Characterization of biliary microbiota dysbiosis in extrahepatic cholangiocarcinoma

Abstract

Extrahepatic cholangiocarcinoma (CCA) accounts for 3% of digestive cancers. The role of biliary microbiota as an environment-related modulator has been scarcely investigated in CCA, and the putative impact of associated diseases has not been yet assessed. We characterized the biliary microbiota in CCA patients in order to identify a specific CCA-related dysbiosis. The biliary effluents were collected through an endoscopic retrograde pancreatic cholangiography (ERCP) examination involving 28 CCA and 47 patients with gallstones, herein considered as controls. The biliary effluents were submitted to bacterial DNA extraction and 16S rRNA sequencing, using Illumina technology. Overall, 32% of CCA and 22% of controls displayed another associated disease, such as diabetes, pancreatitis, inflammatory bowel disease, or primary sclerosing cholangitis. Such associated diseases were considered in the comparisons that were made. Principal coordinate analysis (PCoA) detected a significant disparity of biliary microbiota composition between CCA patients and controls without an associated disease. Amongst the most abundant phyla, Proteobacteria did not significantly differ between CCA patients and controls, whereas Firmicutes levels were lower and Bacteroidetes higher in CCAs' biliary microbiota than in the controls' microbiota. The most abundant genera were Enterococcus, Streptococcus, Bacteroides, Klebsiella, and Pyramidobacter in CCA's biliary microbiota. Additionally, levels of Bacteroides, Geobacillus, Meiothermus, and Anoxybacillus genera were significantly higher in CCA patients' biliary microbiota, without an associated disease, in comparison with controls. A specific CCA-related dysbiosis was identified as compared to controls independently from associated diseases. This suggests that a microorganism community may be involved in CCA pathogenesis.

Fig 1. Relative abundances (%) of genera in patients and controls.

The 12 most abundant genera are illustrated in the cases and controls. A) CCA (n = 28) and controls (n = 47), regardless of associated diseases. B) CCA and controls, with (Yes) or without (NO) associated diseases.

Fig 2. Relative abundances (%) of phyla in patients and controls.

The 12 most abundant phyla in all individuals. A) CCA (n = 28) and controls (n = 47), regardless of associated diseases. B) CCA and controls, with (Yes) or without (NO) associated diseases.

Fig 3. Principal coordinate analysis (P CoA) of biliary microbiota in patients and controls.

A) PCoA according to the diagnosis, in all individuals (permanova test; p = 0.058). B) PCoA according to the diagnosis, limited to patients without associated diseases (permanova test = 0.01).