Idiosyncratic Drug-Induced Liver Injury and Amoxicillin-Clavulanate: Spotlight on Gut Microbiota, Fecal Metabolome and Bile Acid Profile in Patients

Abstract

Several hepatic disorders are influenced by gut microbiota, but its role in idiosyncratic drug-induced liver injury (iDILI), whose main causative agent is amoxicillin-clavulanate, remains unknown. This pioneering study aims to unravel particular patterns of gut microbiota composition and associated metabolites in iDILI and iDILI patients by amoxicillin-clavulanate (iDILI-AC). Thus, serum and fecal samples from 46 patients were divided into three study groups: healthy controls (n = 10), non-iDILI acute hepatitis (n = 12) and iDILI patients (n = 24). To evaluate the amoxicillin-clavulanate effect, iDILI patients were separated into two subgroups: iDILI non-caused by amoxicillin-clavulanate (iDILI-nonAC) (n = 18) and iDILI-AC patients (n = 6). Gut microbiota composition and fecal metabolome plus serum and fecal bile acid (BA) analyses were performed, along with correlation analyses. iDILI patients presented a particular microbiome profile associated with reduced fecal secondary BAs and fecal metabolites linked to lower inflammation, such as dodecanedioic acid and pyridoxamine. Moreover, certain taxa like Barnesiella, Clostridia UCG-014 and Eubacterium spp. correlated with significant metabolites and BAs. Additionally, comparisons between iDILI-nonAC and iDILI-AC groups unraveled unique features associated with iDILI when caused by amoxicillin-clavulanate. In conclusion, specific gut microbiota profiles in iDILI and iDILI-AC patients were associated with particular metabolic and BA status, which could affect disease onset and progression.

Keywords: amoxicillin–clavulanate; bile acids; drug-induced liver disease; fecal metabolome; gut microbiota.

Figure 1

Experimental design. C, healthy control; H, non-iDILI acute hepatitis; BAs, bile acids; iDILI, idiosyncratic drug-induced liver injury; iDILI-AC, idiosyncratic drug-induced liver injury caused by amoxicillin–clavulanate; iDILI-nonAC, idiosyncratic drug-induced liver injury caused by compounds other than amoxicillin–clavulanate; SCFAs, short-chain fatty acids.

Figure 2

Differences in gut microbiota composition associated with idiosyncratic drug-induced liver injury. (A) α-diversity measured by the Shannon and Simpson index. (B) Principal coordinates analysis (PCoA) plot representing β-diversity based on the Bray–Curtis similarity index at the operational taxonomic unit (OTU) level. The percentage of the total variance explained is indicated in parentheses on each axis. (C) Venn diagram at the OTU level. (D) Differences in the relative abundance at the phylum level. (E) Boxplots representing differences in the relative abundance at the family level. (F) Linear discriminant analysis (LDA) at the genus level. p-value cutoff was settled on 0.1. Log LDA score was settled on 2.0. (G) Boxplots representing differences in the relative abundance at the genus level. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. C; ^# p < 0.05; ^## p < 0.01 vs. H by a non-parametric Kruskal–Wallis test followed by the Mann–Whitney U-Test. C, healthy control group (n = 7); iDILI, idiosyncratic drug-induced liver disease group (n = 24); H, non-iDILI acute hepatitis group (n = 12); LDA, linear discriminant analysis; OUT, operational taxonomic unit.

Figure 3

Differences in fecal gut metabolome related to idiosyncratic drug-induced liver injury. (A) Principal component analysis (PCA) based on the Bray–Curtis index showing the differences in the fecal metabolomic profile. The percentage of the total variance explained is indicated in parenthesis in XY axes. (B) Boxplots representing non-targeted metabolites expressed as the peak area of detection. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. C; ^### p < 0.001 vs. H by a non-parametric Kruskal–Wallis test followed by the Mann–Whitney U-Test. (C) Total bile acid concentration and relative levels of primary, secondary, conjugated and unconjugated bile acids in serum. Relative levels of glycocholic and cholic acid. The dashed line represents total bile acid levels relativized to one. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. C by one-way ANOVA and data are presented as mean ± SEM. (D) Boxplots showing significant fecal bile acids. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. C by a non-parametric Kruskal–Wallis test followed by the Mann–Whitney U-Test. BAs, bile acids; C, healthy control group (Figure 2A,B,D: n = 7; Figure 2C: n = 4); CA, cholic acid; GCA, glycocholic acid; iDILI, idiosyncratic drug-induced liver disease group (Figure 2A,B,D: n = 17; Figure 2C: n = 23); H, non-iDILI acute hepatitis group (Figure 2A,B,D: n = 6; Figure 2C: n = 12).

Figure 4

Correlation analysis among gut microbiota composition, fecal metabolome and fecal and serum bile acid profiles. (A) Heatmap of correlations between serum bile acids and gut microbiota composition. (B) Heatmap of correlations between gut microbiota composition and fecal bile acids. Each square represents the Spearman’s correlation coefficient (p < 0.05). Red and blue cells represent positive and negative correlations, respectively. White crosses designate the level of significance. ⁺ p < 0.05; ⁺⁺ p < 0.01; ⁺⁺⁺ p < 0.001. C, healthy control group (Figure 3A: n = 4; Figure 3B: n = 7); iDILI, idiosyncratic drug-induced liver disease group (Figure 3A: n = 23; Figure 3B: n = 17); H, non-iDILI acute hepatitis group (Figure 3A: n = 12; Figure 3B: n = 6).

Figure 5

Correlation analysis of gut microbiota composition and metabolomic profile. Each square represents the Spearman’s correlation coefficient (p < 0.05). Red and blue cells represent positive and negative correlations, respectively. White crosses designate the level of significance. ⁺ p < 0.05; ⁺⁺ p < 0.01. Remarkable bacterial taxa and fecal metabolites are represented in boxplots. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. C; ^# p < 0.05 vs. H by a non-parametric Kruskal–Wallis test followed by the Mann–Whitney U-Test. C, healthy control group (n = 7); iDILI, idiosyncratic drug-induced liver disease group (n = 17); H, non-iDILI acute hepatitis group (n = 6).

Figure 6

Differences in patients with idiosyncratic drug-induced liver injury caused by amoxicillin–clavulanate in gut microbiota composition, compared with iDILI-nonAC. (A) Differences in relative abundance at the phylum level. (B) Differences in relative abundance at the family and genus levels. (C) Linear discriminant analysis (LDA) at the genus level. p-value cutoff was settled on 0.1. Log LDA score was settled on 2.0. (D) α-diversity measured by the Shannon and Simpson index. (E) Principal coordinates analysis (PCoA) plot representing β-diversity based on the Bray-Curtis similarity index at the operational taxonomic unit (OTU) level. The percentage of the total variance explained is indicated in parentheses on each axis. * p < 0.05 vs. iDILI-nonAC by a non-parametric Kruskal–Wallis test followed by the Mann–Whitney U-Test. iDILI-AC, idiosyncratic drug-induced liver injury caused by the amoxicillin–clavulanate group (n = 6); iDILI-nonAC, idiosyncratic drug-induced liver injury caused by compounds other than the amoxicillin–clavulanate group (n = 18).

Figure 7

Differences in patients with idiosyncratic drug-induced liver injury caused by amoxicillin–clavulanate in fecal metabolomic profile and fecal bile acid quantification compared with iDILI-nonAC. (A) Boxplots representing non-targeted metabolites expressed as the peak area of detection. (B) Boxplots representing the concentration of fecal bile acids. (C) Total concentration and relative levels of bile acids in serum. The dashed line represents total bile acid levels relativised to one. * p < 0.05; ** p < 0.01 vs. iDILI-nonAC by a non-parametric Kruskal–Wallis test followed by the Mann–Whitney U-Test Wallis test. Serum bile acids were analyzed by a one-way ANOVA test and data are presented as mean ± SEM. BAs, bile acids; CA, cholic acid; CDCA, chenodeoxycholic acid; iDILI-AC, idiosyncratic drug-induced liver injury caused by the amoxicillin–clavulanate group (Figure 6A,B: n = 5; Figure 6C: n = 6); iDILI-nonAC, idiosyncratic drug-induced liver injury caused by compounds other than the amoxicillin–clavulanate group (Figure 6A,B: n = 12; Figure 6C: n = 17); TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid.