Due to differences in lifestyle, dietary structure, and living environment, primary CBD stones maintain a high incidence in China, which has brought about property damage and health hazards for our country and people. As an important condition for the occurrence of the disease, bacteria have been studied by scholars. Most previous experiments were based on Cholecystolithiasis or in vitro model to infer the relationship between the two. In addition, the intestinal microbiota plays a role in human diseases with its rich functions. There are a number of diseases such as Crohn's disease, colon cancer, and metabolic diseases that have been shown to be related to the intestinal flora[5–7].The duodenal microbiota, as the nearest neighbor to the biliary tract, was considered to be the main source of possible biliary bacterial infections. In this study, bile and duodenal fluid were directly obtained through ERCP techniques and diversity analysis was performed. Through the rigorous screening of the subjects, we obtained a total of 19 samples of patients. It is worth noting that 4 of them were pancreatic-related diseases patients without hepatobiliary system disease. For the first time, the project used normal normal bile duct bile was used as CK to investigate the relationship between CBD and microbiota. The study was ethical and informed consents were obtained from the patients. To ensure that samples were not contaminated with each other was a difficult point in the sampling process. Thus, the duodenal fluid was first taken and then bile was extracted under the premise of strict aseptic processing to ensure the fidelity of this study.
In the field of microbial research, the widespread application of high-throughput sequencing technology has broken the limitations of traditional culture techniques. In our study, the 16S rRNA gene high-throughput sequencing technology was used for biliary and intestinal (duodenal) microbiota in patients with CBD stones and in patients without hepatobiliary system diseases. Through Illumina platform sequencing, a total of 1,811,427 effective sequencing sequences were obtained, and a total of 2801 bacterial OTUs were identified, The Goods coverage (%) of each group was above 99%, that is, the amount of sequencing of each sample is nearly saturated. In addition, the Species accumulation curves indicate that the current sample size was enough to reflect the richness of the community. The above two fully illustrate that this study has a very high reliability and reference value. 748,340 high-quality gene sequences were obtained in B subgroup, and 2062 bacterial OTUs were identified, which is similar to previous studies by Shen et al. Interestingly, at the same time, we obtained a large number of effective high-quality sequences of 172,025 and 1597 OTUs in BCK subgroup (without hepatobiliary system disease). In the past, based on bacterial culture, it was found that there was no bacteria in normal bile which without biliary tract infections, because of its potent antimicrobial properties. This study has broken through the limitations of this conclusion. Some scholars have also demonstrated the existence of bacteria in bile without common bile duct stones. For example, Pereira P et al. found the presence of bile bacterial communities in PSC patients. Liu et al. found bacterial structures in acute liver failure and normal mice. However, none of the patients enrolled in this study had the above-mentioned liver and biliary tract diseases, which provided valuable resources for the study of normal biliary microbiota.
The sequence quantity and the number of OTUs identified indicated that the bile duct bile and duodenal juice are rich in bacteria, regardless of whether the patients have hepatobiliary disease. Our result showed that the Alpha diversity of bile microbiota in the experiment group was lower than that in CK. But there was also study showing that the Alpha diversity of biliary microbiotawas higher than that of normal intestinal flora and had a higher richness. The factors for this difference may be related to the sites of the bile samples (gallbladder or common bile duct) and the sites of the intestinal microbiota samples (fecal or duodenal fluid).At the same time, we observed that in the same group, the Alpha diversity of bile and duodenal microbiota was similar, at least for their respective dominant bacterium.
We found that the most dominant phylum in bile and duodenal fluid was Proteobacteria in obtained 23 phyla. Other phyla with higher abundances included Firmicutes, Actinobacteria, Bacteroidetes, etc., this is similar to previous studies[4, 22].The difference is that Synergistetes (0.63%) with a high abundance in B subgroup were not found in BCK subgroup. Synergistetes, the main pathogens of oral diseases such as periodontitis, may also participate in the formation of CBD stones. The analysis of the composition of the biliary and duodenal microbiota showed that phyla contained in the bile could be found in the duodenal fluid almost. Similar result was also found in previous work of Ye et al., their work showed the biliary microbiota had a comparatively higher similarity with the duodenal microbiota. However, the abundance of Proteobacteria and Cyanobacteria in the bile of the experiment group was significantly higher than that in the duodenal fluid. It shows that although the composition of biliary and duodenal bacterial community is similar, but the composition ratio is different.
At the genus level, the most dominant genus was Pseudomonasof the phylum Proteobacteria, followed by Escherichia-Shigella. This is different from previous research results, Ye et al. found three Enterobacteriaceae genera (Escherichia, Klebsiella, and an unclassified genus) and Pyramidobacter were abundant in bile of six gallstone patients, while Li et al. found Acinetobacter and Prevotella were dominant genera in the duodenum of nine healthy volunteers[4, 24].According to Metastatsand PLS-DA, Pseudomonas (VIP < 1) and Escherichia-Shigella (VIP < 1) were discovered to have significant difference in bile between EG and CK, and were not critical to bile grouping. In our view, the two most abundant genera may only exist as normal flora in bile, not as a risk factor for CBD stones. At the same time, Escherichia-Shigella (VIP > 1) was found to be more abundant in the duodenal fluid of EG than that of CK. It shows that Escherichia-Shigellaplays an important role in the duodenum of patients with CBD stones. The bile or duodenal fluid in EG contained more genera than CK. The results showed that although the floras of the samples were relatively conservative at phylum level, there was dysbacteriosis and the imbalance of the composition ratio in the bile or duodenal fluid on genus level in EG. Meanwhile, the associations among bacterial genera in EG were more complicated by Spearman Association Network Analysis. It further proves that dysbacteriosis occurs in both the biliary tract and the duodenum in the relationship among bacterial genera.
Studies have shown that changes in the bacterial community of the duodenum were associated with various diseases such as small intestinal bacterial overgrowth (SIBO), irritable bowel syndrome (IBS) and celiac disease (CD) . It was found that Clostridium sensu_stricto (VIP > 1), Lachnospiraceae_UCG-008 (VIP > 1), Butyrivibrio (VIP > 1), Roseburia (VIP > 1), which can produce short chain fatty acids (SCFA), were significantly reduced in bile in EG compared with CK. SCFA has the functions of sterilization, bacteriostasis, and provide energy for host antibodies to improve immunity[26, 27]. SCFA not only affects the intestinal epithelium, but has a certain effect on airway epithelium, enteric nervous system (ENS), central nervous system (CNS), and peripheral neurons. The occurrence of multiple diseases is associated with a decrease in the proportion of "good bacteria" that can produce SCFA in the intestine, such as inflammatory bowel disease, type 2 diabetes, Asthma and so on. So we can conclude that the low expression of "good bacteria" in bile has a negative impact on biliary health in patients with common bile duct stones, which ultimately leads to the development of stones. In addition, Klebsiella (VIP > 1) and Helicobacter (VIP > 1) have been reported in previous studies to be associated with CBD stone formation. For example, Klebsiellapneumoniae and Helicobacter pylori have been cultured by bacteria or identified by PCR technique in bile from patients with CBD stones[18, 32].Similar results were obtained in this study. But the two genera were more abundant in BCK subgroup. This phenomenon suggests that the disorder of the biliary bacteria community may be more important than the number of related pathogenic bacteria in the formation of CBD stones.
The comparison of biliary microbiota and duodenum microbiota indicated that gut microbiota showed a higher diversity of genera, both in EG and in CK. At the same time, the specificity and complexity of the two bacterial communities could be seen from as many as 60 differential genera between biliary microbiota and duodenum microbiota in EG. According to the Spearman Association Network Analysis of Dominant genera in EG and the normal group, we found that the relationship between Ralstonia and Ochrobactrum, Streptococcus and Granulicatella, Acinetobacter and Alloprevotella were antagonistic to each other in CK, but they showed cooperative relationships in EG. This shift in bacterial relationship may be the biological basis for the occurrence and development of biliary tract diseases (e g. primary CBD stones). The reasons for this shift are not referenced in the relevant literature, may be related to the elimination or reduction of specific genus, or may be affected by other genera. These speculations need further research to explore.
The relationship between biliary and intestinal microbial communities has been a subject of continuous research and discussion. Bile bacterial infection was an important factor in the formation of CBD stones. Based on human anatomy and related studies, biliary microbiota in patients with CBD stones originated from the intestinal tract[33, 34]. In recent years, research has also tried to confirm this view. Ye et al. provided evidence that the biliary flora in patients with CBD stones originated in the intestine and mainly from the upper gastrointestinal tract by the 16S rRNA gene amplicon sequence.In this study, the beta diversity analysis of the sample community structure showed that the biliary and the duodenum microbiota showed some similarities in both EG and CK, especially in terms of bacterial population abundance (slightly different in bacterial composition), moreover this similarity was more evident in the EG. At the same time, a Venn diagram showed “core microbiome” (1205 shared OTUs), which was first proposed by Turnbaugh et al.Thus we can conclude that the most of biliary microbiota and the duodenal microbiota have a common source. More importantly, this study confirmed the existence of self-microbiota in the normal biliary tract, which constituted a microenvironment of bile together with bile salts, bile acids, and so on. In addition, though the abundance of unique OTUs was low in four subgroups, we still have the possibility to find biomarkers related CBD stones among them.
In order to gain more understanding of the biliary and duodenal microbiota, we performed microbial function prediction by PICTUSt. Both the duodenal and the biliary microbiota have gained rich function pathways. There are differences in some pathways at KEGG level 3 in bile samples between EG and CK, such as cellular processes (Apoptosis, Endocytosis), environmental information processing (Two-component system, CAM ligands) and the immune system (RIG-I-like receptor signaling pathway), of which the abundance in EG decreased. We believe that some genera appeared to have a decline of self-repair ability and environmental adaptability in bile in EG and were in a "pathological state." In duodenal fluid samples, microbiota in EG hadhigher abundance of functions involved in Naphthalene degradation and Bacterial invasion of epithelial cells. Studies have shown that Pseudomonas sp. JLR11 and E. coli were related to pathway of Naphthalene degradation[36, 37].Some bacteria such as Shigella, Streptococcus and Listeria could enter the epithelial cells through pathway of Bacterial invasion of epithelial cells[38–40].It is indicated that the increase of bacteria with invasive ability of duodenal microbiota may indirectly lead to the formation of the stone, which may also explain the increase in the abundance of Escherichia-Shigella in the duodenal fluid of EG. However, in the Infectious Diseases pathway, Shigellosis pathways and pathogenic Escherichia coli infection pathways had no difference in duodenal microbiota between two groups. This is also a good illustration of why the high abundance of Escherichia-Shigellain duodenal flora does not cause related diseases.
This study further explored the composition of the biliary and intestinal microbiota in patients with primary CBD stones and the differences in key genera. And we had a completely new understanding of the biological communities in the normal biliary tract. However, this experiment still has its limitations. The small sample size may lead to certain deviations. As far as research methods are concerned, in the future, metagenomic sequencing, animal experiments and other methods may be needed to study and validate the biliary flora, so as to further clarify the role of bacteria on the formation of primary CBD stones.