Altered gut microbe metabolites in patients with alcohol‑induced osteonecrosis of the femoral head: An integrated omics analysis

Abstract

Excessive alcohol consumption is considered to be a major risk factor of alcohol-induced osteonecrosis of the femoral head (AONFH). The gut microbiota (GM) has been reported to aid in the regulation of human physiology and its composition can be altered by alcohol consumption. The aim of the present study was to improve the understanding of the GM and its metabolites in patients with AONFH. Metabolomic sequencing and 16S rDNA analysis of fecal samples were performed using liquid chromatography-mass spectrometry to characterize the GM of patients with AONFH and healthy normal controls (NCs). Metagenomic sequencing of fecal samples was performed to identify whether GM changes on the species level were associated with the expression of gut bacteria genes or their associated functions in patients with AONFH. The abundance of 58 genera was found to differ between the NC group and the AONFH group. Specifically, Klebsiella, Holdemanella, Citrobacter and Lentilactobacillus were significantly more abundant in the AONFH group compared with those in the NC group. Metagenomic sequencing demonstrated that the majority of the bacterial species that exhibited significantly different abundance in patients with AONFH belonged to the genus Pseudomonas. Fecal metabolomic analysis demonstrated that several metabolites were present at significantly different concentrations in the AONFH group compared with those in the NC group. These metabolites were products of vitamin B6 metabolism, retinol metabolism, pentose and glucuronate interconversions and glycerophospholipid metabolism. In addition, these changes in metabolite levels were observed to be associated with the altered abundance of specific bacterial species, such as Basidiobolus, Mortierella, Phanerochaete and Ceratobasidium. According to the results of the present study, a comprehensive landscape of the GM and metabolites in patients with AONFH was revealed, suggesting the existence of interplay between the gut microbiome and metabolome in AONFH pathogenesis.

Keywords: alcohol; gut microbiota; metabolite; metagenomics; osteonecrosis of the femoral head.

Figure 1

Gut microbiome diversity and structure analysis of patients with AONFH and NCs. (A) Venn diagram of the observed features (amplicon sequence variants) in the AONFH and NC groups. PCoA of the microbiota based on (B) unweighted UniFrac (ANOSIM, R=0.058, P=0.005) and (C) Jaccard (ANOSIM, R=-0.060, P=0.003) distance matrices for the AONFH and NC groups. (D) Heatmap was generated at phylum level based on the relative abundance values. Statistically significant differences in bacterial abundance at the (E) phylum and (F) genus level between the AONFH and NC groups (mean ± SD). ^*P<0.05, ^**P<0.01. AONFH, alcohol-induced osteonecrosis of the femoral head; NC, negative control; PCoA, principal coordinate analysis; ANOSIM, analysis of similarities; p_, at phylum level, g_, at genus level.

Figure 2

Gut microbiome composition and functional analyses. (A) Cladogram indicating the phylogenetic distribution of the microbiota in the AONFH and NC groups. (B) Linear discriminant analysis integrated with effect size demonstrated differences in abundance between the AONFH and NC groups. (C) Predicted functions of the gut microbiota based on KEGG pathway analysis. The extended error bar plot demonstrated the significantly different KEGG pathways between the AONFH and NC groups. AONFH, alcohol-induced osteonecrosis of the femoral head; NC, negative control; KEGG, Kyoto Encyclopedia of Genes and Genomes. p, phylum; c, class; o, order; f, family; g, genus; s, species.

Figure 3

Differences in the gut microbiota in the AONFH and NC groups based on the metagenomic sequencing data. (A) PCoA analysis based on the Bray-Curtis distance matrix between the AONFH and NC groups at the species level (ANOSIM, R=0.02; P=0.06). (B) Relative abundance of the top 20 species enriched in the AONFH and NC groups. Data were presented as median ± interquartile range. (C) Performance of a random forest model classification as assessed using the R ‘random forest’ package. (D) Receiver operating characteristic curve displaying the top five biomarkers for distinguishing between the AONFH and NC groups. ^*P<0.05. AONFH, alcohol-induced osteonecrosis of the femoral head; NC, negative control; PCoA, principal coordinate analysis; ANOSIM, analysis of similarities; AUC, area under the curve.

Figure 4

(A) Partial least squares discriminant analysis score plot in negative ion mode. (B) Permutation plot in negative ion mode. (C) Partial least squares discriminant analysis score plot in positive ion mode. (D) Permutation plot in positive ion mode. AONFH, alcohol-induced osteonecrosis of the femoral head; NC, negative control; PC, principal component; Cor, correlation coefficient.

Figure 5

Fecal metabolomic changes and metabolic pathway in patients with AONFH. (A) The gut metabolites were identified by HMDB superclass analysis. (B) Volcano plot demonstrated the number of dysregulated metabolites in the feces of patients with AONFH compared with the NC group. (C) Computed metabolic pathways as a function of P-value and the pathway impacts of the key metabolites that were differentially expressed between the AONFH and NC groups. AONFH, alcohol-induced osteonecrosis of the femoral head; NC, negative control; HMDB, Human Metabolome Database.

Figure 6

Differentially expressed metabolic pathways in patients with AONFH. (A) The most abundant metabolic pathways in the two groups as identified by Kyoto Encyclopedia of Genes and Genomes enrichment analysis. (B) Correlation between changes in the gut microbiota and the changes in metabolic product concentrations in patients with AONFH. AONFH, alcohol-induced osteonecrosis of the femoral head; NC, negative control.