Microbial Communities

Question 1

What is the microbiome

Answer 1

• The microbiota is the collection of microorganisms in a particular environmental niche
• Includes bacteria, archaea, fungi, and other single-cell organisms
• The microbiome specially refers to the collective genomes of the microbiota
• The human microbiota are the microbes that live in and on the human body
• primary colonization sites for microbes in humans: nose, mouth, skin, gut and microbes
• other sites: eyes, ear, lung. Etc

Question 2

Probing the microbiome

Answer 2

• culturing - grow on media that is selective fro different species - is the species we are looking for present
• imaging - label cells, image through microscopy or other techniques
  - Where are the bacteria
  - what are the relative ratios of different species
  - what genes are being expressed by the community

Question 3

Labelling bacteria

Answer 3

• Green Fluorescence Protein (GFP) and variants
• Bioluminescence
• Fluorescence in-situ hybridisation (FISH)

Question 4

Green Fluorescence Protein (GFP) and variants

Answer 4

• Bacteria are engineered to express proteins that fluoresce
• labels living cells
• requires excitation light (hard to label deep inside tissues)
• requires oxygen to mature (bacteria growing anaerobically are hard to label)
• limited to only culturable/modifiable bacteria
• limited number of colors (only label 2-3 species at the same time)

Question 5

Bioluminescence

Answer 5

• Bacteria are engineered to express an enzyme that emits lights (luminescence)
• labels living cells
• does not require excitation light
• only one color (only pne species of bacteria can be tracked at any time)
• can image through deeper tissue
• limited to only culturable/midfiable bacteria

Question 6

Fluorescence in-situ hybridisation (FISH)

Answer 6

• Fluorescence compounds are linked to single stranded DNA that is complementary to specific bacterial species
• any species with known DNA sequences can be labeled
• can track multiple species at the same time (16-20 different colours)
• requires fixed and permeabilized cells (no dynamic/living systems)

Question 7

Ex-vivo imaging

Answer 7

• image inside tissue by cutting it open and cutting it into thin sections
• GFP and RFP-labeled bacteria inside intestine
• Fluorescent dyes that bind to specific proteins can also be used

Question 8

Biofilm composition

Answer 8

Parental strain

Oxygen

Nitric acid

eDNA

Protein

Polysaccharide

Fatty acid

Amyloid fibre

Filamentous phage

Question 9

Biofilms protect against antibodies

Answer 9

• antibiotic cannot penetrate the biofilm
• nutrient gradient means some bacteria will be growing slowly
• a subset of the population that is resistant can protect the population as a whole
• “differentiation” of the population into a “persister state”
• resistant mutants can be “collected” in the population

Question 10

Biofilm in human disease

Answer 10

• organ transplants
• implanted devices (cosmetic surgery, prosthetic joints, dental fillings etc)
• catheters, piercings
• open wounds (cuts, burns)
• genetic diseases (e.g. Cystic fibrosis)
• tooth decay

Question 11

Sequencing approaches

Answer 11

• use PCR to amplify parts of the 16S ribosomal gene to identify spec
• 16S ribosomal sequences used to redefine the domains of life (Bacteria, archaea, eukaryota)
• these sequences can identify what species are present and determine their relative ratios

Question 12

Human microbiome composition

Answer 12

• each anatomical location contains a different species composition
• what kind of factors can influence microbiome composition?
  • Other microbes
  • pH
  • temp
  • activities etc

Question 13

Gut biome composition changes with age and diet

Question 14

Commensal microbes prevent infection by pathogens

Question 15

Antibiotic treatment can lead to Clostridium difficult infection (CDI)

Answer 15

Look at notes for the fecal transplant treatment

Question 16

Modulating the microbiome

Answer 16

Look at the diagram in the notes