LIFESTYLE-ASSOCIATED ALTERATIONS OF INTESTINAL

Transcription

1 LIFESTYLE-ASSOCIATED ALTERATIONS OF INTESTINAL MICROBIOTA AND THEIR POTENTIAL ROLE IN ETIOLOGY OF IBD: A METAGENOMIC ANALYSIS Stephan J. Ott, University Hospital (UKSH), Campus Kiel Institute for Clinical Molecular Biology (IKMB) Christian-Albrechts-University (CAU) Kiel BMRP ANNUAL MEETING February 2011 Los Angeles, USA / 1

2 Lifestyles Major Objectives Background: Epidemiologic studies point to the critical role of environmental factors in the pathogenesis of chronic inflammatory barrier diseases, e.g. the chronic inflammatory bowel diseases (IBD) or asthma ( civilization diseases ). Lifestyle factors, e.g. nutrition, hygiene, and improved medical services, were accounted for the raising incidence of these disorders over the last decades in the industrialized countries ( hygiene hypothesis ). Intestinal microbiota as an integral part of the enteric mucosal barrier reflects the evolution of lifestyle factors. Aim of the study: The aim of this study is to compare the composition of the intestinal microbiota of rural areas with low incidence of civilization diseases (representing the ancient lifestyle of the pre-industrialization period) and a region in an urban area, which is over decades influenced by multiple environmental factors ( modern lifestyle).

3 Lifestyles Study Design Patients N=90 Lithuania Healthy Controls N=10 Ulcerative Colitis N=10 Crohn Disease N=10 Germany Healthy Controls N=10 Ulcerative Colitis N=10 Crohn Disease N=10 India Healthy Controls N=10 Ulcerative Colitis N=10 Crohn Disease N=10 high [5-7/ ] mod. [1-3/ ] low [< 1/ ] no data Germany Source Material Sigma Biopsies Lithuania India

4 Lifestyles Status of the Project Patient Recruitment Complete set of samples from all 90 individuals DNA/RNA Extraction Completed for all 90 samples. Microbial Molecular Investigation Completed for all cohorts, RNA and DNA, > sequences. Statistical Analysis Analysis on species level for DNA, complete RNA work (transcriptional level), most of the comparative analysis to be done. Publication In preparation.

5 P2 - Percent variation explained 4.94% Lifestyles Experimental Setup Sigma biopsy N=90 16S rrna- Extraction 16S rdna- Extraction High-throughput bar-coded second generation sequencing of V 1-2 regions of 16S rrna gene (RNA and DNA) Statistical analysis - Mothur - Uni Frac PCoA - P3 vs P P3 - Percent variation explained 3.97%

6 Lifestyles Statistical Analysis sequence reads were screened and filtered for quality and length using perl script (Giongo et al., 2010) sequences were trimmed and binned by samples using the specific MIDs (barcode) bad quality sequences were excluded from further analysis Criteria: - no perfect match with MID sequences or specific primers - less than 200 base long reads - quality score less than 20, - ambiguous bases - sequences with more than 6 homopolymers chimeric sequences were removed by MOTHUR chimera slayer command sequences were classified by using RDP multiclassifier Giongo, A. et al., PANGEA: pipeline for analysis of next generation amplicons. ISME J, 4(7), Pruesse, E. et al., SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucl. Acids Res., 35(21),

7 P2 - Percent variation explained 4.94% Lifestyles Unifrac Analysis of All Samples Unweighted fast unifrac analysis PCoA - P3 vs P2 Indian Lithuanian German Total of ~ sequences Regardless of disease status Indian cohort clusters distinctly P3 - Percent variation explained 3.97%

8 P2 - Percent variation explained 7.12% P2 - Percent variation explained 7.86% P2 - Percent variation explained 6.53% Lifestyles Unifrac Analysis Healthy vs. Disease Unweighted fast unifrac analysis 0.4 PCoA - P3 vs P2 0.3 PCoA - P3 vs P2 0.5 PCoA - P1 vs P P3 - Percent variation explained 6.36% Healthy individuals Indian Lithuanian German P3 - Percent variation explained 6.41% Crohn s disease P1 - Percent variation explained 13.53% Ulcerative colitis

9 % of Sequences Lifestyles Major Phyla in Healthy Individuals P < German_healthy Healthy indian Indian Healthy Healthy Lithuanian healthy Healthy 10 0 Bacteroidetes Firmicutes Proteobacteria Actinobacteria Others Major phyla in healthy individuals from three Cohorts.

10 % of sequences Lifestyles Differences on Bacterial Family Level Selected significantly different selected bacterial taxa at family level Healthy_Germans Healthy_Lithuanian Indian Healthy_Indian Lithuanian P <

11 % of Sequences Lifestyles Differences on Bacterial Genus Level Selected significantly different selected bacterial taxa at genus level Healthy_Germans Healthy_Lithuanian Indian Healthy_Indian Lithuanian P <

12 % of sequences Lifestyles Differences in Crohn s Disease Crohn German disease_german CD Crohn Indian disease_lithuania CD Crohn Lithuanian disease_indian CD P < Significantly different taxa in Crohn s disease

13 % of Sequences Lifestyles Differences in Ulcerative Colitis Selected significantly different taxa in Ulcerative Colitis 30 P < German_Ulcerative UC colitis Lithuania_Ulcerative Indian UC Colitis Indian_Ulcerative Lithuanian UC Colitis 0

14 % of Sequences Lifestyles Differences Healthy vs. Disease P < German_healthy German_CD German_UC Bacteroidetes Firmicutes Proteobacteria Actinobacteria Others Major phyla healthy vs. disease in Germans

15 % of Sequences Lifestyles Differences Healthy vs. Disease P < Indian_healthy Indian_CD Indian_UC Bacteroidetes Firmicutes Proteobacteria Actinobacteria Others Major phyla healthy vs. disease in Indians

16 % of Sequences Lifestyles Differences Healthy vs. Disease P < Lithuania_healthy Lithuania_CD Lithuania_UC 10 0 Bacteroidetes Firmicutes ProteobacteriaActinobacteria Others Major phyla healthy vs. disease in Lithuanians

17 Lifestyles Conclusions so far The Indian individuals show significant differences in their intestinal bacterial composition: - analysis of questionnaires might reveal environmental or nutritional (vegetarians) factors to be responsible The intestinal bacterial microbiota is significantly different between the different populations and the disease groups: - further analysis of microbiota on species level will provide more detailed information Future perpectives: To identify characteristics of a healthy, ancient intestinal microbiota, i.e. species that might be protective or harmful for the host as a basis for specific manipulation (e.g. new probiotics).

18 Many Thanks! Kiel - Institute for Clinical Molecular Biology (ICMB)/ Manuela Kramp Sanaz Sabet Birgit Pieper Femke Heisen Henrik Knecht Ateequr Rehman Tanja Kühbacher Anette Friedrichs Stephan J. Ott Stefan Schreiber (Head of all) Kaunas, University Hospital (Lithuania) Aida Zwirbliene Limas Kupszinskas Ameya RBI Employees Co-Op Housing Society Mumbai (India) Deepak Amarapurkar Future Ocean I. Medizinische Klinik, Klinik für Allgemeine Innere Medizin