Infectious agents in inflammatory bowel disease Bharadwaj R1, Walujkar S1,2, Lawate P, Shouche Y2. (1) Department of Medical Microbiology, B.J Govt Medical College, Pune, Maharashtra, India. (2) Molecular Biology Unit, National Centre for Cell Science, Ganeshkhind, Pune - 411 007 Introduction: Crohn’s disease (CD) and Ulcerative colitis (UC) are two separate clinical presentations of Inflammatory Bowel Disease (IBD). They are more commonly reported from the developed world and once thought to be uncommon in India but are now being increasingly reported. Pathogenesis of IBD is a challenge for the clinician Recent studies have shed light on the role of commensal bacteria intrinsic to the gastrointestinal tract in the pathogenesis and aetiology of IBD. The peculiar nature of dysbiosis which occurs in the microbiota of gastrointestinal tract during IBD needs to be explored further. IBD patients are more prone to concomitant C. difficile infection, which increases the morbidity and mortality associated with the disease. Objective: The principal aim of the current study was to study the role of microbes associated with the gut microbiota in the acute phase of IBD and to assess changes in this microbial flora during the remission phase i.e. in the follow-up samples. Special focus of the study was to assess the role of Clostridium difficile in acute exacerbation of IBD. FIGURE 1. PCoA PLOT showing the overall differences in distribution of OTUs FIGURE 2. Box Plots showing the relative abundance of four bacterial genus FIGURE 3. Scattered plot diagram showing the total C. difficile count as estimated by qPCR method, found under detectable levels except for two non IBD-control sample Methods: In this study, gut microbiota of twenty two patients suffering from IBD (exacerbated stage) was compared with gut microbiota of the follow-up samples from same patient (remission stage). Biopsy samples were collected after informed consent via colonoscopy, and were processed immediately upon collection for isolation of DNA. Mucosal microbiota was analyzed by means of 16S rRNA gene-based short gun clone library sequencing. 900 base pair (bp) long 16S rRNA gene sequences were generated during clone library sequencing, only good quality sequences were used for further bioinformatics and ecological analysis. Clostridium difficile co-infection during IBD was analyzed using qPCR method. FIGURE 4. Heat Map represents clustering of bacterial communities at genus level from diseased (acute stage) patients and their follow up group (remission stage) Genus representing commensal bacteria and infectious bacteria Discussion & Conclusion : Previous reports from DN Frank et.al in PNAS, C Huttenhover et.al in Genome Biology and TW Hoffmann et.al in Nature have concluded that mucosal microbiota plays a pivotal role in causation of IBD, but it has rarely been reported at genus level of bacterial classification, our results demonstrates the major bacterial communities/players involved in the exacerbation of IBD at genus level. These bacterial communities may prevail as bacterial biofilms, and single bacterial genus or species may not be involved in exacerbation of IBD. Clostridium difficile bacterium has been previously reported to be existing as a co-infection in IBD patients, in our current study we investigated whether c.difficile is involved in acute exacerbation of IBD, but could not positively correlate the results. Results: Analysis of 6437 good quality sequences demonstrated a significant reduction of bacterial diversity consistently from phylum to species level (p-value < 0.05) in individuals with IBD during the acute phase. Significant increase in abundance of unusual aerobes and facultative anaerobes(p-value=0.031) was also observed. Bacterial communities belonging to genus Prevotella & Dialister were observed at increased levels in the follow up group individuals. Infectious bacterial communities belonging to genus Stenotrophomonas, Parabacteroides, Elizabethkingia, Pseudomonas, Micrococcus, Ochrobactrum and Achromobacter were found to be dominant during the acute phase of IBD as compared to the remission phase. Our qPCR results suggested no significant correlation between C.difficile bacterial infection and IBD in any stage of the disease. Key Message: Probiotics directed towards restoring the intestinal flora especially the anaerobic bacterial communities, could help in prevention of acute exacerbation of IBD. Reference: TW Hoffmann et.al : Microorganisms linked to inflammatory bowel disease. The ISME Journal. (Nature) 2016. Jeremiah J. Faith, Janaki L. Guruge, et al. The Long-Term Stability of the Human Gut Microbiota. Science. 2013. Xochitl C Morgan, Timothy L Tickle, et al. Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment. Genome Biology. 2012. Alan W Walker, Jeremy D Sanderson,et al. High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and noninflamed regions of the intestine in inflammatory bowel disease. BMC Microbiology. 2011. Acknowledgement: SW acknowledges a research fellowship from the Council of Scientific and Industrial Research (CSIR). This work was supported by funding from the Department of Biotechnology, Government of India. Email for Corresponding and presenting author: renu.bharadwaj@gmail.com