Gut flora and the Microbiome Flashcards
What is genomics?
Whole cell gene contents
What is transcriptomics?
Whole cell gene expression
What is proteomics?
Whole cell protein content
What is metabolomics?
Whole cell metabolite content
What is the microbiome?
A characteristic microbial community occupying a reasonably well-defined habitat which has distinct physio-chemical properties
Refers to both microorganisms involved and their theatre of activity
What is the microbiota?
Ecological community of commensal and pathogenic microorganisms
Includes bacteria, archaea, protists, fungi and viruses
What is a biome?
A reasonably well-defined habitat which has distinct physiochemical properties
What is the weight of the human microbiome?
70kg
What is the weight of the bacteria microbiome?
1-3kg
Describe the human microbiome
Not only 1 microbiome
Each area/part of the body has a distinct microbiome with different compositions
Unique to each individual (even twins)
What may play a role in determining the microbiome?
Host genetics
Describe the gut microbiome
Significant variation between healthy individuals
Integral to host digestion
Approximately 400 species
Diversity throughout GI tract - changing pH, adhesion, nutrients
How can species be categorised in the gut microbiome?
Autochthonous
Allochthonous
Describe autochthonous species
Indigenous/resident
Colonise GI tract
Describe allochthonous species
Transient/passenger
Only colonise under abnormal conditions
What do the species in the gut microbiome do?
Provide unique and specific enzymes and biochemical pathways
Competitive exclusion of pathogens, protecting the host
What is the diagnostic role of the gut microbiome?
Can classify individuals as lean or obese with >90% accuracy
Changes in gut microbiome have been associated with multiple human illnesses - IBS, depression, cancer
Early life gut microbiomes linked to development of allergic conditions eg. asthma
How is the gut microbiome targeted for helping IBS?
Altering microbiota through dietary changes, probiotics or antibiotics can show benefit
What is the microbiota-gut-brain axis?
Network of connections, crucial to homeostasis
Changes in the gut may be linked to symptom perception in the brain
What factors can influence the brain?
Metabolic and immunological factors
How does the microbiota modulate behaviour?
Short chain fatty acids
Modulation of neuroendocrine system
Bacteroides, bifidobacterium can producegamma-aminobutyric acid (GABA)
What happens in IBS?
Inflammation in the gut, environmental and genetic risk factors
Alterations can be seen in gut microbiota
How does colorectal cancer differ from IBS?
Higher proportion of Pseudomonas. helicobacer and acinetobacter
Lower richness of beneficial bacteria eg. butyrate producing bacteria
Reduced abundance of Bifidobacterium species
What interventions can be used for gut microbiome?
Prebiotics - non-digestable food ingredient to promote growth of beneficial organism
Probiotics - beneficial organisms
Synbiotics - micture of prebiotics and probiotics
What is most commonly in probiotics?
Lactobacillus
Bifidobacterium
What is an intervention that has proven to be effective for gut microbiome?
Faecal microbiota transplant (FMT) - example of probiotics
What is antibiotic associated diarrhoea?
Related to overgrowth of C.diff
What is susceptibility to C.diff infection associated with?
Decreased microbiota diversity
What cures C.diff infection?
FMT
How is FMT used to treat CDI?
Healthy donor, filter sample and administer by colonoscopy
What happens to the gut microbiota after FMT?
Increased diversity
Increased abundance of various Firmicutes and Bacteroides
Decreased abundance of Proteobacteria
How are psychiatric disease related to gut microbiome?
Dysbiosis increases translocation of gut bacteria into lymphoid tissue
Provokes an immune response
Activation of vagus nerve and spinal afferent neurons
What are other associations to the gut microbiome?
Food allergies
Asthma
Obesity
Diabetes
Rheumatoid arthritis
CVD
Liver diseases
What are technological approaches to assess the microbiome?
Targeted PCR amplification - 16S rRNA bacteria, internal transcribed spacer (ITS) 18S rRNA eukaryotes
Whole genome shotgun sequencing
What is 16S rRNA?
Component of 30S small subunit of prokaryotic ribosome
Describe prokaryotic ribosome
50S subunit - 5S, 23S
30S subunit - 16S
Describe eukaryotic ribosome
60S subunit - 5S, 28S
40S subunit - 18S
How is 16S targeted PCR amplification done?
Sample collection
DNA extraction
16S PCR amplification of whole sample
PCR products are sequenced
DNA sequences are analysed
What 16S databases can be used?
Greengenes
Silva
RDP
What are the 16S databases used for?
To separate samples into species/genus
What is an operational taxonomic unit?
Used to classify groups of closely related individuals
How are OTUs done?
Sequences are clustered according to their similarity
What are the 2 types of diversity?
Alpha
Beta
What is alpha diversity?
Diversity within a sample
What is beta diversity?
Diversity between samples
How can alpha diversity be measured?
Species richness
Species diversity
What is species richness?
OTU count
Measure of how many different species can be detected in a microbial system
How is species diversity measured?
Shannon index
Measure of how the microbes are balanced to each other and if there is species evenness (similar abundance level) or if some species dominate others
How is beta diversity measured?
Bray-Curtis dissimilarity = based on abundance
Jaccard distance = based on presence or absence of species
UniFrac = based on phylogenetic trees
How is a variable region chosen for 16S targeted PCR amplification?
Amplicon length
Phylogenetic signal
What needs to be considered when deciding what controls to use?
16S rRNA gene found in all bacteria
Method is sensitive to contamination - environment, operator, reagents
Contamination is more important to consider for low biomass samples
How can contamination be mitigated?
Randomise samples
Note batch numbers of reagents
Sequence negative controls
How does whole genome shotgun workflow work?
What are problems with Whole genome shotgun workflow?
Host cells often in excess in sample
No amplification step to enrich for bacterial DNA
Sample dependent
How can enrichment be done without amplification?
Pre-extraction - differential lysis of mammalian cells, enrich for intact microbial cells, potential bias towards gram-positive bacteria
Post-extraction - enzymatic degradation of methylated molecules targets mammalian DNA, bias against AT rich bacterial genomes
