technical university of munich chair for …...technical university of munich chair for...

Gut microbiology

Dr. Thomas Clavel

Technical University of MunichChair for Biofunctionality

PRACTICAL COURSE 2011

Table of content

1 – Introduction

2 – Culture-based approaches

3 – 16S rRNA

4 – Sequencing

5 – Gut microbiota in IBD

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

Role of bacteria in IBD

+

1

Onderdonk et al. , 1981

Fecal stream diversion(Rutgeerts et al., 1991)

2

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

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

Culture-dependent analysis of bacterial diversity

- Morphology -

- Growth characteristics -

-Enzymatic tests -

- SCFA -

-Antibiotic resistance -

-Sporulation -

- Motility -

-Cell wall structure -

- CFA / quinones / polar lipids -

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)

Isoflavone conversion by strain Mt1-B8

Matthies et al., AEM, 2008

Gnotobiology

Germfree environment

Simulator of the Gastrointestinal Microbial Ecosystem

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

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

Sanger sequencing

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

Genome sequencer FLX (454 pyrosequencing)

A B

16S-specific PCR1

emPCR

2

Genome sequencer FLX (454 pyrosequencing)

Pyrosequencing

adenosine 5´-phosphosulfate

luciferin

oxy-luciferin + light!!!

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

The core microbiome

http://commonfund.nih.gov/hmp

http://www.metahit.eu

Qin et al., Nature, 2010

Gut microbiota in IBD

Loss of Firmicutes in IBD(Manichanh et al., Gut, 2006)

(Peterson et al., 2008)

(Sokol et al., PNAS, 2008)

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)

Bacteriophages in the gut

Reyes et al., Nature, 2010

Bacteriophages in IBD

(n = 14)(n = 19)

100 nm

Lepage et al., Gut, 2008

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