Bile Microbiome and Metabolic Characteristics in Primary Common Bile Duct Stone Patients with Juxtapapillary Duodenal Diverticula: A Clinical Investigation

Bile Microbiome and Metabolic Characteristics in Primary Common Bile Duct Stone Patients with Juxtapapillary Duodenal Diverticula: A Clinical Investigation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bile Microbiome and Metabolic Characteristics in Primary Common Bile Duct Stone Patients with Juxtapapillary Duodenal Diverticula: A Clinical Investigation Mengying Wang, Hongtao Hou, wei Sang, pingping Li, Xuxu Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6863628/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Objective This study investigates the microbiological and metabolic characteristics of bile in patients with common bile duct stones (CBDs) with and without juxtapapillary duodenal diverticulum (JPDD) to analyze stone formation causes and influencing factors. Methods From January to May 2024, CBDs patients undergoing endoscopic retrograde cholangiopancreatography at our hospital were prospectively enrolled. Bile samples were collected for 16SrRNA sequencing and LC-MS/MS metabolomics analysis. Patients were divided into JPDD (n = 15) and CBDs (n = 15) groups. Results The JPDD group had larger stone and bile duct diameters ( P < 0.05). Proteobacteria dominated the bile microbiota in both groups. The JPDD group showed higher abundances of Escherichia-Shigella, Enterococcus, and Escherichia_coli. Beta diversity differed significantly ( P < 0.05). LEfSe analysis identified 25 differential bacterial species. Enterococcus, Klebsiella, and Gemellaceaeke were enriched in the JPDD group, while Peptococcaceae, Roseburia, and Alistipes were enriched in the CBDs group ( P < 0.05). Enterococcaceae and Enterococcus correlated positively with bile duct and stone size in the JPDD group ( P < 0.05). Peptococcaceae and Acinetobacter showed negative correlations ( P < 0.05). Ten metabolic pathways, including phenylalanine and alanine metabolism, differed significantly ( P < 0.05). Metabolites like bilirubin glucuronide and taurochenodeoxycholic acid were upregulated in the JPDD group. Enterococcus in the JPDD group correlated with bile acid metabolites like chenodeoxycholylasparagine ( P < 0.05). Conclusions JPDD influences CBD stone formation and size. JPDD alters bile microbiota, with Enterococcus and Klebsiella enriched in the JPDD group, and Peptococcaceae in the CBDs group. These microbiota correlate with stone size. JPDD changes bile metabolism, with metabolites like taurochenodeoxycholic acid and altered metabolic pathways influencing stone formation. Juxtapapillary duodenal diverticula common bile duct stones Bile microorganisms 16SrRNA KEGG Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Common bile duct stones (CBDs) are a common digestive disease, with clinical manifestations varying greatly between individuals, including common symptoms such as abdominal pain, fever, and jaundice, and progression of the disease causing a series of biliary tract disorders such as cholangitis, biliary obstruction, and cholestatic pancreatitis. Due to changes in lifestyle and dietary habits, the incidence of bile duct stones and cholangitis is increasing worldwide[ 1 ], which not only increases the medical burden, but also poses a serious threat to the patient's life and health as the disease progresses. At present, endoscopic retrograde cholangiopancreatography (ERCP) is a safe and effective treatment, which can effectively alleviate obstruction and reduce the incidence of severe disease, but the recurrence rate of common bile duct stone is high, according to relevant clinical data, the recurrence rate of stone is as high as 14.3% in the first time of ERCP treatment. According to relevant clinical data, the recurrence rate of stones is as high as 14.3% in the first ERCP treatment group, with an average of 1 out of 7 patients treated having recurrence of stones[ 2 ].Therefore the exploration of the causes of common bile duct stone formation and its prevention is particularly important. Primary common bile duct stones are mainly composed of bilirubin[ 3 ], and are more common in Asia. The exact etiology and overall prevalence of these stones is unknown, and they may be associated with bacterial infections. Cholestasis and infection are important factors in stone formation. Juxtapapillary Duodenal diverticula (JPDD) may affect the bile duct environment through compression of the common bile duct leading to biliary sludge, poor bile elimination by affecting Sphincter of Oddi function, food sludge and bacterial infections, and changes in the course of the bile ducts, thereby affecting stone formation. The cause of common bile duct stone formation is still unclear. In this paper, we investigated the effect of parapapillary duodenal diverticular on common bile duct stone formation by applying 16SrRNA, liquid chromatography mass spectrometry (LC-MS/MS) technology non-targeted metabolomics analysis of bile microorganisms and metabolites. Material and Methods 1. Subject of the study Patients who attended the Department of Gastroenterology of Hebei Provincial People's Hospital with a primary diagnosis of common bile duct stone from January 2024 to May 2024 and were proposed to undergo ERCP were prospectively included. Inclusion criteria:(1) Diagnosed as primary common bile duct stone by clinical symptoms, combined with ultrasound, CT, MRCP and other imaging and endoscopy (new stones in the common bile duct greater than 6 months after cholecystectomy or no clear stone shadow in the gallbladder, the operator considered primary common bile duct stone based on the colour, texture, and nature of the stone combined with imaging data and clinical experience). (2) Fully voluntary and signed informed consent. Exclusion criteria: (1) Patients with obvious non-bile duct stone stenosis and biliary tract tumour confirmed by imaging. (2) History of previous endoscopic sphincterotomy (EST). (3) Patients with the combination of severe infectious shock, severe cardiac, pulmonary and cerebrovascular diseases who cannot cooperate and tolerate ERCP surgery. (4) Patients with combination of other malignant tumours. This study was approved by the Ethics Committee of Hebei Provincial People's Hospital. 2. Data and sample collection A total of 30 patients with common bile duct stones were finally included, of which a total of 15 patients with combined juxtapapillary duodenal diverticula (diverticular group, JPDD group, J) and 15 patients with simple bile duct stones (CBDs group, C). Patient demographic and clinical data, including gender, age, and Body Mass Index (BMI), were collected. Medical history, such as prior cholecystectomy, hypertension, coronary heart disease, diabetes, and previous cerebral infarction, was recorded. Preoperative laboratory tests encompassed inflammatory markers, bilirubin levels, and transaminase levels. Preoperatively, the patient's cholangitis grade was assessed according to the Tokyo Guidelines 2018: diagnostic criteria and severity grading of acute cholangitis[ 4 ]. The presence of diverticular was recorded during ERCP, the presence of intubation difficulties, and recorded the dilated diameter of the common bile duct, and the maximum diameter of the common bile duct stone. After the duodenoscope entered the duodenal department, sterile saline was applied to rinse the scope and the duodenal large papilla, a guide wire was applied to successfully insert the bile duct, and 5 ml of bile was withdrawn with a 20 ml syringe prior to the application of the contrast agent, and was quickly placed into a freezing tube on the sterile operating table and frozen in a -80°C refrigerator for microbial sequencing and non-metabolomics analyses. Gene sequencing and bioinformatics analysis: DNA extraction and PCR amplification were performed using kits to extract genomic DNA from the samples according to the instructions. Universal primers 343F TACGGRAGGCAGCAG and 798R AGGGTATCTAATCCT) were used. After the raw data were down-loaded, quality control analyses such as shearing, quality filtering, noise reduction, splicing and de-chimerisation were performed to obtain representative sequences and ASV abundance tables. Representative sequences for each ASV were selected using the QIIME 2 software package and analysed for α and β diversity. Metabolomics was detected by applying liquid chromatography mass spectrometry (LC-MS/MS) technique, and the raw data were processed by Progenesis QI v3.0 software. Orthogonal partial least squares-discriminant analysis (OPLS-DA) was applied to calculate the value of variable importance (VIP, Variable importance in projection) of each metabolite to differentiate differentially expressed metabolites among groups. Differentially expressed metabolites were screened by P-value < 0.05, FC ≥ 1.2 or FC ≤ 1/1.2 (log2FoldChange: experimental group mean (Average)-control group mean (Average), FC: fold change (2^log2FoldChange) criteria. Metabolic pathway enrichment analysis of differential metabolites was performed based on KEGG database. Gene sequencing and bioinformatics analyses were performed by Shanghai Ouyi Biotechnology Co. 3. Statistical analysis SPSS27.0 was applied for statistical analysis. Quantitative data conforming to normal distribution should be expressed as mean ± standard deviation, and those not conforming to normal distribution should be expressed as median and interquartile spacing, and t-test and Mann-Whitney U-test should be applied to make comparisons, respectively. Quantitative data were expressed as rate and percentage, and χ2 or Fisher's exact test should be applied to determine the significance. Those conforming to normal distribution were analysed by Pearson's correlation, and those not conforming to normality was analysed by Spearman correlation. Differences were considered statistically significant at P < 0.05. Results 1.Analysis of clinical data In 30 patients with primary bile duct stones the male to female ratio was 7:8, in which the mean age of patients in the diverticular group (73.73 years) was greater than that of the simple common bile duct stones group (64.33 years), and the BMI diverticular group was lower than that of the simple common bile duct stones group, but there was no statistically significant difference ( P > 0.05).Previously, literature has reported that as age increases, the incidence of JPDD increases[ 5 ], which may be associated with the small sample size. Consistent with previous reports[ 6 ], the common bile duct diameter and maximum diameter diverticular diameter of stones were larger in the diverticular group than in the group with simple common bile duct stones ( P < 0.05), suggesting that the presence of JPDD affects the size of CBDs. In the preoperative laboratory laboratory indicators, the white blood cell count and cholinesterase diverticular group were higher than that of the simple bile duct stones group ( P < 0.05), and the differences between the two groups in terms of past history, remaining laboratory indicators, grade of cholangitis, and the degree of difficulty in intraoperative intubation were not statistically significant in the two groups ( P > 0.05) (Table 1 ). 2. Overall distribution of biliary flora A total of 30 samples were included in this study, and the data volume of the raw data (Raw reads) obtained from sequencing ranged from 78079 to 81999, while the data volume of the Clean tags obtained after the quality control process was distributed in the interval of 68867 to 76025. Further after eliminating the chimeric sequences, we get the data volume distributed between 63586 and 75167, and the number of ASVs of each sample is distributed between 29 and 270 (Fig. 1 A). The microbial species classification ordinal includes: kingdom, phylum, order, order, family, genus and species. The distribution of the number of species at each level for the 30 bile samples in this study is plotted (Fig. 1 B). Distribution at the phylum level: The four phyla in the diverticular and simple common bile duct stones groups were Proteobacteria (40.25% in the JPDD group vs. 31.83% in the CBDs group, hereinafter referred to as Proteobacteria), Bacteroidota (29.55% vs. 38.21%), and Firmicutes (25.12% vs. 25.25%), Actinobacteriota (2.86% vs 2.69%). Proteobacteria predominated in the JPDD group and Bacteroidota in the group of CBDs (Fig. 1 C). At the genus level the four common genera in the diverticular group were Escherichia-Shigella (31.17%), Muribaculaceae (17.47%), Bacteroides (6.27%), Enterococcus (4.57%), and in the group of simple common bile duct stones were: Muribaculaceae (21.02%), Escherichia-Shigella (17.07%), Bacteroides (9.88%), Lachnospiraceae_NK4A136_group (5.15%). Patients in the diverticular group had higher Escherichia-Shigella (31.17% vs 17.07%), Enterococcus (4.57% vs 0.24%) compared to the simple common bile duct stones group (Fig. 1 D). At the species level, due to the limitations of sequencing technology, the detection of colonies at the species level was limited and the results were significantly individualised, we found that the common organisms in the JPDD group were: Escherichia_coli__g__Escherichia-Shigella (28.45%), Enterococcus_durans__g__ Enterococcus (3.61%), Atopobium_vaginae__g__Atopobium (1.80%), and Klebsiella_pneumoniae__g__Klebsiella (1.79%). Common common bile duct stones group were Escherichia_coli_g__Escherichia-Shigella (12.48%), Shewanella_algae_g__Shewanella (4.82%), Atopobium_vaginae_g__Atopobium (2.18%), and Bacteroides_vulgatus_g__Bacteroides (1.91%). Escherichia_coli__g__Escherichia-Shigella (28.45% vs. 12.48%) were elevated in the diverticular group as compared to the group of simple bile duct stones, and we also found that Shigella_flexneri__g__Escherichia-Shigella (1.42%) was only present in the diverticular group (Fig. 1 E). Alpha diversity analysis of bile microorganisms: there was no significant difference ( P > 0.05) between the two groups in Chao1, shanma, simpson, goods_coverage, and observed_species (Fig. 2 A). We applied Principal co-ordinates analysis (PCoA) and Nonmetric Multidimensional Scaling (NMDS) analyses to illustrate the degree of difference between samples and explore the distribution of Beta diversity between the two groups. The biliary microbial PCoA of the patients in the diverticular group showed different regions of aggregation at the maximum variability of 4.74% and 4.49%, respectively, and were analysed by applying the analysis of differences between the groups (binary_jaccard algorithm, Adonis test, P < 0.05).The NMDS stress function (taking the value of 0 ~ 1) is a measure of the degree of dissimilarity between the results of the objects in the sorting space and the original The stress function (taking value 0 ~ 1) is a measure of the degree of dissimilarity between the object results and the original distance matrix in the sorting space. It is usually considered that stress < 0.2 can be represented by a two-dimensional point plot of NMDS, and the graph has some interpretative significance, and when stress < 0.05, it indicates a good representation. Our result of stress < 0.05 can be considered as a significant difference in bile microbial community composition between the two groups (Fig. 2 B). In this study LDA value distribution was applied to show the results of LEfSe analysis and 25 differential flora were found to be statistically significant between the two groups. The expression of seven key flora, Enterococcaceae, Enterococcus, Klebsiella, Gemellaceaeke, Gemella, Staphylococcales, and Myxococcota, was elevated in the diverticular group, and Peptococcaceae, Roseburia, Alistipes, Acinetobacter, Streptococcus, Alteromonadales, Acidovorax, and 18 other flora with decreased expression, and the results showed that there were significant differences in the flora in bile between the JPDD group and the CBDs group (Fig. 3 A). To further explore the correlation between the characteristic biliary microbial flora and clinical laboratory indicators, we applied Spearman correlation coefficient and used R language packages, etc. to produce Heatmap plots, which responded to the magnitude of the correlation by colour change. The results showed that the abundance of Enterococcacee and Enterococcus, which were significantly enriched in the diverticular group, showed a positive correlation with the diameter of common bile duct and stone size ( P = 0.00045629, P = 0.001764904), and a negative correlation with the cholinesterase level ( P = 0.01532788), and the diverticular group significantly expressed decreased Peptococcaceae, Peptococcales, Moraxellaceae, Acinetobacter, Rikenellaceae, Alistipes, Alphaproteobacteria showed negative correlation with common bile duct diameter and common bile stone size ( P < 0.05), the Streptococcaceae, Streptococcus, Peptococcaceae, Peptococcales showed negative correlation with leukocyte count, Alphaproteobacteria, Streptococcaceae, Streptococcus showed positive correlation with cholinesterase, Alphaproteobacteria showed positive correlation with cholinesterase, Alphaproteobacteria showed positive correlation with cholinesterase. and Alphaproteobacteria also showed positive correlation with bile acids. The biliary microbial signature community of the diverticular group was strongly associated with the formation and size of common bile duct stones (Fig. 3 B). PICRUSt 2 was applied to predict KEGG pathways and abundance values. Ten differential metabolic pathways were statistically different between the diverticular group and the group with simple common bile duct stones ( P < 0.05). These pathways included: Cell growth and death, Transport and catabolism, Nervous system, Biosynthesis of other secondary metabolites, Valine, leucine and isoleucine biosynthesis, Hisditine metabolism, 2-Oxocarboxylic acid metabolism, Glycine, serine and threonine metabolism, Phenylalanine, tyrosine and tryptophan biosynthesis. tryptophan biosynthesis, Alanine, aspartate and glutamate metabolism (Fig. 3 C-D). 3. Metabolomic characteristics of the diverticular group and the group of simple common bile duct stones A total of 4998 metabolites were detected in this study, and the two groups of samples were significantly different on the OPLS-DA score plot (Fig. 4 B). Differential substance-based screening conditions identified 235 metabolites that were significantly up-regulated and 324 that were down-regulated in the diverticular group (Fig. 4 A). We found that among the two groups of differential metabolites associated with bile acid metabolism Lamellosterol A, 3-Dehydroteasterone, 5beta-Cyprinolsulfate, Chenodeoxycholylmet-hionine, and Taurochenodeoxycholic acid were significantly up-regulated; and the metabolites related to bilirubin metabolism, Stercobilin and Bilirubin glucuronide, Mono(glucosyluronic acid) bilirubin and other expressions were also significantly up-regulated, while Stercobilinogen was significantly down-regulated; 5-Methoxytryptophan, which is related to the tryptophan metabolic pathway, was significantly down-regulated in the JPDD group. Further continuing to analyse the KEGG-predicted pathways of metabolites in both groups, the diverticular group showed a significant down-regulation of 5-Methoxytryptophan in Primary bile acid biosynthesis, Taurine and hypotaurine metabolism, Phenylalanine, tyrosine and tryptophan biosynthesis and other metabolic pathways are significantly enriched in the diverticulome, which is mainly involved in Breast cancer, Prostate cancer, and Endocrine resistance at the human disease level (Fig. 5 A). 4. Combined analysis of bile microorganisms and metabolites We further analysed the association of genus-level biliary microorganisms with metabolomic profiles, applying spearman correlation analysis, and found that Enterococcus was associated with 3beta,12alpha-Dihydroxy-5alpha-cholan-24-oic Acid, Chenodeoxycholylasparagine, 7-(3,4-Dimethyl-5-propylfuran-2-yl)heptanoylcarnitine showed linear positive correlation and Streptococcus with PC (P-16_0_0_0), Acinetobacter and PS(18_4(6Z,9Z,12Z,15Z)_20_4(5Z,8Z,11Z,14Z)-OH(19S)) and other microorganisms showed linear correlation with the metabolites as well (Fig. 5 B), which indicated that there was a close association between the dominant genera of diverticular group and the metabolites. Discussion In biliary tract stone disease, common bile duct stones are the most prevalent, with epidemiological characteristics showing significant regional and gender differences. The incidence rate in Asian countries is higher compared to Western countries, and the prevalence of the disease in females exceeds that in males. As age increases, the incidence of stones also rises. The cause of primary common bile duct stones is currently not definitively established, but it may be closely related to bacterial infections, bile stasis, changes in bile composition, or metabolic factors. The presence of juxtapapillary duodenal diverticula significantly increases the incidence of common bile duct stones[ 5 , 7 ]. Juxtapapillary duodenal diverticula are diverticula located near the opening of the common bile duct and pancreatic duct. Their unique position not only affects the function of the sphincter of Oddi but also, in the case of Type I intradiverticular papilla, significantly increases the difficulty of ERCP cannulation and stone extraction[ 8 ]. JPDD exhibits a significant correlation with age, with its incidence markedly increasing among the elderly population, particularly in individuals over the age of 50, where it is more prevalent. Conversely, it is relatively rare in young adults under the age of 30[ 7 ]. The formation of JPDD may be associated with degenerative changes. In the present study involving 30 patients, it was observed that the average age of patients with concomitant periampullary duodenal diverticula was higher. These patients exhibited a significantly increased risk of developing common bile duct stones, and the presence of diverticula also influenced the size of the stones. Our research found that the diameter of common bile duct dilation and the size of the stones were significantly larger in the diverticula group compared to the control group, which is consistent with previous study findings[ 6 ]. Not only was the size of the stones affected, but the white blood cell count was also significantly elevated in the diverticula group, particularly among patients with Type I diverticula. We hypothesize that this may be related to biliary tract infections. Patients with common bile duct stones accompanied by juxtapapillary duodenal diverticula are more prone to concurrent biliary tract infections, which may further exacerbate systemic inflammatory responses. Bacteria and microorganisms are diverse and play a crucial role in maintaining the homeostasis of the human body's internal environment. With the continuous advancement of science and technology, cutting-edge techniques such as next-generation sequencing and metagenomics are increasingly being applied in clinical settings. These technologies have significantly expanded our understanding of the microecological environments within various organs and tissues. In clinical practice, these advanced techniques are predominantly utilized for the diagnosis and treatment of diseases. Numerous studies have shown that bacterial microbiota can significantly influence gastrointestinal cancers and chronic diseases through mechanisms such as microbial translocation, immune regulation, metabolic modulation, and enzymatic degradation. These processes lead to notable alterations in microbial diversity and play a pivotal role in the onset and progression of diseases[ 9 , 10 ]. Current research has revealed that a rich microbiota is also present in the biliary tract of healthy individuals[ 11 ], and dysbiosis of this microbiota is closely associated with biliary tract diseases. In patients with common bile duct stones, the bile commonly harbors microbial phyla such as Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria[ 12 – 14 ]. In our study, we have arrived at similar findings, although the proportions of each bacterial phylum in the bile differ from other research. We discovered that the most common bile microorganisms in the diverticula group were, in order, Proteobacteria (40.25%), Bacteroidota (29.55%), Firmicutes (25.12%), and Actinobacteriota (2.86%). Among these, the proportion of Proteobacteria was higher in the diverticula group compared to the simple common bile duct stone group. At the genus and species levels, the abundances of Escherichia-Shigella, Escherichia_coli, and Shigella_flexneri were also increased. Escherichia-Shigella, Escherichia_coli, and Klebsiella are among the common infectious microbiota in humans[ 15 , 16 ]. The enrichment of these bacterial microorganisms in bile may increase the risk of cholangitis. In bacterial taxonomy, Proteobacteria is the largest phylum, which includes Escherichia_coli, Salmonella, Helicobacter pylori, and others. These bacteria are typical members of the gut microbiota and are also common pathogens found in bile cultures[ 17 ]. An imbalance in the gut microbiota is often caused by a persistent increase in the abundance of Proteobacteria[ 18 ]. Biliary tract infection is a significant contributing factor to the formation of common bile duct stones, and Proteobacteria may play a similar role within the biliary system. Deng and colleagues have suggested that Pseudomonas, a genus within the Proteobacteria phylum, could influence the crystallization and deposition of stones by modulating the metabolism of cholesterol and bilirubin in bile[ 19 ]. Desulfovibrionales, which belong to the Proteobacteria phylum, can also be enriched in the intestines of patients with cholelithiasis, influencing the formation of cholesterol stones by modulating hepatic bile acid and cholesterol metabolism[ 20 ]. These microorganisms may ascend through the biliary tract, causing infections that lead to inflammation and biliary strictures, thereby accelerating the formation of common bile duct stones. Lyu et al.[ 21 ], by comparing the microbiota of bile and duodenal fluid in patients with common bile duct stones, found that Proteobacteria predominated in both bile and duodenal fluid. Similarly, Liu et al.[ 13 ] have suggested that duodenal microbiota resembles bile microbiota. The composition of Proteobacteria from phylum to genus level was found to be essentially the same in the bile and duodenal tissue of recurrent common bile duct stones[ 22 ]. Retrograde migration of duodenal microbiota may lead to alterations in bile microbiota. There exists an entero-biliary reflux between duodenal microbiota and biliary microbiota, where microorganisms migrate from the duodenum into the biliary tract through the sphincter of Oddi or reach the liver via the bloodstream and then enter the bile, influencing stone formation by altering the biliary microenvironment. The function of the sphincter of Oddi is to regulate bile outflow and separate the bile duct from the intestine; its dysfunction can lead to bile stasis and even infection, promoting the development of common bile duct stones[ 23 ]. The close relationship between JPDD and the duodenal papilla may cause sphincter of Oddi dysfunction, allowing intestinal microorganisms to enter the biliary tract retrogradely, increasing the risk of infection and promoting the formation of common bile duct stones. Therefore, we hypothesize that juxtapapillary duodenal diverticula influence the formation of common bile duct stones by altering the biliary microenvironment. In patients with concomitant juxtapapillary duodenal diverticula, the composition and abundance of bile microbiota exhibit significant differences. Our analysis revealed that while the richness of biliary microbiota is comparable between the two groups, Beta diversity results indicate a notable distinction in bile microbiota between them. In the diverticula group, the abundance of pathogenic bacteria such as Enterococcaceae, Enterococcus, Klebsiella, Enterococcus_durans, Enterococcus_faecalis, and Bacteroides_stercoris was elevated. The variation in bacterial microbial abundance may be closely related to the formation of common bile duct stones. The current mechanisms by which bacterial microbiota contribute to the formation of common bile duct stones primarily include beta-glucuronidase, bile salt hydrolase, phospholipase, mucin, and glycocalyx. Certain bacteria in bile produce beta-glucuronidase, which hydrolyzes bilirubin glucuronide into bilirubin and glucuronic acid, precipitating with calcium molecules to form calcium bilirubinate crystals, thereby promoting the formation of pigment stones[ 24 , 25 ]. Enterococcus is a common pathogenic bacterium in biliary tract infections, belonging to the typical Gram-positive cocci. It can tolerate a wide range of pH levels and high concentrations of sodium chloride, exhibiting strong environmental resilience, and plays a significant role particularly when the biliary tract structure is compromised or in the presence of obstruction[ 26 ]. Klebsiella, a common Gram-negative bacterium that colonizes the respiratory tract and intestines, can increase the incidence and severity of cholangitis. Both of these primary biliary pathogens are capable of producing enzymes such as beta-glucuronidase and phospholipase[ 27 ]. which in our findings were significantly more abundant in the diverticula group and are closely linked to the formation of common bile duct stones. Additionally, bile microbiota can modulate the formation of common bile duct stones by altering bile composition. Studies have shown that microorganisms like Enterococcus, Klebsiella, Bacteroides, Clostridium, and Lactobacillus possess bile salt hydrolase activity; these bacteria can catalyze the dissociation of bile acids into free bile acids and amino acids within the small intestine, affecting bile acid and cholesterol metabolism[ 28 ], thereby altering bile composition and promoting the formation of common bile duct stones. Mucin in bile also plays a role in the formation of pigment stones. Chronic inflammation of the gallbladder and bile ducts can increase the production of mucin in epithelial cells, such as elevated levels of MUC5AC expression. Mucin can accelerate the formation and progression of pigment stones by providing a matrix, promoting precipitation, and interacting with bacteria associated with chronic inflammation and biliary infections (such as Klebsiella and Escherichia coli)[ 29 ]. When the intestinal mucosal barrier function is compromised and entero-biliary reflux occurs, there is a disturbance in the biliary microecology, leading to elevated levels of LPS in the serum and an increase in LPS levels within the bile ducts. This further enhances the endogenous expression of beta-glucuronidase and mucin in the bile duct epithelial cells[ 30 ], akin to the role of bacterial beta-glucuronidase and glycocalyx in stone formation. Our findings indicate that the abundance of bacteria associated with biliary infections (such as Enterococcus, Klebsiella, Enterococcus faecalis, etc.) in the bile microbiota of the diverticula group is significantly higher than in the simple common bile duct stone group, potentially promoting stone formation by modulating mucin expression. Controlling the expression of these factors may help prevent the formation and recurrence of bile duct stones. Microbiota may also influence the progression of stones through the formation of biofilms and the production of metabolites, indirectly affecting energy intake, altering intestinal permeability, and leading to chronic inflammation of the biliary tract, thereby promoting the formation of primary common bile duct stones. Research has found that in patients with pigment gallstones, genes extracted from Klebsiella and Enterococcus are involved in the arrangement of biofilms[ 31 ], contributing to the formation and development of stones. Notably, in our results, the abundance of Enterococcaceae and Enterococcus showed a significant positive correlation with the diameter of the common bile duct and the size of the stones, indicating a close relationship between juxtapapillary duodenal diverticula, bile microbiota, and common bile duct stones. Similarly, low expression of some beneficial bacteria in bile is detrimental to the health of the biliary tract and may contribute to stone formation and development. We found that in the diverticular group, Roseburia, Acinetobacter, Alistipes, Rikenellaceae, Peptococcales, Helicobacter_typhlonius, Nesterenkonia_sp. The abundance of Peptococcales, Helicobacter_typhlonius, and Nesterenkonia_sp. decreased significantly. Roseburia is a common genus localized in the digestive tract and plays a major protective role in the progression of many diseases. Roseburia has been shown to ameliorate alcohol-associated liver disease by strengthening intestinal tight junctions[ 32 ]. In the intestines of patients with inflammatory bowel disease, genetic risk scores were significantly associated with a reduction in Roseburia in healthy control subjects, leading to a decrease in butyrate conversion[ 33 ]. Keren et al. found that Roseburia, a beneficial bacterium in the gut microflora, was also significantly reduced in patients with gallstones and could serve as a biomarker for symptomatic gallstone formation[ 34 ]. A significant reduction in the number of Roseburia capable of producing short-chain fatty acids has also been found in patients with primary common bile duct stones[ 21 ]. In addition, Li et al. found that short-chain fatty acid-producing bacteria Clostridium butyricum (C. butyricum) reduced common bile duct stone formation and preserved gut microbiome balance in mice, reducing the risk of gastrointestinal and biliary infections in a basal mouse study[ 35 ]. In our results, the abundance of short-chain fatty acid-producing microorganisms such as Acinetobacter, Alistipes, Rikenellaceaeza, and Peptococcales was reduced in the diverticular group compared with the group with simple common bile duct stones and was negatively correlated with the diameter of the common bile ducts and the size of the stones, so we hypothesized that Roseburia, Therefore, we hypothesized that the decrease in the abundance of Roseburia, Acinetobacter, Alistipes, Rikenellaceaeza, and Peptococcales may contribute to the formation and development of common bile duct stones. The reduction in the abundance of these “beneficial” bacteria may contribute to stone formation, but their role in the biliary tract has been poorly investigated and needs to be further confirmed by a number of studies. It is worth noting that the formation and development of primary common bile duct stones is not the result of the action of a single microorganism, but is a complex process of correlation and interaction between microbiota. Biliary and intestinal microorganisms are involved in almost all aspects of bile formation, including lipid and cholesterol metabolism, biotransformation, and enterohepatic circulation. The presence of parapapillary duodenal diverticula significantly alters the balance of bile metabolism. Predictions of KEGG pathways in microbiologically sequenced samples showed that the diverticular group was significantly different from the group with simple common bile duct stones in terms of 2-Oxocarboxylic acid metabolism, Glycine, serine and threonine metabolism, Alanine, aspartate and glutamate metabolism and lipid and cholesterol metabolism. Ten pathways were differentially enriched for 2-Oxocarboxylic acid metabolism, Glycine, serine and threonine metabolism, Alanine, aspartate and glutamate metabolism, which cover bile acid synthesis and metabolism, energy conversion mechanisms in the liver, oxidative stress, and inflammatory responses, with specific metabolic pathways involved. Further LC-MS biliary metabolomics revealed that metabolites such as Taurochenodeoxycholic acid (TCDCA), Stercobilin, Bilirubin glucuronide, Mono(glucosyluronic acid) bilirubin and other metabolites were significantly up-regulated in the diverticular group. were significantly up-regulated in the diverticulum group. Enrichment of KEGG pathway revealed that these metabolites were mainly involved in primary bile acid biosynthesis, taurine and hypotaurine metabolism, Phenylalanine, tyrosine and tryptophan biosynthesis Phenylalanine, tyrosine and tryptophan biosynthesis. Taurochenodeoxycholic Acid has anti-inflammatory and antifibrotic effects, protects the biliary system, and is involved in the regulation of cholesterol metabolism and bile acid metabolism, leading to alterations in the composition of bile acids, which affects bile solubility and fluidity, thereby increasing the risk of stone formation[ 36 ]. In our results, the elevated biliary component TCDCA in the diverticular group may be related to cholestasis and altered biliary composition, a finding that still needs to be further validated in large samples.Stercobilin is the end product of bilirubin metabolism, which is converted from bilirubin by bacterial action in the intestine[ 37 ]. Imbalances in the ratio of Stercobilin may lead to disorders of bilirubin metabolism leading to calcium bilirubin oversaturation, which may indirectly contribute to stone formation.Taurine and hypotaurine metabolism Metabolic pathways are associated with bile acid metabolism. Several studies have shown that bacterial microorganisms can regulate body health by influencing bile acid metabolism. For example, the primary bile acid biosynthesis, taurine and hypotaurine metabolism pathways are also significantly enriched in the blood of pregnant women with intrahepatic cholestasis[ 38 ]. Compound woodchuck granules may ameliorate hepatic fibrosis and attenuate hepatic oxidative stress and cell apoptosis by modulating taurine and taurine metabolism as well as bile acid metabolism[ 39 ]. The potential probiotic Lactobacillus gasseri LA39 can activate hepatic primary bile acid biosynthesis and promote intestinal secondary bile acid biotransformation[ 40 ]. A positive correlation between the concentration of TCDCA and the abundance of Microbacterium, Lutibacterium, Sphingomonas genera, and Prevotella intermedia species has been found[ 41 ]. When microorganisms are dysregulated and disorganized, secondary metabolic imbalances of bile acids, amino acids, and other metabolites may lead to the development of choledocholithiasis, cholestasis, and steatohepatitis, among other disorders. Therefore, we believe that the presence of juxtapapillary duodenal diverticula may affect the formation of common bile duct stone by regulating metabolite composition and influencing bile acid and lipid metabolism. We also found a clear correlation between some dominant genera, such as Enterococcus spp. and partially bound bile acids. Further studies are needed to investigate the potential relationship between bile metabolism and microbial characteristics. Conclusions The relationship between juxtapapillary duodenal diverticula and primary common bile duct stones is complex, and the role of altered biliary microbiology and metabolites in stone formation and the mechanisms involved need to be further explored. We analyzed bile microorganisms and metabolites in patients with primary common bile duct stones by 16SrRNA microbial sequencing and metabolomics to investigate the effect of parapapillary duodenal diverticular on stone formation. The relationship between microorganisms, metabolites, and stones requires more experimental evidence in future studies. Abbreviations CBDs Common bile duct stones PD Primary common bile duct stones MRCP Magnetic resonance cholangiopancreatography ERCP Endoscopic retrograde cholangio-pancreatography JPDD Juxtapapillary duodenal diverticula 16SrRNA 16S ribosomal ribonucleic acid LC-MS/MS Liquid chromatography mass spectrometry EST Endoscopic sphincterotomy BMI Body mass index LEfSe Linear discriminant analysis coupled with effect size measurements. Declarations Ethics approval and consent to participate This study was approved by the Ethics Clerkship of Hebei General Hospital, Grant No. 2024002. Our study adheres to the principles of the Helsinki Declaration. Informed consent was obtained from all subjects involved in the study. Consent for publication Not Applicable Availability of data and materials The raw data are available from the corresponding author on receipt of reasonable request and have been deposited in the publicly accessible NAME database. Competing Interests All authors have no conflict of interest. Funding 2023 Hebei Provincial Government Sponsored Medical Talent Project (No. ZF2023188). Author contributions Mengying Wang and Hongtao Hou were involved in the conception and design of the data; Mengying Wang wrote the first draft of the paper, and Pingping LI, Wei Sang, Xuxu Yang, Ping Qi were involved in the collection of the experimental specimens; Hou Hongtao revised and finally approved the version to be published; and all the authors agreed to the publication of the article. Acknowledgements We thank Professor Hongtao Hou and the Hebei Provincial Government Sponsored Medical Talent Project for their financial support. 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Bile Microbiome in Patients with Recurrent Common Bile Duct Stones and Correlation with the Duodenal Microbiome. Life (Basel). 2022 Oct 3;12(10). Yang Y, Zhao Z, Wu S, et al. Structural or functional abnormality of sphincter of Oddi: an important factor for the recurrence of choledocholithiasis after endoscopic treatment. Ann Med. 2025 Dec;57(1):2440119. Maki T. Pathogenesis of calcium bilirubinate gallstone: role of E. coli, beta-glucuronidase and coagulation by inorganic ions, polyelectrolytes and agitation. Ann Surg. 1966 Jul;164(1):90-100. Skar V, Skar AG, Midtvedt T, et al. Beta-glucuronidase-producing bacteria in bile from the common bile duct in patients treated with endoscopic papillotomy for gallstone disease. Scand J Gastroenterol. 1986 Mar;21(2):253-6. Mussa M, Martinez Perez-Crespo PM, Lopez-Cortes LE, et al. Risk Factors and Predictive Score for Bacteremic Biliary Tract Infections Due to Enterococcus faecalis and Enterococcus faecium: a Multicenter Cohort Study from the PROBAC Project. Microbiol Spectr. 2022 Aug 31;10(4):e0005122. Leung JW, Liu YL, Leung PS, et al. Expression of bacterial beta-glucuronidase in human bile: an in vitro study. Gastrointest Endosc. 2001 Sep;54(3):346-50. Kriaa A, Bourgin M, Potiron A, et al. Microbial impact on cholesterol and bile acid metabolism: current status and future prospects. J Lipid Res. 2019 Feb;60(2):323-332. Zhao Z, Yang Y, Wu S, et al. Role of Secretory Mucins in the Occurrence and Development of Cholelithiasis. Biomolecules. 2024 Jun 10;14(6). Ma WJ, Wu ZR, Yang Q, et al. Biliary antibiotics irrigation for E. coli-induced chronic proliferative cholangitis and hepatolithiasis: A pathophysiological study in rabbits. Clin Res Hepatol Gastroenterol. 2020 Jun;44(3):356-367. Kose SH, Grice K, Orsi WD, et al. Metagenomics of pigmented and cholesterol gallstones: the putative role of bacteria. Sci Rep. 2018 Jul 25;8(1):11218. Seo B, Jeon K, Moon S, et al. Roseburia spp. Abundance Associates with Alcohol Consumption in Humans and Its Administration Ameliorates Alcoholic Fatty Liver in Mice. Cell Host Microbe. 2020 Jan 8;27(1):25-40 e6. Imhann F, Vich Vila A, Bonder MJ, et al. Interplay of host genetics and gut microbiota underlying the onset and clinical presentation of inflammatory bowel disease. Gut. 2018 Jan;67(1):108-119. Keren N, Konikoff FM, Paitan Y, et al. Interactions between the intestinal microbiota and bile acids in gallstones patients. Environ Microbiol Rep. 2015 Dec;7(6):874-80. Li G, Yu T, Du H, et al. Effect of Clostridium butyricum on the formation of primary choledocholithiasis based on intestinal microbiome and metabolome analysis. J Appl Microbiol. 2023 Aug 1;134(8). Bao L, Hao D, Wang X, et al. Transcriptome investigation of anti-inflammation and immuno-regulation mechanism of taurochenodeoxycholic acid. BMC Pharmacol Toxicol. 2021 Apr 29;22(1):23. Hamoud AR, Weaver L, Stec DE, et al. Bilirubin in the Liver-Gut Signaling Axis. Trends Endocrinol Metab. 2018 Mar;29(3):140-150. Li GH, Huang SJ, Li X, et al. Response of gut microbiota to serum metabolome changes in intrahepatic cholestasis of pregnant patients. World J Gastroenterol. 2020 Dec 14;26(46):7338-7351. Men L, Gu Z, Wang E, et al. Fufang Muji Granules Ameliorate Liver Fibrosis by Reducing Oxidative Stress and Inflammation, Inhibiting Apoptosis, and Modulating Overall Metabolism. Metabolites. 2024 Aug 11;14(8). Hu J, Hou Q, Zheng W, et al. Lactobacillus gasseri LA39 promotes hepatic primary bile acid biosynthesis and intestinal secondary bile acid biotransformation. J Zhejiang Univ Sci B. 2023 Aug 15;24(8):734-748. Petrov VA, Fernandez-Peralbo MA, Derks R, et al. Biliary Microbiota and Bile Acid Composition in Cholelithiasis. Biomed Res Int. 2020;2020:1242364. Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 06 Oct, 2025 Reviews received at journal 30 Sep, 2025 Reviews received at journal 16 Sep, 2025 Reviewers agreed at journal 25 Aug, 2025 Reviewers agreed at journal 23 Aug, 2025 Reviewers invited by journal 22 Jul, 2025 Editor assigned by journal 09 Jul, 2025 Editor invited by journal 07 Jul, 2025 Submission checks completed at journal 06 Jul, 2025 First submitted to journal 06 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6863628","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":489214206,"identity":"8925640f-2398-494e-be11-be780da0ee0c","order_by":0,"name":"Mengying Wang","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mengying","middleName":"","lastName":"Wang","suffix":""},{"id":489214207,"identity":"01314bb2-da43-4101-a888-30c1b8f00ed6","order_by":1,"name":"Hongtao Hou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAl0lEQVRIiWNgGAWjYBACPnYgkVAhIcdPtBY2ZpCWMxbGkg0kaWFsq0jcQIIWHuMPD+dJMG5gYH746AaRWgwMErdJMJszsBkb5xCnhS0hAaiFzbKBh02aaC0HEudI8BgcIF4L88GGxAYJCZK0HGZIOCZhINlMrF/42RubP/6oqavvZ29++JgoLQjATJryUTAKRsEoGAX4AADg/yVoO4KEXAAAAABJRU5ErkJggg==","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":true,"prefix":"","firstName":"Hongtao","middleName":"","lastName":"Hou","suffix":""},{"id":489214208,"identity":"79df712f-7c76-4c92-a526-a3e4aa058646","order_by":2,"name":"wei Sang","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"wei","middleName":"","lastName":"Sang","suffix":""},{"id":489214209,"identity":"a1acc4af-9970-43f4-b7a8-95d4c0c10f17","order_by":3,"name":"pingping Li","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"pingping","middleName":"","lastName":"Li","suffix":""},{"id":489214211,"identity":"69064720-4211-48e9-85ac-0186ab65b414","order_by":4,"name":"Xuxu Yang","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xuxu","middleName":"","lastName":"Yang","suffix":""},{"id":489214212,"identity":"ecb67514-944f-4221-bceb-f303c13f422a","order_by":5,"name":"ping Qi","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"ping","middleName":"","lastName":"Qi","suffix":""},{"id":489214213,"identity":"baf1956b-c333-4af4-8f91-726fa81b7feb","order_by":6,"name":"yizhuo Ma","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"yizhuo","middleName":"","lastName":"Ma","suffix":""}],"badges":[],"createdAt":"2025-06-10 13:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6863628/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6863628/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87720688,"identity":"f7281bd6-3234-4b51-bbf7-5a85c923b319","added_by":"auto","created_at":"2025-07-28 09:49:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":810649,"visible":true,"origin":"","legend":"\u003cp\u003eA. Petal plot of ASV distribution (Fig. Numbers in Core represent ASVs common to all samples, and numbers on petals represent total ASVs minus the number of common ASVs in individual samples B. Distribution of species detected in all samples C. Differential distribution of microbial compositions at the portal level between JPDD and CBDs groups D. Differences between the two groups at the genus level E. Distribution of microorganisms at the species level between the two groups (C-S simple common bile duct stones group, J-S diverticular group).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6863628/v1/681fc5337c2c6a07c5e3ca9c.png"},{"id":87720686,"identity":"26e490a4-445e-4f1f-8f96-b09cd474be4a","added_by":"auto","created_at":"2025-07-28 09:49:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":102726,"visible":true,"origin":"","legend":"\u003cp\u003eResults of Alpha diversity and β diversity analysis between the two groups (B_Left:PCoA: binary_jaccard algorithm, Adonis test, B_Right:NMDS: stress \u0026lt; 0.2 can be represented by the two-dimensional dot plot of NMDS, and its graph has some interpretative significance; when stress \u0026lt; 0.1, it can be regarded as a good sorting; when stress \u0026lt; 0.05, it is well representative. stress \u0026lt; 0.05, it is well represented. C-S: simple common bile duct stones group, J-S: diverticular group)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6863628/v1/7723faa8d248d049145c1400.png"},{"id":87722119,"identity":"48e8c941-541e-4b44-90fa-a068ae01ee98","added_by":"auto","created_at":"2025-07-28 10:05:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":982371,"visible":true,"origin":"","legend":"\u003cp\u003eA: LEfSe differential species in both groups (default LDA \u0026gt; 2), B: Clinical data and differential microbiological correlation analysis (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001), C and D: Prediction of KEGG pathway function (C-S: simple common bile duct stones group, J-S: diverticular group).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6863628/v1/886f2fd112ce1f11f09ed21b.png"},{"id":87721962,"identity":"38ad0818-81a7-4960-b029-0784b812485d","added_by":"auto","created_at":"2025-07-28 09:57:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":772963,"visible":true,"origin":"","legend":"\u003cp\u003eA: Histogram of differential metabolite distribution in the two groups after differential screening. B: OPLS-DA plot. C: Volcano plot of significantly up-regulated and down-regulated differential metabolites in the diverticular group compared to the simple common bile duct stones group. Red dots represent significantly up-regulated differential metabolites in diverticular group and blue dots are significantly down-regulated differential metabolites (screening condition: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, and FC\u0026gt;1). D: boxplot distribution of differential metabolites between the two groups (C-D: simple common bile duct stones group, J-D: diverticular group).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6863628/v1/2bd5821dbb967054db37e328.png"},{"id":87720696,"identity":"5f684dea-c44e-41f5-b2d0-ed38f5c5bba5","added_by":"auto","created_at":"2025-07-28 09:49:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":771978,"visible":true,"origin":"","legend":"\u003cp\u003eA: KEGG enrichment analysis of diverticular group significantly up-regulated pathways at Level3 level (horizontal coordinates are -log10 p-values for each pathway, vertical coordinates are names of different pathways, numbers on the bars are the number of differential metabolites annotated to the pathway, different colours of the bars represent different KEGG Level1 information). B: Microbial correlation of differential species and metabolites at genus level Analytical line graph (C-D :simple common bile duct stones group, J-D: diverticular group).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6863628/v1/958e6643184d43d2c2e04fad.png"},{"id":87723256,"identity":"0cc999a0-c905-4d73-a715-73796e75d2ac","added_by":"auto","created_at":"2025-07-28 10:13:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4107587,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6863628/v1/23da860f-3ee6-4847-a043-96a4e481c8e1.pdf"},{"id":87720684,"identity":"bdbab1cc-5c0c-406c-9807-519dd0a94e1f","added_by":"auto","created_at":"2025-07-28 09:49:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19438,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6863628/v1/495bfefa1c93b48d92194432.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bile Microbiome and Metabolic Characteristics in Primary Common Bile Duct Stone Patients with Juxtapapillary Duodenal Diverticula: A Clinical Investigation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCommon bile duct stones (CBDs) are a common digestive disease, with clinical manifestations varying greatly between individuals, including common symptoms such as abdominal pain, fever, and jaundice, and progression of the disease causing a series of biliary tract disorders such as cholangitis, biliary obstruction, and cholestatic pancreatitis. Due to changes in lifestyle and dietary habits, the incidence of bile duct stones and cholangitis is increasing worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], which not only increases the medical burden, but also poses a serious threat to the patient's life and health as the disease progresses. At present, endoscopic retrograde cholangiopancreatography (ERCP) is a safe and effective treatment, which can effectively alleviate obstruction and reduce the incidence of severe disease, but the recurrence rate of common bile duct stone is high, according to relevant clinical data, the recurrence rate of stone is as high as 14.3% in the first time of ERCP treatment. According to relevant clinical data, the recurrence rate of stones is as high as 14.3% in the first ERCP treatment group, with an average of 1 out of 7 patients treated having recurrence of stones[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].Therefore the exploration of the causes of common bile duct stone formation and its prevention is particularly important.\u003c/p\u003e\u003cp\u003ePrimary common bile duct stones are mainly composed of bilirubin[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and are more common in Asia. The exact etiology and overall prevalence of these stones is unknown, and they may be associated with bacterial infections. Cholestasis and infection are important factors in stone formation. Juxtapapillary Duodenal diverticula (JPDD) may affect the bile duct environment through compression of the common bile duct leading to biliary sludge, poor bile elimination by affecting Sphincter of Oddi function, food sludge and bacterial infections, and changes in the course of the bile ducts, thereby affecting stone formation. The cause of common bile duct stone formation is still unclear. In this paper, we investigated the effect of parapapillary duodenal diverticular on common bile duct stone formation by applying 16SrRNA, liquid chromatography mass spectrometry (LC-MS/MS) technology non-targeted metabolomics analysis of bile microorganisms and metabolites.\u003c/p\u003e"},{"header":"Material and Methods","content":"\n\u003ch3\u003e1. Subject of the study\u003c/h3\u003e\n\u003cp\u003ePatients who attended the Department of Gastroenterology of Hebei Provincial People's Hospital with a primary diagnosis of common bile duct stone from January 2024 to May 2024 and were proposed to undergo ERCP were prospectively included. Inclusion criteria:(1) Diagnosed as primary common bile duct stone by clinical symptoms, combined with ultrasound, CT, MRCP and other imaging and endoscopy (new stones in the common bile duct greater than 6 months after cholecystectomy or no clear stone shadow in the gallbladder, the operator considered primary common bile duct stone based on the colour, texture, and nature of the stone combined with imaging data and clinical experience). (2) Fully voluntary and signed informed consent. Exclusion criteria: (1) Patients with obvious non-bile duct stone stenosis and biliary tract tumour confirmed by imaging. (2) History of previous endoscopic sphincterotomy (EST). (3) Patients with the combination of severe infectious shock, severe cardiac, pulmonary and cerebrovascular diseases who cannot cooperate and tolerate ERCP surgery. (4) Patients with combination of other malignant tumours. This study was approved by the Ethics Committee of Hebei Provincial People's Hospital.\u003c/p\u003e\n\u003ch3\u003e2. Data and sample collection\u003c/h3\u003e\n\u003cp\u003eA total of 30 patients with common bile duct stones were finally included, of which a total of 15 patients with combined juxtapapillary duodenal diverticula (diverticular group, JPDD group, J) and 15 patients with simple bile duct stones (CBDs group, C). Patient demographic and clinical data, including gender, age, and Body Mass Index (BMI), were collected. Medical history, such as prior cholecystectomy, hypertension, coronary heart disease, diabetes, and previous cerebral infarction, was recorded. Preoperative laboratory tests encompassed inflammatory markers, bilirubin levels, and transaminase levels.\u003c/p\u003e\u003cp\u003ePreoperatively, the patient's cholangitis grade was assessed according to the Tokyo Guidelines 2018: diagnostic criteria and severity grading of acute cholangitis[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The presence of diverticular was recorded during ERCP, the presence of intubation difficulties, and recorded the dilated diameter of the common bile duct, and the maximum diameter of the common bile duct stone.\u003c/p\u003e\u003cp\u003eAfter the duodenoscope entered the duodenal department, sterile saline was applied to rinse the scope and the duodenal large papilla, a guide wire was applied to successfully insert the bile duct, and 5 ml of bile was withdrawn with a 20 ml syringe prior to the application of the contrast agent, and was quickly placed into a freezing tube on the sterile operating table and frozen in a -80\u0026deg;C refrigerator for microbial sequencing and non-metabolomics analyses.\u003c/p\u003e\u003cp\u003eGene sequencing and bioinformatics analysis: DNA extraction and PCR amplification were performed using kits to extract genomic DNA from the samples according to the instructions. Universal primers 343F TACGGRAGGCAGCAG and 798R AGGGTATCTAATCCT) were used. After the raw data were down-loaded, quality control analyses such as shearing, quality filtering, noise reduction, splicing and de-chimerisation were performed to obtain representative sequences and ASV abundance tables. Representative sequences for each ASV were selected using the QIIME 2 software package and analysed for α and β diversity. Metabolomics was detected by applying liquid chromatography mass spectrometry (LC-MS/MS) technique, and the raw data were processed by Progenesis QI v3.0 software. Orthogonal partial least squares-discriminant analysis (OPLS-DA) was applied to calculate the value of variable importance (VIP, Variable importance in projection) of each metabolite to differentiate differentially expressed metabolites among groups. Differentially expressed metabolites were screened by P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, FC\u0026thinsp;\u0026ge;\u0026thinsp;1.2 or FC\u0026thinsp;\u0026le;\u0026thinsp;1/1.2 (log2FoldChange: experimental group mean (Average)-control group mean (Average), FC: fold change (2^log2FoldChange) criteria. Metabolic pathway enrichment analysis of differential metabolites was performed based on KEGG database. Gene sequencing and bioinformatics analyses were performed by Shanghai Ouyi Biotechnology Co.\u003c/p\u003e\n\u003ch3\u003e3. Statistical analysis\u003c/h3\u003e\n\u003cp\u003eSPSS27.0 was applied for statistical analysis. Quantitative data conforming to normal distribution should be expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, and those not conforming to normal distribution should be expressed as median and interquartile spacing, and t-test and Mann-Whitney U-test should be applied to make comparisons, respectively. Quantitative data were expressed as rate and percentage, and χ2 or Fisher's exact test should be applied to determine the significance. Those conforming to normal distribution were analysed by Pearson's correlation, and those not conforming to normality was analysed by Spearman correlation. Differences were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\n\u003ch3\u003e1.Analysis of clinical data\u003c/h3\u003e\n\u003cp\u003eIn 30 patients with primary bile duct stones the male to female ratio was 7:8, in which the mean age of patients in the diverticular group (73.73 years) was greater than that of the simple common bile duct stones group (64.33 years), and the BMI diverticular group was lower than that of the simple common bile duct stones group, but there was no statistically significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).Previously, literature has reported that as age increases, the incidence of JPDD increases[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], which may be associated with the small sample size. Consistent with previous reports[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], the common bile duct diameter and maximum diameter diverticular diameter of stones were larger in the diverticular group than in the group with simple common bile duct stones (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting that the presence of JPDD affects the size of CBDs. In the preoperative laboratory laboratory indicators, the white blood cell count and cholinesterase diverticular group were higher than that of the simple bile duct stones group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the differences between the two groups in terms of past history, remaining laboratory indicators, grade of cholangitis, and the degree of difficulty in intraoperative intubation were not statistically significant in the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003e2. Overall distribution of biliary flora\u003c/h3\u003e\n\u003cp\u003eA total of 30 samples were included in this study, and the data volume of the raw data (Raw reads) obtained from sequencing ranged from 78079 to 81999, while the data volume of the Clean tags obtained after the quality control process was distributed in the interval of 68867 to 76025. Further after eliminating the chimeric sequences, we get the data volume distributed between 63586 and 75167, and the number of ASVs of each sample is distributed between 29 and 270 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The microbial species classification ordinal includes: kingdom, phylum, order, order, family, genus and species. The distribution of the number of species at each level for the 30 bile samples in this study is plotted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDistribution at the phylum level: The four phyla in the diverticular and simple common bile duct stones groups were Proteobacteria (40.25% in the JPDD group vs. 31.83% in the CBDs group, hereinafter referred to as Proteobacteria), Bacteroidota (29.55% vs. 38.21%), and Firmicutes (25.12% vs. 25.25%), Actinobacteriota (2.86% vs 2.69%). Proteobacteria predominated in the JPDD group and Bacteroidota in the group of CBDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). At the genus level the four common genera in the diverticular group were Escherichia-Shigella (31.17%), Muribaculaceae (17.47%), Bacteroides (6.27%), Enterococcus (4.57%), and in the group of simple common bile duct stones were: Muribaculaceae (21.02%), Escherichia-Shigella (17.07%), Bacteroides (9.88%), Lachnospiraceae_NK4A136_group (5.15%). Patients in the diverticular group had higher Escherichia-Shigella (31.17% vs 17.07%), Enterococcus (4.57% vs 0.24%) compared to the simple common bile duct stones group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eAt the species level, due to the limitations of sequencing technology, the detection of colonies at the species level was limited and the results were significantly individualised, we found that the common organisms in the JPDD group were: Escherichia_coli__g__Escherichia-Shigella (28.45%), Enterococcus_durans__g__ Enterococcus (3.61%), Atopobium_vaginae__g__Atopobium (1.80%), and Klebsiella_pneumoniae__g__Klebsiella (1.79%). Common common bile duct stones group were Escherichia_coli_g__Escherichia-Shigella (12.48%), Shewanella_algae_g__Shewanella (4.82%), Atopobium_vaginae_g__Atopobium (2.18%), and Bacteroides_vulgatus_g__Bacteroides (1.91%). Escherichia_coli__g__Escherichia-Shigella (28.45% vs. 12.48%) were elevated in the diverticular group as compared to the group of simple bile duct stones, and we also found that Shigella_flexneri__g__Escherichia-Shigella (1.42%) was only present in the diverticular group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eAlpha diversity analysis of bile microorganisms: there was no significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between the two groups in Chao1, shanma, simpson, goods_coverage, and observed_species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We applied Principal co-ordinates analysis (PCoA) and Nonmetric Multidimensional Scaling (NMDS) analyses to illustrate the degree of difference between samples and explore the distribution of Beta diversity between the two groups. The biliary microbial PCoA of the patients in the diverticular group showed different regions of aggregation at the maximum variability of 4.74% and 4.49%, respectively, and were analysed by applying the analysis of differences between the groups (binary_jaccard algorithm, Adonis test, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).The NMDS stress function (taking the value of 0\u0026thinsp;~\u0026thinsp;1) is a measure of the degree of dissimilarity between the results of the objects in the sorting space and the original The stress function (taking value 0\u0026thinsp;~\u0026thinsp;1) is a measure of the degree of dissimilarity between the object results and the original distance matrix in the sorting space. It is usually considered that stress\u0026thinsp;\u0026lt;\u0026thinsp;0.2 can be represented by a two-dimensional point plot of NMDS, and the graph has some interpretative significance, and when stress\u0026thinsp;\u0026lt;\u0026thinsp;0.05, it indicates a good representation. Our result of stress\u0026thinsp;\u0026lt;\u0026thinsp;0.05 can be considered as a significant difference in bile microbial community composition between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn this study LDA value distribution was applied to show the results of LEfSe analysis and 25 differential flora were found to be statistically significant between the two groups. The expression of seven key flora, Enterococcaceae, Enterococcus, Klebsiella, Gemellaceaeke, Gemella, Staphylococcales, and Myxococcota, was elevated in the diverticular group, and Peptococcaceae, Roseburia, Alistipes, Acinetobacter, Streptococcus, Alteromonadales, Acidovorax, and 18 other flora with decreased expression, and the results showed that there were significant differences in the flora in bile between the JPDD group and the CBDs group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further explore the correlation between the characteristic biliary microbial flora and clinical laboratory indicators, we applied Spearman correlation coefficient and used R language packages, etc. to produce Heatmap plots, which responded to the magnitude of the correlation by colour change. The results showed that the abundance of Enterococcacee and Enterococcus, which were significantly enriched in the diverticular group, showed a positive correlation with the diameter of common bile duct and stone size (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00045629, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001764904), and a negative correlation with the cholinesterase level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01532788), and the diverticular group significantly expressed decreased Peptococcaceae, Peptococcales, Moraxellaceae, Acinetobacter, Rikenellaceae, Alistipes, Alphaproteobacteria showed negative correlation with common bile duct diameter and common bile stone size (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the Streptococcaceae, Streptococcus, Peptococcaceae, Peptococcales showed negative correlation with leukocyte count, Alphaproteobacteria, Streptococcaceae, Streptococcus showed positive correlation with cholinesterase, Alphaproteobacteria showed positive correlation with cholinesterase, Alphaproteobacteria showed positive correlation with cholinesterase. and Alphaproteobacteria also showed positive correlation with bile acids. The biliary microbial signature community of the diverticular group was strongly associated with the formation and size of common bile duct stones (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003ePICRUSt 2 was applied to predict KEGG pathways and abundance values. Ten differential metabolic pathways were statistically different between the diverticular group and the group with simple common bile duct stones (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These pathways included: Cell growth and death, Transport and catabolism, Nervous system, Biosynthesis of other secondary metabolites, Valine, leucine and isoleucine biosynthesis, Hisditine metabolism, 2-Oxocarboxylic acid metabolism, Glycine, serine and threonine metabolism, Phenylalanine, tyrosine and tryptophan biosynthesis. tryptophan biosynthesis, Alanine, aspartate and glutamate metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D).\u003c/p\u003e\n\u003ch3\u003e3. Metabolomic characteristics of the diverticular group and the group of simple common bile duct stones\u003c/h3\u003e\n\u003cp\u003eA total of 4998 metabolites were detected in this study, and the two groups of samples were significantly different on the OPLS-DA score plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Differential substance-based screening conditions identified 235 metabolites that were significantly up-regulated and 324 that were down-regulated in the diverticular group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We found that among the two groups of differential metabolites associated with bile acid metabolism Lamellosterol A, 3-Dehydroteasterone, 5beta-Cyprinolsulfate, Chenodeoxycholylmet-hionine, and Taurochenodeoxycholic acid were significantly up-regulated; and the metabolites related to bilirubin metabolism, Stercobilin and Bilirubin glucuronide, Mono(glucosyluronic acid) bilirubin and other expressions were also significantly up-regulated, while Stercobilinogen was significantly down-regulated; 5-Methoxytryptophan, which is related to the tryptophan metabolic pathway, was significantly down-regulated in the JPDD group. Further continuing to analyse the KEGG-predicted pathways of metabolites in both groups, the diverticular group showed a significant down-regulation of 5-Methoxytryptophan in Primary bile acid biosynthesis, Taurine and hypotaurine metabolism, Phenylalanine, tyrosine and tryptophan biosynthesis and other metabolic pathways are significantly enriched in the diverticulome, which is mainly involved in Breast cancer, Prostate cancer, and Endocrine resistance at the human disease level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e4. Combined analysis of bile microorganisms and metabolites\u003c/h3\u003e\n\u003cp\u003eWe further analysed the association of genus-level biliary microorganisms with metabolomic profiles, applying spearman correlation analysis, and found that Enterococcus was associated with 3beta,12alpha-Dihydroxy-5alpha-cholan-24-oic Acid, Chenodeoxycholylasparagine, 7-(3,4-Dimethyl-5-propylfuran-2-yl)heptanoylcarnitine showed linear positive correlation and Streptococcus with PC (P-16_0_0_0), Acinetobacter and PS(18_4(6Z,9Z,12Z,15Z)_20_4(5Z,8Z,11Z,14Z)-OH(19S)) and other microorganisms showed linear correlation with the metabolites as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), which indicated that there was a close association between the dominant genera of diverticular group and the metabolites.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn biliary tract stone disease, common bile duct stones are the most prevalent, with epidemiological characteristics showing significant regional and gender differences. The incidence rate in Asian countries is higher compared to Western countries, and the prevalence of the disease in females exceeds that in males. As age increases, the incidence of stones also rises. The cause of primary common bile duct stones is currently not definitively established, but it may be closely related to bacterial infections, bile stasis, changes in bile composition, or metabolic factors. The presence of juxtapapillary duodenal diverticula significantly increases the incidence of common bile duct stones[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Juxtapapillary duodenal diverticula are diverticula located near the opening of the common bile duct and pancreatic duct. Their unique position not only affects the function of the sphincter of Oddi but also, in the case of Type I intradiverticular papilla, significantly increases the difficulty of ERCP cannulation and stone extraction[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. JPDD exhibits a significant correlation with age, with its incidence markedly increasing among the elderly population, particularly in individuals over the age of 50, where it is more prevalent. Conversely, it is relatively rare in young adults under the age of 30[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The formation of JPDD may be associated with degenerative changes. In the present study involving 30 patients, it was observed that the average age of patients with concomitant periampullary duodenal diverticula was higher. These patients exhibited a significantly increased risk of developing common bile duct stones, and the presence of diverticula also influenced the size of the stones. Our research found that the diameter of common bile duct dilation and the size of the stones were significantly larger in the diverticula group compared to the control group, which is consistent with previous study findings[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Not only was the size of the stones affected, but the white blood cell count was also significantly elevated in the diverticula group, particularly among patients with Type I diverticula. We hypothesize that this may be related to biliary tract infections. Patients with common bile duct stones accompanied by juxtapapillary duodenal diverticula are more prone to concurrent biliary tract infections, which may further exacerbate systemic inflammatory responses.\u003c/p\u003e\u003cp\u003eBacteria and microorganisms are diverse and play a crucial role in maintaining the homeostasis of the human body's internal environment. With the continuous advancement of science and technology, cutting-edge techniques such as next-generation sequencing and metagenomics are increasingly being applied in clinical settings. These technologies have significantly expanded our understanding of the microecological environments within various organs and tissues. In clinical practice, these advanced techniques are predominantly utilized for the diagnosis and treatment of diseases. Numerous studies have shown that bacterial microbiota can significantly influence gastrointestinal cancers and chronic diseases through mechanisms such as microbial translocation, immune regulation, metabolic modulation, and enzymatic degradation. These processes lead to notable alterations in microbial diversity and play a pivotal role in the onset and progression of diseases[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Current research has revealed that a rich microbiota is also present in the biliary tract of healthy individuals[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and dysbiosis of this microbiota is closely associated with biliary tract diseases. In patients with common bile duct stones, the bile commonly harbors microbial phyla such as Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In our study, we have arrived at similar findings, although the proportions of each bacterial phylum in the bile differ from other research. We discovered that the most common bile microorganisms in the diverticula group were, in order, Proteobacteria (40.25%), Bacteroidota (29.55%), Firmicutes (25.12%), and Actinobacteriota (2.86%). Among these, the proportion of Proteobacteria was higher in the diverticula group compared to the simple common bile duct stone group. At the genus and species levels, the abundances of Escherichia-Shigella, Escherichia_coli, and Shigella_flexneri were also increased. Escherichia-Shigella, Escherichia_coli, and Klebsiella are among the common infectious microbiota in humans[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The enrichment of these bacterial microorganisms in bile may increase the risk of cholangitis. In bacterial taxonomy, Proteobacteria is the largest phylum, which includes Escherichia_coli, Salmonella, Helicobacter pylori, and others. These bacteria are typical members of the gut microbiota and are also common pathogens found in bile cultures[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. An imbalance in the gut microbiota is often caused by a persistent increase in the abundance of Proteobacteria[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Biliary tract infection is a significant contributing factor to the formation of common bile duct stones, and Proteobacteria may play a similar role within the biliary system. Deng and colleagues have suggested that Pseudomonas, a genus within the Proteobacteria phylum, could influence the crystallization and deposition of stones by modulating the metabolism of cholesterol and bilirubin in bile[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Desulfovibrionales, which belong to the Proteobacteria phylum, can also be enriched in the intestines of patients with cholelithiasis, influencing the formation of cholesterol stones by modulating hepatic bile acid and cholesterol metabolism[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These microorganisms may ascend through the biliary tract, causing infections that lead to inflammation and biliary strictures, thereby accelerating the formation of common bile duct stones. Lyu et al.[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], by comparing the microbiota of bile and duodenal fluid in patients with common bile duct stones, found that Proteobacteria predominated in both bile and duodenal fluid. Similarly, Liu et al.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] have suggested that duodenal microbiota resembles bile microbiota. The composition of Proteobacteria from phylum to genus level was found to be essentially the same in the bile and duodenal tissue of recurrent common bile duct stones[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Retrograde migration of duodenal microbiota may lead to alterations in bile microbiota. There exists an entero-biliary reflux between duodenal microbiota and biliary microbiota, where microorganisms migrate from the duodenum into the biliary tract through the sphincter of Oddi or reach the liver via the bloodstream and then enter the bile, influencing stone formation by altering the biliary microenvironment. The function of the sphincter of Oddi is to regulate bile outflow and separate the bile duct from the intestine; its dysfunction can lead to bile stasis and even infection, promoting the development of common bile duct stones[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The close relationship between JPDD and the duodenal papilla may cause sphincter of Oddi dysfunction, allowing intestinal microorganisms to enter the biliary tract retrogradely, increasing the risk of infection and promoting the formation of common bile duct stones. Therefore, we hypothesize that juxtapapillary duodenal diverticula influence the formation of common bile duct stones by altering the biliary microenvironment.\u003c/p\u003e\u003cp\u003eIn patients with concomitant juxtapapillary duodenal diverticula, the composition and abundance of bile microbiota exhibit significant differences. Our analysis revealed that while the richness of biliary microbiota is comparable between the two groups, Beta diversity results indicate a notable distinction in bile microbiota between them. In the diverticula group, the abundance of pathogenic bacteria such as Enterococcaceae, Enterococcus, Klebsiella, Enterococcus_durans, Enterococcus_faecalis, and Bacteroides_stercoris was elevated. The variation in bacterial microbial abundance may be closely related to the formation of common bile duct stones. The current mechanisms by which bacterial microbiota contribute to the formation of common bile duct stones primarily include beta-glucuronidase, bile salt hydrolase, phospholipase, mucin, and glycocalyx. Certain bacteria in bile produce beta-glucuronidase, which hydrolyzes bilirubin glucuronide into bilirubin and glucuronic acid, precipitating with calcium molecules to form calcium bilirubinate crystals, thereby promoting the formation of pigment stones[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Enterococcus is a common pathogenic bacterium in biliary tract infections, belonging to the typical Gram-positive cocci. It can tolerate a wide range of pH levels and high concentrations of sodium chloride, exhibiting strong environmental resilience, and plays a significant role particularly when the biliary tract structure is compromised or in the presence of obstruction[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Klebsiella, a common Gram-negative bacterium that colonizes the respiratory tract and intestines, can increase the incidence and severity of cholangitis. Both of these primary biliary pathogens are capable of producing enzymes such as beta-glucuronidase and phospholipase[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. which in our findings were significantly more abundant in the diverticula group and are closely linked to the formation of common bile duct stones. Additionally, bile microbiota can modulate the formation of common bile duct stones by altering bile composition. Studies have shown that microorganisms like Enterococcus, Klebsiella, Bacteroides, Clostridium, and Lactobacillus possess bile salt hydrolase activity; these bacteria can catalyze the dissociation of bile acids into free bile acids and amino acids within the small intestine, affecting bile acid and cholesterol metabolism[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], thereby altering bile composition and promoting the formation of common bile duct stones. Mucin in bile also plays a role in the formation of pigment stones. Chronic inflammation of the gallbladder and bile ducts can increase the production of mucin in epithelial cells, such as elevated levels of MUC5AC expression. Mucin can accelerate the formation and progression of pigment stones by providing a matrix, promoting precipitation, and interacting with bacteria associated with chronic inflammation and biliary infections (such as Klebsiella and Escherichia coli)[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. When the intestinal mucosal barrier function is compromised and entero-biliary reflux occurs, there is a disturbance in the biliary microecology, leading to elevated levels of LPS in the serum and an increase in LPS levels within the bile ducts. This further enhances the endogenous expression of beta-glucuronidase and mucin in the bile duct epithelial cells[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], akin to the role of bacterial beta-glucuronidase and glycocalyx in stone formation. Our findings indicate that the abundance of bacteria associated with biliary infections (such as Enterococcus, Klebsiella, Enterococcus faecalis, etc.) in the bile microbiota of the diverticula group is significantly higher than in the simple common bile duct stone group, potentially promoting stone formation by modulating mucin expression. Controlling the expression of these factors may help prevent the formation and recurrence of bile duct stones. Microbiota may also influence the progression of stones through the formation of biofilms and the production of metabolites, indirectly affecting energy intake, altering intestinal permeability, and leading to chronic inflammation of the biliary tract, thereby promoting the formation of primary common bile duct stones. Research has found that in patients with pigment gallstones, genes extracted from Klebsiella and Enterococcus are involved in the arrangement of biofilms[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], contributing to the formation and development of stones. Notably, in our results, the abundance of Enterococcaceae and Enterococcus showed a significant positive correlation with the diameter of the common bile duct and the size of the stones, indicating a close relationship between juxtapapillary duodenal diverticula, bile microbiota, and common bile duct stones.\u003c/p\u003e\u003cp\u003eSimilarly, low expression of some beneficial bacteria in bile is detrimental to the health of the biliary tract and may contribute to stone formation and development. We found that in the diverticular group, Roseburia, Acinetobacter, Alistipes, Rikenellaceae, Peptococcales, Helicobacter_typhlonius, Nesterenkonia_sp. The abundance of Peptococcales, Helicobacter_typhlonius, and Nesterenkonia_sp. decreased significantly. Roseburia is a common genus localized in the digestive tract and plays a major protective role in the progression of many diseases. Roseburia has been shown to ameliorate alcohol-associated liver disease by strengthening intestinal tight junctions[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In the intestines of patients with inflammatory bowel disease, genetic risk scores were significantly associated with a reduction in Roseburia in healthy control subjects, leading to a decrease in butyrate conversion[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Keren et al. found that Roseburia, a beneficial bacterium in the gut microflora, was also significantly reduced in patients with gallstones and could serve as a biomarker for symptomatic gallstone formation[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. A significant reduction in the number of Roseburia capable of producing short-chain fatty acids has also been found in patients with primary common bile duct stones[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In addition, Li et al. found that short-chain fatty acid-producing bacteria Clostridium butyricum (C. butyricum) reduced common bile duct stone formation and preserved gut microbiome balance in mice, reducing the risk of gastrointestinal and biliary infections in a basal mouse study[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In our results, the abundance of short-chain fatty acid-producing microorganisms such as Acinetobacter, Alistipes, Rikenellaceaeza, and Peptococcales was reduced in the diverticular group compared with the group with simple common bile duct stones and was negatively correlated with the diameter of the common bile ducts and the size of the stones, so we hypothesized that Roseburia, Therefore, we hypothesized that the decrease in the abundance of Roseburia, Acinetobacter, Alistipes, Rikenellaceaeza, and Peptococcales may contribute to the formation and development of common bile duct stones. The reduction in the abundance of these \u0026ldquo;beneficial\u0026rdquo; bacteria may contribute to stone formation, but their role in the biliary tract has been poorly investigated and needs to be further confirmed by a number of studies. It is worth noting that the formation and development of primary common bile duct stones is not the result of the action of a single microorganism, but is a complex process of correlation and interaction between microbiota.\u003c/p\u003e\u003cp\u003eBiliary and intestinal microorganisms are involved in almost all aspects of bile formation, including lipid and cholesterol metabolism, biotransformation, and enterohepatic circulation. The presence of parapapillary duodenal diverticula significantly alters the balance of bile metabolism. Predictions of KEGG pathways in microbiologically sequenced samples showed that the diverticular group was significantly different from the group with simple common bile duct stones in terms of 2-Oxocarboxylic acid metabolism, Glycine, serine and threonine metabolism, Alanine, aspartate and glutamate metabolism and lipid and cholesterol metabolism. Ten pathways were differentially enriched for 2-Oxocarboxylic acid metabolism, Glycine, serine and threonine metabolism, Alanine, aspartate and glutamate metabolism, which cover bile acid synthesis and metabolism, energy conversion mechanisms in the liver, oxidative stress, and inflammatory responses, with specific metabolic pathways involved. Further LC-MS biliary metabolomics revealed that metabolites such as Taurochenodeoxycholic acid (TCDCA), Stercobilin, Bilirubin glucuronide, Mono(glucosyluronic acid) bilirubin and other metabolites were significantly up-regulated in the diverticular group. were significantly up-regulated in the diverticulum group. Enrichment of KEGG pathway revealed that these metabolites were mainly involved in primary bile acid biosynthesis, taurine and hypotaurine metabolism, Phenylalanine, tyrosine and tryptophan biosynthesis Phenylalanine, tyrosine and tryptophan biosynthesis. Taurochenodeoxycholic Acid has anti-inflammatory and antifibrotic effects, protects the biliary system, and is involved in the regulation of cholesterol metabolism and bile acid metabolism, leading to alterations in the composition of bile acids, which affects bile solubility and fluidity, thereby increasing the risk of stone formation[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In our results, the elevated biliary component TCDCA in the diverticular group may be related to cholestasis and altered biliary composition, a finding that still needs to be further validated in large samples.Stercobilin is the end product of bilirubin metabolism, which is converted from bilirubin by bacterial action in the intestine[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Imbalances in the ratio of Stercobilin may lead to disorders of bilirubin metabolism leading to calcium bilirubin oversaturation, which may indirectly contribute to stone formation.Taurine and hypotaurine metabolism Metabolic pathways are associated with bile acid metabolism. Several studies have shown that bacterial microorganisms can regulate body health by influencing bile acid metabolism. For example, the primary bile acid biosynthesis, taurine and hypotaurine metabolism pathways are also significantly enriched in the blood of pregnant women with intrahepatic cholestasis[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Compound woodchuck granules may ameliorate hepatic fibrosis and attenuate hepatic oxidative stress and cell apoptosis by modulating taurine and taurine metabolism as well as bile acid metabolism[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The potential probiotic Lactobacillus gasseri LA39 can activate hepatic primary bile acid biosynthesis and promote intestinal secondary bile acid biotransformation[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. A positive correlation between the concentration of TCDCA and the abundance of Microbacterium, Lutibacterium, Sphingomonas genera, and Prevotella intermedia species has been found[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. When microorganisms are dysregulated and disorganized, secondary metabolic imbalances of bile acids, amino acids, and other metabolites may lead to the development of choledocholithiasis, cholestasis, and steatohepatitis, among other disorders. Therefore, we believe that the presence of juxtapapillary duodenal diverticula may affect the formation of common bile duct stone by regulating metabolite composition and influencing bile acid and lipid metabolism. We also found a clear correlation between some dominant genera, such as Enterococcus spp. and partially bound bile acids. Further studies are needed to investigate the potential relationship between bile metabolism and microbial characteristics.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe relationship between juxtapapillary duodenal diverticula and primary common bile duct stones is complex, and the role of altered biliary microbiology and metabolites in stone formation and the mechanisms involved need to be further explored. We analyzed bile microorganisms and metabolites in patients with primary common bile duct stones by 16SrRNA microbial sequencing and metabolomics to investigate the effect of parapapillary duodenal diverticular on stone formation. The relationship between microorganisms, metabolites, and stones requires more experimental evidence in future studies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCBDs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCommon bile duct stones\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePrimary common bile duct stones\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMRCP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMagnetic resonance cholangiopancreatography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eERCP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEndoscopic retrograde cholangio-pancreatography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eJPDD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eJuxtapapillary duodenal diverticula\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e16SrRNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e16S ribosomal ribonucleic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLC-MS/MS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLiquid chromatography mass spectrometry\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEST Endoscopic sphincterotomy\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBMI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBody mass index\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLEfSe\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLinear discriminant analysis coupled with effect size measurements.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Clerkship of Hebei General Hospital, Grant No. 2024002.\u0026nbsp;Our study adheres to the principles of the Helsinki Declaration.\u0026nbsp;Informed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data are available from the corresponding author on receipt of reasonable request and have been deposited in the publicly accessible NAME database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2023 Hebei Provincial Government Sponsored Medical Talent Project (No. ZF2023188).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMengying Wang and Hongtao Hou were involved in the conception and design of the data; Mengying Wang wrote the first draft of the paper, and Pingping LI, Wei Sang, Xuxu Yang, Ping Qi were involved in the collection of the experimental specimens; Hou Hongtao revised and finally approved the version to be published; and all the authors agreed to the publication of the article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Professor Hongtao Hou and the Hebei Provincial Government Sponsored Medical Talent Project for their financial support.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi S, Guizzetti L, Ma C, et al. Epidemiology and outcomes of choledocholithiasis and cholangitis in the United States: trends and urban-rural variations. BMC Gastroenterol. 2023 Jul 27;23(1):254.\u003c/li\u003e\n\u003cli\u003eKozyk M, Giri S, Harindranath S, et al. Recurrence of common bile duct stones after endoscopic clearance and its predictors: A systematic review. DEN Open. 2024 Apr;4(1):e294.\u003c/li\u003e\n\u003cli\u003eTazuma S. Gallstone disease: Epidemiology, pathogenesis, and classification of biliary stones (common bile duct and intrahepatic). Best Pract Res Clin Gastroenterol. 2006;20(6):1075-83.\u003c/li\u003e\n\u003cli\u003eKiriyama S, Kozaka K, Takada T, et al. Tokyo Guidelines 2018: diagnostic criteria and severity grading of acute cholangitis (with videos). J Hepatobiliary Pancreat Sci. 2018 Jan;25(1):17-30.\u003c/li\u003e\n\u003cli\u003eMohammad Alizadeh AH, Afzali ES, Shahnazi A, et al. ERCP features and outcome in patients with periampullary duodenal diverticulum. ISRN Gastroenterol. 2013;2013:217261.\u003c/li\u003e\n\u003cli\u003eKim CW, Chang JH, Kim JH, et al. Size and type of periampullary duodenal diverticula are associated with bile duct diameter and recurrence of bile duct stones. J Gastroenterol Hepatol. 2013 May;28(5):893-8.\u003c/li\u003e\n\u003cli\u003eJakubczyk E, Pazurek M, Mokrowiecka A, et al. The position of a duodenal diverticulum in the area of the major duodenal papilla and its potential clinical implications. Folia Morphol (Warsz). 2021;80(1):106-113.\u003c/li\u003e\n\u003cli\u003eYue P, Zhu KX, Wang HP, et al. Clinical significance of different periampullary diverticulum classifications for endoscopic retrograde cholangiopancreatography cannulation. World J Gastroenterol. 2020 May 21;26(19):2403-2415.\u003c/li\u003e\n\u003cli\u003eDepommier C, Everard A, Druart C, et al. Supplementation with Akkermansia muciniphila in overweight and obese human volunteers: a proof-of-concept exploratory study. Nat Med. 2019 Jul;25(7):1096-1103.\u003c/li\u003e\n\u003cli\u003eFernandes MR, Aggarwal P, Costa RGF, et al. Targeting the gut microbiota for cancer therapy. Nat Rev Cancer. 2022 Dec;22(12):703-722.\u003c/li\u003e\n\u003cli\u003eMolinero N, Ruiz L, Milani C, et al. The human gallbladder microbiome is related to the physiological state and the biliary metabolic profile. Microbiome. 2019 Jul 4;7(1):100.\u003c/li\u003e\n\u003cli\u003eFeng R, Zhang T, Kayani MUR, et al. Patients with Primary and Secondary Bile Duct Stones Harbor Distinct Biliary Microbial Composition and Metabolic Potential. Front Cell Infect Microbiol. 2022;12:881489.\u003c/li\u003e\n\u003cli\u003eLiu Q, Zheng L, Wang Y, et al. Primary choledocholithiasis occurrence and recurrence is synergetcally modulated by the bile microbiome and metabolome alternations. Life Sci. 2023 Oct 15;331:122073.\u003c/li\u003e\n\u003cli\u003eXiao M, Zhou Y, Wang Z, et al. The dysregulation of biliary tract microflora is closely related to primary choledocholithiasis: a multicenter study. Sci Rep. 2024 Apr 18;14(1):9004.\u003c/li\u003e\n\u003cli\u003eGromski MA, Gutta A, Lehman GA, et al. Microbiology of bile aspirates obtained at ERCP in patients with suspected acute cholangitis. Endoscopy. 2022 Nov;54(11):1045-1052.\u003c/li\u003e\n\u003cli\u003eShin JH, Tillotson G, MacKenzie TN, et al. Bacteroides and related species: The keystone taxa of the human gut microbiota. Anaerobe. 2024 Feb;85:102819.\u003c/li\u003e\n\u003cli\u003eZheng X, Yan Y, Li X, et al. Microbial characteristics of bile in gallstone patients: a comprehensive analysis of 9,939 cases. Front Microbiol. 2024;15:1481112.\u003c/li\u003e\n\u003cli\u003eShin NR, Whon TW, Bae JW. Proteobacteria: microbial signature of dysbiosis in gut microbiota. Trends Biotechnol. 2015 Sep;33(9):496-503.\u003c/li\u003e\n\u003cli\u003eDeng C, Pan J, Zhu H, et al. Effect of Gut Microbiota on Blood Cholesterol: A Review on Mechanisms. Foods. 2023 Nov 29;12(23).\u003c/li\u003e\n\u003cli\u003eHu H, Shao W, Liu Q, et al. Gut microbiota promotes cholesterol gallstone formation by modulating bile acid composition and biliary cholesterol secretion. Nat Commun. 2022 Jan 11;13(1):252.\u003c/li\u003e\n\u003cli\u003eLyu Z, Yu T, Zhang L, et al. Analysis of the relationship between bile duct and duodenal microbiota reveals that potential dysbacteriosis is the main cause of primary common bile duct stones. Synth Syst Biotechnol. 2021 Dec;6(4):414-428.\u003c/li\u003e\n\u003cli\u003eLee J, Park JS, Bae J, et al. Bile Microbiome in Patients with Recurrent Common Bile Duct Stones and Correlation with the Duodenal Microbiome. Life (Basel). 2022 Oct 3;12(10).\u003c/li\u003e\n\u003cli\u003eYang Y, Zhao Z, Wu S, et al. Structural or functional abnormality of sphincter of Oddi: an important factor for the recurrence of choledocholithiasis after endoscopic treatment. Ann Med. 2025 Dec;57(1):2440119.\u003c/li\u003e\n\u003cli\u003eMaki T. Pathogenesis of calcium bilirubinate gallstone: role of E. coli, beta-glucuronidase and coagulation by inorganic ions, polyelectrolytes and agitation. Ann Surg. 1966 Jul;164(1):90-100.\u003c/li\u003e\n\u003cli\u003eSkar V, Skar AG, Midtvedt T, et al. Beta-glucuronidase-producing bacteria in bile from the common bile duct in patients treated with endoscopic papillotomy for gallstone disease. Scand J Gastroenterol. 1986 Mar;21(2):253-6.\u003c/li\u003e\n\u003cli\u003eMussa M, Martinez Perez-Crespo PM, Lopez-Cortes LE, et al. Risk Factors and Predictive Score for Bacteremic Biliary Tract Infections Due to Enterococcus faecalis and Enterococcus faecium: a Multicenter Cohort Study from the PROBAC Project. Microbiol Spectr. 2022 Aug 31;10(4):e0005122.\u003c/li\u003e\n\u003cli\u003eLeung JW, Liu YL, Leung PS, et al. Expression of bacterial beta-glucuronidase in human bile: an in vitro study. Gastrointest Endosc. 2001 Sep;54(3):346-50.\u003c/li\u003e\n\u003cli\u003eKriaa A, Bourgin M, Potiron A, et al. Microbial impact on cholesterol and bile acid metabolism: current status and future prospects. J Lipid Res. 2019 Feb;60(2):323-332.\u003c/li\u003e\n\u003cli\u003eZhao Z, Yang Y, Wu S, et al. Role of Secretory Mucins in the Occurrence and Development of Cholelithiasis. Biomolecules. 2024 Jun 10;14(6).\u003c/li\u003e\n\u003cli\u003eMa WJ, Wu ZR, Yang Q, et al. Biliary antibiotics irrigation for E. coli-induced chronic proliferative cholangitis and hepatolithiasis: A pathophysiological study in rabbits. Clin Res Hepatol Gastroenterol. 2020 Jun;44(3):356-367.\u003c/li\u003e\n\u003cli\u003eKose SH, Grice K, Orsi WD, et al. Metagenomics of pigmented and cholesterol gallstones: the putative role of bacteria. Sci Rep. 2018 Jul 25;8(1):11218.\u003c/li\u003e\n\u003cli\u003eSeo B, Jeon K, Moon S, et al. Roseburia spp. Abundance Associates with Alcohol Consumption in Humans and Its Administration Ameliorates Alcoholic Fatty Liver in Mice. Cell Host Microbe. 2020 Jan 8;27(1):25-40 e6.\u003c/li\u003e\n\u003cli\u003eImhann F, Vich Vila A, Bonder MJ, et al. Interplay of host genetics and gut microbiota underlying the onset and clinical presentation of inflammatory bowel disease. Gut. 2018 Jan;67(1):108-119.\u003c/li\u003e\n\u003cli\u003eKeren N, Konikoff FM, Paitan Y, et al. Interactions between the intestinal microbiota and bile acids in gallstones patients. Environ Microbiol Rep. 2015 Dec;7(6):874-80.\u003c/li\u003e\n\u003cli\u003eLi G, Yu T, Du H, et al. Effect of Clostridium butyricum on the formation of primary choledocholithiasis based on intestinal microbiome and metabolome analysis. J Appl Microbiol. 2023 Aug 1;134(8).\u003c/li\u003e\n\u003cli\u003eBao L, Hao D, Wang X, et al. Transcriptome investigation of anti-inflammation and immuno-regulation mechanism of taurochenodeoxycholic acid. BMC Pharmacol Toxicol. 2021 Apr 29;22(1):23.\u003c/li\u003e\n\u003cli\u003eHamoud AR, Weaver L, Stec DE, et al. Bilirubin in the Liver-Gut Signaling Axis. Trends Endocrinol Metab. 2018 Mar;29(3):140-150.\u003c/li\u003e\n\u003cli\u003eLi GH, Huang SJ, Li X, et al. Response of gut microbiota to serum metabolome changes in intrahepatic cholestasis of pregnant patients. World J Gastroenterol. 2020 Dec 14;26(46):7338-7351.\u003c/li\u003e\n\u003cli\u003eMen L, Gu Z, Wang E, et al. Fufang Muji Granules Ameliorate Liver Fibrosis by Reducing Oxidative Stress and Inflammation, Inhibiting Apoptosis, and Modulating Overall Metabolism. Metabolites. 2024 Aug 11;14(8).\u003c/li\u003e\n\u003cli\u003eHu J, Hou Q, Zheng W, et al. Lactobacillus gasseri LA39 promotes hepatic primary bile acid biosynthesis and intestinal secondary bile acid biotransformation. J Zhejiang Univ Sci B. 2023 Aug 15;24(8):734-748.\u003c/li\u003e\n\u003cli\u003ePetrov VA, Fernandez-Peralbo MA, Derks R, et al. Biliary Microbiota and Bile Acid Composition in Cholelithiasis. Biomed Res Int. 2020;2020:1242364.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-gastroenterology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmge","sideBox":"Learn more about [BMC Gastroenterology](http://bmcgastroenterol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmge/default.aspx","title":"BMC Gastroenterology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Juxtapapillary duodenal diverticula, common bile duct stones, Bile microorganisms, 16SrRNA, KEGG","lastPublishedDoi":"10.21203/rs.3.rs-6863628/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6863628/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eThis study investigates the microbiological and metabolic characteristics of bile in patients with common bile duct stones (CBDs) with and without juxtapapillary duodenal diverticulum (JPDD) to analyze stone formation causes and influencing factors.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eFrom January to May 2024, CBDs patients undergoing endoscopic retrograde cholangiopancreatography at our hospital were prospectively enrolled. Bile samples were collected for 16SrRNA sequencing and LC-MS/MS metabolomics analysis. Patients were divided into JPDD (n\u0026thinsp;=\u0026thinsp;15) and CBDs (n\u0026thinsp;=\u0026thinsp;15) groups.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe JPDD group had larger stone and bile duct diameters (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Proteobacteria dominated the bile microbiota in both groups. The JPDD group showed higher abundances of Escherichia-Shigella, Enterococcus, and Escherichia_coli. Beta diversity differed significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). LEfSe analysis identified 25 differential bacterial species. Enterococcus, Klebsiella, and Gemellaceaeke were enriched in the JPDD group, while Peptococcaceae, Roseburia, and Alistipes were enriched in the CBDs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Enterococcaceae and Enterococcus correlated positively with bile duct and stone size in the JPDD group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Peptococcaceae and Acinetobacter showed negative correlations (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Ten metabolic pathways, including phenylalanine and alanine metabolism, differed significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Metabolites like bilirubin glucuronide and taurochenodeoxycholic acid were upregulated in the JPDD group. Enterococcus in the JPDD group correlated with bile acid metabolites like chenodeoxycholylasparagine (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eJPDD influences CBD stone formation and size. JPDD alters bile microbiota, with Enterococcus and Klebsiella enriched in the JPDD group, and Peptococcaceae in the CBDs group. These microbiota correlate with stone size. JPDD changes bile metabolism, with metabolites like taurochenodeoxycholic acid and altered metabolic pathways influencing stone formation.\u003c/p\u003e","manuscriptTitle":"Bile Microbiome and Metabolic Characteristics in Primary Common Bile Duct Stone Patients with Juxtapapillary Duodenal Diverticula: A Clinical Investigation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 09:49:11","doi":"10.21203/rs.3.rs-6863628/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-06T11:03:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T09:59:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-16T12:53:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96341976402144734506409081030320749950","date":"2025-08-25T09:40:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145672346918275860739516948067607434664","date":"2025-08-23T05:37:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-22T13:23:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-09T22:56:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-07T11:51:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-06T10:16:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Gastroenterology","date":"2025-07-06T10:13:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-gastroenterology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmge","sideBox":"Learn more about [BMC Gastroenterology](http://bmcgastroenterol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmge/default.aspx","title":"BMC Gastroenterology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"971b146d-a40e-474b-b26f-cd27d2f6b9b1","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2025-12-12T11:53:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-28 09:49:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6863628","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6863628","identity":"rs-6863628","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}