Technical University of Munich Chair for Biofunctionality. Gut microbiology PRACTICAL COURSE Dr. Thomas Clavel
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1 Technical University of Munich Chair for Biofunctionality Gut microbiology PRACTICAL COURSE 2011 Dr. Thomas Clavel
2 Table of content 1 Introduction 2 Culture-based approaches 3 16S rrna 4 Sequencing 5 Gut microbiota in IBD
3 MICROBIOLOGY: Study of microscopic organisms - Bacteria (BACTERIOLOGY) - Fungi (including yeasts) - Protozoa - Viruses CELLULAR MICROBIOLOGY Metabolism; Biochemistry SYSTEMATICS Classification; e.g., Akkermansia muciniphila CIP T MOLECULAR MICROBIOLOGY Phylogeny; Genomics MICROBIAL ECOLOGY Interactions
4 Role of bacteria in IBD Fecal stream diversion (Rutgeerts et al., 1991) Onderdonk et al., 1981
5
6 Diversity and distribution of intestinal microbiota Stomach Small intestine Large intestine (colon) Stomach ( CFU g -1 ) Helicobacter; Lactobacillus; Streptococcus Ileum ( CFU g -1 ) Enterobacteriaceae; Lactobacillus; Streptococcus Colon ( CFU g -1 ); > species Alistipes; Bacillus; Bacteroides; Bifidobacterium; Collinsella; Clostridium; Dorea; Enterococcus; Eubacterium; Faecalibacterium; Parabacteroides; Roseburia; Ruminococcus; Streptococcus
7 Culture-dependent analysis of bacterial diversity Complex ecosystem Plating of serial dilutions Anaerobic box N 2 + CO 2 + H 2 Rich or selective agar Growth Various colonies Counting and phenotyping
8 Culture-dependent analysis of bacterial diversity - Morphology - - Growth characteristics - -Enzymatic tests - - SCFA - -Antibiotic resistance - -Sporulation - - Motility - -Cell wall structure - - CFA / quinones / polar lipids -
9 0.1 Olsenella 0.1 Isolation and characterization of bacteria do03 equoli obob2aaa03g05 Clone obob2_aaa03g05 (EF096493) Uncultured Clone H75N2_66f08 (EU453459) B7 Enterorhabdus caecimuris DSM T (DQ789120) sinensishku14 NPOH1 Slackia Coriobacterium SWPT5aaa04e11 Clone SWPT5_aaa04e11 (EF100091) mucosicola Enterorhabdus mucosicola DSM T (AM747811) C16E16 Clone C16_E16 (AY992290) SWPT12aaa04f04 Clone SWPT12_aaa04f04 (EF098044) M3f063 Clone M3_f06_3 (DQ015649) RL176aah44c05 Clone RL176_aah44c05 (DQ793846) Asaccharobacter celatus DSM T (AB266102) Adlercreutzia equolifaciens DSM T (AB306661) Julong732 Human intestinal bacterium SNU-Julong732 (AY310748) lentam2 Eggerthella lenta DSM 2243 T (AF292375) Eggerthella sinensis DSM T (AY321958) HKU Paraeggerthella hongkongensis DSM T (AY288517) Gordonibacter pamelaeae b T (AM886059) Denitrobacterium detoxificans CCUG T (U43492) Slackia exigua ATCC T (AF101240) isoflavoniconvertens Slackia isoflavoniconvertens DSM T (EU826403) Cryptobacterium curtum DSM T (AB019260) Eubacterium Collinsella aerofaciens DSM 3979 T (AB011816) Coriobacterium glomerans DSM T (X79048) A.minutum Atopobium minutum DSM T (X67148) Olsenella uli DSM 7084 T (AF292373) Eubacterium limosum DSM T (M59120) limo Strain Mt1B8 10 µm Clavel et al., IJSEM, 2009
10 Isoflavone conversion by strain Mt1-B8 Matthies et al., AEM, 2008
11 Gnotobiology Germfree environment
12 Simulator of the Gastrointestinal Microbial Ecosystem
13 The 16S ribosomal RNA Noller and Woese, 1981, Science S 50S 16S 5S + 23S Conserved Variable Highly variable Specific structure - Ubiquitous and abundant
14 Clone libraries 1 Complex ecosystem DNA extraction DNA mixture Specific PCR bp Numerous copies of different 16S rdna amplicons lacz Ligation Transformation Sequencing Lactose- and Antibioticcontaining medium 5 ACGTAGCTAGCCCATGCGGTTTTACGC ATCACTACGAGGTTTTTGCGTACGTAGC ATCGACGGGCATGCATGCTGACTGCGTC TACGTAGCTAG Alignment Similarity % Suau et al., AEM, 1999
15 Sanger sequencing
16 High-throughput sequencing Roche (454) Illumina SOLiD Chemie Pyrosequenzierung Polymerase-basiert Ligations-basiert Amplifikation Emulsions PCR Brücken Amplifikation Mb/Lauf 100 Mb 1300 Mb Emulsions PCR 3000 Mb Zeit/Lauf 7 h 4 Tage 5 Tage Länge der reads bp bp 35 bp Kosten pro Lauf (gesamt) $ $ $ Kosten pro Mb $ 84,4 $ 5,97 $ 5,81
17 Genome sequencer FLX (454 pyrosequencing) 1 16S-specific PCR A B 2 empcr
18 Genome sequencer FLX (454 pyrosequencing)
19 Pyrosequencing adenosine 5 -phosphosulfate luciferin oxy-luciferin + light!!!
20 Functional metagenomic Assessment of the intestinal microbiome ( genes) 1 Density gradient Cell lysis 40,000 bp Complex ecosystem Bacteria Size exclusion DNA Screening of activities E. coli Cloning 25,000 Fosmid
21 The core microbiome Qin et al., Nature, 2010
22 Gut microbiota in IBD Loss of Firmicutes in IBD (Manichanh et al., Gut, 2006) (Peterson et al., 2008) (Sokol et al., PNAS, 2008)
23 Gut microbiota in IBD Fluorescence in situ hybridization Higher density of mucosa -associated bacteria in IBD (Swidsinski et al., 2005) Adherent-invasive E. coli (AIEC) (Darfeuille-Michaud et al., Gastroenterol 1998)
24 Bacteriophages in the gut Reyes et al., Nature, 2010
25 Bacteriophages in IBD (n = 14) (n = 19) 100 nm Lepage et al., Gut, 2008
26 Culture (Large-scale) selective isolation and characterization of (novel) bacteria Molecular HTS and screening approaches to identify (novel) bacterial signals associated with diseases Host-Bacteria Relevance of selected bacteria or products thereof
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Eugonagar [Becton Dickinson, Cockeysville, Md.], 10 g of maltose, 5 mg of hemin, 400 ml of tomato juice [autoclaved separately]).
![Eugonagar [Becton Dickinson, Cockeysville, Md.], 10 g of maltose, 5 mg of hemin, 400 ml of tomato juice [autoclaved separately]).](/thumbs/94/118303061.jpg "Eugonagar [Becton Dickinson, Cockeysville, Md.], 10 g of maltose, 5 mg of hemin, 400 ml of tomato juice [autoclaved separately]).")
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2000, p. 4523 4527 Vol. 66, No. 10 0099-2240/00/$04.00 0 Copyright 2000, American Society for Microbiology. All Rights Reserved. Development of 16S rrna-based