technical university of munich chair for …...technical university of munich chair for...
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Gut microbiology
Dr. Thomas Clavel
Technical University of MunichChair for Biofunctionality
PRACTICAL COURSE 2011
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Table of content
1 – Introduction
2 – Culture-based approaches
3 – 16S rRNA
4 – Sequencing
5 – Gut microbiota in IBD
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MICROBIOLOGY: Study of microscopic organisms
MICROBIAL ECOLOGY Interactions
MOLECULAR MICROBIOLOGY Phylogeny; Genomics
CELLULAR MICROBIOLOGY Metabolism; Biochemistry
- Fungi (including yeasts)
- Viruses
- Bacteria (BACTERIOLOGY)
- Protozoa
SYSTEMATICS Classification; e.g., Akkermansia muciniphila CIP 107961T
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Role of bacteria in IBD
+
1
Onderdonk et al. , 1981
Fecal stream diversion(Rutgeerts et al., 1991)
2
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Diversity and distribution of intestinal microbiota
Colon (1011-1013 CFU g-1); > 1 000 speciesAlistipes; Bacillus; Bacteroides; Bifidobacterium; Collinsella; Clostridium; Dorea; Enterococcus; Eubacterium; Faecalibacterium; Parabacteroides;Roseburia; Ruminococcus; Streptococcus
Ileum (107-108 CFU g-1)Enterobacteriaceae; Lactobacillus; Streptococcus
Stomach (101-103 CFU g-1)Helicobacter; Lactobacillus; Streptococcus
Small intestine
Large intestine(colon)
Stomach
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Plating of serial dilutions
Rich or selective agar
Complex ecosystem
Counting and phenotyping
Culture-dependent analysis of bacterial diversity
Anaerobic box
Various colonies
Growth
N2 + CO2 + H2
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Culture-dependent analysis of bacterial diversity
- Morphology -
- Growth characteristics -
-Enzymatic tests -
- SCFA -
-Antibiotic resistance -
-Sporulation -
- Motility -
-Cell wall structure -
- CFA / quinones / polar lipids -
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Isolation and characterization of bacteria
10 µm
Strain Mt1B8
Clavel et al., IJSEM, 2009
0.1
SWPT5aaa04e11
obob2aaa03g05
mucosicola
C16E16
SWPT12aaa04f04
M3f063
RL176aah44c05
Uncultured
B7
do03
equoli
Julong732
lentaM2
sinensisHKU14
HKU
Gordonibacter
NPOH1
Slackia
isoflavoniconvertens
Cryptobacterium
Eubacterium
Coriobacterium
A.minutum
Olsenella
limosum
0.1
Clone SWPT5_aaa04e11 (EF100091)
Clone obob2_aaa03g05 (EF096493)
Clone C16_E16 (AY992290)Enterorhabdus mucosicola DSM 19490T (AM747811)
Clone SWPT12_aaa04f04 (EF098044)
Enterorhabdus caecimuris DSM 21839T (DQ789120)
Clone RL176_aah44c05 (DQ793846)
Clone M3_f06_3 (DQ015649)
Human intestinal bacterium SNU-Julong732 (AY310748)
Adlercreutzia equolifaciens DSM 19450T (AB306661)Asaccharobacter celatus DSM 18785T (AB266102)
Paraeggerthella hongkongensis DSM 16106T (AY288517)
Eggerthella lenta DSM 2243T (AF292375)Eggerthella sinensis DSM 16107T (AY321958)
Denitrobacterium detoxificans CCUG 56741T (U43492)
Slackia isoflavoniconvertens DSM 22006T (EU826403)
Cryptobacterium curtum DSM 15641T (AB019260)
Collinsella aerofaciens DSM 3979T (AB011816)
Coriobacterium glomerans DSM 20642T (X79048)
Atopobium minutum DSM 20586T (X67148)Olsenella uli DSM 7084T (AF292373)
Eubacterium limosum DSM 20543T (M59120)
Slackia exigua ATCC 700122T (AF101240)
Gordonibacter pamelaeae 7-10-1-bT (AM886059)
Clone H75N2_66f08 (EU453459)
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Isoflavone conversion by strain Mt1-B8
Matthies et al., AEM, 2008
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Gnotobiology
Germfree environment
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Simulator of the Gastrointestinal Microbial Ecosystem
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30S
50S 5S + 23S
16S
5’
3’
- Specific structure
Conserved
Variable
Highly variable
- Ubiquitous and abundant
Noller and Woese, 1981, Science 212
The 16S ribosomal RNA
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5‘ACGTAGCTAGCCCATGCGGTTTTACGCATCACTACGAGGTTTTTGCGTACGTAGCATCGACGGGCATGCATGCTGACTGCGTCTACGTAGCTAG…
Sequencing
Complexecosystem
DNAextraction
DNA mixture
SpecificPCR
Numerouscopies ofdifferent16S rDNAamplicons
Ligation
Transformation
AlignmentSimilarity %
1 500 bp
1
Lactose- and Antibiotic-containing medium
lacZ
Suau et al., AEM, 1999
Clone libraries
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Sanger sequencing
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High-throughput sequencing
3000 Mb1300 Mb100 MbMb/Lauf
5 Tage4 Tage7 hZeit/Lauf
35 bp32 - 40 bp250 - 500 bpLänge der reads
$ 17 447$ 8 950$ 8 439Kosten pro Lauf(gesamt)
$ 5,81$ 5,97$ 84,4Kosten pro Mb
Roche (454) Illumina SOLiD
Chemie Pyrosequenzierung Polymerase-basiert Ligations-basiert
Amplifikation Emulsions PCR BrückenAmplifikation
Emulsions PCR
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Genome sequencer FLX (454 pyrosequencing)
A B
16S-specific PCR1
emPCR
2
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Genome sequencer FLX (454 pyrosequencing)
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Pyrosequencing
adenosine 5´-phosphosulfate
luciferin
oxy-luciferin + light!!!
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40,000 bp
Complex ecosystem
Densitygradient
Bacteria
Cell lysis
Sizeexclusion
CloningE. coli
Screening of activities
Fosmid
1
DNA
× 25,000
Assessment of the intestinal microbiome (3 × 106 genes)
Functional metagenomic
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The core microbiome
http://commonfund.nih.gov/hmp
http://www.metahit.eu
Qin et al., Nature, 2010
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Gut microbiota in IBD
Loss of Firmicutes in IBD(Manichanh et al., Gut, 2006)
(Peterson et al., 2008)
(Sokol et al., PNAS, 2008)
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Higher density of mucosa-associated bacteriain IBD (Swidsinski et al., 2005)
Fluorescence in situ hybridization
Gut microbiota in IBD
Adherent-invasive E. coli (AIEC)(Darfeuille-Michaud et al., Gastroenterol 1998)
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Bacteriophages in the gut
Reyes et al., Nature, 2010
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Bacteriophages in IBD
(n = 14)(n = 19)
100 nm
Lepage et al., Gut, 2008
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Culture Molecular
HTS andscreening approaches
to identify(novel) bacterial signalsassociated with diseases
(Large-scale) selectiveisolation and characterization
of (novel) bacteria
Host-Bacteria
Relevance of selected bacteria or products thereof