Preliminary study on the correlation between diversity of biliary flora and recurrence of common bile duct stones

Objective To investigate the correlation between the diversity of biliary bacterial flora and the recurrence of common bile duct stones. Methods From September 2018 to March 2019, 48 patients underwent endoscopic retrograde cholangiopancreatography (ERCP) at the First Affiliated Hospital of Nanjing Medical University were enrolled and divided into primary choledocholithiasis (primary group, 38 patients) and recurrent choledocholithiasis group (recurrent group, 10 patients). The bile of the patients of the two groups was collected and analyzed with high-throughput sequencing of 16S rRNA. The Chao1 estimator indicated the richness of the biliary bacterial flora, and Shannon index and Simpson index demonstrated the diversity of biliary bacterial flora. The bacterial flora distribution was explored from different levels of phylum, class, order, family, genus and species. Chi-square test and t test were performed for statistical analysis. Results The Chao1 estimator of the primary group was higher than that of the recurrent group (419.413±118.704 vs. 396.000±70.483), and the difference was statistically significant (t=3.130, P=0.003). At the level of the phylum, the highest abundance of bacteria in the primary group was Firmicute (56.48%), the highest abundance of bacteria in the recurrent group was Proteobacteria (57.79%). The dominant bacteria of the recurrent group was Proteobacteria, which was inconsistent with that of the primary group, and there were significant differences in the distribution of Proteobacteria and Firmicute between two groups (χ2=0.962 and -2.619, both P<0.05). At the genus level, the abundance of Bacillus and Lactococcus of the recurrent group were both lower than those of the primary group (9.75% vs. 20.77%, 10.86% vs. 22.01%, respectively), and the differences were statistically significant (χ2=0.354 and 0.503, both P<0.05). The abundance of Morganella of the recurrent group was higher than that of the primary group (9.00% vs. 0.44%), and the difference was statistically significant (χ2=-2.025, P=0.049). At the species level, the abundance of Bacillus unclassified, Carnobacterium maltaromaticum and Bacillus circulans of the primary group were all higher than those of the recurrent group (17.78% vs. 8.84%, 2.39% vs. 1.11%, 2.59% vs. 0.74%, respectively), and the differences were all statistically significant (χ2=2.540, 2.643 and 2.515, all P<0.05). The abundance of Aeromonas veronii of recurrent group was higher than that of the primary group (2.04% vs. 0.01%), and the difference was statistically significant (χ2=-2.397, P=0.021). The bacteria that had significant effects in the primary group included Lactococcus (P=0.012), Lactobacillus (P=0.033) and Geobacillus (P=0.021), while in the recurrent group, which included Enterobacter (P=0.007), Aeromonadaceae (P=0.001), Actinomycetes (P=0.009), and Aeromonas (P=0.001). Conclusions There are differences in abundance and composition of biliary bacterial flora between the recurrent group and the primary group. It remains to be further studied whether the changes of some bacterial flora correlated with the recurrence of common bile duct stones. Key words: Choledocholithiasis; Recurrence of choledocholithiasis; Biliary flora; 16S rRNA