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PWE-068 The analysis of bile salt resistance of bacteria isolated from the human biliary tract
  1. V Tank

Abstract

Introduction The gastrointestinal tract is home to 10 trillion bacteria1 and dysbiosis within this microbiota has been linked with autoimmune diseases such as Inflammatory Bowel Disease, Rheumatoid Arthritis, type I Diabetes Mellitus and Hasimoto’s Thyroiditis2, 3. The biliary tree is intrinsically linked to the liver and the gastrointestinal tract with 70% of the livers blood supply coming directly from the gastrointestinal tract via the portal vein, resulting in continual exposure to gut bacteria and bacterial cell contents4. Bile is produced in the liver and drains into the small bowel via the biliary system to aid digestion. The pathogenesis of hepato-biliary diseases such as Primary Biliary Cirrhosis (PBC) and Primary Sclerosing Cholangitis (PSC) are thought to have an infectious trigger although studies have failed to show any significant bacteraemia in mesenteric and peripheral blood samples5. However the immune response in PBC is restricted to the epithelial cells of the intra-hepatic ducts6. It is therefore possible that bile may contain bacteria, which could trigger disease in genetically susceptible patients, especially if the bacteria are able to survive within the biliary tract and alter bile salts, which are now thought to be essential in homeostasis of the microbiota and therefore gut health7. PBC and PSC are two of the leading causes of liver death and liver transplantation7. Both conditions present with a multitude of unpleasant symptoms and currently there is no cure for either of these conditions with the exact causes remaining unclear. Conventional wisdom dictates that bile is sterile due to the anti-microbial effects of bile salts. No studies have assessed bile from patients not known to have biliary infection or biliary disease. Studying bacteria isolated from bile samples, and seeing whether they contain any resistant properties against bile salts, helps gain an improved understanding in the pathophysiology of both conditions that could then be further investigated to implement a possible treatment pathway.

Method Samples were obtained from the following three surgical interventions; liver resection, pancreatectomy or laparoscopic cholecystectomy. Samples were acquired from the gallbladder and biliary tract in both normal and diseased participants.The identity of the isolated bacteria was established by sequencing of their 16S ribosomal RNA genes. This resulted in 25 different bacterial strains being identified. The bacteria were stored as glycerols at −80ćC to maintain the integrity of each strain. They were then thawed and grown in Brain Heart Infusion (BHI) media containing 0.15% bile salt concentration in order to activate bile resistance genes, which may have been switched off during storage. The bacterial strains where then exposed to bile resilience studies and then plated into quadrants using BHI medium and were counted at time 0, 24 and 48 hours. Serial dilutions were completed ranging from 0 to 10–7. In the strains that had grown in 0.15% Bile Salts, growth in the presence of increasing concentrations of bile salts was measured using the Bio-screen C equipment.

Results In total 72 different colonies were isolated from 22 biliary samples. The majority of bacteria (49) isolated from the biliary tract were unable to replicate in the presence of bile salts. However they did survive in bile salt concentrations found within the normal biliary tract (0.15%)1,2 for up to 48 hours, albeit in reduced numbers (table 1). The remaining 23 samples, both commensal and pathogenic, were exposed to 0, 0.2, 0.5, 1, 3, 5 and 10% bile salt concentrations. As the concentrations increased growth was inhibited, with the minimal inhibitory concentration occurring between 5% and 10%. The numbers of surviving bacteria at 10% were measured at 48 hours and although no growth was detected, the majority of bacteria had survived (table 2).

Conclusion The human biliary tract has its own unique microbiota. Bacteria are able to survive and thrive within this environment. The findings from this study may have important implications in the pathogenesis of liver disease. Further work such as metabolite and protein analysis needs to be done to understand the interactions of the microbiota with the host immune system and its role

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Disclosure of Interest None Declared

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