Biliary microbiota associated with early postoperative stone reappearance under T-Tube surveillance: an exploratory 16 S rRNA sequencing study

Early postoperative stone reappearance after biliary surgery may reflect residual stones, early re-formation, or bile duct recolonization within an altered biliary environment. Although mechanical and anatomical factors are traditionally implicated in recurrent common bile duct stones, they do not fully explain early stone reappearance after apparently successful duct clearance. Biliary dysbiosis has been linked to recurrent stone disease, but its features during routine T-tube surveillance remain unclear. This single-center exploratory study included 50 patients who underwent biliary surgery with T-tube drainage and completed 3-month reassessment by T-tube cholangiography and magnetic resonance cholangiopancreatography. According to concordant imaging findings, 18 patients were assigned to the stone reappearance group and 32 to the non-reappearance group. Bile samples collected at T-tube opening were analyzed using 16 S rRNA V3–V4 sequencing. Clinical variables, microbial diversity, taxonomic composition, LEfSe, the exploratory random-forest classifier, and PICRUSt2 functional prediction were evaluated. Recorded clinical variables, including age, sex, smoking, diabetes, hyperlipidemia, common bile duct dilatation, distal stricture, biliary infection, and post-discharge antibiotic use, did not differ significantly between groups. Compared with the non-reappearance group, the stone reappearance group showed reduced alpha diversity and a trend toward beta-diversity separation. Proteobacteria and Firmicutes dominated both groups, with greater Proteobacteria enrichment in the reappearance group. Enterobacteriaceae- and Enterococcus-related taxa, including Escherichia-Shigella, Klebsiella, Enterococcus, and Acinetobacter, were more abundant in patients with stone reappearance. LEfSe and the exploratory random-forest classifier consistently identified these taxa as candidate discriminatory microbial features, and the exploratory random-forest classifier yielded an apparent AUC of 0.80 in this dataset. PICRUSt2 predicted differences in glucuronate/glucuronide metabolism, membrane transport, biofilm formation, lipopolysaccharide-associated pathways, and selected carbohydrate and amino acid metabolism. Early postoperative stone reappearance under T-tube surveillance is associated with bile microbial dysbiosis characterized by reduced diversity and enrichment of enteric opportunistic taxa. These findings suggest that bile microbiota may provide candidate markers for early stone reappearance and warrant validation in larger longitudinal studies.