Sepsis in preterm infants causes alterations in mucosal gene expression and microbiota profiles compared to non-septic twins

Abstract

Sepsis is a life-threatening condition in preterm infants. Neonatal microbiota plays a pivotal role in the immune system maturation. Changes in gut microbiota have been associated to inflammatory disorders; however, a link with sepsis in the neonatal period has not yet been established. We aimed to analyze gut microbiota and mucosal gene expression using non-invasively obtained samples to provide with an integrative perspective of host-microbe interactions in neonatal sepsis. For this purpose, a prospective observational case-control study was conducted in septic preterm dizygotic twins and their non-septic twin controls. Fecal samples were used for both microbiota analysis and host genome-wide expression using exfoliated intestinal cells. Gene expression of exfoliated intestinal cells in septic preterm showed an induction of inflammatory and oxidative stress pathways in the gut and pro-oxidant profile that caused dysbiosis in the gut microbiota with predominance of Enterobacteria and reduction of Bacteroides and Bifidobacterium spp.in fecal samples, leading to a global reduction of beneficial anaerobic bacteria. Sepsis in preterm infants induced low-grade inflammation and oxidative stress in the gut mucosa, and also changes in the gut microbiota. This study highlights the role of inflammation and oxidative stress in neonatal sepsis on gut microbial profiles.

Figure 1

(Panel a) Principal component analysis (PCA) of the gene expression profiles of exfoliated intestinal cells of septic preterm twins (in red) and their non-septic controls (in green). The 3D plot shows the correlation of each sample with respect to the first three principal components. (Panel b) Represents the unsupervised hierarchical cluster analysis. Significantly regulated genes (p < 0.05) were used for 2-D hierarchical clustering of sepsis and control samples. Each row represents the expression of an individual probe set and each column of an individual sample, summarizing control samples with a green bar and sepsis samples with a red bar. Upregulated genes are represented in red and downregulated genes in blue.

Figure 2. Pathway Studio Analysis Protein-protein interaction network.

(Panel a) Genes correlated with Oxidative Stress Pathway. (Panel b) Genes linked with the NF-кB canonical pathway.

Figure 3

IPA analysis: (Panel a) Top Network Cell Death and Survival, Inflammatory Response, Cancer. (Panel b) Upstream regulation: evidence of the activation of IL1B pathway. IL1B predicted to be activated (z-score 2.368) p-value = 1.84E-01: 6 out of 6 genes have expression direction consistent with activation of IL1B.

Figure 4. Microbiota composition obtained by pyrosequencing.

(Panel a) Relative abundance of bacterial distribution at class level in the Sepsis and Control groups. (Panel b) Rarefaction curves at level of 90% for families. The graphs shows rarefaction curve relating the sequencing effect compared with an estimate of the number of bacterial families, as inferred by the number of OTUs.

Figure 5

Depicts the colored representation of the correlation between bacterial groups (reads of pyrosequencing of numbers estimated by qPCR) and expression (signal intensity) of the differentially expressed genes in the Oxidative Stress pathway (panel a) and in the NF-κB pathway and IL-1β pathways (panel b). Red represents significant inverse correlation and blue cells’ significant direct correlation. Genes with white cells gave no significant correlation with the corresponding bacterial taxon and genes not shown had no significant correlation with any bacterial taxa.

Figure 6. Model explaining changes in gene expression and the gut.

Circulating lymphocytes expressed high levels of inflammatory markers (TNF-α, NF-κB, IL-1β and SP1). High amounts of innate immune mediators secreted to the bloodstream would be reaching the mucosae of the small and large intestine of VLBW infants with sepsis. This would activate master regulators TNF-α and IL-1β, inducing the expression of pro-inflammatory signaling and innate immune defense systems, such as oxidative stress pathway in the gut mucosal cells. The secretion of ROS and NOS would then correlate with bacterial profiles richer in Enterobacteriaceae and with the lower presence of Bifidobacteriaceae and Bacteroides, due to the lack in the latter of the enzymatic armor to survive in the presence of ROS, RNS and derived toxic compounds.