THE MICROBIOME

Question 1

when considering the microbiome, what is more important to consider than whether a species is beneficial or detrimental?

Answer 1

the relationships between species and how they are interacting within the microbiome

Question 2

some crazy microbiome facts?

Answer 2

50% of our cells are not our cells they are microbes

genes in our microbiome outnumber the genes in our genome 100 to 1

99.9% genome similarity between humans but microbiome between people often varies up to 80-90%

up to 90% disease can be reached back in some way to gut/microbiome health

Question 3

how do microbiomes and the host co-evolve?

Answer 3

selective pressure: climate change, switch from herbivore to carnivore habits, famines, infections, industrialisation

Question 4

how are microbiomes dynamic?

Answer 4

infants have low alpha-diversity microbiome but high beta diversity

as we grow beta diversity tends to decrease somewhat (people interact etc.) and alpha diversity increase

microbiome stabilises sometime during adulthood and then age related dysbiosis starts due to losing immune activity and diet change

Question 5

what is a well-established role of the microbiome and how has it been tested?

Answer 5

competition by commensal microbes protects from pathogens

tested lethal doses of salmonella on mice and was 10 mill in normal mice and 10 in germ free and 10 in normal with streptomycin

Question 6

how do commensal microbes protect from pathogens?

Answer 6

prevent it from being successful: block colonisation niches, competing for nutrients, modifying environment to change virulence factor expression

making environment actively hostile: bacteriocin and SCFA production, lowering pH, causes host to thicken mucus layer, causes host to upreg antimicrobial peptides, primes host neutrophils and macrophages

so we have to think about how bacteria are interacting w eachother to change environment not just obvious things like antagonism/competition

Question 7

what are some outcomes we see when gut microbiome health is favourable?

Answer 7

SCFA production

antioxidants production

low gut inflammation

improved lipid metabolism

reduced risk of infection

Question 8

what are some things good for microbiome health and what are some bad things?

Answer 8

good - probiotic intake, dietary fibre intake

bad - sugar intake, excessive protein consumption, saturated fatty acid intake, antibiotics, altered pH

Question 9

outline how gut microbiome benefits health by synthesising vitamins?

Answer 9

we don’t encode enough genes to synthesise all the vitamins we need for growth and development

a lot come from microbes response to the food we eat

approximately 80% of our vitB6 from gut microbes

this is cause gut microbiome has a lot more biosynthetic capability than us cause way more genes

Question 10

what happens when you dont have a microbiome?

Answer 10

germ-free and gnotobiotic mice have constant diarrhoea, require vitamin K supplements, have increased activity and reduced anxiety

it wont kill you but more susceptible to pathogens and also vitamin deficiency - but longer lifespan that SPF mice (pathogen free)

cant do any germ-free studies on humans tho

Question 11

outline short chain fatty acid (SCFA) production by microbiome?

Answer 11

SCFA produced by digestion of cellulose/starches and other fibres and important role in health

provide about 10% of our energy and some gut epithelial cells getting 70% from this

key SCFAs: butyrate, methane, acetate, propionate

ratio of these also important for immune function and different gut microbes synthesise different SCFAs

so theres a soup of metabolic intermediates and metabolites in the gut and microbes compete for these and drive reactions in favourable directions or competitors driving them back the other way by producing some intermediate etc.

Question 12

what is the link between SCFA and neurological disease?

Answer 12

SCFA produced from diet help produce key neurotransmitters like serotonin

proportions ofd SCFA important e.g. acetate stimulates serotonin more

other SCFAs important for tight junctions preventing pathogen invasion

Question 13

how do we study the microbiome?

Answer 13

early studies focused on composition, functional capacity and current function of community - problem is microbial species behave differently in different microbiomes

traditional culture based methods of studying limited, ribosomal RNA was a breakthrough and then genomics

Question 14

how do we study microbiome function?

Answer 14

understanding community composition didn’t do much to understand function in relation to human health

need better understanding of interactions between species (ecology) and if it changes species function

can use shotgun metagenomics to understand different genes (but we dont know what’s being transcribed or upregulated)

Question 15

despite there being huge community variation between microbiomes, what did one study find?

Answer 15

that despite the variation they still had similar functional capacity

but 50% of the genes functions we don’t know

Question 16

what gap in our understanding of the microbiome could protein-based and metabolite-based approaches help fill?

Answer 16

huge gap in understanding of what the microbiome is doing and how what it is producing is affecting human health

Question 17

what is dysbiosis?

Answer 17

microbial imbalance or maladaptation

can be caused by antibiotics

Question 18

how do species respond differently to antibiotics?

Answer 18

when you take antibiotics opportunists spike but then drop back down as survivors make a come back

de novo colonisers that are not normally present in microbiome but antibiotic treatment opens up a niche for them

can also eliminate species from microbiome

Question 19

when antibiotic usage fucks up SCFA production?

Answer 19

certain byproducts can build up which can get in the way of other shit and fucks everything up

e.g. decrease of propionate causes increase of precursors which indirectly cause increase of precursors for butyrate which causes increased butyrate

basically the whole balance just gets thrown off and if byproducts build up we might not be able to absorb them all

Question 20

how can antibiotic usage lead to osmotic diarrhea?

Answer 20

antibiotics can cause build up of precursors or solutes cause SCFA transport coupled to transport of many other ions

after treatment microbiome usually returns to normal but sometimes causes longer problems (e.g. c. difficile)

Question 21

what is C. difficile?

Answer 21

associated with antibiotics which allow it to proliferate - extensive multiplication only occurs when commensals depleted

produces toxins and virulence factors that cause colitis and diarrhea

can form spores so good at persisting and spreading in environment - stomach acid kills vegetative cells but not spores

spores germinate in small intestine and colonise colon/large intestine

hard as fuck to remove/treat once infections

Question 22

what toxins and conditions does it cause?

Answer 22

produces toxin A, toxin B, binary toxin

results in antibiotic-associated diarrhea, pseudomembranous colitis, toxic megacolon

these hard to treat

Question 23

discuss the relationship between bile salts and microbes?

Answer 23

liver produces primary conjugated bile acids

microbiota deconjugates these with bile salt hydrolase and then modifies primary to secondary bile acid with bile salt 7 alpha-dehydroxylase

this way 95% of conjugated bile acids are reabsorbed

theres different microbiomes throughout the intestine

Question 24

outline bile salts and the lifecycle of clostridium difficile?

Answer 24

germination of spores promoted by most bile salts

secondary bile salts inhibit germination and vegetative growth

so the concentration of these metabolites keeps c. diff under control

key player in microbiome is c. scindens; not much living in microbiome but important role in converting primary to secondary

so everything kept in check then throw in antibiotics and your fucked

Question 25

specifically how do antibiotics exacerbate c. difficile infections?

Answer 25

c. scindens (converts primary to secondary bile salts) very sensitive to antibiotics (especially vancomycin) so population gets knocked down during treatment for other shit

means there a lot less secondary bile salts and thus less suppression of c. diff (isn’t very sensitive to vancomycin either)

Question 26

why is it hard for c. scindens to recover following c. difficile infection?

Answer 26

competition

Question 27

why is vancomycin a bit fucked for the gut microbiome?

Answer 27

fucks up c. scindens (primary to secondary) and also fucks up bacteroidales (super common in our gut)

cause its quite broad acting so fucks the whole microbiome up making c. difficile infection easier

Question 28

outline fecal transplant therapy?

Answer 28

used to treat antibiotic side effects by transplanting microbial communities

some cases pre-treatment with antibiotics to really knock back microbiota for better establishment of a good one

shown to be effective against c. difficile - robust clinical outcomes

Question 29

why is enterococcus particularly dangerous for stem cell transplant patients (SCTP)?

Answer 29

enterococcus intrinsically resistant to a lot of antibiotics - ability to acquire vancomycin resistance which is last resort antibiotic

SCTP often receive prophylactic antibiotic pre-treatment to knock back immune system so that new stuff can be added

this makes them severely immunocompromised

VRE can cause fatal bacteraemia and colonisation can persist for years - microbiome may go back to homeostasis but future antibiotic treatment will knock everything back leaving the resistant enterococcus to colonise

Question 30

how do c. difficile and enterococcus infections cause us to rethink Koch’s postulates?

Answer 30

two of the postulates are:

the microorganism must be found found in abundance in all organisms suffering from the the disease but should not be found in healthy organisms

the cultured microorganism should cause disease when introduced to a healthy organism

Question 31

what is inflammatory bowel disease?

Answer 31

covers CD (deep damage - fistulas and leakage) and UC (predominantly affects colon - superficial tissue damage)

increased incidence over the last 30 years but unclear if increased diagnostics or disease incidence - big shift in diet so could be either

both genetic and environmental factors

Question 32

what is the evidence for an IBD-microbiome link?

Answer 32

some studies have shown antibiotics can help keep people in remission

studies on ileostomys as treatment for severe cases have shown exposure to lumenal contents influences inflammation

IBD cunts have distinct microbiomes - few species; cause or symptomatic tho?

many IBD-associated genes relate to sensing bacteria

new-onset IBD associated with changes in microbiota

Question 33

outline the how IBD can result in a negative feedback loop further exacerbating it?

Answer 33

in IBD we see immune system overeacting in the form of pro-inflammatory response

inflammation damages microbiome which further exacerbates inflammation and gets worse and worse

cause healthy microbiome important for production of mucin and AMPs and shit that maintains normal barrier and stops things causing inflammation

Question 34

what is the evidence mouse models are highly influenced by bacteria?

Answer 34

knocking out IL-10 gene in mice meant they got UC but if raised germ free they don’t

Question 35

does dysbiosis cause disease or does disease cause dysbiosis?

Answer 35

option 1 - dysbiosis causes disease; changes in microbiome lead to sickness (this is how crohns works in mouse models)

option 2 - dysbiosis is caused by disease; sickness>inflammation>microbiome changes (definitely happens in people with crohns)

option 3 - dysbiosis not the root of disease but may perpetuate it

Question 36

what are some microbiome based interventions?

Answer 36

fecal microbiota transplants

fecal filtrates (filter out bacteria but phages and molecules e.g. SCFA still there) (less chance giving someone c. difficile)

specific diets for IBD fucks e.g. fermented foods, pro-biotics

phage therapy - kill problematic members of community

post-biotics - manipulate host immune response to return to homeostasis

Question 37

outline the recent study that found Klebsiella pneumoniae to be a potential culprit behind IBD?

Answer 37

study look at specific species causing disease by comparing diversity and composition of microbiomes in IBD vs healthy

then looks for specific members of microbial community that changes between IBD and healthy patients

found K. pneumoniae highly enriched in IBD patients but few others

K. pneumoniae numbers decreased when patients went into remission

put identified strains in germ-free mice and showed upregulated IFN-gamma and CD4+ T cells after a couple weeks and also saw inflammatory repsonse in intestinal tissue