Tingting Xia,1,* Shuo Feng,1,* Zigui Zou,2 Jikai Zhou,1 Xiaodi Cai,1 Jianxin Ye,1 Chenguang Dai1 

1Department of Gastroenterology, First Affiliated Hospital of Soochow University, Suzhou, 215006, People’s Republic of China; 2Department of Pathology, First Affiliated Hospital of Soochow University, Suzhou, 215006, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Chenguang Dai, Email daicg_soochow@163.com Jianxin Ye, Email yjx13906210479@sina.com

Background: Endoscopic retrograde cholangiopancreatography (ERCP) is widely used in the treatment of choledocholithiasis, while successful extraction of common bile duct stone (CBDS) is commonly hampered by the number of stones. Biliary microbiota has a profound influence on the occurrence of CBDS. In this study, we aimed to investigate the characteristics and metabolic potential of biliary microbiota in patients with multiple CBDS.
Methods: Eligible patients were prospectively enrolled in this study at First Affiliated Hospital of Soochow University between December 2022 and October 2023. Bile samples were collected through ERCP. The samples were tested for biliary microbiota and bile acids using 16S rRNA sequencing and ultra-performance liquid chromatography-tandem mass spectrometry, respectively. Metabolic functions were predicted by PICRUSt 2.0 calculation based on MetaCyc database.
Results: A total of 31 patients were enrolled, including 17 in multiple stone (MS) group and 14 in single stone (SS) group. Distinct biliary microbial composition was identified in MS group, with a significantly higher abundance of Proteobacteria at phylum level and Enterococcus at genus level, respectively. Klebsiella, Aquabacterium, Morganella and Diaphorobacter were significantly abundant in MS group. Both Morganella and Aeromonas were exclusively found in MS group, along with the absence of Metaprevotella. Chenodeoxycholic acid was significantly enriched in MS group. It was negatively correlated with Enhydrobacter, Massilia and Neglecta that were abundant in SS group. Several metabolic pathways that could increase the risk of CBDS were also enriched in MS group, including L-methionine biosynthesis, aspartate superpathway, glucose and glucose-1-phosphate degradation and superpathway of glycolysis and the Entner-Doudoroff pathway.
Conclusion: This study illustrated the microbial structure and metabolic potential of biliary flora in patients with multiple CBDS. The unique biliary microbial community holds the predictive value for clinical conditions. The findings provide new insights about biliary microbiota into the etiology of multiple CBDS.

Keywords: biliary microbiota, multiple common bile duct stones, 16S rRNA sequencing, metabolic function, bile acid