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

BACKGROUND: Patients 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. METHODS: Patients 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. RESULTS: PDAC 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. CONCLUSION: PDAC 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.

Endoscopic retrograde cholangiopancreatography-obtained bile culture in acute cholangitis: retrospective analysis of bile cultures and risk factors in a tertiary care center.

BACKGROUND: Collection of bile aspirate during endoscopic retrograde cholangiopancreatography (ERCP) is essential to identify pathogens responsible for acute cholangitis. Limited data are available on the risk factors for the presence of multidrug-resistant organisms (MDRO) in bile. METHODS: We conducted this retrospective, single-center study to assess the prevalence and susceptibility rates of bacteria in bile cultures, and the risk factors for the presence of pathogens, MDRO, and fungi in bile. All consecutive patients who underwent biliary drainage for acute cholangitis from January 2017 to December 2019 were included. RESULTS: 443/1610 ERCPs were performed for acute cholangitis. Bile culture was collected in 91.4% (405/443), of which 86.7% were positive. Most common isolates were Enterococcus faecalis (37.6%) and Escherichia coli (32.8%). Vancomycin resistance was found in 9.9% of Enterococcus species (spp.); extended-spectrum beta-lactamases (ESBL) and carbapenemases in 11.2% and 0.9% of Enterobacteriaceae, respectively. The empiric antimicrobial therapy was changed in 26.4% (n = 107) of cases, with a clinical response in 90.7%. In multivariate analysis, biliary stenting was an independent risk factor for positive bile culture (odds ratio [OR] 9.43; P < 0.01). Independent risk factors for MDRO in bile were patient age>60 years (OR 2.51; P = 0.03), previous sphincterotomy (OR 2.57; P = 0.02), and biliary stenting (OR 2.80; P < 0.01). Previous sphincterotomy was the only risk factor for isolation of fungi in bile (OR 1.61; P = 0.04). CONCLUSIONS: Our study showed an increasing prevalence of Enterococcus spp. and MDRO. Bile cultures should be routinely collected in cholangitis and in patients with repeated ERCPs to allow more efficient antimicrobial treatment.

Bile Culture May Guide Antibiotic Stewardship in Acute Bacterial Cholangitis.

BACKGROUND: Bile cultures are often sent with blood cultures in patients with acute bacterial cholangitis. AIMS: To assess the yield of blood and bile cultures in patients with cholangitis and the clinical utility of bile cultures in guiding therapy. METHODS: All patients diagnosed with cholangitis, based on the Tokyo 2013/2018 guidelines were recruited retrospectively over ten years. The clinical and investigation details were recorded. The results of bile and blood cultures including antibiotic sensitivity patterns were noted. The concordance of microorganisms grown in blood and bile cultures and their sensitivity pattern were assessed. RESULTS: A total of 1063 patients with cholangitis were included. Their mean age was 52.7 ± 14 years and 65.4% were males. Blood cultures were positive in 372 (35%) patients. Bile culture was performed in 384 patients with 84.4% being positive, which was significantly higher than the yield of blood culture (p < 0.001). Polymicrobial growth was more in bile (59.3%) than in blood cultures (13.5%, p < 0.001). E.coli, Klebsiella, Enterococcus and Pseudomonas were the four most common organisms isolated from both blood and bile. Extended spectrum betalactamase producing organisms were isolated in 57.7% and 58.8% of positive blood and bile cultures, respectively. Among 127 patients with both blood and bile cultures positive, complete or partial concordance of organisms was noted in about 90%. CONCLUSION: Bile and blood cultures have a similar microbial profile in most patients with cholangitis. As bile cultures have a significantly higher yield than blood cultures, they could effectively guide antimicrobial therapy, especially in those with negative blood cultures.

Comparison of Cefazolin and Ceftriaxone as Antimicrobial Prophylaxis in Pancreatoduodenectomy with Preoperative Drainage: Incidence of Surgical Site Infection and Susceptibility of Bacteria in Bile.

BACKGROUND: The optimal perioperative antimicrobial agent for preventing surgical site infection (SSI) in pancreatoduodenectomy (PD) with preoperative biliary drainage (PBD) remains unclear. METHODS: We retrospectively reviewed 288 patients who underwent PD after PBD between 2010 and 2020 at our institution. Patients were classified into two groups according to the perioperative antimicrobial agent used (cefazoline [CEZ] group [n = 108] and ceftriaxone [CTRX] group [n = 180]). The incidence of SSI, type of bacteria in intraoperative bile culture (IBC), and antimicrobial susceptibility to prophylactic antimicrobial agents were analyzed. RESULTS: The incidence of incisional SSI was significantly lower in the CTRX group than in the CEZ group (18% vs. 31%, P = 0.021), whereas the incidence of organ/space SSI in the two groups did not differ to a statistically significant extent (35% vs. 44%, P = 0.133). Gram-negative rod (GNR) bacteria in the IBC showed better antimicrobial susceptibility in the CTRX group than in the CEZ group. In multivariate analysis, antimicrobial resistance due to GNR was a significant risk factor for incisional SSI (odds ratio, 3.50; P < 0.001). CONCLUSIONS: CTRX had better antimicrobial coverage than CEZ for GNR cultured from intraoperative bile samples. In addition, CTRX provides better antimicrobial prophylaxis than CEZ against superficial SSI in patients with PD after PBD. TRIAL REGISTRATION NUMBER: This study was not a clinical trial and had no registration numbers.

Suggested use of empirical antibiotics in acute cholecystitis based on bile microbiology and antibiotic susceptibility.

BACKGROUND: Bacterial infection is common in acute cholecystitis (AC). To identify appropriate empirical antibiotics, we investigated AC-associated microorganisms and their susceptibilities to antibiotics. We also compared preoperative clinical findings of patients grouped according to specific microorganisms. METHODS: Patients who underwent laparoscopic cholecystectomy for AC between 2018 and 2019 were enrolled. Bile cultures and antibiotic susceptibility tests were performed, and clinical findings of patients were noted. RESULTS: A total of 282 patients were enrolled (147 culture-positive and 135 culture-negative). The most frequent microorganisms were Escherichia (n = 53, 32.7%), Enterococcus (n = 37, 22.8%), Klebsiella (n = 28, 17.3%), and Enterobacter (n = 18, 11.1%). For Gram-negative microorganisms, second-generation cephalosporin (cefotetan: 96.2%) was more effective than third-generation cephalosporin (cefotaxime: 69.8%). Vancomycin and teicoplanin (83.8%) were the most effective antibiotics for Enterococcus. Patients with Enterococcus had higher rates of CBD stones (51.4%, p = 0.001) and biliary drainage (81.1%, p = 0.002), as well as higher levels of liver enzymes, than patients with other microorganisms. Patients with ESBL-producing bacteria had higher rates of CBD stones (36.0% vs. 6.8%, p = 0.001) and biliary drainage (64.0% vs. 32.4%, p = 0.005) than those without. DISCUSSION: Preoperative clinical findings of AC are related to microorganisms in bile samples. Periodic antibiotic susceptibility tests should be conducted to select appropriate empirical antibiotics.

Antibiotic prophylaxis for acute cholecystectomy: PEANUTS II multicentre randomized non-inferiority clinical trial.

BACKGROUND: Guidelines recommending antibiotic prophylaxis at emergency cholecystectomy for cholecystitis were based on low-quality evidence. The aim of this trial was to demonstrate that omitting antibiotics is not inferior to their prophylactic use. METHODS: This multicentre, randomized, open-label, non-inferiority clinical trial randomly assigned adults with mild-to-moderate acute calculous cholecystitis (immediate cholecystectomy indicated) to 2 g cefazolin administered before incision or no antibiotic prophylaxis. The primary endpoint was a composite of all postoperative infectious complications in the first 30 days after surgery. Secondary endpoints included all individual components of the primary endpoint, other morbidity, and duration of hospital stay. RESULTS: Sixteen of 226 patients (7.1 per cent) in the single-dose prophylaxis group and 29 of 231 (12.6 per cent) in the no-prophylaxis group developed postoperative infectious complications (absolute difference 5.5 (95 per cent c.i. -0.4 to 11.3) per cent). With a non-inferiority margin of 10 per cent, non-inferiority of no prophylaxis was not proven. The number of surgical-site infections was significantly higher in the no-prophylaxis group (5.3 versus 12.1 per cent; P = 0.010). No differences were observed in the number of other complications, or duration of hospital stay. CONCLUSION: Omitting antibiotic prophylaxis is not recommended.

Microbiology of bile aspirates obtained at ERCP in patients with suspected acute cholangitis.

BACKGROUND: The cornerstone of treatment for acute cholangitis is source control with biliary drainage and early antibiotics. The primary aim of this study was to describe the microbiology of bile aspirate pathogens obtained at the time of endoscopic retrograde cholangiopancreatography (ERCP) in patients suspected of having acute cholangitis. METHODS: In this single-center retrospective study, patients were included if a bile aspirate was collected at ERCP for suspicion of acute cholangitis, from 1 January 2010 to 31 December 2016. RESULTS: There were 721 ERCP procedures for suspected acute cholangitis with bile culture results, with 662 positive bile cultures (91.8 %). Pathogens included: Enterococcus species (spp.) 448 (67.7 %); Klebsiella spp. 295 (44.6 %); Escherichia coli 269 (40.6 %); Pseudomonas spp. 52 (7.9 %); and anaerobes 64 (9.7 %). Susceptibility of Klebsiella pneumoniae and E.coli isolates to ciprofloxacin was 88 % and 64 %, respectively. Extended-spectrum beta-lactamases and carbapenem resistance were found in 7.9 % and 3.6 % of Enterobacteriaceae, respectively. There were 437 concurrent blood cultures, of which 174 were positive (39.8 % of cultures drawn). Prior biliary endoscopic sphincterotomy (ES) was evident in 459 ERCP cases (63.7 %), and was associated with increased frequency of Klebsiella spp., Pseudomonas aeruginosa, Enterobacter spp., and Enterococcus spp. Prior biliary ES significantly increased the probability of vancomycin-resistant Enterococcus (VRE). CONCLUSIONS: The vast majority of bile cultures (91.8 %) were positive. The susceptibilities of E.coli and K.pneumoniae to ciprofloxacin are lower than historically noted. A notable portion of cultures contained pathogenic drug-resistant organisms. Prior biliary ES is associated with a higher frequency of certain organisms and higher frequency of VRE.

Microorganisms isolated from the bile of the patients who have undergone cholecystectomy and their antibiotic resistance pattern: multicenter prospective study.

BACKGROUND: Gallbladder and biliary tract infections are diseases with high mortality rates if they are not treated properly. Microbiological evaluation of perioperatively collected samples both ensures proper treatment of patients and guides empirical treatment due to the determination of microorganism susceptibility. AIMS: This study aimed to isolate the microorganisms in bile cultures from patients who underwent cholecystectomy and to determine sensitivity results of these microorganisms. METHODS: This study was a multi-center and prospective design, included 360 patients, and was performed between 2019 and 2020. Culture results of bile taken during cholecystectomy were evaluated. RESULTS: Bacterial growth was found in the bile cultures of 84 out of 360 (23.3%) patients. Patients were divided into two groups according to whether they had risk factors for resistant microorganisms or not. While Escherichia coli (n = 11, 13%), Enterococcus spp. (n = 8, 9.5%), and Enterobacter spp. (n = 4, 4.7%) were detected most frequently in patients without risk. Staphylococcus spp. (n = 17, 20.2%), Enterococcus spp. (n = 16, 19%), and E. coli (n = 8, 9.5%) were the most frequently found microorganism at-risk patients. In multivariate analysis, bile culture positivity was found higher in patients who had history of biliary disease (p = 0.004), operation performed concurrently with a cholecystectomy (p = 0.035), and high rate of polymorphonuclear leukocytes (PNL) in total leukocyte count (p = 0.001). CONCLUSIONS: Our study shows that when starting empirical antibiotic treatment for bile ducts, whether patients are at risk for the development of resistant bacterial infection should be evaluated after which antibiotic selection should be made accordingly.

Antibiotic resistance patterns of bacterial bile cultures during pancreatic surgery-a single center analysis and systematic review.

BACKGROUND: Septic complications after pancreatic surgery are common. However, it remains unclear if and how a shift of the microbiological spectrum affects morbidity. The aim of the present study was to assess the microbiological spectrum and antibiotic resistance patterns and their impact on outcome. METHODS: We conducted a retrospective study including patients undergoing pancreatic surgery at our center between 2005 and 2018. A systematic literature review and descriptive meta-analysis of the published and original data was performed according to the PRISMA guidelines. RESULTS: A total of 318 patients were included in the analysis. Patients with biliary drainage had a significantly higher incidence of bacterobilia (93% vs. 25%) and received preoperative antibiotics (46% vs. 12%). The analyzed bile cultures showed no resistance to piperacillin/tazobactam, fluoroquinolones, or carbapenems. Resistance to cefuroxime was seen in 58% of the samples of patients without biliary drainage (NBD) and 93% of the samples of those with drainage (BD). In general, there was no significant difference in overall postoperative morbidity. However, superficial surgical site infections (SSIs) were significantly more common in the BD group. We included a total of six studies and our own data (1627 patients) in the descriptive meta-analysis. The percentage of positive bile cultures ranged from 53 to 81%. In patients with BD, the most frequent microorganisms were Enterococcus spp. (58%), Klebsiella spp. (29%), and E. coli (27%). Almost all studies demonstrated resistance to first- and second-generation cephalosporins and to third- and fourth-generation cephalosporins for patients with BD. CONCLUSION: A change in perioperative antibiotic strategy according to local resistance patterns, especially after BD, might be useful for patients undergoing pancreatic surgery. Appropriate perioperative antibiotic coverage may help to prevent abdominal infectious complications and especially superficial SSIs.

The association between bacterobilia and the risk of postoperative complications following pancreaticoduodenectomy.

BACKGROUND: Pre-operative biliary bacterial colonisation (bacterobilia) is considered a risk factor for infectious complications after pancreaticoduodenectomy (PD). This study aimed to investigate the role of the PD biliary microbiome grown in the development of post-PD complications. METHODS: In a retrospective study of 162 consecutive patients undergoing PD (2008-2018), intra-operative bile cultures were analyzed and sensitivities compared to pre-anesthetic antibiotics and thirty-day post-surgery complications. RESULTS: Bacterobilia was present in 136 patients (84%). Most bile cultures grew bacteria resistant to pre-operative antibiotics (n = 112, 82%). Patients with bacterobilia had significantly higher rates of major complication than patients without (P = 0.017), as well as higher rates of surgical-site infections (SSI) (P = 0.010). Patients with negative bile cultures (n = 26) had significantly lower rates of major complication and SSI than those growing sensitive (n = 24) or non-sensitive (n = 112) bacteria (major complication P = 0.029 and SSI P = 0.011). CONCLUSION: Positive bile cultures were associated with a higher incidence of major complications and SSI. Patients with sterile bile cultures had the lowest risk of post-operative complications and efforts to reduce rates of bacterobilia, such as limitation of biliary instrumentation, should be considered. Sensitivity to antibiotics had no effect upon the rate of post-operative complications, but this may reflect low cohort numbers.

Impact of Qualitative and Quantitative Biliary Contamination Status on the Incidence of Postoperative Infection Complications in Patients Undergoing Pancreatoduodenectomy.

BACKGROUND: Bacterial contamination status may differ under different biliary drainage conditions. The purpose of this study was to determine the impact of qualitative and quantitative biliary bacterial contamination on the incidence of infection complications in patients undergoing pancreatoduodenectomy. METHODS: Patients undergoing pancreatoduodenectomy for periampullary diseases with different biliary drainage conditions, such as external drainage (ED), internal drainage (ID), and no drainage (ND), were included. Bile was collected intraoperatively, and biliary contamination status was qualified and quantified using bacterium-specific ribosomal RNA-targeted reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The impact of biliary contamination status on infection complications was analyzed. RESULTS: A total of 152 patients were included (38 with ED, 40 with ID, and 74 with ND). The positive rate of microorganisms in bile was higher in the ID group (98%) compared with the ED group (82%, p = 0.021) and the ND group (65%, p < 0.001). The number of microorganisms detected in bile samples was higher in the ID group compared with the ED group (median 489,788 vs. 5375 bacteria/mL of bile, p < 0.001). With multivariate analysis, soft pancreas, intraoperative bleeding (> 600 mL), and biliary contamination by Atopobium cluster were identified as independent risk factors for infection complications. Biliary contamination by Atopobium cluster was significantly higher in the ID group compared with the other groups. CONCLUSIONS: Biliary bacterial contamination is more frequently induced by ID than either ED or ND. In addition to the previously known risk factors, biliary contamination with Atopobium cluster may be one of the risk factors of infection complications following pancreatoduodenectomy.

Influence of Klebsiella pneumoniae and quinolone treatment on prognosis in patients with pancreatic cancer.

BACKGROUND: An increasing body of evidence suggests that microbiota may promote progression of pancreatic ductal adenocarcinoma (PDAC). It was hypothesized that gammaproteobacteria (such as Klebsiella pneumoniae) influence survival in PDAC, and that quinolone treatment may attenuate this effect. METHODS: This was a retrospective study of patients from the Massachusetts General Hospital (USA) and Ludwig-Maximilians-University (Germany) who underwent preoperative treatment and pancreatoduodenectomy for locally advanced or borderline resectable PDAC between January 2007 and December 2017, and for whom a bile culture was available. Associations between tumour characteristics, survival data, antibiotic use and results of intraoperative bile cultures were investigated. Survival was analysed using Kaplan-Meier curves and Cox regression analysis. RESULTS: Analysis of a total of 211 patients revealed that an increasing number of pathogen species found in intraoperative bile cultures was associated with a decrease in progression-free survival (PFS) (-1·9 (95 per cent c.i. -3·3 to -0·5) months per species; P = 0·009). Adjuvant treatment with gemcitabine improved PFS in patients who were negative for K. pneumoniae (26·2 versus 15·3 months; P = 0·039), but not in those who tested positive (19·5 versus 13·2 months; P = 0·137). Quinolone treatment was associated with improved median overall survival (OS) independent of K. pneumoniae status (48·8 versus 26·2 months; P = 0·006) and among those who tested positive for K. pneumoniae (median not reached versus 18·8 months; P = 0·028). Patients with quinolone-resistant K. pneumoniae had shorter PFS than those with quinolone-sensitive K. pneumoniae (9·1 versus 18·8 months; P = 0·001). CONCLUSION: K. pneumoniae may promote chemoresistance to adjuvant gemcitabine, and quinolone treatment is associated with improved survival.

Ruminococcoides bili gen. nov., sp. nov., a bile-resistant bacterium from human bile with autolytic behavior.

A strictly anaerobic, resistant starch-degrading, bile-tolerant, autolytic strain, IPLA60002T, belonging to the family Ruminococcaceae, was isolated from a human bile sample of a liver donor without hepatobiliary disease. Cells were Gram-stain-positive cocci, and 16S rRNA gene and whole genome analyses showed that Ruminococcus bromii was the phylogenetically closest related species to the novel strain IPLA60002T, though with average nucleotide identity values below 90 %. Biochemically, the new isolate has metabolic features similar to those described previously for gut R. bromii strains, including the ability to degrade a range of different starches. The new isolate, however, produces lactate and shows distinct resistance to the presence of bile salts. Additionally, the novel bile isolate displays an autolytic phenotype after growing in different media. Strain IPLA60002T is phylogenetically distinct from other species within the genus Ruminococcus. Therefore, we propose on the basis of phylogenetic, genomic and metabolic data that the novel IPLA60002T strain isolated from human bile be given the name Ruminococcoides bili gen. nov., sp. nov., within the new proposed genus Ruminococcoides and the family Ruminococcaceae. Strain IPLA60002T (=DSM 110008T=LMG 31505T) is proposed as the type strain of Ruminococcoides bili.

Proton Pump Inhibitors Increase the Risk of Early Biliary Infection After Placement of Percutaneous Transhepatic Biliary Stents.

PURPOSE: To investigate the association between the use of proton pump inhibitors (PPIs) and the risk of early biliary infection (EBI) after the placement of percutaneous transhepatic biliary stents (PTBS) in patients with unresectable malignant biliary obstruction (MBO). MATERIALS AND METHODS: A total of 136 patients with unresectable MBO (82 males and 54 females) treated with PTBS were included in this multicenter retrospective study. PPIs were prescribed to MBO patients with dyspepsia. The risk factors for EBI were identified by univariate and multivariate analyses. The association between the use of PPIs and EBI was assessed by logistic analyses. RESULTS: A total of 72 (53%) patients were regular users of PPIs, and 33 (24%) patients developed EBI after PTBS. Univariate and multivariate analyses revealed that diabetes (hazard ratio [HR], 20.3; 95% confidence interval [CI], 5.6-72.9; P <.001), biliary stones (HR, 20.3; 95% CI, 5.6-72.9; P <.001) and PPIs (HR, 4.0; 95% CI, 1.2-12.8; P =.020) were risk factors for EBI. Further analyses of the correlation between the duration of PPIs use and EBI demonstrated that a prolonged use of PPIs significantly increased the risk of EBI (PPIs for <15 days vs 15-30 days: HR, 10.2; 95% CI, 3.1-33.3; P <.001; and PPIs <15 days vs ≥30 days; HR, 20.4; 95% CI, 2.2-192.3; P <.001). CONCLUSION: The use of PPIs increased the risk of EBI after PTBS in patients with unresectable MBO. Furthermore, the risk of EBI increased with a prolonged duration of PPIs use.

Prevalence, Resistance Rates, and Risk Factors of Pathogens in Routine Bile Cultures Obtained during Endoscopic Retrograde Cholangiography.

INTRODUCTION AND OBJECTIVE: Acute cholangitis is a life-threatening condition. The early initiation of antibiotic therapy significantly impacts the course of disease. Only few data are available on distribution and resistance profiles of bile pathogens. Here, we report on an analysis of routinely acquired bile specimens and provide an overview of the prevalence, resistance rates, and risk factors for the presence of pathogens in bile. METHODS: Bile cultures obtained from 388 endoscopic retrograde cholangiographies (ERCs) with corresponding clinical data were analysed in 208 patients. RESULTS: The majority (84.8%) of cultures yielded positive for at least 1 organism. Abundance was highest for Enterococcus faecalis, Enterococcus faecium, and Escherichia coli. Multiresistant organisms were present in 14.9%. The initial antibiotic regimen was changed in 44.1%, which increased the length of hospital stay significantly (***p < 0.001). Pre-existing papillotomy (EPT) or biliary drainage was associated with higher frequency of bile pathogens (**p < 0.01) in a univariate analysis. Multivariate analysis confirmed these results for EPT and revealed significantly more positive results for pathogens, gram-negative bacteria, and fungi in patients with biliary drainage. Significant differences in the prevalence of pathogens were observed between relevant subgroups of ERC indications. The highest susceptibility rates were observed for linezolid and tigecycline in gram-positive bacteria and for meropenem and gentamicin in gram-negative bacteria. CONCLUSIONS: Our study provides a comprehensive analysis of the distribution, resistance profiles, and risk factors for the detection of bile pathogens. The frequent change in initial antibiotic treatment highlights the importance of routine bile culture and indicates that current schemas of empirical treatment might not cover the contemporary spectrum of pathogens in bile.

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

BACKGROUND: Cholecystitis 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. METHODS: We 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. RESULTS: No 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. CONCLUSION: We 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.

Quick detection of causative bacteria in cases of acute cholangitis and cholecystitis using a multichannel gene autoanalyzer.

PURPOSES: Acute cholangitis and cholecystitis can become severe conditions as a result of inappropriate therapeutic administration and thereafter become increasingly resistant to antimicrobial treatment. The simultaneous detection of the bacterial nucleic acid and antimicrobial resistance gene is covered by the national health insurance program in Japan for sepsis. In this study, we evaluate the use of a multichannel gene autoanalyzer (Verigene system) for the quick detection of causative bacteria in cases of acute cholangitis and cholecystitis. METHODS: This study included 108 patients diagnosed with acute cholangitis or cholecystitis between June 2015 and November 2018. A bacterial culture test and Verigene assay were used to evaluate the bile samples. RESULTS: The most commonly isolated bacteria were Escherichia coli, which includes six extended-spectrum beta-lactamase (ESBL)-producing E. coli. Among the patients with positive bile cultures, bacteria were detected in 35.7% of cases via the Verigene system. The detection rates of the Verigene system significantly increased when the number of bacterial colonies was ≥ 106 colony-forming unit (CFU)/mL (58.1%). Cases with a maximum colony quantity of ≥ 106 CFU/mL exhibited higher inflammation, suggesting the presence of a bacterial infection. CONCLUSIONS: The Verigene system might be a new method for the quick detection of causative bacteria in patients with infectious acute cholangitis and cholecystitis.

Biliary microbiota and mucin 4 impact the calcification of cholesterol gallstones.

BACKGROUND: Cholesterol gallstones account for over 80% of gallstones, and the pathogenesis of gallstone formation involves genetic and environmental factors. However, data on the evolution of cholesterol gallstones with various densities are limited. This study aimed to determine the roles of microbiota and mucins on the formation of calcified cholesterol gallstones in patients with cholelithiasis. METHODS: Paired gallbladder tissues and bile specimens were obtained from cholelithiasis patients who were categorized into the isodense group and calcified group according to the density of gallstones. The relative abundance of microbiota in gallbladder tissues was detected. Immunohistochemistry and enzyme-linked immunosorbent assay were performed to detect the expression levels of MUC1, MUC2, MUC3a, MUC3b, MUC4, MUC5ac and MUC5b in gallbladder tissues and bile. The correlation of microbiota abundance with MUC4 expression was evaluated by linear regression. RESULTS: A total of 23 patients with gallbladder stones were included. The density of gallstones in the isodense group was significantly lower than that of the calcified group (34.20 ± 1.50 vs. 109.40 ± 3.84 HU, P < 0.0001). Compared to the isodense group, the calcified group showed a higher abundance of gram-positive bacteria at the fundus, in the body and neck of gallbladder tissues. The concentrations of MUC1, MUC2, MUC3a, MUC3b, MUC5ac and MUC5b in the epithelial cells of gallbladder tissues showed no difference between the two groups, while the concentrations of MUC4 were significantly higher in the calcified group than that in the isodense group at the fundus (15.49 ± 0.69 vs. 10.23 ± 0.54 ng/mL, P < 0.05), in the body (14.54 ± 0.94 vs. 11.87 ± 0.85 ng/mL, P < 0.05) as well as in the neck (14.77 ± 1.04 vs. 10.85 ± 0.72 ng/mL, P < 0.05) of gallbladder tissues. Moreover, the abundance of bacteria was positively correlated with the expression of MUC4 (r = 0.569, P < 0.05) in the calcified group. CONCLUSIONS: This study showed the potential clinical relevance among biliary microbiota, mucins and calcified gallstones in patients with gallstones. Gram-positive microbiota and MUC4 may be positively associated with the calcification of cholesterol gallstones.

[Pathogens' distribution and changes of antimicrobial resistance in the bile of acute biliary tract infection patients].

Objective: To investigate the pathogens' distribution and antimicrobial resistance in the bile of acute biliary tract infection patients. Methods: The data of bile bacterial culture and drug sensitivity test of 223 acute biliary tract infection patients who underwent gallbladder puncture or endoscopic retrograde cholangiopancreatography drainage from July 2009 to July 2019 were analyzed retrospectively at Department of General Surgery,Xinhua Hospital,Affiliated to Shanghai Jiao Tong University School of Medicine.There were 141 males and 82 females with age of 67.3 years(range:28 to 93 years).Three to five milliliter of bile was extracted from each patient and sent to the laboratory for bacterial culture,identification and drug sensitivity test.The patients were divided into two groups according to the visiting time: the former group (n=124) was admitted from July 2009 to July 2014,and the latter group(n=99) was admitted from August 2014 to July 2019.The distribution of pathogenic bacteria and the changing trend of drug resistance rate of common bacteria in the two groups were compared.The results of drug sensitivity test were analyzed by WHONET software provided by WHO bacterial surveillance network.The drug resistance rates in different time periods were compared by χ2 test. Results: In this study,there were 147 cases of acute cholangitis and 76 cases of acute cholecystitis.A total of 376 strains of pathogenic bacteria were cultured.Among them,98 strains(26.1%) were gram-positive bacteria,269 strains(71.5%) were gram-negative bacteria and 9 strains(2.4%) were fungi.The top three gram-positive bacteria were Enterococcus faecium (49.0%,48/98),Enterococcus faecalis(20.4%,20/98),and Enterococcus luteus(7.1%,7/98).The top 5 gram-negative bacteria were Escherichia coli(33.5%,90/269),Klebsiella pneumoniae(13.8%,37/269),Pseudomonas aeruginosa(13.0%,35/269),Acinetobacter baumannii (12.6%,34/269),and Enterobacter cloacae(4.8%,13/269).From 2009 to 2019,there was no significant change in the proportion of gram-positive bacteria (former group vs. latter group: 25.3% vs. 28.2%) and gram-negative bacteria(former group vs.latter group: 74.7% vs. 71.8%) in the bile of patients with acute biliary tract infection.Gram-positive bacteria were mainly Enterococci(85.7%,84/98) and gram-negative bacteria were Escherichia coli(33.5%,90/269).Acinetobacter baumannii accounted for 7.8%(11/142) of gram-negative bacteria in the former group and 18.1%(23/127) in the latter group,an increase of 10.3% over previous five years.Pseudomonas aeruginosa had a downward trend,16.9% in the former group(24/142) and 8.7% in the latter group (11/127),the proportion decreased by 8.2%,and the other changes were not significant.The drug resistance rates of common gram-positive bacteria were relatively stable,and the drug resistance rates of Enterococcus faecium to many antibiotics were higher than those of Enterococcus faecalis.The resistance rates of gram-negative bacteria to most antibiotics showed an upward trend,among which Klebsiella pneumoniae showed an upward trend to most of antibiotics(former group: 0/15－4/13, latter group: 55.0%－70.0%; χ2=3.996－16.942, P=0.000－0.046).The drug resistance rates of Acinetobacter baumannii was generally higher,but there were no significant changes in the drug resistance rates of Acinetobacter baumannii between the two groups.The drug resistance rates of Pseudomonas aeruginosa to most antibiotics increased,and the overall drug resistance rates of Escherichia coli were stable and showed a slight upward trend. Conclusions: The main pathogens in bile of patients with acute biliary tract infection are gram-negative bacteria.The constituent ratio of various gram-negative bacteria had no significant change from 2009 to 2019,but the drug resistance rates shows an upward trend.Among the gram-negative bacteria, Escherichia coli is the most important pathogen,and the proportion has no significant change.The proportion of Acinetobacter baumannii in the former group was significantly higher than that in the former group.And the proportion of Pseudomonas aeruginosa has a decreased trend.

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.