Human & Oral Microbiome

• What is a eukaryote?

  An organism consisting of a cell or cells in which the genetic material is contained within a distinct nucleus, and has membrane-bound organelles: animalia, plantae, protista, fungi
• What is a prokaryote?

  Single cell microorganism that lacks a distinct nucleus & membrane-bound organelles: bacteria, archaea
• What is a habitat?

  Specific site of organism growth
• What does microbial community mean?

  The microorganisms that are present in a given habitat
• What does microbiota mean?

  The total collection of microorganisms within a microbial community
• What is a microbiome?

  The microbiota and all its associated genes
• What is a metagenome?

  The total genomic DNA of all the organisms within a community
• What is a biofilm?

  A physically (often temporarily) structured aggregate of microorganisms, adhered to each other and/or a defined substrate (i.e. dental plaque attached to a tooth/gum margin)
• Why is the human microbiome important?

  1. Specific micro-organisms are protective against disease and pathogenic bacterial species.

  
  

  2.Changes in the composition of the microbiome are associated with disease

  
  

  3. Microbial genes modulate fundamental human physiological processes.

  
• What were the aims of the HMP (human microbiome project)?

  Generate resources to help study the human microbiome

  Characterise the microbiome associated with human health & disease

  Determine whether individuals share a core human microbiome

  To understand whether changes in the human microbiome results in changes to human health
• What 5 fundamental questions did the HMP lead to?

  What microbes populate the human host?

  What are these microbes doing?

  How does the host respond to these microbes?

  What are the forces that maintain equilibrium among the microbial communities?

  What are the unique characteristics of each individual's microbiome?
• How can you study the microbiome?

  1. The traditional culture approach

  2. The new molecular approach
• What is the traditional culture approach?

  Grow microbes directly from samples

  Requires phenotypic identification of isolates: can be done by looking at morphology/motility, biochemistry, what antibodies they respond to (serological), and what their metabolism is
• What is the new molecular approach?

  Identifies organisms by gene sequence homology:

  Extract microbial DNA from samples

  Sequence analysis of DNA (targeted sequencing, shotgun metagenomics sequencing)
• What are the positives & negatives of the traditional culture approach?

  
• What are the positives & negatives of the new molecular approach?

  
• What are the key features of the bacterial 16S rRNA gene?


  -1542 bp gene

  -found in all bacteria and archaea

  -encodes the small subunit for the ribosomal complex, necessary for protein synthesis. Therefore v. Important. Due to this, there are conserved regions which dont change.

  -variable genes are available which allow us to see differences between microorganisms

  
• What is the workflow for 16S rRNA sequencing?

  
• What is shotgun metagenomics?

  The study of uncultured microbial communities, typically relying on high-throughout experimental data & bioinformatic techniques

  You sequence everything as opposed to targeted sequencing of 1 particular gene so it covers all kingdoms, not just Bacteria & Archaea

  Gives both functional & taxonomic information

  More technical & expensive
• What is the workflow you'd follow for shotgun metagenomics?

  Phylogeny - what is present there.

  
• What is the common result you get from both 16S rRNA sequencing & shotgun metagenomics?

  Phylogenetic view of community composition (what is present/taxonomy of the community is).

  
• How do you make sense of sequencing data?

  Compare the A, T, C & G's you get to a curated database for identification
• What are the limitations of making sense of sequencing data?


  1. Can only identify sequences present in database

  2. Need to perform whole genome sequencing of more organisms so this genetic information can be added to databases

  3. Need to annotate the sequences e.g. what do ATCGs code for, what's their function?
• What is the importance of annotation?

  Makes sense of A, T, C, Gs

  Identifies the open reading frame (OFR) of genes

  Predicts putative functions for genes & gene products
• What are the problems with annotation?

  Only a prediction- needs confirmation experiments

  Takes about 100 hrs per genome (1 min/gene)

  Mistakes are made

  Various software available to help predict function of gene, some are better than others

  Genes shared & vary between species
• What are the important genes and what do they do?

  Diagnostic markers:

  
  

  Genes for biotechnology applications:

  Novel production processes
• What are virulence genes?

  Genes that contribute to pathogenicity of organism (i.e. endotoxin gene, fimbriae gene)
• What are resistance genes?


  Genes that induce antibiotic resistance (i.e. Erm genes, mecA gene)
• What are diagnostic markers?

  Genes that aid in the rapid diagnosis of disease by using diagnostic markers (i.e. Hepatitis antibodies for HepC)
• What are genes for biotechnology applications?

  Novel production processes

  Genes that may produce a certain compound may be used further downstream. Can help produce a new antimicrobial drug
• What are the findings regarding the microbiome?

  Colonisation begins at birth

  Microbiome changes over time (most dramatically over the first 3 yrs of life)

  Influenced by diet, lifestyle, environment

  Microbiomes are characteristic of each body site

  Microbiome plays a role in disease e.g. obesity, type II diabetes

  The individual is the primary determinant for composition- everyone's microbiome is different
• Describe the microbiome of the stomach.

  
• Describe the microbiome of the small intestine.

  -Lower number of organisms but number increases closer to the colon.

  -Microbes found are ones which survived the stomach acid

  -More aerobic bacteria found since there are aerobic conditions

  
• Describe the microbiome of the colon/large intestine.

  -Highest variety of bacterial genera

  -More anaerobic microbes

  
• Describe the microbiome of the skin.

  -Variation between different sites

  -low numbers of microbes on exposed areas

  -large numbers of microbes in protected areas

  -large numbers also present around orifices (openings)

  
• Describe the oral microbiome.

  -High number of bacteria in mouth

  -very diverse

  -several different microbial habitats

  -Associated with oral diseases in most ppl

  -This microbiota is not in full harmony with the host

  
• What is the most dominant species in salivary microbiome?

  Streptococcus
• Which bacteria genera are commonly found in the mouth?

  Cocci:

  Gram positive- Streptococcus, peptostreptococcus

  Gram negative- Neisseria, veillonella, haemophilus

  
  

  Bacilli:

  Gram positive- Actinomyces, lactobacillus, eubacterium, propionibacterium

  Gram negative- prevotella, fusobacterium, porphyromonas, spirochaetes
• How does the oral microbiota change over time?

  There's not just a simple build-up of microbes

  There's a shift in the species seen as plaque develops

  Microbiome-host interactions play a key role in this process

  
• Give an example of the dynamic changes in plaque microbiota & disease.