shift from a microbiota dominated by the genus Streptococcus (50%) in conditions with low SpO2 towards the genus Enterococcus (61%) in conditions with high SpO2. We identified three distinct compositional microbial clusters, corresponding neonates differed significantly in intestinal blood flow and global gut perfusion.

Conclusion: Early life differences in gut microbiota of CHD neonates versus HCs are possibly linked to oxygen levels. Delivery method may affect microbiota stability. However, further studies are needed to assess the effect of potential interventions including probiotics or fecal transplants on early life microbiota perturbations in neonates with CHD.

Keywords: O2C; congenital heart disease; gut microbiome; intestinal microbiota; next-generation sequencing; oxygen to see; oxygenation.

Figure 1

Description of cohort. Samples from 43 neonates were analyzed within this study. Groups were divided into healthy controls (HC) and neonates with congenital heart disease (CHD). Two other independent subgroups were created separately according to mode of delivery (HC and CHD) and according to oxygen saturation (SpO2) of ≤90% or > 90% (CHD group only).

Figure 2

(A–C) Relative abundance of 10 most frequent genera by group and mode of delivery. Relative abundance (0–1.0) of the 10 most frequent genera are shown according to group (A), mode of delivery (B) and the combination of group and mode of delivery (C). HC, healthy control; CHD, congenital heart disease.

Figure 3

Diversities of all genera by type of birth according to patient cohort (CHD or HC). Box-Whisker Plots with median and interquartile range (IQR) of alpha diversity metrics (Shannon’s, Simpson’s and InvSimpson indices) of the intestinal microbiota composition according to mode of delivery and divided by patient group (CHD, HC). All values are displayed on the same scale to allow for comparison of different constellations. p-values for comparison are not given due to small numbers and lack of inference toward a general population.

Figure 4

(A,B) Model of the effect of SpO2 on intestinal microbiota composition of congenital heart disease (CHD) patients under consideration of mode of delivery. Intestinal core microbiota composition data and oxygen saturation (SpO2) of neonates with CHD were used to create this visual, statistical model showing shifts in the intestinal core microbiota composition (y-axis: relative frequencies, 0–1.0) according to gradual alterations in SpO2 (%). Model for neonates with CHD born by vaginal delivery (A), neonates with CHD born by cesarean section (B) was assessed by MANOVA.

Figure 5

Hypothesis free cluster analysis of microbiota composition of patients with congenital heart disease (CHD). Heat map of relative abundances of the dominating genera in the total cohort of neonates with CHD (n = 13) in combination with the dendrogram resulting from an unsupervised hierarchical cluster analysis. Plotted genera were limited to those that resulted in a relative abundance of at least 0.1. The assignment of cases to final clusters followed essentially the results from the cluster analysis except case “P04.” This was reassigned to a different cluster due to its content of the genus Streptococcus. Patient numbers in italic are those born by cesarean section, others were born by vaginal delivery. Cluster 1 (red) was either dominated by the phylum Firmicutes and the genera Staphylococcus or Enteroccocus with low interindividual diversity. Cluster 2 (black) was mainly dominated by the phylum Proteobacteria, and the genera Pseudomonas, Escherichia, Klebsiella and Enterobacter, but showed high interindividual diversity. Cluster 3 (blue) consisted of 3 patients and was mainly characterized by a more diverse microbiota composition and presence of the genus Streptococcus in all patients. Other genera found in these patients were Clostridium and Corynebacteria, but no Bacteroides or Bifidobacterium at all.

Figure 6

(A–E) Boxplots of oxygen saturation and perfusion parameters according to the three identified different microbiota composition clusters. Box whisker plots for oxygen saturation (SpO2, %) (A), intestinal fractional tissue oxygen extraction (iFTOE, fraction) (B), relative blood flow velocity in the liver (rlVelocityAU) (C) and umbilical region (ruVelocityAU) (D), and global gut perfusion per birth weight (E) according to grouping by the three identified different microbiota composition clusters. Cluster 1 (red), Cluster 2 (black), Cluster 3 (blue). p-values result from Wilcoxon tests and applied oxygenation and perfusion parameters according to the three clusters. p-values here give rather the probability to see a more extreme result of the statistical test function within our sample subset than that actually proving the null hypothesis to be true. Thus, the result gives hardly any clue toward generalization.