The human gallbladder microbiome is related to the physiological state and the biliary metabolic profile

Abstract

Background: The microbial populations of the human intestinal tract and their relationship to specific diseases have been extensively studied during the last decade. However, the characterization of the human bile microbiota as a whole has been hampered by difficulties in accessing biological samples and the lack of adequate methodologies to assess molecular studies. Although a few reports have described the biliary microbiota in some hepatobiliary diseases, the bile microbiota of healthy individuals has not been described. With this in mind, the goal of the present study was to generate fundamental knowledge on the composition and activity of the human bile microbiota, as well as establishing its potential relationship with human bile-related disorders.

Results: Human bile samples from the gallbladder of individuals from a control group, without any record of hepatobiliary disorder, were obtained from liver donors during liver transplantation surgery. A bile DNA extraction method was optimized together with a quantitative PCR (qPCR) assay for determining the bacterial load. This allows the selection of samples to perform functional metagenomic analysis. Bile samples from the gallbladder of individuals suffering from lithiasis were collected during gallbladder resection and the microbial profiles assessed, using a 16S rRNA gene-based sequencing analysis, and compared with those of the control group. Additionally, the metabolic profile of the samples was analyzed by nuclear magnetic resonance (NMR). We detected, for the first time, bacterial communities in gallbladder samples of individuals without any hepatobiliary pathology. In the biliary microecosystem, the main bacterial phyla were represented by Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. Significant differences in the relative abundance of different taxa of both groups were found. Sequences belonging to the family Propionibacteriaceae were more abundant in bile samples from control subjects; meanwhile, in patients with cholelithiasis members of the families Bacteroidaceae, Prevotellaceae, Porphyromonadaceae, and Veillonellaceae were more frequently detected. Furthermore, the metabolomics analysis showed that the two study groups have different metabolic profiles.

Conclusions: Our results indicate that the gallbladder of human individuals, without diagnosed hepatobiliary pathology, harbors a microbial ecosystem that is described for the first time in this study. Its bacterial representatives and metabolites are different from those detected in people suffering from cholelithiasis. In this regard, since liver donors have been subjected to the specific conditions of the hospital's intensive care unit, including an antibiotic treatment, we must be cautious in stating that their bile samples contain a physiologically normal biliary microbiome. In any case, our results open up new possibilities to discover bacterial functions in a microbial ecosystem that has not previously been explored.

Keywords: Bile microbiota; Cholelithiasis; Gallstones patients; Microbial bile metabolites.

Conflict of interest statement

The authors declared that they have no competing interests.

Figures

Fig. 1

Boxplots representing 16S rRNA gene and 18S rRNA gene levels in the control group (n = 13). The central rectangles represent interquartile ranges (IQR), the lines inside the rectangles show the median, and the whiskers indicate the maximum and minimum values. The dots outside the rectangles are suspected outliers (> 1.5 × IQR). Statistically significant differences (p value < 0.05) between the two variables (16S rRNA gene and 18S rRNA gene) were found (Mann-Whitney U test)

Fig. 2

Distribution of Clusters of Orthologous Groups (COGs) in three bile samples from control group (H-04, H-05, and H-06). The results show the percentage of sequences assigned to different metabolic functions (relative to all sequenced microbes). Secondary metabolite biosynthesis includes antibiotics, pigments, and non-ribosomal peptides. Inorganic ion transport and metabolism includes phosphate, sulfate, and various cation transporters

Fig. 3

Boxplots representing 16S rRNA gene levels between cholelithiasis (n = 14) and control group (n = 13). The central rectangles represent interquartile ranges (IQR), the lines inside the rectangles show the median, and the whiskers indicate the maximum and minimum values. The dots outside the rectangles are suspected outliers (> 1.5 × IQR). Statistically significant differences (p value < 0.05) between the two groups of the study were not found (Mann-Whitney U test)

Fig. 4

Comparison of Shannon’s diversity indices in cholelithiasis (n = 14) and control (n = 13) groups. The central rectangles represent interquartile ranges (IQR), the lines inside the rectangles show the median, and the whiskers indicate the maximum and minimum values. The dots outside the rectangles are suspected outliers (> 1.5 × IQR). Statistically significant differences (p value < 0.05) between groups were found (Mann-Whitney U test)

Fig. 5

Principal Coordinate Analysis (PCoA) plot of weighted UniFrac distances, comparing the bacterial communities among samples from cholelithiasis (red circles, n = 14) and control group (blue circles, n = 13). Percentages shown in the axes represent the proportion of dissimilarities. Analysis of molecular variance (AMOVA) was used to assess the statistical significance of the spatial separation between both groups (p value < 0.001)

Fig. 6

Principal component analysis (PCA) plot of bile metabolites. PC1 versus PC2 obtained for the whole spectra is represented. Control samples are represented in black. Blue dots belong to bile samples from cholelithiasis patients