Gut flora and the Microbiome

Question 1

What is genomics?

Answer 1

Whole cell gene contents

Question 2

What is transcriptomics?

Answer 2

Whole cell gene expression

Question 3

What is proteomics?

Answer 3

Whole cell protein content

Question 4

What is metabolomics?

Answer 4

Whole cell metabolite content

Question 5

What is the microbiome?

Answer 5

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

Question 6

What is the microbiota?

Answer 6

Ecological community of commensal and pathogenic microorganisms
Includes bacteria, archaea, protists, fungi and viruses

Question 7

What is a biome?

Answer 7

A reasonably well-defined habitat which has distinct physiochemical properties

Question 8

What is the weight of the human microbiome?

Answer 8

70kg

Question 9

What is the weight of the bacteria microbiome?

Answer 9

1-3kg

Question 10

Describe the human microbiome

Answer 10

Not only 1 microbiome
Each area/part of the body has a distinct microbiome with different compositions
Unique to each individual (even twins)

Question 11

What may play a role in determining the microbiome?

Answer 11

Host genetics

Question 12

Describe the gut microbiome

Answer 12

Significant variation between healthy individuals
Integral to host digestion
Approximately 400 species
Diversity throughout GI tract - changing pH, adhesion, nutrients

Question 13

How can species be categorised in the gut microbiome?

Answer 13

Autochthonous
Allochthonous

Question 14

Describe autochthonous species

Answer 14

Indigenous/resident
Colonise GI tract

Question 15

Describe allochthonous species

Answer 15

Transient/passenger
Only colonise under abnormal conditions

Question 16

What do the species in the gut microbiome do?

Answer 16

Provide unique and specific enzymes and biochemical pathways
Competitive exclusion of pathogens, protecting the host

Question 17

What is the diagnostic role of the gut microbiome?

Answer 17

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

Question 18

How is the gut microbiome targeted for helping IBS?

Answer 18

Altering microbiota through dietary changes, probiotics or antibiotics can show benefit

Question 19

What is the microbiota-gut-brain axis?

Answer 19

Network of connections, crucial to homeostasis
Changes in the gut may be linked to symptom perception in the brain

Question 20

What factors can influence the brain?

Answer 20

Metabolic and immunological factors

Question 21

How does the microbiota modulate behaviour?

Answer 21

Short chain fatty acids
Modulation of neuroendocrine system
Bacteroides, bifidobacterium can producegamma-aminobutyric acid (GABA)

Question 22

What happens in IBS?

Answer 22

Inflammation in the gut, environmental and genetic risk factors
Alterations can be seen in gut microbiota

Question 23

How does colorectal cancer differ from IBS?

Answer 23

Higher proportion of Pseudomonas. helicobacer and acinetobacter
Lower richness of beneficial bacteria eg. butyrate producing bacteria
Reduced abundance of Bifidobacterium species

Question 24

What interventions can be used for gut microbiome?

Answer 24

Prebiotics - non-digestable food ingredient to promote growth of beneficial organism
Probiotics - beneficial organisms
Synbiotics - micture of prebiotics and probiotics

Question 25

What is most commonly in probiotics?

Answer 25

Lactobacillus Bifidobacterium

Question 26

What is an intervention that has proven to be effective for gut microbiome?

Answer 26

Faecal microbiota transplant (FMT) - example of probiotics

Question 27

What is antibiotic associated diarrhoea?

Answer 27

Related to overgrowth of C.diff

Question 28

What is susceptibility to C.diff infection associated with?

Answer 28

Decreased microbiota diversity

Question 29

What cures C.diff infection?

Answer 29

FMT

Question 30

How is FMT used to treat CDI?

Answer 30

Healthy donor, filter sample and administer by colonoscopy

Question 31

What happens to the gut microbiota after FMT?

Answer 31

Increased diversity Increased abundance of various Firmicutes and Bacteroides Decreased abundance of Proteobacteria

Question 32

How are psychiatric disease related to gut microbiome?

Answer 32

Dysbiosis increases translocation of gut bacteria into lymphoid tissue Provokes an immune response Activation of vagus nerve and spinal afferent neurons

Question 33

What are other associations to the gut microbiome?

Answer 33

Food allergies Asthma Obesity Diabetes Rheumatoid arthritis CVD Liver diseases

Question 34

What are technological approaches to assess the microbiome?

Answer 34

Targeted PCR amplification - 16S rRNA bacteria, internal transcribed spacer (ITS) 18S rRNA eukaryotes Whole genome shotgun sequencing

Question 35

What is 16S rRNA?

Answer 35

Component of 30S small subunit of prokaryotic ribosome

Question 36

Describe prokaryotic ribosome

Answer 36

50S subunit - 5S, 23S 30S subunit - 16S

Question 37

Describe eukaryotic ribosome

Answer 37

60S subunit - 5S, 28S 40S subunit - 18S

Question 38

How is 16S targeted PCR amplification done?

Answer 38

Sample collection DNA extraction 16S PCR amplification of whole sample PCR products are sequenced DNA sequences are analysed

Question 39

What 16S databases can be used?

Answer 39

Greengenes Silva RDP

Question 40

What are the 16S databases used for?

Answer 40

To separate samples into species/genus

Question 41

What is an operational taxonomic unit?

Answer 41

Used to classify groups of closely related individuals

Question 42

How are OTUs done?

Answer 42

Sequences are clustered according to their similarity

Question 43

What are the 2 types of diversity?

Answer 43

Alpha Beta

Question 44

What is alpha diversity?

Answer 44

Diversity within a sample

Question 45

What is beta diversity?

Answer 45

Diversity between samples

Question 46

How can alpha diversity be measured?

Answer 46

Species richness Species diversity

Question 47

What is species richness?

Answer 47

OTU count Measure of how many different species can be detected in a microbial system

Question 48

How is species diversity measured?

Answer 48

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

Question 49

How is beta diversity measured?

Answer 49

Bray-Curtis dissimilarity = based on abundance Jaccard distance = based on presence or absence of species UniFrac = based on phylogenetic trees

Question 50

How is a variable region chosen for 16S targeted PCR amplification?

Answer 50

Amplicon length Phylogenetic signal

Question 51

What needs to be considered when deciding what controls to use?

Answer 51

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

Question 52

How can contamination be mitigated?

Answer 52

Randomise samples Note batch numbers of reagents Sequence negative controls

Question 53

How does whole genome shotgun workflow work?

Question 54

What are problems with Whole genome shotgun workflow?

Answer 54

Host cells often in excess in sample No amplification step to enrich for bacterial DNA Sample dependent

Question 55

How can enrichment be done without amplification?

Answer 55

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