Alterations in the gut microbiota in individuals with infantile cholestasis: a comparison of high- and low-γ-glutamyltransferase subtypes

Abstract

Background: There are two subtypes of infantile cholestasis (IC): high-γ-glutamyltransferase (GGT) and low-GGT (LG) cholestasis. This study aims to investigate the differences in the gut microbiota between infants with cholestasis and healthy infants and the correlations between the gut microbiota and blood or urine metabolites associated with high-GGT (HG) and LG IC subtypes.

Methods: A total of 37 infants were recruited into the cholestasis (Cho) group, including 21 infants with HG and 16 infants with LG levels, with 23 infants included in the control (Con) group. The gut microbiota composition was comprehensively analyzed, as were blood and urine metabolites, clinical indicators, and their correlations. The study was registered in both the Chinese Clinical Trial Registry (www.chictr.org.cn, ChiCTR2300072387) and the National Medical Research Registration & Filing Information System (www.medicalresearch.org.cn, MR-35-24-056613).

Results: The Lactobacillus, Streptococcus, Bacteroides, and Lactococcus abundances were greater and the Lachnoclostridium abundance was lower in the Cho group than in the Con group. The Bacteroides abundance was significantly greater in the HG group than in the LG group. The characteristic bacterial taxa in the HG group were Bacteroides, Lactococcus, Streptococcus thermophilus TH1435, Haemophilus, Parabacteroides and Subgroup_6, whereas those in the LG group were Gammaproteobacteria, Streptococcus, Blautia, Pasteurellaceae, Staphylococcus, Megamonas, Helicobacter and Bacillus. The HG group presented higher concentrations of alkapton-3 and lower concentrations of methylfumaric acid-2, 2-hydroxyglutaric acid-3 and malic acid-3 than the LG group. The abundances of Pasteurellaceae and Parabacteroides were negatively correlated with GGT levels and pediatric end-stage liver disease (PELD) scores, respectively.

Conclusions: Infants with cholestasis exhibit gut microbiota dysbiosis. Notably, distinct differences in the microbiota profiles were observed between the HG and LG cholestasis groups. Bacteroides and Parabacteroides may play roles in the HG group, whereas Gammaproteobacteria and Pasteurellaceae did so in the LG group. Our research provides new insights into the relationships between gut microbiota and different subtypes of IC, but the causal relationships and specific mechanisms need to be characterized further and verified.

Keywords: Infantile; cholestasis; gamma-glutamyl transpeptidase; gut microbiota; metabolite.

Figure 1

Abundance and structural changes in gut microbiota at the phylum and genus levels. (A) Distribution of the top 5 species at the phylum level in the Cho and Con groups. (B) Distributions of the top 10 species at the genus level in the Cho and Con groups. (C) Distribution of the top 5 species at the phylum level in the HG and LG groups. (D) Distribution of the top 10 species at the genus level in the HG and LG groups. Cho, cholestasis; Con, control; GGT, γ-glutamyltransferase; HG, high-GGT; LG, low-GGT.

Figure 2

Comparison of the abundances of the gut microbiota in the Cho and Con groups at the phylum and genus levels. The Mann-Whitney U test was used to compare the abundances of the gut microbiota between the Cho and Con groups. *, P<0.05, **, P<0.01, and the y-axis represents the abundance (log₂). (A-E) Violin plots showing the abundances of Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria in the Cho and Con groups at the phylum level. (F-O) Violin plots comparing the abundances of Bacteroides, Bifidobacterium, Clostridium_sensu_stricto_1, Enterococcus, Escherichia-Shigella, Lachnoclostridium, Lactobacillus, Lactococcus, Streptococcus and Veillonella in the Cho and Con groups at the genus level. Cho, cholestasis; Con, control.

Figure 3

Diversity analysis and a Circos plot of the microbial composition. (A,B) Alpha diversity of the gut microbiota in the Con, HG and LG groups expressed by the Richness and Chao1 indices. The x-axis represents different groups, while the y-axis represents the Richness and Chao1 indices. The Wilcoxon test was performed for pairwise comparisons; ****, P<0.0001. Beta diversity of the gut microbiota in the Cho and Con groups (C) and in the HG and LG groups (D) expressed according to PCoA based on Bray-Curtis distance. The P value was tested with ANOSIM; each point corresponds to a sample shaped and colored by diagnosis. (E) Circos plot of the top 8 most abundant samples in the Con, HG, and LG groups. The first circle: sample grouping information; the second circle: the percentage of OTU relative abundance in each sample; the third circle: OTUs and the main block of the sample are distinguished by different colors, and the scale outside the block is the absolute abundance of OTUs; the fourth circle: OTUs and the sample subblock, corresponding to the main block (the third circle), showing the abundance of OTUs in each sample and the abundance of each OTU contained in each sample; the fifth circle: the connection of the OTUs and the sample association information, corresponding to the OTUs and the sample subblock (the fourth circle). ANOSIM, analysis of similarities; Cho, cholestasis; Con, control; GGT, γ-glutamyltransferase; HG, high-GGT; LG, low-GGT; OTU, operational taxonomic unit; PCoA, principal coordinate analysis.

Figure 4

LEfSe analysis of the gut microbiota and KOs. Statistical significance was determined via LEfSe, with a P value of <0.05 (Wilcoxon test) and a LDA score (log₁₀) of >2.5 considered significant. (A) Taxonomic differences in the Con, HG and LG groups. (B) Significantly different KOs were detected in the Con, HG and LG groups. Con, control; GGT, γ-glutamyltransferase; HG, high-GGT; LG, low-GGT; LDA, linear discriminant analysis; LEfSe, linear discriminant analysis effect size; KOs, functional categories.

Figure 5

Correlation analysis of gut microbiota and metabolites or clinical indicators. Spearman correlation analysis between the characteristic bacteria of LEfSe analysis and differentially abundant metabolites (A) and clinical indicators (B) in the HG and LG groups was employed. Red and green represent positive and negative correlations, respectively. (A) Heatmap of correlation analysis of the characteristic bacteria and differentially abundant metabolites in the HG and LG groups. (B) Heatmap of correlation analysis of the characteristic bacteria and clinical indicators in the HG and LG groups. *, P<0.05; **, P<0.01. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Dbil, direct bilirubin; GGT, γ-glutamyltransferase; HG, high-GGT; Ibil, indirect bilirubin; INR, international normalized ratio; LEfSe, linear discriminant analysis effect size; LG, low-GGT; PELD, pediatric end-stage liver disease; PT, prothrombin time; TBA, total bile acid; Tbil, total serum bilirubin.