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Akram Najafi, Bushehr University
of Medical Science, Iran
Gut 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

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Gut microbiota

  • 1. Akram Najafi, Bushehr University of Medical Science, Iran Gut Microbiota Presented by: Akram Najafi
  • 2. 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
  • 3. Akram Najafi, Bushehr University of Medical Science, Iran
  • 4. 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…
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  • 13. 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)
  • 14. 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
  • 15. 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
  • 16. 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
  • 17. 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
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  • 19. 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.
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  • 21. 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
  • 22. 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
  • 23. 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
  • 24. 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.
  • 25. 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
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  • 27. 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.
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  • 30. Akram Najafi, Bushehr University of Medical Science, Iran Thanks for your attention