Objective: In recent years, the incidence of gallstones and their complications has increased, bringing a heavy burden to patients, emphasizing the need to explore the pathogenesis of gallstones. Evidences suggest that the formation of gallstones is closely related to the biliary tract and the gut flora. This study aims to reveal the diversity and abundance of intestinal flora in patients with biliary stones, investigate the relationship between the structure of gallstone formation and its flora, and preliminarily research gene function annotation and metabolic pathways. Methods: The subjects were 21 eligible gallstone patients undergoing surgery and 20 eligible gallstone-free patients admitted to Beijing Tiantan Hospital, Capital Medical University, from November 2019 to November 2020. Gallstones (GSS group), bile (GSZ group), gallbladder mucosa (GSN group), feces (GSF group) samples were collected from the gallstone group, as well as feces from the control group (HF group). High-throughput sequencing of the V3-V4 regions of the 16S rRNA gene was performed by the Illumina HiSeq platform, bioinformatics analysis was performed on the sequencing results. Results: 1. The age, body mass index (BMI) and indirect bilirubin (IBil) of gallstone patients were higher than gallstone-free patients (P < 0.05). 2. A total of 23 427 Operational Taxonomic Units (OTUs) were identified in this study, with a mean ± standard deviation of 340±93, including 4 095 from gallstones (GSS group), 3 065 from bile (GSZ group), 4 687 from gallbladder mucosa (GSN group), and 5 203 from feces (GSF group). 6 377 OTUs were identified from the feces of the gallstone-free control group (HF group). 3. There was no significant difference in the diversity and phylum composition of intestinal flora between gallstone patients and the control group (P > 0.05); however, at the genus level, Achromobacter (P=0.010), Faecalibacterium (P=0.042), Lachnospira (P=0.011) were significantly reduced, while Enterococcus (P=0.001) was significantly increased. 4. The diversity and composition of biliary flora (stone, bile, mucosa) among patients with gallstones have no statistical differences (P > 0.05). The diversity and composition between the biliary and intestinal microflora in gallstones patients have statistical differences: (1) The diversity of biliary flora was significantly higher than the intestinal flora (Simpson index, P < 0.05). (2) At the phylum level, the abundance of Proteobacteria in the bile duct (stone, bile and mucosa) was significantly higher, while Firmicutes and Bacteroidetes were significantly lower than in the intestinal tract (P < 0.05). (3) At the genus level, the abundance of Acinetobacter in the biliary tract was significantly higher, while Bacteroides, Faecalibacterium, Lachnoclostridium and Subdoligranulumbacteria were significantly lower than in the intestinal tract (P < 0.05). 5. The patient's stone, bile and gallbladder mucosa shared more than 90% of OTUs. The shared OTUs of intestinal flora between gallstones patients and the control group was greater than 85%, while the five groups of samples shared more than 60% of OTUs. 6. LefSe showed that LDA > 4 in the biliary tract was Gammaproteobacteria, Pseudomonadales, Moraxellaceae, Acinetobacter, Betaproteobacteria, Burkholderiales and Prevotella that all belong to Proteobacteria. Conclusion: The intestinal flora of patients with gallstones and without gallstones exhibited significant bacterial heterogeneity at the genus level. Compared with the intestinal flora of patients with gallstones, the biliary flora exhibited higher diversity. There were significant differences in the bacterial community structure at the phylum and genus levels. The biliary tract (stone, bile, mucosa) and intestinal flora of patients with gallstones have overlaps and differences, which provides the foothold for future studies on the biliary tract flora.
