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PWE-061 Dysbiosis in Patients with Bile Acid Diarrhoea (BAD) Demonstrated Using 16S RNA Gene Sequencing
  1. N Sagar1,
  2. G Kay2,
  3. N O’Connell3,
  4. C Nwokolo3,
  5. K Bardhan4,
  6. C Quince2,
  7. M Pallen2,
  8. R Arasaradnam4
  1. 1School of Medicine
  2. 2School of Microbiology, University of Warwick
  3. 3Gastroenterology, University Hospitals Coventry and Warwickshire
  4. 4Clinical Sciences Research Institute, University of Warwick, Coventry, UK

Abstract

Introduction The gastrointestinal tract harbours a diverse community of approximately 1014 microorganisms comprising 500 to 1000 bacterial species.1 Dysbiosis occurs when pathological imbalances in the gut bacterial community precipitate disease and has been linked to the dysmetabolism of bile acids (BAs) in the gut. 1–2% of primary BAs escape the enterohepatic circulation and undergo microbial biotransformation in the large bowel to form the secondary BAs, deoxycholic acid (DCA) and lithocholic acid (LCA).2 BA metabolites as a result of microbial transformations act as signalling molecules via the Farnesoid X receptor (FXR). Activation of the ileal FXR stimulates expression of Fibroblast Growth Factor 19 (FGF-19), which binds hepatic FGF-4 and activates JNK1/2 and ERK1/2 to impede the physiological feedback of BA synthesis and maintain a functional BA pool.3 The degree of activation of BA receptors is influenced primarily by the gut microbiota and therefore dysbiosis may result in abnormal BA modification leading to the development of gastrointestinal disease.

To enhance our understanding of gut dysbiosis in patients with BAD, 16 S rRNA gene sequencing was undertaken to investigate bacterial communities from faecal samples in patients with BAD and IBS.

Methods 35 patients (20 with BAD and 15 with IBS) were recruited from the nuclear medicine department at University Hospitals Coventry and Warwickshire NHS Trust after referral from the gastroenterology clinic for a SeHCAT scan to investigate chronic diarrhoea. The patients with BAD had either Type 1 with a diagnosis of Crohn’s disease, type 2 or type 3 post cholecystectomy. Stool samples were stored at -80oC within two hours of collection and then defrosted for 45 minutes at room temperature prior to analysis. 16 s RNA sequencing was undertaken by isolating DNA from stool samples using the QIAamp Fast DNA Stool Extraction kit. V3-V4 primers and extensor ready mix (Thermo scientific) were used to amplify the 16 s rRNA gene sequences from isolated metagenomic DNA. The eluted DNA was then quantified using the broad-range Qubit kit. DNA was analysed by electrophoresis on 1% agarose gels and purified post-PCR. The DNA was then denatured and sequenced on a MiSeq using the Illumina Miseq V2 2x250 bp paired end protocol. Bioinformatic statistical analysis was undertaken using QUIIME and the UPARSE pipeline.

Results The rarefaction plots showed reduced diversity in BAD patients compared with IBS; p = 0.0007216. This was also confirmed using the Shannon’s diversity index. The abundance of certain taxa namely Lachnospiraceae (p = 0.00134), Rumiinococcus (p = 0.0034) and Prevotella (p = 0.0046) were reduced in patients with BAD.

Conclusion We have shown that diversity in those with BAD is significantly reduced compared to those with IBS with certain phyla predominating.

References 1 Gérard P. Metabolism of Cholesterol and Bile Acids by the Gut Microbiota. Pathogens 2014;3:14–24.

2. Vlahcevic ZR, Heuman DM, Hylemon PB. Physiology and pathophysiology of enterohepatic circulation of bile acids. In D. Zakim and T. Boyer, editors. Hepatology: A Textbook of Liver Disease. 1996, 3rd edition. Vol. 1. Saunders, Philadelphia, PA. 376–417

3 Tsuei J, Chau T, Mills D, et al. Bile acid dysregulation, gut dysbiosis, and gastrointestinal cancer. Exp Biol Med (Maywood). 2014 Nov;239(11):1489–504.

Disclosure of Interest None Declared

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