A Comparative Investigation of the Bile Microbiome in Patients with Choledocholithiasis and Cholecystolithiasis through Metagenomic Analysis

Abstract

While the precise triggers of gallstone formation remain incompletely understood, it is believed to arise from a complex interplay of genetic and environmental factors. The bile microbiome is being increasingly recognized as a possible contributor to the onset of gallstone disease. The primary objective of this study was to investigate distinctions in the microbial communities within bile specimens from patients with choledocholithiasis (common bile duct stones) and cholecystolithiasis (gallbladder stones). We employed massively parallel sequencing of the 16S rRNA gene to examine the microbial communities within bile samples obtained from 28 patients with choledocholithiasis (group DS) and cholecystolithiasis (group GS). The taxonomic composition of the bile microbial communities displayed significant disparities between the group DS and the group GS. Within the 16 prevalent genera, only Streptococcus, Ralstonia, Lactobacillus, and Enterococcus were predominantly found in the group GS. In contrast, the group DS displayed a more diverse range of genera. The alpha diversity of bile specimens was also notably lower in the group GS compared to the group DS (p = 0.041). Principal coordinate analysis unveiled distinct clustering of bile microbial communities depending on the location of the gallstone. Linear discriminant analysis effect size analysis, with a score threshold of >3 and the Kruskall-Wallis test (α < 0.05), recognized Bacilli and Lactobacillales as potential taxonomic markers for distinguishing patients with cholecystolithiasis limited to the gallbladder. Significant variations were found in the distribution and diversity of bile microbial communities between patients with choledocholithiasis and cholecystolithiasis. This observation suggests that alterations in the bile microbiome may contribute to the development of gallstones in these patients.

Keywords: 16S rRNA gene sequencing; bile microbial communities; choledocholithiasis; cholelithiasis; common bile duct; gallbladder; metagenomic analysis.

Figure 1

Representation of the averaged taxonomic composition proportions within the bile microbial communities at the phylum and genus levels for the group DS and the group GS. Stacked bar charts illustrating the taxonomic composition at the phylum (a) and genus (b) levels. ETC (et cetera) refers to the population of identified phylum or genus strains less than 1%. The taxonomic profiling of the microbiome at the genus level reveals that 1% of the composition in higher taxonomic ranks is classified as unassigned in group DS, whereas none is observed in group GS. At the phylum level, unassigned taxa were not identified in either group DS or GS. The x-axis represents the value in percentage.

Figure 2

Comparative analysis of taxonomic composition within four selected taxa known for their significance in the human gastrointestinal tract. The group DS exhibited a higher relative taxonomic abundance of Bacteroides (a), Enterobacteriaceae (b), Prevotella (c), and Proteobacteria (d) compared to the group GS.

Figure 3

Visualization of rarefaction and rank abundance curves for the DS and group GS. Rarefaction curves and species richness indices indicate the extent of comprehensive sampling in group DS (a) and group GS (b). The broader span of rank abundance curves reflects higher relative species abundance, and the smoother curve on the Y-axis signifies greater evenness in the bacterial distribution in group DS (c) and group GS (d).

Figure 4

Analysis of alpha diversity in bile microbial communities in the DS and group GS at the genus level using massively parallel 16S rRNA gene sequencing. Evaluation of alpha diversity for species richness (a–d) and diversity index (e–h) within bile microbial communities collected from the common bile duct in the choledocholithiasis (group DS) and the gall bladder in the cholelithiasis (group GS).

Figure 5

Beta diversity assessment within bile microbial communities in the DS and group GS at the genus level using massively parallel 16S rRNA gene sequencing. (a) Principal coordinate analysis (PCoA) highlighting distinct clustering, indicative of differences in overall bile microbial communities between the group DS (blue solid dot) and the group GS (green solid dot) based on gallstone location. (b,c) Hierarchical clustering analysis using the unweighted pair group method with arithmetic mean (UPGMA) revealed variations in abundance and diversity between the group DS (blue empty box) and the group GS (green empty box) based on generalized UniFrac (b) and UniFrac (c). (d,e) Calculation of diversity index differences between the group DS and the group GS presented in a representative box plot using permutational multivariate analysis of variance (PERMANOVA).

Figure 6

Illustration of the taxonomic distribution in the DS and group GS generated by linear discriminant analysis effect size (LEfSe) with an LDA score threshold > 3 and the Kruskall–Wallis test set at a significance level of 0.05.