The biliary microbiome in symptomatic cholelithiasis: a prospective comparison across distinct bacterial niches

Abstract Background Cholelithiasis is highly prevalent in the Western world and frequently requires surgical or endoscopic intervention. However, the composition and role of the biliary microbiome remain poorly understood. This study aimed to characterize and compare bacterial communities of the biliary niche with saliva and faeces in patients with cholelithiasis. Methods In this prospective observational study, patients with symptomatic cholecystolithiasis, chronic or acute cholecystitis, and choledocholithiasis were included. Bacterial community composition was characterized across bile, gallstone, saliva, and faecal samples using 16 S rRNA-based sequencing. Community-level and genus-level differences were assessed according to anatomical niche and clinical diagnosis, complemented by compositional sensitivity analyses and predicted functional profiling. Results A total of 227 samples from 81 patients were analyzed. Bacterial community composition was strongly determined by anatomical niche (PERMANOVA, R² = 0.273, P < 0.001), with bile and gallstone communities being most closely related but remaining significantly distinct (R² = 0.146, q < 0.001). Diagnosis-associated differences were predominantly confined to the biliary compartments and remained significant after adjustment for age, explaining 7.9% of variation in bile and 14.1% in gallstones, whereas no significant associations were observed in saliva or faeces. Compositionally aware analysis with continuous age adjustment identified robust diagnosis - associated signals in bile, including Escherichia/Shigella , Leptotrichia , Serratia , and Pseudomonas . Biliary communities were characterized by taxa including Enterococcus , Escherichia/Shigella , Serratia , and Pseudomonas . In bile, Escherichia/Shigella increased with inflammatory disease, while Pseudomonas decreased; compositionally aware analysis confirmed diagnosis-associated differences for Escherichia/Shigella , Leptotrichia , and Serratia . Predicted bacterial functions showed even stronger anatomical compartmentalization (R² = 0.507, P = 0.001), whereas diagnosis-associated functional differences were limited to two MetaCyc pathways in gallstones. Sensitivity analyses provided no evidence that prolonged antibiotic exposure significantly affected biliary bacterial diversity or community composition. Conclusion This comprehensive multi-niche analysis demonstrates pronounced taxonomic and predicted functional compartmentalization of bacterial communities across saliva, bile, gallstones, and faeces in patients with cholelithiasis. Diagnosis-associated alterations were predominantly observed within biliary niches, with Escherichia/Shigella emerging as a consistent taxon associated with inflammatory biliary disease. These findings identify anatomical niche as a major determinant of bacterial community structure and support disease-associated alterations within the biliary microbiome. Trial registration German Clinical Trials Register (Deutsches Register Klinische Studien) DRKS00030566 (retrospectively registered 20250818).