"Answers in hours": A prospective clinical study using nanopore sequencing for bile duct cultures.

BACKGROUND: Surgical site infection is a major source of morbidity in patients undergoing pancreatic head resection and is often from organisms in intraoperative bile duct cultures. As such, many institutions use prolonged prophylactic antibiotics and tailor based on bile duct cultures. However, standard cultures take days, leaving many patients unnecessarily on prolonged antibiotics. Nanopore sequencing can provide data in hours and, thus, has the potential to improve antibiotic stewardship. The present study investigates the feasibility of nanopore sequencing in intraoperative bile samples. METHODS: Patients undergoing pancreatic head resection were included. Intra-operative bile microbial profiles were determined with standard cultures and nanopore sequencing. Antibiotic recommendations were generated, and time-to-results determined for both methods. Organism yields, resistance patterns, antibiotic recommendations, and costs were compared. RESULTS: Out of 42 patients, 22 (52%) had samples resulting in positive standard cultures. All positive standard cultures had microbes detected using nanopore sequencing. All 20 patients with negative standard cultures had negative nanopore sequencing. Nanopore sequencing detected more bacterial species compared to standard cultures (10.5 vs 4.4, p < 0.05) and more resistance genotypes (10.3 vs 2.7, p < 0.05). Antimicrobial recommendations based on nanopore sequencing provided coverage for standard cultures in 27 out of 44 (61%) samples, with broader coverage recommended by nanopore sequencing in 13 out of 27 (48%) of these samples. Nanopore sequencing results were faster (8 vs 98 hours) than standard cultures but had higher associated costs ($165 vs $38.49). CONCLUSION: Rapid microbial profiling with nanopore sequencing is feasible with broader organism and resistance profiling compared to standard cultures. Nanopore sequencing has perfect negative predictive value and can potentially improve antibiotic stewardship; thus, a randomized control trial is under development.

Intraoperative bile duct cultures in patients undergoing pancreatic head resection: Prospective comparison of bile duct swab versus bile duct aspiration.

BACKGROUND: Postoperative surgical site infection is a major source of morbidity after pancreatic head resections, and data suggest bacterobilia as a leading cause. Some centers use intraoperative bile duct cultures to guide postoperative antimicrobial prophylaxis. This prospective study evaluates culture differences between traditional bile duct swab versus bile duct aspiration intraoperative samples. METHODS: Prospective patients undergoing pancreatic head resection with both bile duct swab and bile duct aspiration were included. Cultures were reviewed for organism characteristics. Any growth of organisms was considered a positive culture. Bile duct swab yield and characteristics were compared with bile duct aspiration. Postoperative surgical site infection complications were compared to bile duct culture results. RESULTS: Fifty patients were included. Bile duct aspiration resulted in a significantly higher median number of organisms compared to bile duct swab (6 vs 3; P < .001). There were no differences in the number of patients (37 vs 33) having positive bile duct aspiration and bile duct swab cultures (P = .385). Anaerobic cultures (not possible with bile duct swab) were positive in 21 patients with bile duct aspiration. A total of 37 (74%) patients had preoperative biliary stenting, which highly associated (P < .001) with positive cultures. Bile duct culture organisms correlated with postoperative surgical site infection in 12/17 (71%) patients. CONCLUSION: Use of bile duct aspiration improves intraoperative bile duct culture organism yield over bile duct swab and may improve tailoring of antibiotics in patients undergoing pancreatic head resection.

Biliary Microbiota and Bile Acid Composition in Cholelithiasis.

BACKGROUND: A functional interplay between BAs and microbial composition in gut is a well-documented phenomenon. In bile, this phenomenon is far less studied, and with this report, we describe the interactions between the BAs and microbiota in this complex biological matrix. Methodology. Thirty-seven gallstone disease patients of which twenty-one with Opisthorchis felineus infection were enrolled in the study. The bile samples were obtained during laparoscopic cholecystectomy for gallstone disease operative treatment. Common bile acid composition was measured by LC-MS/MS. Gallbladder microbiota were previously analyzed with 16S rRNA gene sequencing on Illumina MiSeq platform. The associations between bile acid composition and microbiota were analyzed. RESULTS: Bile acid signature and Opisthorchis felineus infection status exert influence on beta-diversity of bile microbial community. Direct correlations were found between taurocholic acid, taurochenodeoxycholic acid concentrations, and alpha-diversity of bile microbiota. Taurocholic acid and taurochenodeoxycholic acid both show positive associations with the presence of Chitinophagaceae family, Microbacterium and Lutibacterium genera, and Prevotella intermedia. Also, direct associations were identified for taurocholic acid concentration and the presence of Actinomycetales and Bacteroidales orders, Lautropia genus, Jeotgalicoccus psychrophilus, and Haemophilus parainfluenzae as well as for taurochenodeoxycholic acid and Acetobacteraceae family and Sphingomonas genus. There were no differences in bile acid concentrations between O. felineus-infected and noninfected patients. Conclusions/Significance. Associations between diversity, taxonomic profile of bile microbiota, and bile acid levels were evidenced in patients with cholelithiasis. Increase of taurochenodeoxycholic acid and taurocholic acid concentration correlates with bile microbiota alpha-diversity and appearance of opportunistic pathogens.

Association of Chronic Opisthorchis Infestation and Microbiota Alteration on Tumorigenesis in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a common hepatobiliary cancer in East and Southeast Asia. The data of microbiota contribution in CCA are still unclear. Current available reports have demonstrated that an Opisthorchis viverrini (OV) infection leads to dysbiosis in the bile duct. An increase in the commensal bacteria Helicobacter spp. in OV-infected CCA patients is associated with bile duct inflammation, severity of bile duct fibrosis, and cholangiocyte proliferation. In addition, secondary bile acids, major microbial metabolites, can mediate cholangiocyte inflammation and proliferation in the liver. A range of samples from CCA patients (stool, bile, and tumor) showed different degrees of dysbiosis. The evidence from these samples suggests that OV infection is associated with alterations in microbiota and could potentially have a role in CCA. In this comprehensive review, reports from in vitro, in vivo, and clinical studies that demonstrate possible links between OV infection, microbiota, and CCA pathogenesis are summarized and discussed. Understanding these associations may pave ways for novel potential adjunct intervention in gut microbiota in CCA patients.

Alterations of the bile microbiome in primary sclerosing cholangitis.

BACKGROUND: Patients with primary sclerosing cholangitis (PSC) display an altered colonic microbiome compared with healthy controls. However, little is known on the bile duct microbiome and its interplay with bile acid metabolism in PSC. METHODS: Patients with PSC (n=43) and controls without sclerosing cholangitis (n=22) requiring endoscopic retrograde cholangiography were included prospectively. Leading indications in controls were sporadic choledocholithiasis and papillary adenoma. A total of 260 biospecimens were collected from the oral cavity, duodenal fluid and mucosa and ductal bile. Microbiomes of the upper alimentary tract and ductal bile were profiled by sequencing the 16S-rRNA-encoding gene (V1-V2). Bile fluid bile acid composition was measured by high-performance liquid chromatography mass spectrometry and validated in an external cohort (n=20). RESULTS: The bile fluid harboured a diverse microbiome that was distinct from the oral cavity, the duodenal fluid and duodenal mucosa communities. The upper alimentary tract microbiome differed between PSC patients and controls. However, the strongest differences between PSC patients and controls were observed in the ductal bile fluid, including reduced biodiversity (Shannon entropy, p=0.0127) and increase of pathogen Enterococcus faecalis (FDR=4.18×10-5) in PSC. Enterococcus abundance in ductal bile was strongly correlated with concentration of the noxious secondary bile acid taurolithocholic acid (r=0.60, p=0.0021). CONCLUSION: PSC is characterised by an altered microbiome of the upper alimentary tract and bile ducts. Biliary dysbiosis is linked with increased concentrations of the proinflammatory and potentially cancerogenic agent taurolithocholic acid.

Gut microbiota translocation promotes autoimmune cholangitis.

Gut microbiota and bacterial translocation have been implicated as significant contributors to mucosal immune responses and tolerance; alteration of microbial molecules, termed pathogen-associated molecular patterns (PAMP) and bacterial translocation are associated with immune pathology. However, the mechanisms by which dysregulated gut microbiota promotes autoimmunity is unclear. We have taken advantage of a well-characterized murine model of primary biliary cholangitis, dnTGFßRII mice, and an additional unique construct, toll-like receptor 2 (TLR2)-deficient dnTGFßRII mice coined dnTGFßRIITLR2-/- mice to investigate the influences of gut microbiota on autoimmune cholangitis. Firstly, we report that dnTGFßRII mice manifest altered composition of gut microbiota and that alteration of this gut microbiota by administration of antibiotics significantly alleviates T-cell-mediated infiltration and bile duct damage. Second, toll-like receptor 2 (TLR2)-deficient dnTGFßRII mice demonstrate significant exacerbation of autoimmune cholangitis when their epithelial barrier integrity was disrupted. Further, TLR2-deficiency mediates downregulated expression of tight junction-associated protein ZO-1 leading to increased gut permeability and bacterial translocation from gut to liver; use of antibiotics reduces microbiota translocation to liver and also decreases biliary pathology. In conclusion, our data demonstrates the important role of gut microbiota and bacterial translocation in the pathogenesis of murine autoimmune cholangitis.

Alterations in Intestinal Microbiota Lead to Production of Interleukin 17 by Intrahepatic γδ T-Cell Receptor-Positive Cells and Pathogenesis of Cholestatic Liver Disease.

BACKGROUND & AIMS: Variants at the ABCB4 or MDR2 locus, which encodes a biliary transport protein, are associated with a spectrum of cholestatic liver diseases. Exacerbation of liver disease has been linked to increased hepatic levels of interleukin (IL) 17, yet the mechanisms of this increase are not understood. We studied mice with disruption of Mdr2 to determine how defects in liver and alteration in the microbiota contribute to production of IL17 by intrahepatic γδ T cells. METHODS: We performed studies with Mdr2-/- and littermate FVB/NJ (control) mice. IL17 was measured in serum samples by an enzyme-linked immunosorbent assay. Mice were injected with neutralizing antibodies against the γδ T-cell receptor (TCR; anti-γδ TCR) or mouse IL17A (anti-IL17A). Livers were collected and bacteria were identified in homogenates by culture procedures; TCRγδ+ cells were isolated by flow cytometry. Fecal samples were collected from mice and analyzed by 16S ribosomal DNA sequencing. Cells were stimulated with antibodies or bacteria, and cytokine production was measured. We obtained tissues from 10 patients undergoing liver transplantation for primary sclerosing cholangitis or chronic hepatitis C virus infection. Tissues were analyzed for cytokine production by γδ TCR+ cells. RESULTS: Mdr2-/- mice had collagen deposition around hepatic bile ducts and periportal-bridging fibrosis with influx of inflammatory cells and increased serum levels of IL17 compared with control mice. Administration of anti-IL17A reduced hepatic fibrosis. Livers from Mdr2-/- mice had increased numbers of IL17A+ γδTCR+ cells-particularly of IL17A+ Vγ6Jγ1 γδ TCR+ cells. Fecal samples from Mdr2-/- mice were enriched in Lactobacillus, and liver tissues were enriched in Lactobacillus gasseri compared with control mice. Mdr2-/- mice also had increased intestinal permeability. The γδ TCR+ cells isolated from Mdr2-/- livers produced IL17 in response to heat-killed L gasseri. Intraperitoneal injection of control mice with L gasseri led to increased serum levels of IL17 and liver infiltration by inflammatory cells; injection of these mice with anti-γδ TCR reduced serum level of IL17. Intravenous injections of Mdr2-/- mice with anti-γδ TCR reduced fibrosis; liver levels of IL17, and inflammatory cells; and serum levels of IL17. γδTCR+ cells isolated from livers of patients with primary sclerosing cholangitis, but not hepatitis C virus infection, produced IL17. CONCLUSIONS: In Mdr2-/- mice, we found development of liver fibrosis and inflammation to require hepatic activation of γδ TCR+ cells and production of IL17 mediated by exposure to L gasseri. This pathway appears to contribute to development of cholestatic liver disease in patients.

"Primary bacterial culture of bile and pancreatic juice in tumor related jaundice (TROJ) - is ascending cholangitis always our fault?"

INTRODUCTION: TROJ (tumor-related obstructive jaundice) is one of the most common indications for endoscopic retrograde choleopancreatography (ERCP) with endoscopic biliary stenting. Despite the effectiveness of this procedure, especially in palliative patients, it is not without flaws. Ascending bacterial cholangitis, a common stenting complication, occurs in about 0.5-1.7% of cases. The authors' intention was to investigate whether this complication occurs solely due to the procedure or whether it is a result of an underlying bacterial infection in the dilated, obstructed bile and pancreatic ducts. METHODS: Sixteen patients with painless obstructive jaundice related to a tumor located in or in the proximity of the bile duct were enrolled for this study. Prior to endoscopic palliative stenting we harvested bile and pancreatic fluid and the proceeded with the initial procedure. RESULTS: In 14 cases (87.5%) we managed to restore the patency of the bile duct endoscopically. Additionaly, we observed that in 13 cases (81.25%) bacteria were present in the bile and/or pancreatic fluid. The most common pathogen was Streptococcus mitis - present in 7 cases (43.75%). The most effective antibiotics for discovered S. mitis strains were cefuroxime and vancomycin. CONCLUSION: Primal bacterial pathogenes may be present in obstructed bile and pancreatic ducts prior to endoscopic intervention. The connection between Streptocccus mitis and TROJ needs further investigation.

Role of inflammation and proinflammatory cytokines in cholangiocyte pathophysiology.

Cholangiocytes, the epithelial cells lining the bile ducts, are an important subset of liver cells. They are involved in the modification of bile volume and composition, and respond to endogenous and exogenous stimuli. Along the biliary tree, two different kinds of cholangiocytes exist: small and large cholangiocytes. Each type has different features and biological role in physiologic and pathologic conditions, and their immunobiology is important for understanding biliary diseases. Cholangiocytes provide the first line of defence against luminal microbes in the hepatobiliary system. Indeed, they express a variety of pattern recognition receptors and may start an antimicrobial defence activating a set of intracellular signalling cascades. In response to injury, cholangiocytes that are normally quiescent become reactive and acquire a neuroendocrine-like phenotype with the release of proinflammatory mediators and antimicrobial peptides, which support biliary epithelial integrity. These molecules act in an autocrine/paracrine manner to modulate cholangiocyte biology and determine the evolution of biliary damage. Failure or dysregulation of such mechanisms may influence the progression of cholangiopathies, a group of diseases that selectively target biliary cells. In this review, we focus on the response of cholangiocytes in inflammatory conditions, with a particular focus on the mechanism driving cholangiocytes adaptation to damage. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

How the biliary tree maintains immune tolerance?

The liver is a vital organ with distinctive anatomy, histology and heterogeneous cell populations. These characteristics are of particular importance in maintaining immune homeostasis within the liver microenvironments, notably the biliary tree. Cholangiocytes are the first line of defense of the biliary tree against foreign substances, and are equipped to participate through various immunological pathways. Indeed, cholangiocytes protect against pathogens by TLRs-related signaling; maintain tolerance by expression of IRAK-M and PPARγ; limit immune response by inducing apoptosis of leukocytes; present antigen by expressing human leukocyte antigen molecules and costimulatory molecules; recruit leukocytes to the target site by expressing cytokines and chemokines. However, breach of tolerance in the biliary tree results in various cholangiopathies, exemplified by primary biliary cholangitis, primary sclerosing cholangitis and biliary atresia. Lessons learned from immune tolerance of the biliary tree will provide the basis for the development of effective therapeutic approaches against autoimmune biliary tract diseases. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Colonization, resistance to bile, and virulence properties of Escherichia coli strains: Unusual characteristics associated with biliary tract diseases.

Escherichia coli is the species that is most frequently isolated from bile of patients with biliary tract diseases. This study was aimed to investigate any association between resistance and virulence properties of these isolates with occurrence of the diseases. A total of 102 bile samples were obtained from patients subjected to endoscopic retrograde cholangiopancreatography for different biliary diseases. Clinical data were collected and culture of the bile samples was done on selective media. Resistance of characterized Escherichia coli isolates to deoxycholate sodium (0-7%) and nineteen antibiotics was determined and PCR using 16 pairs of primers targeting stx1, stx2, exhA, eae, bfp, agg, pcvd432, lt, st, ipaH, pic, pet, ast, set, sen, and cdtB genes was done. Our results showed a statistically significant association between E. coli colonization and existence of common bile duct and gallbladder stones (p value 0.028). Out of the 22 E. coli strains (22/102) multidrug resistance phenotype was present in 95.45%. None of the strains belonged to common E. coli pathotypes. However, bfp + EhxA-hly, bfp + astA, bfp + EhxA-hly + pic, and EhxA-hly + pic + astA, bfp, and astA genotypes were detected in these strains. bfp (7/22, 31.8%) and astA (5/22, 22.7%) were among most frequent virulence factors in these strains. Results of this study showed significant association between colonization of E. coli and choledocholithiasis. Unusual existence of virulence gene combinations in these strains and their resistance to DOC and multiple classes of antibiotics could be considered as possible causes of their persistence in this harsh microenvironment.

Bile duct regeneration and immune response by passenger lymphocytes signals biliary recovery versus complications after liver transplantation.

This study aimed to elucidate the impact of epithelial regenerative responses and immune cell infiltration on biliary complications after liver transplantation. Bile duct (BD) damage after cold storage was quantified by a BD damage score and correlated with patient outcome in 41 patients. Bacterial infiltration was determined by fluorescence in situ hybridization (FISH). BD samples were analyzed by immunohistochemistry for E-cadherin, cytokeratin, CD56, CD14, CD4, CD8, and double-immunofluorescence for cytokine production and by messenger RNA (mRNA) microarray. Increased mRNA levels of adherens junctions (P < 0.01) were detected in damaged BDs from patients without complications compared with damaged BDs from patients with biliary complications. Immunohistochemistry showed increased expression of E-cadherin and cytokeratin in BDs without biliary complications (P = 0.03; P = 0.047). FISH analysis demonstrated translocation of bacteria in BDs. However, mRNA analysis suggested an enhanced immune response in BDs without biliary complications (P < 0.01). Regarding immune cell infiltration, CD4+ and CD8+ cells were significantly increased in patients without complications compared with those with complications (P = 0.02; P = 0.01). In conclusion, following BD damage during cold storage, we hypothesize that the functional regenerative capacity of biliary epithelium and enhanced local adaptive immune cell infiltration are crucial for BD recovery. Such molecular immunological BD analyses therefore could help to predict biliary complications in cases of "major" epithelial damage after cold storage.Liver Transplantation 23 1422-1432 2017 AASLD.

Association of Helicobacter pylori with hepatobiliary stone disease, a prospective case control study.

BACKGROUND: Hepatobiliary stone disease is one of the most common surgical conditions worldwide. There are multiple causative agents responsible for the formation of hepatobiliary stones, and bacterial infection is one of them. The presence of Helicobacter DNA species has been investigated in the biliary epithelium of patients with biliary diseases. However, conflicting results have been observed that may have been due to the small number of subjects studied, difficulty in obtaining a healthy control group, absence of controlling for confounding factors, or ethical and regional differences among populations. METHODS: We investigated the presence of Helicobacter pylori species by PCR of 26-kDa surface antigen specific to H. pylori in bile samples from 50 cases with hepatobiliary stones and 25 controls without hepatobiliary stones. The control group comprised of 20 patients of hydatid cyst disease of liver and 5 patients of right colonic growth. RESULT: H. pylori was present in 20 bile samples among cases and was absent in 30 bile samples among cases. Among controls, H. pylori could not be detected. A significant association of the presence of H. pylori with hepatobiliary stone disease was seen (p < 0.001). CONCLUSION: There is an association between bile infection with H. pylori and hepatobiliary stone disease.

[FORMATION OF BILIODIGESTIVE AND INTERINTESTINAL ANASTOMOSES IN ENVIRONMENT OF BILIARY PERITONITIS, USING A HIGH FREQUENCY ELECTRIC WELDING IN EXPERIMENT].

In experiment on 20 rabbits a diffuse biliary peritonitis was simulated, using intraabdominal injection of a laboratory culture of E. coli suspension and a medicinal bile. In 24 h on background of peritonitis on excluded loop of a small bowel in accordance to method of Roux, using a high frequency electric welding with the help of apparatus Patonmed EKB3-300 a one-layered everting cholecystoenteroanastomosis and enteroenteroanastomosis was formated. In a 6 mo postoperatively a connection line was not revealed from outside or from inside, the signs of stenosis were absent. In environment of a diffuse biliary peritonitis a welding technologies have permitted to form a hermetic and competent biliodigestive and interintestinal anastomoses, the processes of a welding suture regeneration postoperatively have a typical course. Using a high frequency-electric welding it is possible to perform a one-staged reconstructive interventions in environment of a pronounced inflammation of tissues due to subsequent precise conjunction of mucosal sheets of connected organs, preventing the anastomotic stricture formation.

Bacteriobilia: un problema sin resolver / Bacteriobilia: a non-resolved problem

La vía biliar habitualmente es estéril, y el aislamiento de microorganismos (bacteriobilia) se ha relacionado con diversos factores, como la edad, el drenaje biliar previo a la cirugía de páncreas o la litiasis biliar. Los gramnegativos continúan siendo los microorganismos más frecuentes, especialmente Escherichia coli. Entre los grampositivos cabe destacar a Enterococcus spp. Actualmente, existe controversia acerca de si la presencia de bacteriobilia tiene impacto en la mala evolución de la enfermedad biliar o de los procedimientos quirúrgicos o en las tasas de mortalidad, con complicaciones como infecciones del sitio quirúrgico o bacteriemia. En los pacientes de mayor riesgo, como los inmunosuprimidos o en los que se practica duodenopancreatectomía, los cultivos sistemáticos de bilis, aunque no existan datos clínicos de infección, pueden ser necesarios para iniciar tratamiento antibiótico o para reducir su espectro (AU)

Bile duct is usually sterile, and the isolating of microorganisms (bacteriobilia) has been related to some factors, such as age, biliary drainage before pancreatic surgery or bile duct stones. Gramnegative strains remain the most frequent pathogens, especially Escherichia coli. Among grampositives Enterococcus spp should be mentioned. Currently, there is controversy about whether the presence of bacteriobilia has an impact on unfavorable outcome of biliary disease or surgical procedures or mortality rates, with complications such as surgical site infections or bacteremia. In high-risk patients, such as immunosuppressed or those underwent pancreaticoduodenectomy, bile duct cultures performed routinely, even if there are not clinical data of infection, could be necessary in order to start antibiotic treatment or to reduce its spectrum (AU)

Cytopathology of Hepatobiliary-Related Actinomycosis.

BACKGROUND: The clinical diagnosis of hepatobiliary-related actinomycosis can be challenging owing to its rarity and variable presentation. Moreover, actinomycotic pseudotumors may mimic malignancy and result in unnecessary surgical resection. Herein, we present the clinical and cytopathological features of 3 cases with hepatobiliary-related actinomycosis. CASES: The first patient was a 73-year-old man who presented with an exophytic liver lesion. The second patient was a 78-year-old woman who presented with a biliary stricture. The third patient was an 88-year-old woman with a right flank mass extending to the liver. The aspirate smears in these 3 cases demonstrated 'cotton ball' clusters of filamentous microorganisms and abscesses. The cell blocks of 2 of the patients showed sulfur granules with peripheral filamentous microorganisms positive with a Gram stain but negative with an acid fast stain, consistent with Actinomyces species. All patients were elderly and shared a past surgical history of laparoscopic cholecystectomy. CONCLUSION: These cases demonstrate the complementary role of cytology in the diagnosis of hepatobiliary actinomycosis. A cytologic diagnosis of actinomycosis is minimally invasive and quick. It can prompt proper culture medium selection and avoid unnecessary or extensive surgery. Based on our experience, laparoscopic cholecystectomy may be a precipitating factor for the subsequent development of hepatobiliary-related actinomycosis.

[Bacteriobilia: a non-resolved problem]. / Bacteriobilia: un problema sin resolver.

Bile duct is usually sterile, and the isolating of microorganisms (bacteriobilia) has been related to some factors, such as age, biliary drainage before pancreatic surgery or bile duct stones. Gramnegative strains remain the most frequent pathogens, especially Escherichia coli. Among grampositives Enterococcus spp should be mentioned. Currently, there is controversy about whether the presence of bacteriobilia has an impact on unfavorable outcome of biliary disease or surgical procedures or mortality rates, with complications such as surgical site infections or bacteremia. In high-risk patients, such as immunosuppressed or those underwent pancreaticoduodenectomy, bile duct cultures performed routinely, even if there are not clinical data of infection, could be necessary in order to start antibiotic treatment or to reduce its spectrum.

Roles of Sphincter of Oddi Laxity in Bile Duct Microenvironment in Patients with Cholangiolithiasis: From the Perspective of the Microbiome and Metabolome.

BACKGROUND: Bile duct microenvironment plays several key roles in cholangiolithiasis occurrence. Sphincter of Oddi laxity (SOL) is associated with cholangiolithiasis, probably due to enhanced reflux of intestinal contents that changes the microenvironment. However, the microenvironment has not been investigated comprehensively. STUDY DESIGN: Patients with cholangiolithiasis were consecutively recruited and their bile was collected intraoperatively for high-throughput experiments. Pyrosequencing of 16S ribosomal RNA gene was performed to characterize the microbiota in the bile. A liquid chromatography mass spectrometry-based method was used to profile bile composition. Clinical manifestation, microbiome, and bile composition were compared between patients with and without SOL. RESULTS: Eighteen patients with SOL and 27 patients without SOL were finally included. Patients with SOL showed more severe inflammation. Bacteria in the bile duct were overwhelmingly aerobes and facultative anaerobes. Proteobacteria and Firmicutes were the most widespread phylotypes, especially Enterobacteriaceae. Compared with those without SOL, patients with SOL possessed more varied microbiota. In the SOL group, pathobionts, such as Bilophila and Shewanella algae had richer communities, and harmless bacteria were reduced. Metabolomics analysis showed the differences in bile composition between groups were mainly distributed in lipids and bile acids. Particularly, the increased abundance of Bilophila involved in taurine metabolism was associated with reduced contents of taurine derivatives in the bile of patients with SOL. CONCLUSIONS: A bile duct microenvironment with more severe bacterial infection and stronger lithogenicity was found in patients with SOL. The findings suggest a possible mechanism of cholangiolithiasis and provide the basis for future strategies for prevention of cholangiolithiasis recurrence.

Microbiota studies in the bile duct strongly suggest a role for Helicobacter pylori in extrahepatic cholangiocarcinoma.

Biliary tract cancer or extrahepatic cholangiocarcinoma (ECCA) represents the sixth commonest cause of cancer in the gastrointestinal tract in western countries. We aimed to characterize the microbiota and its predicted associated functions in the biliary tract of ECCA and benign biliary pathology (BBP). Samples were taken from 100 patients with ECCA and 100 patients with BBP by endoscopic cholangio-pancreatography for DNA extraction. Ten patients with ECCA and ten with BBP were selected for microbiota studies using the V4-16S rRNA gene and sequenced in Illumina platform. Microbiota analyses included sample-to-sample distance metrics, ordination/clustering and prediction of functions. Presence of Nesterenkonia sp. and Helicobacter pylori cagA and vacA genes were tested in the 100 ECCA and 100 BBP samples. Phylum Proteobacteria dominated all samples (60.4% average). Ordination multicomponent analyses showed significant microbiota separation between ECCA and BBP (p 0.010). Analyses of 4002 operational taxonomic units with presence variation in at least one category probed a separation of ECCA from BBP. Among these, Nesterenkonia decreased, whereas Methylophilaceae, Fusobacterium, Prevotella, Actinomyces, Novosphingobium and H. pylori increased in ECCA. Predicted associated functions showed increased abundance of H. pylori virulence genes in ECCA. cagA and vacA genes were confirmed by PCR in ECCA and BBP samples. This is the first microbiota report in ECCA and BBP to show significant changes in microbial composition. Bacterial species unusual for human flora were found: Methylophilaceae and Nesterenkonia are reported in hypersaline soils, and Mesorhizobium is a nitrogen-fixing bacterium. Enrichment of virulence genes confirms previous studies suggesting that H. pylori might be associated with ECCA.