Bile Microbiome Research Articles

Alterations of the bile microbiome is associated with progression-free survival in pancreatic ductal adenocarcinoma patients

BackgroundPatients with pancreatic ductal adenocarcinoma (PDAC) display an altered oral, gastrointestinal, and intra-pancreatic microbiome compared to healthy individuals. However, knowledge regarding the bile microbiome and its potential impact on progression-free survival in PDACs remains limited.MethodsPatients with PDAC (n = 45), including 20 matched pairs before and after surgery, and benign controls (n = 16) were included prospectively. The characteristics of the microbiomes of the total 81 bile were revealed by 16 S-rRNA gene sequencing. PDAC patients were divided into distinct groups based on tumor marker levels, disease staging, before and after surgery, as well as progression free survival (PFS) for further analysis. Disease diagnostic model was formulated utilizing the random forest algorithm.ResultsPDAC patients harbor a unique and diverse bile microbiome (PCoA, weighted Unifrac, p = 0.038), and the increasing microbial diversity is correlated with dysbiosis according to key microbes and microbial functions. Aliihoeflea emerged as the genus displaying the most significant alteration among two groups (p < 0.01). Significant differences were found in beta diversity of the bile microbiome between long-term PFS and short-term PFS groups (PCoA, weighted Unifrac, p = 0.005). Bacillota and Actinomycetota were identified as altered phylum between two groups associated with progression-free survival in all PDAC patients. Additionally, we identified three biomarkers as the most suitable set for the random forest model, which indicated a significantly elevated likelihood of disease occurrence in the PDAC group (p < 0.0001). The area under the receiver operating characteristic (ROC) curve reached 80.8% with a 95% confidence interval ranging from 55.0 to 100%. Due to the scarcity of bile samples, we were unable to conduct further external verification.ConclusionPDAC is characterized by an altered microbiome of bile ducts. Biliary dysbiosis is linked with progression-free survival in all PDACs. This study revealed the alteration of the bile microbiome in PDACs and successfully developed a diagnostic model for PDAC.

A Comparative Investigation of the Bile Microbiome in Patients with Choledocholithiasis and Cholecystolithiasis through Metagenomic Analysis.

While the precise triggers of gallstone formation remain incompletely understood, it is believed to arise from a complex interplay of genetic and environmental factors. The bile microbiome is being increasingly recognized as a possible contributor to the onset of gallstone disease. The primary objective of this study was to investigate distinctions in the microbial communities within bile specimens from patients with choledocholithiasis (common bile duct stones) and cholecystolithiasis (gallbladder stones). We employed massively parallel sequencing of the 16S rRNA gene to examine the microbial communities within bile samples obtained from 28 patients with choledocholithiasis (group DS) and cholecystolithiasis (group GS). The taxonomic composition of the bile microbial communities displayed significant disparities between the group DS and the group GS. Within the 16 prevalent genera, only Streptococcus, Ralstonia, Lactobacillus, and Enterococcus were predominantly found in the group GS. In contrast, the group DS displayed a more diverse range of genera. The alpha diversity of bile specimens was also notably lower in the group GS compared to the group DS (p = 0.041). Principal coordinate analysis unveiled distinct clustering of bile microbial communities depending on the location of the gallstone. Linear discriminant analysis effect size analysis, with a score threshold of >3 and the Kruskall-Wallis test (α < 0.05), recognized Bacilli and Lactobacillales as potential taxonomic markers for distinguishing patients with cholecystolithiasis limited to the gallbladder. Significant variations were found in the distribution and diversity of bile microbial communities between patients with choledocholithiasis and cholecystolithiasis. This observation suggests that alterations in the bile microbiome may contribute to the development of gallstones in these patients.

Microbiome Dysbiosis with Enterococcus Presence in the Upper Gastrointestinal Tract is A Risk Factor for Mortality in Patients Undergoing Surgery for Pancreatic Cancer.

Recent retrospective studies suggest a role for distinct microbiota in the perioperative morbidity and mortality of pancreatic head resections. We aimed to prospectively investigate the microbial colonization of critical operative sites of pancreatic head resections to identify microbial stratification factors for surgical and long-term oncologic outcomes. Prospective biomarker study applying 16S rRNA sequencing and microbial culturing to samples collected from various sites of the GI tract and surgical sites of patients during pancreatic head resections at a German single high-volume pancreatic center. A total of 101 patients were included (38 non-cancer, 63 cancer patients [50 PDAC patients]) in the study. In a first data analysis series, 16S rRNA sequencing data were utilized from 96 patients to assess associations of microbiome profiles with clinical parameters and outcomes. In general, microbiome composition varied according to sampling site, cancer, age or preoperative ERCP intervention, notably for the bile microbiome. In the PDAC subcohort, compositional variance of the bile or periampullary microbiome was significantly associated with postoperative complications such as ICU admission; on a taxonomic level we observed Enterococcus spp. to be significantly more abundant in patients developing deep or organ-space surgical site infections (SSI). Elevated Enterococcus relative abundances in the upper GI tract, in turn, were associated with 6-months mortality rates. In a second step, we focused on microbiological cultures collected from bile aspirates during surgery and investigated associations with perioperative complications and long-term survival. Notably, Enterococcus spp. were among the most prevalent pathobiont isolates observed in cancer patient bile specimens that were associated with severe SSIs, and thereby elevated mortality rates up to 24 months. Clinically relevant postoperative pancreatic fistulas or severe SSI were found as other major variables determining short-term mortality in this cancer patient cohort. In the context of adverse microbiological factors, a preoperative ERCP was also observed to segregate long-term survival, and it appeared to interact with the presence of Enterococcus spp. as highest mortality rates were observed in PDAC patients with both preoperative ERCP and presence of E. faecalis in bile aspirates. The presence of Enterococcus spp. in bile ducts of PDAC patients undergoing pancreatic surgery represents a significant risk factor for perioperative infections and, thereby, elevated postoperative and long-term mortality. This finding supports previous data on the use of the antibiotic drug piperacillin-tazobactam as appropriate perioperative antibiotic prophylaxis for preventing adverse outcomes after pancreatoduodenectomy.

The effects of photoactivated ciprofloxacin and bile acids on biofilms on bile duct catheters

ObjectivesThis study examined the potential of a novel photoactivatable ciprofloxacin to act against bacterial infections and microbiomes related to biliary diseases. It also evaluated treatment by combining the impact of bile acids and antibiotics on biofilms. Innovative strategies were evaluated to address the elusive bile duct microbiome resulting in biofilm-related infections linked to biliary catheters. The healthy biliary system is considered sterile, but bile microbiomes can occur in disease, and these correlate with hepatobiliary diseases. Causes include biofilms that form on internal-external biliary drainage catheters. These biliary catheters were used to noninvasively study the otherwise elusive bile microbiome for a pilot study. MethodsA new photoactivatable antibiotic was tested for efficacy against human-derived pathogenic bacterial isolates – Salmonella enterica and Escherichia coli – and catheter-derived bile duct microbiomes. In addition, the effect of bile acids on the antibiotic treatment of biofilms was quantified using crystal violet staining, confocal laser scanning microscopy, and biofilm image analysis. Two novel approaches for targeting biliary biofilms were tested. ResultsA photoactivated antibiotic based on ciprofloxacin showed efficacy in preventing biofilm formation and reducing bacterial viability without harming eukaryotic cells. Furthermore, combination treatment of antibiotics with bile acids, such as ursodesoxycholic acid, mildly influenced biofilm biomass but reduced bacterial survival within biofilms. ConclusionBile acids, in addition to their endocrine and paracrine functions, may enhance antibiotic killing of bacterial biofilms compared with antibiotics alone. These approaches hold promise for treating biliary infections such as cholangitis.

Bile microbiome signatures associated with pancreatic ductal adenocarcinoma compared to benign disease: a UK pilot study

Bile microbiome signatures associated with pancreatic ductal adenocarcinoma compared to benign disease: a UK pilot study

Bile Microbiome Signatures as Biomarkers for Differentiating Pancreatic Ductal Adenocarcinoma from Benign Disease: Discovery of novel microbial signatures in a UK pilot study

Bile Microbiome Signatures as Biomarkers for Differentiating Pancreatic Ductal Adenocarcinoma from Benign Disease: Discovery of novel microbial signatures in a UK pilot study

Insight into the mechanism of gallstone disease by proteomic and metaproteomic characterization of human bile.

Cholesterol gallstone disease is a prevalent condition that has a significant economic impact. However, the role of the bile microbiome in its development and the host's responses to it remain poorly understood. In this study, we conducted a comprehensive analysis of microbial and human bile proteins in 40 individuals with either gallstone disease or gallbladder polyps. We employed a combined proteomic and metaproteomic approach, as well as meta-taxonomic analysis, functional pathway enrichment, and Western blot analyses. Our metaproteomic analysis, utilizing the lowest common ancestor algorithm, identified 158 microbial taxa in the bile samples. We discovered microbial taxa that may contribute to gallstone formation, including β-glucuronidase-producing bacteria such as Streptococcus, Staphylococcus, and Clostridium, as well as those involved in biofilm formation like Helicobacter, Cyanobacteria, Pseudomonas, Escherichia coli, and Clostridium. Furthermore, we identified 2,749 human proteins and 87 microbial proteins with a protein false discovery rate (FDR) of 1% and at least 2 distinct peptides. Among these proteins, we found microbial proteins crucial to biofilm formation, such as QDR3, ompA, ndk, pstS, nanA, pfIB, and dnaK. Notably, QDR3 showed a gradual upregulation from chronic to acute cholesterol gallstone disease when compared to polyp samples. Additionally, we discovered other microbial proteins that enhance bacterial virulence and gallstone formation by counteracting host oxidative stress, including sodB, katG, rbr, htrA, and ahpC. We also identified microbial proteins like lepA, rtxA, pckA, tuf, and tpiA that are linked to bacterial virulence and potential gallstone formation, with lepA being upregulated in gallstone bile compared to polyp bile. Furthermore, our analysis of the host proteome in gallstone bile revealed enhanced inflammatory molecular profiles, including innate immune molecules against microbial infections. Gallstone bile exhibited overrepresented pathways related to blood coagulation, folate metabolism, and the IL-17 pathway. However, we observed suppressed metabolic activities, particularly catabolic metabolism and transport activities, in gallstone bile compared to polyp bile. Notably, acute cholelithiasis bile demonstrated significantly impaired metabolic activities compared to chronic cholelithiasis bile. Our study provides a comprehensive metaproteomic analysis of bile samples related to gallstone disease, offering new insights into the microbiome-host interaction and gallstone formation mechanism.

Bile Microbiome Signatures Associated with Pancreatic Ductal Adenocarcinoma Compared to Benign Disease: A UK Pilot Study

Pancreatic ductal adenocarcinoma (PDAC) has a very poor survival. The intra-tumoural microbiome can influence pancreatic tumourigenesis and chemoresistance and, therefore, patient survival. The role played by bile microbiota in PDAC is unknown. We aimed to define bile microbiome signatures that can effectively distinguish malignant from benign tumours in patients presenting with obstructive jaundice caused by benign and malignant pancreaticobiliary disease. Prospective bile samples were obtained from 31 patients who underwent either Endoscopic Retrograde Cholangiopancreatography (ERCP) or Percutaneous Transhepatic Cholangiogram (PTC). Variable regions (V3–V4) of the 16S rRNA genes of microorganisms present in the samples were amplified by Polymerase Chain Reaction (PCR) and sequenced. The cohort consisted of 12 PDAC, 10 choledocholithiasis, seven gallstone pancreatitis and two primary sclerosing cholangitis patients. Using the 16S rRNA method, we identified a total of 135 genera from 29 individuals (12 PDAC and 17 benign). The bile microbial beta diversity significantly differed between patients with PDAC vs. benign disease (Permanova p = 0.0173). The separation of PDAC from benign samples is clearly seen through unsupervised clustering of Aitchison distance. We found three genera to be of significantly lower abundance among PDAC samples vs. benign, adjusting for false discovery rate (FDR). These were Escherichia (FDR = 0.002) and two unclassified genera, one from Proteobacteria (FDR = 0.002) and one from Enterobacteriaceae (FDR = 0.011). In the same samples, the genus Streptococcus (FDR = 0.033) was found to be of increased abundance in the PDAC group. We show that patients with obstructive jaundice caused by PDAC have an altered microbiome composition in the bile compared to those with benign disease. These bile-based microbes could be developed into potential diagnostic and prognostic biomarkers for PDAC and warrant further investigation.

Primary choledocholithiasis occurrence and recurrence is synergetcally modulated by the bile microbiome and metabolome alternations

AimsPrimary choledocholithiasis is a common digestive disease with high morbidity and relapse. However, the compositions and functions of the bile microbial ecosystem and the pathogenesis of microfloral regulation of host metabolism resulting in stone formation are poorly understood. Main methodsBiliary samples collected from patients with acute cholangitis induced by benign biliary stricture (nonlithiasis group, n = 17) and primary choledocholithiasis (lithiasis group, n = 33) were subjected to multiomics analyses. Furthermore, clinicopathological features collected over a 24-month follow-up period were examined to evaluate the predictive value of candidate microbes. Key findingsFive alpha diversity indices of the bile microbiome were significantly decreased in the lithiasis group. Furthermore, we identified 49 differential bile flora between the two groups, and the relative abundances of 6 bacteria, Actinobacteria, Actinobacteriota, Staphylococcales, Micrococcales, Altererythrobacter and Carnobacteriaceae, were associated with primary choledocholithiasis relapse conditions. Multiomics analyses showed that specific changes in disease-related bacterial taxa were closely related to metabolite variation (low-molecular weight carboxylic acids, sterol liquid and acylcarnitine), which might reflect disease prognosis. According to microbiomic and metabolomic pathway analyses, we revealed that bacterial infections, microbiota-derived amino acid metabolites and secondary bile acid-related pathways were significantly enriched in the stone-formation group, suggesting a novel host-microbial metabolic mechanism of primary choledocholithiasis. SignificanceOur study first indicates bile host-microbial dysbiosis modulates the abnormal accumulation of metabolites might further disrupt calcium homeostasis and generate insoluble saponification. Additionally, we determined the predictive value of Actinomycetes phylum reduction for recurrence in primary common bile duct stone patients.

The Role of the Bile Microbiome in Common Bile Duct Stone Development.

Common bile duct (CBD) stones are a health concern for 10-20% of individuals with symptomatic gallstones, leading to health complications and placing a burden on healthcare systems. This study was initiated to investigate the changes in microbiome compositions and the metabolic signature associated with CBD stones. The research approach integrated taxonomic and functional data with metabolomics data, complemented by in vivo experiments. In a single tertiary healthcare institution, a total of 25 patients were enrolled who had undergone endoscopic retrograde cholangiopancreatography (ERCP) between February 2019 and January 2021. We harvested DNA from bile samples acquired from these individuals. The amplification of the bacterial 16S rRNA gene V3-V4 region was conducted through polymerase chain reaction (PCR), followed by sequencing. We utilized QIIME2 for a comprehensive data analysis. Furthermore, we performed a metabolomic analysis of the bile samples using nuclear magnetic resonance (NMR) spectroscopy. For the assessment of functional gene enrichment, we employed MetaboAnalyst 5.0. Lastly, we executed in vivo experiments on C57BL/6 mice and undertook histological examinations of tissue samples. Out of the 25 study subjects, 17 underwent ERCP due to CBD stones (the CBD stone group), while the remaining 8 had the procedure for different reasons (the non-CBD stone group). An alpha diversity analysis showed a significantly greater microbial diversity in the bile samples of the non-CBD stone group (p < 0.01), and a beta diversity analysis confirmed the greater microbial compositional abundance in the gut microbiomes in this group (p = 0.01). A taxonomic analysis revealed that the abundances of Enterococcaceae and Enterococcus were higher in the bile microbiomes of the CBD stone group. A metabolic profile analysis showed that the acetate, formate, and asparagine levels were higher in the CBD stone group. A pathway enrichment analysis showed the metabolic pathways (Arginine and Proline Metabolism, Aspartate Metabolism, Glycine, and Serine Metabolism, and Ammonia Recycling pathways) that were associated with these differences. Preclinical experiments demonstrated systemic inflammation and extracellular trap formation in the CBD stone group. Our study highlights the importance of biliary dysbiosis and bile metabolites, specifically acetate and formate, in CBD stone development and progression. These findings have implications for the development of diagnostic and therapeutic strategies using microbiomes for CBD stones.

Opisthorchis viverrini, Clonorchis sinensis and Opisthorchis felineus liver flukes affect mammalian host microbiome in a species-specific manner.

Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis are epidemiologically significant food-borne trematodes endemic to diverse climatic areas. O. viverrini and C. sinensis are both recognized to be 1A group of biological carcinogens to human, whereas O. felineus is not. The mechanisms of carcinogenesis by the liver flukes are studied fragmentarily, the role of host and parasite microbiome is an unexplored aspect. Specific pathogen free Mesocricetus auratus hamsters were infected with C. sinensis, O. viverrini and O. felineus. The microbiota of the adult worms, colon feces and bile from the hamsters was investigated using Illumina-based sequencing targeting the prokaryotic 16S rRNA gene. The analysis of 43 libraries revealed 18,830,015 sequences, the bacterial super-kingdom, 16 different phyla, 39 classes, 63 orders, 107 families, 187 genera-level phylotypes. O. viverrini, a fluke with the most pronounced carcinogenic potential, has the strongest impact on the host bile microbiome, changing the abundance of 92 features, including Bifidobacteriaceae, Erysipelotrichaceae, [Paraprevotellaceae], Acetobacteraceae, Coriobacteraceae and Corynebacteriaceae bacterial species. All three infections significantly increased Enterobacteriaceae abundance in host bile, reduced the level of commensal bacteria in the gut microbiome (Parabacteroides, Roseburia, and AF12). O. felineus, O. viverrini, and C. sinensis infections cause both general and species-specific qualitative and quantitative changes in the composition of microbiota of bile and colon feces of experimental animals infected with these trematodes. The alterations primarily concern the abundance of individual features and the phylogenetic diversity of microbiomes of infected hamsters.

Bile Microbiome Signatures as Biomarkers for Differentiating Pancreatic Ductal Adenocarcinoma from Benign Disease

Bile Microbiome Signatures as Biomarkers for Differentiating Pancreatic Ductal Adenocarcinoma from Benign Disease

Bile Microbiome in Patients with Recurrent Common Bile Duct Stones and Correlation with the Duodenal Microbiome.

Background: Common bile duct (CBD) stone recurrence is a common late adverse event after CBD stone treatment. In this preliminary study, we compared the bile fluid and duodenum microbial profiles of patients with or without recurrent CBD stones to identify risk factors associated with recurrence. Methods: Bile fluid samples of 47 consecutive patients who underwent ERCP for biliary diseases were subjected to microbiome analysis. Nineteen patients were stone-recurrent (SR), and 28 patients were non-stone-recurrent (NSR). Paired samples (duodenum biopsy tissue and bile fluid samples) from five SR patients were used to compare microbiome compositions in the biliary system and duodenum. In addition, we compared the microbiome compositions of these duodenal tissue samples with those 12 controls (gastric ulcer patients without recurrent CBD stones). Results: Enterococcaceae_unclassified and enterococcus were more abundant in bile fluid in the SR group than in the NSR group (p = 0.002 and p = 0.003, respectively). A comparison of the microbiome compositions of duodenum tissue and bile fluid samples of the five recurrent CBD stone patients revealed proteobacteria compositions were almost identical from the phylum to genus level. In these five patients, alpha and beta diversities were no different in bile fluid and duodenal tissues. Furthermore, a comparison of the microbiome compositions of duodenal mucosa in patients with recurrent CBD stone patients (n = 5) and controls (n = 12) revealed significant differences between microbiome compositions. Conclusions: Enterococcus seems to contribute to CBD stone development. Furthermore, our results indicate that retrograde migration of the duodenal microbiome may contribute to bile microbiome alterations. We recommend that more research be conducted to confirm this hypothesis.

Microbiologic risk factors of recurrent choledocholithiasis post-endoscopic sphincterotomy.

BACKGROUNDCholedocholithiasis is a severe disorder that affects a significant portion of the world’s population. Treatment using endoscopic sphincterotomy (EST) has become widespread; however, recurrence post-EST is relatively common. The bile microbiome has a profound influence on the recurrence of choledocholithiasis in patients after EST; however, the key pathogens and their functions in the biliary tract remain unclear. AIMTo investigate the biliary microbial characteristics of patients with recurrent choledocholithiasis post-EST, using next-generation sequencing.METHODSThis cohort study included 43 patients, who presented with choledocholithiasis at the Guangdong Second Provincial General Hospital between May and June 2020. The patients had undergone EST or endoscopic papillary balloon dilation and were followed up for over a year. They were divided into either the stable or recurrent groups. We collected bile samples and extracted microbial DNA for analysis through next-generation sequencing. Resulting sequences were analyzed for core microbiome and statistical differences between the diagnosis groups; they were examined using the Kyoto Encyclopedia of Genes and Genomes pathway hierarchy level using analysis of variance. Correlation between the key genera and metabolic pathways in bile, were analyzed using Pearson’s correlation test. RESULTSThe results revealed distinct clustering of biliary microbiota in recurrent choledocholithiasis. Higher relative abundances (RAs) of Fusobacterium and Neisseria (56.61% ± 14.81% vs 3.47% ± 1.10%, 8.95% ± 3.42% vs 0.69% ± 0.32%, respectively) and the absence of Lactobacillus were observed in the bile of patients with recurrent disease, compared to that in stable patients. Construction of a microbiological co-occurrence network revealed a mutual relationship among Fusobacterium, Neisseria, and Leptotrichia, and an antagonistic relationship among Lactobacillales, Fusobacteriales, and Clostridiales. Functional prediction of biliary microbiome revealed that the loss of transcription and metabolic abilities may lead to recurrent choledocholithiasis. Furthermore, the prediction model based on the RA of Lactobacillales in the bile was effective in identifying the risk of recurrent choledocholithiasis (P = 0.03).CONCLUSIONWe demonstrated differences in the bile microbiome of patients with recurrent choledocholithiasis compared to that in patients with stable disease, thereby adding to the current knowledge on its microbiologic etiology.

The Microbiome in Autoimmune Liver Diseases: Metagenomic and Metabolomic Changes.

Recent studies have identified the critical role of microbiota in the pathophysiology of autoimmune liver diseases (AILDs), including autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC). Metagenomic studies reveal significant decrease of gut bacterial diversity in AILDs. Although profiles of metagenomic vary widely, Veillonella is commonly enriched in AIH, PBC, and PSC. Apart from gut microbiome, the oral and bile microbiome seem to be associated with these diseases as well. The functional analysis of metagenomics suggests that metabolic pathways changed in the gut microbiome of the patients. Microbial metabolites, including short-chain fatty acids (SCFAs) and microbial bile acid metabolites, have been shown to modulate innate immunity, adaptive immunity, and inflammation. Taken together, the evidence of host–microbiome interactions and in-depth mechanistic studies needs further accumulation, which will offer more possibilities to clarify the mechanisms of AILDs and provide potential molecular targets for the prevention and treatment in the future.

Association of Microbial Dysbiosis with Gallbladder Diseases Identified by Bile Microbiome Profiling.

BackgroundCholecystitis is an important risk factor for gallbladder cancer, but the bile microbiome and its association with gallbladder disease has not been investigated fully. We aimed to analyze the bile microbiome in normal conditions, chronic cholecystitis, and gallbladder cancer, and to identify candidate bacteria that play an important role in gallbladder carcinogenesis.MethodsWe performed metagenome sequencing on bile samples of 10 healthy individuals, 10 patients with chronic cholecystitis, and 5 patients with gallbladder cancer, and compared the clinical, radiological, and pathological characteristics of the participants.ResultsNo significant bacterial signal was identified in the normal bile. The predominant dysbiotic bacteria in both chronic cholecystitis and gallbladder cancer were those belonging to the Enterobacteriaceae family. Klebsiella increased significantly in the order of normal, chronic cholecystitis, and gallbladder cancer. Patients with chronic cholecystitis and dysbiotic microbiome patterns had larger gallstones and showed marked epithelial atypia, which are considered as precancerous conditions.ConclusionWe investigated the bile microbiome in normal, chronic cholecystitis, and gallbladder cancer. We suggest possible roles of Enterobacteriaceae, including Klebsiella, in gallbladder carcinogenesis. Our findings reveal a possible link between a dysbiotic bile microbiome and the development of chronic calculous cholecystitis and gallbladder cancer.

Profiling of Bile Microbiome Identifies District Microbial Population between Choledocholithiasis and Cholangiocarcinoma Patients.

Objective:Choledocholithiasis (CDL), a potential risk for cholangiocarcinoma (CCA) development, is often a consequence of bacterial infection. Thus, the microbial population that contributes to CDL might also be involved in CCA development. We compared the microbiome in bile fluid of CDL patients and CCA patients. Methods:Bile samples were collected from CDL (n = 30) and CCA (n =30) patients. Microbial profiling was performed individually by the sequencing of V3-V4 regions of the 16S rRNA gene. Results: Enterobacter, Pseudomonas, and Stenotrophomonas species were much more abundant in bile samples from CCA compared to CDL (p<0.05). However, Cetobacterium, Pyramidobacter, and Streptococcus species were less abundant in bile samples of CCA compared to CDL (p<0.05). Although Escherichia was predominant in the CCA, Escherichia coli itself was more abundant in CDL than in CCA. One CDL case (3.3%) harbored genotoxin-producing E. coli as confirmed by PCR. Enterobacter and Pseudomonas also predominated in CCA according to linear discriminant-analysis effect size. Conclusion:we demonstrated vast differences between microbial communities in bile of CDL and CCA patients. These bacteria might be partly involved in CCA genesis and may provide novel biomarkers for CCA.

Effect of bacterial contamination in bile on pancreatic cancer cell survival

BackgroundIntroduction of gut flora into the biliary system is common owing to biliary stenting in patients with obstructing pancreatic head cancer. We hypothesize that alteration of biliary microbiome modifies bile content that modulates pancreatic cancer cell survival. MethodsHuman bile samples were collected during pancreaticoduodenectomy. Bacterial strains were isolated from contaminated (stented) bile and identified using 16S ribosomal RNA sequencing. Human pancreatic cancer cells (AsPC1, CFPAC, Panc1) were treated for 24 hours with sterile (nonstented) bile, contaminated (stented) bile, and sterile bile preincubated with 106 colony forming unit of live bacteria isolated from contaminated bile or a panel of bile acids for 24 hours at 37°C, and evaluated using CellTiter-Blue Cell Viability Assay (Promega Corp. Madison, WI). Human bile (30–50 μl/mouse) was coinjected intraperitoneally with 105 Panc02 mouse pancreatic cancer cells in C57BL6/N mice to evaluate the impact of bile on peritoneal metastasis 3 to 4 weeks after tumor challenge. ResultsWhile all bile samples significantly reduced peritoneal metastasis of Panc02 cells in mice, some contaminated bile samples had diminished antitumor effect. All sterile bile (n = 4) reduced pancreatic cancer cell survival in vitro. Only 40% (2/5) of contaminated bile samples had significant effect. Preincubation of sterile bile with live Enterococcus faecalis or Streptococcus oralis modified the antitumor effect of sterile bile. These changes were not observed with culture media preincubated with live bacteria, suggesting live gut bacteria can modify the antitumor components present in bile. Conjugated bile acids were more potent than unconjugated cholic acid in reducing pancreatic cancer cell survival. ConclusionAlteration of bile microbiome from biliary stenting has a direct impact on pancreatic cancer cell survival. Further study is warranted to determine if this microbiome shift alters tumor microenvironment.

Alterations of the Bile Microbiome in Recurrent Common Bile Duct Stone.

Objective Common bile duct stone (CBDS) recurrence is associated with bile microbial structure. This study explored the structure of bile microbiome in patients with recurrent CBDS, and its relationship with the recurrence of CBDS. Methods Patients with recurrent CBDS (recurrence group) and controls without CBDS (control group) requiring endoscopic retrograde cholangiopancreatography (ERCP) were prospectively included. The control group was noncholelithiasis patients, mainly including benign and malignant biliary stenosis. Bile samples were collected, and bile microbiome structure was analyzed by the 16S rRNA encoding gene (V3–V4). Results A total of 27 patients in the recurrence group and 19 patients in the control group were included. The diversity of bile microbiome in the recurrence group was significantly lower than that in the control group (Shannon index: 2.285 vs. 5.612, P = 0.001). In terms of bile microbial distribution, patients with recurrent CBDS had significantly higher Proteobacteria (86.72% vs. 64.92%, P = 0.037), while Bacteroidetes (3.16% vs. 8.53%, P = 0.001) and Actinobacteria (0.29% vs. 6.74%, P = 0.001) are significantly lower compared with the control group at the phylum level. At the genus level, the recurrence group was mainly the Escherichia, and there was a variety of more evenly distributed microbiome in the control group, with significant differences between the two groups. Conclusion The diversity of bile microbiome in patients with recurrent CBDS is lower. Patients with recurrent CBDS may have bile microbial imbalance, which may be related to the repeated formation of CBDS.

45 The Role of the Biliary and Gut Microbiome in the Progression of Cholangiocarcinoma and Primary Sclerosing Cholangitis

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory, cholestatic liver disease of unknown etiopathogenesis which is recognized as a major risk factor for cholangiocarcinoma (CCA). We aimed to investigate the roles of the biliary and intestinal microflora in the inflammatory process of PSC and to determine if the microflora is associated with progression to CCA. METHODS: We studied the bile and stool microbiomes in samples obtained from patients with PSC (31 stool and 35 bile samples), sporadic CCA (11 stool and 26 bile samples), and PSC coexisting with CCA (16 stool and bile 17 samples). Control bile was obtained from patients with cholelithiasis or choledocholithiasis (n = 17). DNA extraction was followed by amplification and high-throughput next generation sequencing (MiSeq) of the 16S rDNA (V3-V5) region to identify the microbiota present. We performed: 1. analysis of the stool microbiome between PSC and CCA patients; 2. analysis of the biliary microbiome in PSC and CCA patients; 3. comparison of the biliary microbiome collected before and after biliary stent placement. RESULTS: Although bile samples from study patients had significantly lower sequencing depths than stool, their sequencing depths were much higher than bile from negative controls. The microbiomes of stool and bile samples from the same patients were significantly correlated (P = 0.022). CCA bile samples had significant difference of alpha diversity from control bile samples with less Streptococcaceae and Desulfovibrionaceae (FDR &lt; 10%) than PSC whereas no significant change of stool microbiome was identified between CCA and PSC. Fusobacterium and Gemella were correlated to PSC duration. An increased abundance of Fusobacteria after stent placement was revealed. Fusobacteria, which is inflammation-promoting, was also increased with the number of stents in the bile duct. CONCLUSION: Our study suggests that the bile microbiome is potentially a more distinctive biomarker than the stool microbiome for PSC and CCA development. There is increased inhabitance of inflammation-promoting bacteria in the bile of CCA patients and after biliary stent placement. Increased PSC duration also increases the microbial burden in the bile, which may also promote CCA development. We further detected specific bile microbes statistically associated with CCA or stent presence in the bile ducts, which raises the possibility of their role in the CCA progression or stent obstruction. Additional analyses with more samples are needed.