Akram Najafi, Bushehr University of Medical Science, Iran

Gut MicrobiotaGut Microbiota

Presented by:

Akram Najafi

Akram Najafi, Bushehr University of Medical Science, Iran

What is Gut Microbiota?

• Skin, mouth, and gut act as host to an enormous variety of microbes, bacterial, archaeal, fungal, and viral.

• The human gut microbiota is estimated to be composed of approximately 1014 bacterial cells.

• Approximately 400-500 bacterial species

• Their total genome capacity is 150 times larger than the human gene complement, with an estimated 3.3 million microbial genes

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

• These microbes help us:

- To digest our food

- To harvest energy from the diet

- To stimulate of the proliferation of the intestinal epithelium

- To regulate of fat storage in the host

- To maintain our immune systems

• More recently, studies strongly suggest that dysbiosis contributes to:

- IBS, intestinal cancers, obesity, type 1 diabetes…

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

Techniques used to characterize the gut microbiota:

• Culture

• qPCR (real time PCR)

• Fluorescence in situ hybridization (FISH)

• Denaturing gradient gel electrophoresis (DGGE)

• Terminal restriction fragment length polymorphism (T-RFLP)

• DNA micro-arrays

• Direct sequencing of 16S rRNA (Pyrosequencing)

Akram Najafi, Bushehr University of Medical Science, Iran

Culture:

• Isolation of bacteria on selective media • Advantages:

- Cheap, semi-quantitative • Disadvantages:

- <30% of gut microbiota have been cultured to date

Akram Najafi, Bushehr University of Medical Science, Iran

16 SrRNA is able to demonstrate the following:

• The microbial diversity of the gut microbiota

• Qualitative & quantitative information on bacterial species

• Changes in the gut microbiota in relation to disease

Akram Najafi, Bushehr University of Medical Science, Iran

qPCR (real time PCR):

• Amplification and quantification of 16S rRNA. Reaction

mixture contains a compound that fluoresces when it binds to double-stranded DNA.

• Advantages:

- Phylogenetic identification, quantitative, fast• Disadvantages:

- PCR bias, unable to identify unknown species

Akram Najafi, Bushehr University of Medical Science, Iran

Fluorescence in situ hybridization (FISH):

• Fluorescently labelled oligonucleotide probes hybridize complementary target 16S rRNA sequences. When hybridization occurs, fluorescence can be enumerated using flow cytometry.

• Advantages: Phylogenetic identification, semi-quantitative, no PCR bias, fast• Disadvantages: Dependent on probe sequences, unable to identify unknown

species

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

DNA micro-arrays (DNA chip):

• Fluorescently labelled oligonucleotide probes hybridize with complementary nucleotide sequences. Fluorescence detected with a laser.

• Mainly used in studies to compare the microbiota between different populations.

• Advantages: Phylogenetic identification, semi-quantitative, fast

• Disadvantages: Cross hybridization, PCR bias, species present in low levels can be difficult to detect.

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

Denaturing gradient gel electrophoresis (DGGE):

• Gel separation of 16S rRNA amplicons using denaturant/ temperature

• Advantages: Fast, semi-quantitative, bands can be excised for further analysis

• Disadvantages: No phylogenetic identification, PCR bias

Akram Najafi, Bushehr University of Medical Science, Iran

Terminal restriction fragment length polymorphism (T-RFLP):

• Fluorescently labelled primers are amplified and then restriction enzymes are used to digest the 16S rRNA amplicon.

• Advantages: Fast, semi-quantitative, cheap • Disadvantages: No phylogenetic identification, PCR bias, low

resolution

Akram Najafi, Bushehr University of Medical Science, Iran

Direct sequencing of 16S rRNA amplicons:

• Massive parallel sequencing of partial 16S rRNA amplicons for example, 454 Pyrosequencing® (Roche Diagnostics GMBH Ltd, Mannheim, Germany)

• Amplicon immobilized on beads, amplified by emulsion PCR, addition of luciferase results in a chemoluminescent signal

• Advantages: Phylogenetic identification, quantitative, fast, identification of unknown bacteria

• Disadvantages: PCR bias, expensive, laborious

Akram Najafi, Bushehr University of Medical Science, Iran

Advantages:

• Pyrosequencing can sequence 500 million bases, at 99% or better accuracy, in a single run.

• It represents an approximately 2,000-fold increase in throughput over Sanger sequencing.

• As shorter sequences are read (approximately one half of

the read lengths generated in Sanger sequencing), thus bacteria that are in low abundance can be detected.

• Phylogenetic identification, quantitative, fast, identification of unknown bacteria.

Akram Najafi, Bushehr University of Medical Science, Iran

Sampling:

• The majority of published studies have actually been based on results from stool samples rather than mucosal biopsy samples or luminal content analysis.

• Stool samples are used as a proxy for the study of the gut

microbiota as these samples are easier to collect than biopsy samples, especially in healthy volunteers.

• Questionnaire

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

• The DNA qualities and concentrations in the samples will apply using the gel electrophoresis and spectrophotometer.

• Pyrosequencing of Barcoded 16S rRNA Gene Amplicons:

- PCR reactions will run in a thermal controller using the

following cycling parameters:

- 5 min of denaturation at 95C, - 30 s at 95 C (denaturing), - 30 s at 56 C (annealing), - 90 s at 72 C (elongation), - A final extension at 72C for 7 min.

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

Akram Najafi, Bushehr University of Medical Science, Iran

Thanks

for your attention