A Preliminary Study of Biliary Microbiota in Patients with Bile Duct Stones or Distal Cholangiocarcinoma

Abstract

Background and objective: The distal cholangiocarcinoma (dCCA) is associated with many factors: genes, environment, infection, etc. The current changes in biliary flora are thought to be involved in the formation of many gastrointestinal tract (GIT) diseases, like colon adenocarcinoma. Therefore we want to investigate whether the dCCA has a certain correlation with biliary microecology, and to detect specific strains.

Methods: A total of 68 adults were enrolled, of whom 8 with dCCA, 16 with recurrent choledocholithiasis, and 44 with the onset of common bile duct stones. Endoscopic Retrograde Cholangiopancretography (ERCP) was utilized to collect bile samples for DNA extraction and 16S rRNA gene sequencing, followed by analysis of bile microbiota composition.

Results: First, Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria are the most dominant phyla in the bile of patients with dCCA and the onset of common bile duct stoes. Secondly, compared with the onset of common bile duct stones patients, we got a significant increase in the phylum Gemmatimonadetes, Nitrospirae, Chloroflexi, Latescibacteria, and Planctomycetes in dCCA patients. Finally, at the genus level, we obtained sequencing results of 252 bacterial genera from patients with dCCA, recurrent choledocholithiasis, and the new onset of common bile duct stones, revealing heterogeneity among individuals.

Conclusion: To the best of our knowledge, this is the first study of the dysbiosis of bile flora in patients with dCCA. This micro-ecological disorder may be a decisive factor in the formation of dCCA. At the same time, for the first time, this study provides a test chart of biliary microbial populations that may be associated with recurrent choledocholithiasis. The compositional changes of the core microbial group of the biliary tract have potentially important biological and medical significance for the microbiological biliary disorders of dCCA.

Figure 1

The richness and diversity of biliary microbiota in patients with dCCA and the onset of common bile duct stones(a&b). Richness rerefaction plot from all samples,the abscissa indicates the number of sequences randomly extracted from the sample, and the ordinate indicates the number of OTUs obtained by clustering the corresponding sequence numbers (c). The dCCA group (Tumor group, T) and the new onset of CBD stones group (CBD stones group, C) denoted as “Group T” and “Group C”, respectively, in the figure.

Figure 2

Relative taxa abundance and OTUs comparison in patients with dCCA and the onset of common bile duct stones. The overall microbial composition at the phylum (a) and genus (b) level illustrated that the taxonomic composition varies widely among individuals, but Proteobacteria and Firmicutes were the dominant phylum among all individual, and Escherichia/Shigella and Halomonas were the major genus. It indicated that the core OTUs from patients with dCCA and the new onset of common bile duct stones were composed of 536 and 703 OTUs, respectively, of which 490 were shared between the two groups (c). The dCCA group (Tumor group, T) and the new onset of CBD stones group (CBD stones group, C) denoted as “Group T” and “Group C”, respectively, in the figure.

Figure 3

Relative abundances of dominant phyla in patients with dCCA (a), and the onset of common bile duct stones (b), patients from both groups contained five dominant phyla: Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Unclassified_Bacteria, which accounted for 97% of the biliary microbes. Relative abundance of Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria is similar between patients with dCCA and the new onset of common bile duct stones. The dCCA group (Tumor group, T) and the new onset of CBD stones group (CBD stones group, C) denoted as “Group T” and “Group C”, respectively, in the figure.

Figure 4

Heatmap of bile microbial from all samples. We tested bile in patients with biliary tract tumors and compared all bile microbial organisms in patients with biliary tract tumors, recurrent choledocholithiasis, and the new onset of common bile duct stones. Analysis of bile microbial colonies reveals heterogeneity between individuals. The dCCA group (Tumor group, T), the new onset of CBD stones group (CBD stones group, C), and recurrent CBD stones group (Post-ERCP CBD stones group, P) denoted as “Group T”, “Group C”, and “Group P”, respectively, in the figure.

Figure 5

Characteristics of microbial community composition in patients with recurrent choledocholithiasis and the onset of common bile duct stones. (a) Cladogram representing taxa enriched in biliary microbiota community of the two groups detected by the LEfSe tool. Differences were represented by the color of the most abundant class (red, patients with the onset of common bile duct stones; green, patients with recurrent choledocholithiasis). The diameter of each circle is proportional to the taxon's abundance. The central point represents the root of the tree (Bacteria), and each ring represents the next lower taxonomic level. (b) Histogram of the LDA scores computed for different abundance levels between patients with recurrent choledocholithiasis and the onset of common bile duct stones, as detected by the LEfSe tool. (c–j) Relative abundance of Prevotella, Alloprevotella, Nesterenkonia, Pyramidobacter, Aeromonas, Enterococcus, Unclassified_Enterobacteriaceae, and Citrobacter in biliary microbiota community of patients with recurrent choledocholithiasis and the onset of common bile duct stones.