Gallbladder microbial species and host bile acids biosynthesis linked to cholesterol gallstone comparing to pigment individuals.

Gallstones are crystalline deposits in the gallbladder that are traditionally classified as cholesterol, pigment, or mixed stones based on their composition. Microbiota and host metabolism variances among the different types of gallstones remain largely unclear. Here, the bile and gallstone microbial species spectra of 29 subjects with gallstone disease (GSD, 24 cholesterol and 5 pigment) were revealed by type IIB restriction site-associated DNA microbiome sequencing (2bRAD-M). Among them (21 subjects: 18 cholesterol and 3 pigment), plasma samples were subjected to liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics. The microbiome yielded 896 species comprising 882 bacteria, 13 fungi, and 1 archaeon. Microbial profiling revealed significant enrichment of Cutibacterium acnes and Microbacterium sp005774735 in gallstone and Agrobacterium pusense and Enterovirga sp013044135 in the bile of cholesterol GSD subjects. The metabolome revealed 2296 metabolites, in which malvidin 3-(6''-malonylglucoside), 2-Methylpropyl glucosinolate, and ergothioneine were markedly enriched in cholesterol GSD subjects. Metabolite set enrichment analysis (MSEA) demonstrated enriched bile acids biosynthesis in individuals with cholesterol GSD. Overall, the multi-omics analysis revealed that microbiota and host metabolism interaction perturbations differ depending on the disease type. Perturbed gallstone type-related microbiota may contribute to unbalanced bile acids metabolism in the gallbladder and host, representing a potential early diagnostic marker and therapeutic target for GSD.

The role of bacteria in gallstone formation.

Gallstones are a prevalent biliary system disorder that is particularly common in women. They can lead to various complications, such as biliary colic, infection, cholecystitis, and even gallbladder cancer. However, the etiology of gallstones remains incompletely understood. The significant role of bacteria in gallstone formation has been demonstrated in recent studies. Certain bacteria not only influence bile composition and the gallbladder environment but also actively participate in stone formation by producing enzymes such as ß-glucuronidase and mucus. Therefore, this review aimed to analyze the mechanisms involving the types and quantities of bacteria involved in gallstone formation, providing valuable references for understanding the etiology and clinical treatment of gallstones.

Bacteriophage Cocktail Can Effectively Control Salmonella Biofilm on Gallstone and Tooth Surfaces.

INTRODUCTION: Salmonellosis, which is typically distinguished by an immediate onset of fever, abdominal pain, diarrhea, nausea, and vomiting, is a bacterial infection caused by Salmonella. The rising incidence of antibiotic resistance in Salmonella Typhimurium is a major worldwide problem, and a better knowledge of the distribution of antibiotic resistance patterns in Salmonella Typhimurium is critical for selecting the best antibiotic for infection treatment. In this work, the efficiency of bacteriophage therapy of vegetative cells and biofilms of S. Typhimurium was investigated. METHODS: Based on their host ranges, five Bacteriophages were chosen for therapy against 22 Salmonella isolates collected from various sources. PSCs1, PSDs1, PSCs2, PSSr1, and PSMc1 phages were found to exhibit potent anti-S. Typhimurium properties. In a 96-well microplate, the efficacy of bacteriophage therapy (105-1011 PFU/mL) against S. Typhimurium biofilm formers was first tested. A bacteriophage treatment (109 PFU/mL) was subsequently applied in the laboratory for 24 hours to minimize Salmonella adhering to the surfaces of gallstones and teeth. In 96-well microplate experiments, bacteriophage treatment inhibited biofilm development and reduced biofilm by up to 63.6% (P ≤ 0.05). RESULT: When compared to controls, bacteriophages (PSCs1, PSDs1, PSCs2, PSSr1, PSMc1) demonstrated a rapid drop in the populations of S. Typhimurium biofilms generated on the surfaces of gallstones and teeth where the structure of the Salmonella bacteria in the biofilm was broken and holes were created. CONCLUSION: Clearly, this study indicated that phages might be employed to eliminate S. Typhimurium biofilms on gallstone and tooth surfaces.

Patients with Primary and Secondary Bile Duct Stones Harbor Distinct Biliary Microbial Composition and Metabolic Potential.

Introduction: Cholelithiasis has a high incidence worldwide and limited treatment options due to its poorly understood pathogenesis. Furthermore, the role of biliary microbiota in cholelithiasis remains understudied. To address these questions, we performed microbial sequencing from biliary samples from primary bile duct stone (PBDS) and secondary bile duct stone (SBDS) patients. Results: We analyzed in total 45 biliary samples, including those from cholelithiasis patients with PBDS or SBDS and people with other digestive diseases. 16S rRNA sequencing showed the bacteria family Alcaligenaceae increased in relative abundance in the lithiasis group compared with the non-lithiasis group. In addition, the PBDS group showed significantly lower bacterial diversity than SBDS, with Propionibacteriaceae, Sphingomonadaceae, and Lactobacillaceae as the most significant bacteria families decreased in relative abundance. We further performed whole metagenomic shotgun sequencing (wMGS) and found increased ability of biofilm synthesis and the ability to sense external stimuli in PBDS based on functional annotation of mapped reads. From genome-resolved analysis of the samples, we identified 36 high-quality draft bacterial genome sequences with completion ≥70% and contamination ≤10%. Most of these genomes were classified into Proteobacteria, Firmicutes, or Actinobacteria. Conclusions: Our findings indicated that there is a subtle impact on biliary microbiome from cholelithiasis while the difference is more pronounced between the PBDS and SBDS. It was revealed that the diversity of biliary microbiota in PBDS is lower, while some metabolic pathways are up-regulated, including those linked to higher incidence of different types of cancer, providing new insights for the understanding of cholelithiasis with different origin.

Circadian Rhythm Disruption Influenced Hepatic Lipid Metabolism, Gut Microbiota and Promoted Cholesterol Gallstone Formation in Mice.

Background: Hepatic lipid metabolism regulates biliary composition and influences the formation of cholesterol gallstones. The genes Hmgcr and Cyp7a1, which encode key liver enzymes, are regulated by circadian rhythm-related transcription factors. We aimed to investigate the effect of circadian rhythm disruption on hepatic cholesterol and bile acid metabolism and the incidence of cholesterol stone formation. Methods: Adult male C57BL/6J mice were fed either a lithogenic diet (LD) only during the sleep phase (time-restricted lithogenic diet feeding, TRF) or an LD ad libitum (non-time-restricted lithogenic diet feeding, nTRF) for 4 weeks. Food consumption, body mass gain, and the incidence of gallstones were assessed. Circulating metabolic parameters, lipid accumulation in the liver, the circadian expression of hepatic clock and metabolic genes, and the gut microbiota were analyzed. Results: TRF caused a dysregulation of the circadian rhythm in the mice, characterized by significant differences in the circadian expression patterns of clock-related genes. In TRF mice, the circadian rhythms in the expression of genes involved in bile acid and cholesterol metabolism were disrupted, as was the circadian rhythm of the gut microbiota. These changes were associated with high biliary cholesterol content, which promoted gallstone formation in the TRF mice. Conclusion: Disordered circadian rhythm is associated with abnormal hepatic bile acid and cholesterol metabolism in mice, which promotes gallstone formation.

The Abundance and Organization of Salmonella Extracellular Polymeric Substances in Gallbladder-Mimicking Environments and In Vivo.

Salmonella enterica serovar Typhi causes chronic infections by establishing biofilms on cholesterol gallstones. The production of extracellular polymeric substances (EPSs) is key to biofilm development, and biofilm architecture depends on which EPSs are made. The presence and spatial distribution of Salmonella EPSs produced in vitro and in vivo were investigated in Salmonella enterica serovar Typhimurium and S. Typhi biofilms by confocal microscopy. Comparisons between serovars and EPS-mutant bacteria were carried out by examining growth on cholesterol-coated surfaces, with human gallstones in ox or human bile, and in mice with gallstones. On cholesterol-coated surfaces, no major differences in EPS biomass were found between serovars. Cocultured biofilms containing wild-type (WT) and EPS-mutant bacteria demonstrated WT compensation for EPS mutations. Analysis of biofilm EPSs from gallbladder-mimicking conditions found that culture in human bile more consistently replicated the relative abundance and spatial organization of each EPS on gallstones from the chronic mouse model than culture in ox bile. S. Typhimurium biofilms cultured in vitro on gallstones in ox bile exhibited colocalized pairings of curli fimbriae/lipopolysaccharide and O-antigen capsule/cellulose, while these associations were not present in S. Typhi biofilms or in mouse gallstone biofilms. In general, the inclusion of human bile with gallstones in vitro replicated biofilm development on gallstones in vivo, demonstrating the strength of this model for studying biofilm parameters or EPS-directed therapeutic treatments.

Characterization of biliary microbiota dysbiosis in extrahepatic cholangiocarcinoma.

Extrahepatic cholangiocarcinoma (CCA) accounts for 3% of digestive cancers. The role of biliary microbiota as an environment-related modulator has been scarcely investigated in CCA, and the putative impact of associated diseases has not been yet assessed. We characterized the biliary microbiota in CCA patients in order to identify a specific CCA-related dysbiosis. The biliary effluents were collected through an endoscopic retrograde pancreatic cholangiography (ERCP) examination involving 28 CCA and 47 patients with gallstones, herein considered as controls. The biliary effluents were submitted to bacterial DNA extraction and 16S rRNA sequencing, using Illumina technology. Overall, 32% of CCA and 22% of controls displayed another associated disease, such as diabetes, pancreatitis, inflammatory bowel disease, or primary sclerosing cholangitis. Such associated diseases were considered in the comparisons that were made. Principal coordinate analysis (PCoA) detected a significant disparity of biliary microbiota composition between CCA patients and controls without an associated disease. Amongst the most abundant phyla, Proteobacteria did not significantly differ between CCA patients and controls, whereas Firmicutes levels were lower and Bacteroidetes higher in CCAs' biliary microbiota than in the controls' microbiota. The most abundant genera were Enterococcus, Streptococcus, Bacteroides, Klebsiella, and Pyramidobacter in CCA's biliary microbiota. Additionally, levels of Bacteroides, Geobacillus, Meiothermus, and Anoxybacillus genera were significantly higher in CCA patients' biliary microbiota, without an associated disease, in comparison with controls. A specific CCA-related dysbiosis was identified as compared to controls independently from associated diseases. This suggests that a microorganism community may be involved in CCA pathogenesis.

Oral Administration of Nanoiron Sulfide Supernatant for the Treatment of Gallbladder Stones with Chronic Cholecystitis.

Cholelithiasis with chronic cholecystitis is prevalent and threatens human health. Most cholecystitis caused by bacterial infection or biofilms is accompanied by gallstones in the clinic, making gallbladder removal the only effective solution. Here, we provide a strategy to eliminate gallstone biofilms and dissolve gallstones by oral administration of a supernatant derived from nanoscale iron sulfide (nFeS supernatant). First, by using gallstones obtained from the clinic, we simulated biofilm formation on gallstones and tested the antibacterial activity of a nFeS supernatant in vitro. We found that the supernatant kills bacteria with a 5-log reduction in viability and destroys the biofilm structure. Smashed gallstones coincubated with E. coli biofilms promote gallstone formation, while nFeS supernatant can inhibit this process. Second, by using a murine (C57BL/6) model of cholelithiasis and cholecystitis, we tested the antibacterial efficacy and therapeutic effects of nFeS supernatant on cholelithiasis in vivo. Animal experimental data show that oral administration of nFeS supernatant can reduce 60% of bacteria in the gallbladder and, remarkably, remove gallstones with 2 days of treatment compared with clinical drug combinations (chenodeoxycholid acid and ciprofloxacin). Third, by performing protein abundance analysis of L02 cells and mouse livers, we observed the changes in CYP7a1, HMGCR, and SCP2 expression, indicating that the nFeS supernatant can also regulate cholesterol metabolism to prevent gallstone formation. Finally, hematologic biochemistry analysis and high-throughput sequencing technology show that the nFeS supernatant possesses high biocompatibility. Therefore, our work demonstrates that the nFeS supernatant may be a potential regimen for the treatment of cholelithiasis and cholecystitis by oral administration.

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.

Gallbladder stone formation in Iraqi patients is associated with bacterial infection and HLA class II-DRB1 antigens.

BACKGROUND: Gallbladder stone is recently increased among the Iraqi society due to many risk factors such as bacterial infection and some HLA class II antigens. AIM(S): This study investigates the types of bacterial infection and HLA-DRB1 antigens' ratio that may be correlated with gallbladder stone formation. Setting and Design: The study included 45 patients and the same number of healthy individuals as a control group. Patients were with multiple gallstones. Gallstone bacterial culture was demonstrated to diagnose viable bacteria. HLA-DRB1 alleles' frequency was investigated using sequence-specific oligonucleotide probes (PCR-SSOP). RESULTS: Irrespective of gallstone type and size, different types of living viable bacteria were isolated from the cores of the studied gallstones in 80% of the studied cases versus 20% of sterile gallstones. Gram-negative bacteria cultures were the dominant (89.3%), including Escherichia coli, Klebsiella spp., Proteus spp., Acinetobacter spp., and Enterobacter spp. Mixed infection of Gram-positive and negative bacteria was noted: Escherichia coli and Enterococus spp. and the others of Escherichia coli and Acitobacter spp., and Klebsiella spp. and Pseudomonas spp. Gram-positive bacteria cultures were also detected at lower rate (10.7%) including Staphylococci spp. The frequency of HLA-DRB1*03:01, HLA-DRB1*4:03, HLA-DRB1*13:22, and HLA-DRB1*15:10 alleles was significantly elevated in patients compared to the healthy control group. CONCLUSION: Results ensured the viability of the bacteria isolated from the core of gallstones and showed positive correlation between gallbladder stone and different bacterial infection. In addition, HLA-DRB1 alleles were significantly high in patients compared to healthy control group suggesting them as risk factors (P < 0.05).

In vitro analysis of gallstone formation in the presence of bacteria.

AIM: In our previous study, we have isolated different genera of bacteria from gallstones and this intrigued us to study their role in gallstone formation. The isolates exhibited certain biliary activities like urease activity, slime production, and ß-glucuronidase production. We aimed to investigate the role of these factors in the formation of gallstone in in vitro conditions at a supersaturated concentration of cholesterol. METHODOLOGY: To mimic bile in in vitro state, Brilliant Green Bile Broth (BGBB) media having a composition similar to human bile was used. Four different experimental sets were prepared, each having nine flasks with varying concentrations of cholesterol and CaCO3 (calcium carbonate). Test sets I, II, III, and IV were inoculated with Salmonella, Enterococcus, Helicobacter, and Neisseria respectively, which were isolated from gallstone itself. Out of these four bacteria, only Helicobacter did not possess slime activity. A control set was also established which was devoid of bacteria. The control also had nine flasks with different concentrations of cholesterol and CaCO3. All the sets were incubated in the incubator shaker at 37 °C and 80 revolution per minutes (RPM) for 20 days. RESULT: It was observed that the sets having bacteria had a less nucleation time as compared to the control (F = 5.274; p < 0.001). Solidification of gallstone was observed only in the set with bacteria having slime activity (sets I, II, and IV). CONCLUSION: The slime activity of bacteria leads to solidification of gallstones, whereas the other activities accelerate the nucleation of gallstone formation enhancing the severity of the disease.

Intestinal flora imbalance affects bile acid metabolism and is associated with gallstone formation.

BACKGROUND: The gut microbiota participates in the metabolism of substances and energy, promotes the development and maturation of the immune system, forms the mucosal barrier, and protects the host from pathogen attacks. Although the pathogenesis of cholesterol gallstones is still not clear, studies have suggested that gut microbiota dysbiosis plays an important role in their formation. METHODS: Microbial DNA from faeces of normal control patients and those of patients with calculi was subjected to 16S rRNA gene sequencing to detect gene expression changes in intestinal microbes. ELISA kits were used to measure free bile acids, secondary bile acids and coprostanol according to the manufacturer's instructions. The relationship between flora and their metabolites was then analysed. RESULTS: In the gallstone group, the diversity of intestinal bacteria and the abundances of certain phylogroups were significantly decreased (p < 0.05), especially Firmicutes (p < 0.05), the largest phylum represented by the gut microbiota. This study found an increase in free bile acids (p < 0.001) and secondary bile acids (p < 0.01) in the enterohepatic circulation. Bile salt hydrolase activity was not related to the abundances of BSH-active bacteria. 7a-dehydroxylating gut bacteria were significantly increased (p < 0.01), whereas cholesterol-lowering bacteria were significantly reduced (p < 0.05). The Ruminococcus gnavus group could be used as a biomarker to distinguish the gallstone group from the control group. CONCLUSION: We conclude that intestinal flora imbalance affects bile acid and cholesterol metabolism and is associated with gallstone formation.

A Preliminary Study of Biliary Microbiota in Patients with Bile Duct Stones or Distal Cholangiocarcinoma.

BACKGROUND AND OBJECTIVE: The distal cholangiocarcinoma (dCCA) is associated with many factors: genes, environment, infection, etc. The current changes in biliary flora are thought to be involved in the formation of many gastrointestinal tract (GIT) diseases, like colon adenocarcinoma. Therefore we want to investigate whether the dCCA has a certain correlation with biliary microecology, and to detect specific strains. METHODS: A total of 68 adults were enrolled, of whom 8 with dCCA, 16 with recurrent choledocholithiasis, and 44 with the onset of common bile duct stones. Endoscopic Retrograde Cholangiopancretography (ERCP) was utilized to collect bile samples for DNA extraction and 16S rRNA gene sequencing, followed by analysis of bile microbiota composition. RESULTS: First, Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria are the most dominant phyla in the bile of patients with dCCA and the onset of common bile duct stoes. Secondly, compared with the onset of common bile duct stones patients, we got a significant increase in the phylum Gemmatimonadetes, Nitrospirae, Chloroflexi, Latescibacteria, and Planctomycetes in dCCA patients. Finally, at the genus level, we obtained sequencing results of 252 bacterial genera from patients with dCCA, recurrent choledocholithiasis, and the new onset of common bile duct stones, revealing heterogeneity among individuals. CONCLUSION: To the best of our knowledge, this is the first study of the dysbiosis of bile flora in patients with dCCA. This micro-ecological disorder may be a decisive factor in the formation of dCCA. At the same time, for the first time, this study provides a test chart of biliary microbial populations that may be associated with recurrent choledocholithiasis. The compositional changes of the core microbial group of the biliary tract have potentially important biological and medical significance for the microbiological biliary disorders of dCCA.

Non-typhoidal salmonellosis presenting as acute calculus cholecystitis.

Non-typhoidal Salmonella spp.are Gram-negative bacilli, which typically cause a clinical picture of gastroenteritis and, less commonly, patients may become a chronic carrier of the pathogen within their gallbladder. We describe a rare clinical presentation of a non-typhoidal Salmonella spp. infection as acute calculus cholecystitis in an adult patient. Salmonella enterica subsp. Salamae (ST P4271) was grown from cholecystostomy fluid, and the patient subsequently underwent a laparoscopic cholecystectomy that demonstrated a necrotic gallbladder fundus. We advise that microbiological sampling of bile is essential, especially in the context of foreign travel, to detect unusual pathogens as in this case or common pathogens that may have unusual antimicrobial resistance. Given the necrotic gallbladder as in this case, we also advise that early cholecystectomy should be strongly considered in these patients.

Pathoadaptive Alteration of Salmonella Biofilm Formation in Response to the Gallbladder Environment.

Typhoid fever, a human-specific disease, is primarily caused by the pathogen Salmonella enterica serovar Typhi. It is estimated that 3 to 5% of people infected with typhoid fever become chronic carriers. Studies have demonstrated that a mechanism of chronic carriage involves biofilm formation on gallstone surfaces. In the course of a previous study using a chronic carriage mouse model, a Salmonella enterica serovar Typhimurium isolate was recovered from a mouse gallstone that exhibited a 2-fold increase in biofilm formation over the wild type. In order to identify the gene(s) responsible for the phenotype, the genomic sequences of this isolate and others were determined and compared. These sequences identified single nucleotide polymorphisms (SNPs) in 14 genes. Mutations in the most promising candidates, envZ and rcsB, were created, but neither showed increased biofilm-forming ability separately or in combination. The hyperbiofilm isolate did, however, present variations in cellular appendages observable using different techniques and a preferential binding to cholesterol. The isolate was also examined for systemic virulence and the ability to colonize the gallbladder/gallstones in a mouse model of chronic infection, demonstrating a systemic virulence defect and decreased gallbladder/gallstone colonization. Finally, to determine if the appearance of hyperbiofilm isolates could be replicated in vitro and if this was a common event, wild-type Salmonella spp. were grown long term in vitro under gallbladder-mimicking conditions, resulting in a high proportion of isolates that replicated the hyperbiofilm phenotype of the original isolate. Thus, Salmonella spp. acquire random mutations under the gallbladder/gallbladder-simulating conditions that may aid persistence but negatively affect systemic virulence.IMPORTANCE Chronic carriers are the main reservoirs for the spread of typhoid fever in regions of endemicity. Salmonella Typhi forms biofilms on gallstones in order to persist. A strain with enhanced biofilm-forming ability was recovered after a nine-month chronic-carriage mouse study. After sequencing this strain and recreating some of the mutations, we could not duplicate the phenotype. The isolate did show a difference in flagella, a preference to bind to cholesterol, and a systemic virulence defect. Finally, gallbladder conditions were simulated in vitro After 60 days, there was a 4.5-fold increase in hyperbiofilm isolates when a gallstone was present. These results indicate that Salmonella spp. can undergo genetic changes that improve persistence in gallbladder albeit at the cost of decreased virulence.

Metagenomics of pigmented and cholesterol gallstones: the putative role of bacteria.

There is growing evidence for bacteria playing a role in the pathogenesis and formation of pigmented gallstones from humans. These studies mainly involved cultivation of gallstone-associated bacteria and 16S rRNA profiling, providing an indirect link between processes involved in gallstone formation by the bacteria in-situ. Here, we provide functional metagenomic evidence of a range of genes involved in bile stress response, biofilm formation, and anaerobic energy metabolism by Gram-negative Klebsiella in pigmented gallstones from a 76-year-old male patient. Klebsiella was also present in one cholesterol-type stone in a 30-year-old female patient who had additional cholesterol gallstones characterised by Gram-positive bacteria. Pigmented stones further revealed a predominance of genes involved in carbohydrate metabolism, whilst cholesterol stones indicated a profile dominanted by protein metabolism possibly reflecting known chemical differences between Gram-negative and Gram-positive biofilm matrices. Archaeal genes were not detected. Complementary carbon and hydrogen isotopic analyses of cholesterol within the patients' stones revealed homogeneity, suggesting a common diet or cholesterol biosynthesis pathway that has little influence on microbial composition. This pilot study provides a framework to study microbial processes that play a potential role in gallstone formation across markedly different types of stones and patient backgrounds.

The characteristics of small intestinal bacterial overgrowth in patients with gallstone diseases.

BACKGROUND AND AIM: Small intestinal bacterial overgrowth (SIBO) might be prevalent in gallstone disease, including cases involving cholecystectomy and gallstones. The study aimed to investigate the prevalence and characteristics of SIBO in patients with gallstone disease. METHODS: This prospective study evaluated 265 patients for gallstone disease (200, gallstones; 65, cholecystectomy) and 39 healthy controls. Laboratory data, abdominal ultrasonography, and glucose breath test (GBT) with bowel symptom questionnaire were performed. RESULTS: Glucose breath test positivity (+) in patients with gallstone disease (36.6%) was significantly higher than that in controls (20.5%). GBT+ in the gallstone group (40.5%) was significantly higher than that in the control or cholecystectomy group (24.6%). The number of patients with gallstone, tend to be higher in the GBT (H2 )+, (CH4 )+, and (mixed)+ groups (56 [28.0%], 11 [5.5%], and 14 [7.00%]), respectively. Gallbladder disease was independently associated with fatty liver, metabolic syndrome, and SIBO. Of 97 GBT+ patients, 70 (72.1%), 12 (12.4%), and 15 (15.5%) were in the GBT (H2 )+, (CH4 )+, and (mixed)+ groups, respectively. GBT (CH4 )+ or GBT (mixed)+ were significantly associated with the gallstone group compared with the cholecystectomy group. The GBT (mixed)+ group had higher total symptom scores than the GBT- group for hard stool and urgency tendency, or the GBT (H2 )+ group in hard stool and loose stool tendency. Gallstone was the only independent factor for SIBO in patients with gallstone diseases. CONCLUSIONS: Small intestinal bacterial overgrowth is common among patients with gallstone. Especially, CH4 or mixed-type SIBO seems to be prevalent and to worsen intestinal symptoms.

Biofilm Formation Protects Salmonella from the Antibiotic Ciprofloxacin In Vitro and In Vivo in the Mouse Model of chronic Carriage.

Typhoid fever is caused by the human-restricted pathogen Salmonella enterica sv. Typhi. Approximately 5% of people that resolve the disease become chronic carriers, with the gallbladder as the main reservoir of the bacteria. Of these, about 90% present with gallstones, on which Salmonella form biofilms. Because S. Typhi is a human-restricted pathogen, these carriers are the main source of dissemination of the disease; unfortunately, antibiotic treatment has shown to be an ineffective therapy. This is believed to be caused by the inherent antibiotic resistance conferred by Salmonella biofilms growing on gallstones. The gallstone mouse model with S. Typhimurium has proven to be an excellent surrogate for S. Typhi chronic infection. In this study, we test the hypothesis that the biofilm state confers Salmonella with the increased resistance to antibiotics observed in cases of chronic carriage. We found that, in the biofilm state, Salmonella is significantly more resistant to ciprofloxacin, a common antibiotic used for the treatment of Salmonella, both in vitro (p < 0.001 for both S. Typhi and S. Typhimurium with respect to planktonic cells) and in vivo (p = 0.0035 with respect to control mice).

In vitro Salmonella typhi biofilm formation on gallstones and its disruption by Manuka honey.

Biofilm is a complex community of single or different types of microorganisms (bacteria, viruses, fungi, protozoa) attached to a surface and stick to each other through production of extracellular matrix. Salmonella typhi forms biofilm on cholesterol gallstones resulting in carrier state. Once formed, biofilm is difficult to treat. To date cholecystectomy is the only cure for this condition. Manuka honey is known to have tremendous antibiofilm activity against various organisms. S. typhi biofilm was grown in vitro on clinical samples of human cholesterol gallstones by Gallstone tube assay method for 12 days. Biofilm mass was quantified on day 1, 5, 7, 9 and 12 by crystal violet assay and was also examined by scanning electron microscope. Three concentrations w/v of Manuka honey (40%, 60% and 80%) were used, each one at 24, 48 and 72 hours. The most effective concentration (80% w/v) was repeated on two sets of gallstones. Biofilm mass was re quantified by crystal violet assay and was examined by scanning electron microscope. S. typhi formed uniform biofilm on cholesterol gallstone surface. The optical density measurements exhibited a rising pattern with time thereby indicating an increase in biofilm mass. It was 0.2 on day 1 and 0.9 on day 12. With 80% w/v Manuka honey, biofilm mass decreased most effectively with 0.5 OD after 72 hours. Biofilm formation by S, typhi on gallstones is surface specific and bile dependant. Either increasing the duration (beyond 72 hours) of the effective concentration (80% w/v) of honey or increasing the concentration (above 80%) of honey for a specific duration (72 hour) may cause complete disruption of the S. typhi biofilm on gallstone. S. typhi forms biofilm on cholesterol gallstones surface in vitro and it can be visualized by scanning electron microscopy. Biofilm mass can be quantified using crystal violet assay. Among various concentrations 80% Manuka honey for 72 hours is most effective in disrupting S. typhi biofilm on gallstones in vitro as evident from crystal violet assay.