Pediatric intensive care unit treatment alters the diversity and composition of the gut microbiota and antimicrobial resistance gene expression in critically ill children

Abstract

Introduction: Common critical illnesses are a growing economic burden on healthcare worldwide. However, therapies targeting the gut microbiota for critical illnesses have not been developed on a large scale. This study aimed to investigate the changes in the characteristics of the gut microbiota in critically ill children after short-term pediatric intensive care unit (PICU) treatments.

Methods: Anal swab samples were prospectively collected from March 2021 to March 2022 from children admitted to the PICU of Xinhua Hospital who received broad-spectrum antibiotics on days 1 (the D1 group) and 7 (the D7 group) of the PICU treatment. The structural and functional characteristics of the gut microbiota of critically ill children were explored using metagenomic next-generation sequencing (mNGS) technology, and a comparative analysis of samples from D1 and D7 was conducted.

Results: After 7 days of PICU admission, a significant decrease was noted in the richness of the gut microbiota in critically ill children, while the bacterial diversity and the community structure between groups remained stable to some extent. The relative abundance of Bacilli and Lactobacillales was significantly higher, and that of Campylobacter hominis was significantly lower in the D7 group than in the D1 group. The random forest model revealed that Prevotella coporis and Enterobacter cloacae were bacterial biomarkers between groups. LEfSe revealed that two Gene Ontology entries, GO:0071555 (cell wall organization) and GO:005508 (transmembrane transport), changed significantly after the short-term treatment in the PICU. In addition, 30 KEGG pathways were mainly related to the activity of enzymes and proteins during the processes of metabolism, DNA catabolism and repair, and substance transport. Finally, 31 antimicrobial resistance genes had significantly different levels between the D7 and D1 groups. The top 10 up-regulated genes were Erm(A), ErmX, LptD, eptB, SAT-4, tetO, adeJ, adeF, APH(3')-IIIa, and tetM.

Conclusion: The composition, gene function, and resistance genes of gut microbiota of critically ill children can change significantly after short PICU treatments. Our findings provide a substantial basis for a better understanding of the structure and function of gut microbiota and their role in critical illnesses.

Keywords: children; critical illness; drug resistance genes; gut microbiota; infection.

Figure 1

Flowchart of patient enrollment.

Figure 2

OTUs and β-diversity analysis of the microbiota in the D1 and D7 groups. (A) Venn diagram of the number of OTUs in the gut microbiota of children in groups D1 and D7. (B–E) PCA and NMDS analyses of β-diversity.

Figure 3

Overall composition of the bacteria at the phylum level before and after PICU treatments in critically ill children. Changes in the relative abundance of bacteria at the phylum level in the gut microbiota of critically ill children in the D1 (A) and D7 (B) groups. Based on the metagenomic sequencing results, the top four microbiota communities at the phylum taxonomic level in groups D1 and D7 were selected, and the remaining were categorized as “others.” The results are presented as a relative abundance histogram. The vertical coordinate represents the sequence number of the anal swab sample (sample name), and the horizontal coordinate represents the relative abundance of the community.

Figure 4

Relative abundances of the top 20 bacteria at the class, order, and species levels in the D1 and D7 groups. Distribution of the top 20 bacterial communities at the (A) class, (B) order, and (C) species levels. *p < 0.05.

Figure 5

Random forest analysis of the D1 and D7 groups at the species level. Random forest analysis is a machine-learning algorithm that can effectively classify and predict grouped samples. The Accuracy (A) and Gini (B) indices are common evaluation metrics, with higher values indicating greater importance of the variable. The horizontal coordinates represent the values of Accuracy and Gini indices, and the vertical coordinates represent the strain names. The figure shows the strains that played major roles in the D1 and D7 groups, with Prevotella coporis and Enterobacter cloacae having the most significant classification effects.

Figure 6

Venn diagram and Box diagram of the number of genes detected in the D1 and D7 groups. (A) Venn diagram of the number of unique or shared genes detected in the gut microbiota of the D1 and D7 groups. (B) Box plot of the number of genes detected in the gut microbiota of the D1 and D7 groups.

Figure 7

Gene function prediction of KEGG pathways. The histograms on the left represent the names of KEGG pathways and their relative abundances, and the dot plots on the right represent the corrected p-values. Corrected p < 0.05 was considered significant and retained.

Figure 8

Gene function prediction and LEfSe analysis of GO entries. (A) The histograms on the left represent the names of GO entries and their relative abundances, and the dot plots on the right represent the corrected p-values. A corrected p < 0.05 was considered significant and retained. (B) The specific changed GO entries identified by linear discriminant analysis (LDA) and effect size (LEfSe) analysis were presented, and LDA scores >2.0 were considered significant.

Figure 9

Volcano plots of antimicrobial resistance gene expression in the D1 and D7 groups. The horizontal coordinates in the graph represent the log₂ fold change, and the vertical coordinates represent the-log10 (p-value). The horizontal dashed line in the figure represents the p-value threshold (0.05), and the two vertical dashed lines represent log₂FC = 1/−1. The red dots represent p < 0.05 and log₂ fold change ≥1, which means that the expression of resistance genes is significantly upregulated. The blue dots represent p < 0.05 and log₂ fold change ≤ − 1, which means that the expression of resistance genes is significantly down-regulated. While the gray dots mean that there is no significant change. The top ten up-regulated resistance genes are labeled in the figure.