Microbiome Flashcards
(34 cards)
Animals harbour complex microbial community in intestines. What type of animal is it particularly important for? What two different kinds of this type of animal are there?
Herbivores (Foregut fermenters [ruminants: large fermentation chamber in stomach, regurgitates food for further chewing], Hindgut fermenters [fermentation takes place in large intestine, often eats poop to extract more nutrients])
Where do you find microbes in humans?
Which bacteria are most common in the gut?
Skin (1 million/cm3), oral cavity (1 billion/mL), stomach and duodenum (10-1000/mL), jejunum and ileum (0.01-100 million/mL), colon (10-100 billion/mL)
Firmicutes (60-70% of fecal bacteria), bacteroides (20-30% of fecal bacteria), actinobacteria, proteobacteria
Oral cavity, stomach/duodenum, small intestine, large intestine: pH and transit time differences?
Bacteria in the colon are _______ _________
Main sites of interaction with immune system?
Oral cavity (pH5-7, sec-min), stomach/duo (pH1-3, 1-2h), small inestine (pH 6-7.5, 1-5h), colon (pH5-7, 10h-days)
strictly anaerobic (less oxygen as you go through system)
Oral cavity, small intestine, and colon
At birth, gut is _______. First bacteria are from where?
What else tends to affect microbiota development?
How long does it take to develop adult microbiota?
What can affect microbiota overall? Which of these is most important?
Sterile. Vaginal/fecal natural, skin bacteria from caesarean
Breast feeding (bifidobacteria) or formula feeding (more complex)
Several years (and diet can make big varieties)
Genetic makeup, immune system, and environment (cleanliness, pets, diet, medications, etc) - environment seems to be most important
Early childhood is crucial for proper maturation of the immune system. What does it enable it to distinguish? Why is the microbiota important?
What is the hygiene hypothesis?
Resident microbiota and harmless environmental agents (tolerance), and pathogens (bacteria, viruses, fungi, parasites, etc - attack). We don’t know why exactly it is important, but we do know it is necessary.
Changes in microbiota due to lifestyle alterations + genetic predispositions = increases in allergies, asthma, autoimmune, etc.
What health conditions have been potentially linked to microbiota?
_____ overall microbial diversity is associated with several disease states
IBS, IBD, colorectal cancer, obesity, metabolic syndrome/diabetes, non-alcoholic fatty liver disease, depression, autism
Lower [NB: with all these things it could either be a causal relationship, or just a coincidence, so be aware when reading papers]
Beneficial and negative effects of microbiota? [4/4]
Benefit: barrier function against pathogens, immune maturation/stimulation, release and transformation of dietary molecules, production of metabolites (with positive end results)
Negative: opportunistic pathogens (that were commensal), may contribute to inflammatory diseases, release and transformation of toxic compounds from diet/environment, production of metabolites (with negative end results)
Microbiota-Diet-Host Interactions [picture summary]
What’s the main source of food for the microbiota?
What happens if we don’t get enough plant material and have too much proteins/fat?
Carbohydrates that we can’t digest (structural polysaccharides and non-digestible oligosaccharides) and remaining starch/sugars
Not much gets through to microbiome = toxins, carcinogens, and lower bacterial activity (lower supply of good metabolites, weaker barrier function)
Main substrates of dietary [4] and intestinal [1] origin for microbiota?
Resistant starch (fraction of dietary starch–amylose/amylopectin–that resists upper gut digestion and enters colon), non-starch polysaccharides (mainly plant cell wall components - cellulose, hemicellulose, pectins, gums, etc), resistant oligosaccharides (inulin, fructan from onions/artichoke/etc, etc), dietary protein
Mucus from the gut wall
What happens to the non-digestible carbs when they reach the microbiota? What three things are they mostly broken down to?
Acetate, butyrate, and propionate
Which bacteria converts starch to acetate?
Which bacteria converts acetate (or lactate) to butyrate?
Which converts lactate to propionate?
What converts oligo/monosaccharides to butyrate?
Bifidobacterium adolescentis
Eubacterium hallii
Coprococcus catus
Roseburia hominis
Short chain fatty acids (acetate, butyrate, and propionate) produced by gut bacteria give how much of our energy? Where is each one mainly metabolized?
What are the dual actions of butyrate (“butyrate paradox”)? WHat other effects does it have?
10% (Acetate = muscle and organs, propionate = liver, butyrate = colon)
Promotes proper function of healthy colon cells (main source of energy), but seems to inhibit growth of cancer cells [Others: anti-inflammatory, regulation of hunger/satiety]
What causes the pH differences in the proximal and distal colon?
Why might more diseases (eg: UC, colon cancer) start in distal colon?
[See pic]
Due to lower levels of bacterial activity (and fewer fermentation acids being formed)
What are phytochemicals?
What are xenobiotics?
How are the microbiota involved with these?
Different plant derived compounds also present in diet (includes many phenols - seem to be health promoting)
Anything not normally found in organism (drugs, pollutants, etc)
Organisms can release compounds (eg: fibre-bound phenolics) or biotransform them into other compounds - they can then operate differently with host machinery, and effect physiology
Microbiota can transform the following into what…
… proteins? [3]
… primary bile acids? [1]
And what else are they involved in the production of? [2]
Phenolic acids, polyamines, N-nitroso compounds
Secondary bile acids
Neurotransmitters (eg: gamma-aminobutyric acid) and vitamins
What is the exchange between metabolism of microbiota and host called? Give an example
Enterohepatic circulation (dietary polyphenols with sugar attached - bacterial glycosidase cleaves off sugar, turning it into an aglycone - this is taken up by the liver, and has B-glucaronic acid attached, and returned to the gut - bacteria then slices off acid (to also use as food stuff, etc)
What is the culturability of microbes partially dependent on? Give examples of each, and how this impacts their culturability.
Even in a culturable system, what might still be difficult?
Natural environment: soil microbes can be difficult (dormant or slow-growing due to nutrient-scarce, frequently-changing environment), gut microbes can mostly be cultured (stable environment, nutrient-rich, open system with turnover).
Establishing the requirements of all the different microbes present
Lay out the steps of effectively culturing gut bacteria. What might affect media selection?
1) Get poop
2) Dilute poop so that it will grow single colonies on the plate
3) Select media: complex media (can feed many microbes, but it can be difficult to ID individual bacteria, and it’s difficult to obtain rare bacteria) or selective media (growth of particular microbe, suitable for rare bacteria)
Culture independent analysis: what gene is often used for genetic analysis of samples? What does it do? Why is it used? How big is it?
What are primes and probes in relation to genetics? What does FISH stand for?
16S rRNA gene (structural component of ribosomes, 15kb long): present in all living organisms (18S in Eukaryotes), has conserved regions that are largely shared, and 9 variable regions that demonstrate more differences between organisms
Primers = short oligonucleotide chain required for amplification of DNA (in Polymerase Chain Reaction), Probe = short oligonucleotide containing marker (usually fluorophore) that can bind to DNA (for techniques such as Fluorescence In Situ Hybridization)
Explain PCR occurring on the 16S rRNA gene…
Double stranded DNA melted to single strands (95 degrees), primers (about 20 nucleotides long) coded to particular conserved regions at the ends of the gene bind (50-60 degrees), DNA polymerase (from thermophile) is allowed to copy each strand (72 degrees), and then strands are heated to break them apart (and the process begins again)
Using the 16S rRNA gene for PCR analysis, how would you broaden or narrow your analysis? How might these different levels of analysis be useful?
Why might a broader analysis be more difficult?
Different primers: you would use primers targeted at conserved regions to capture a broader range of microbes (investigating whole community, changes within it, and overall diversity), and those targeted at variable regions to look at more specific groupings (looking at alterations in the specific population of one group before/after an intervention)
Different microbes have different extraction methods (too gentle will not lyse some cells, whereas some DNases may degrade the DNA of others, etc)
Name DNA-based methods that can be used to ID specific microbes, and those that use a “profiling method” (fingerprint of community) [4/2]
Can you determine the ID of microbes at all using profiling methods?
ID: sequencing, qPCR, FISH, phylogenetic microarrays, Profiling: DGGE, tRFLP
No. Identity remains unknown without further downstream analysis.
What does DGGE stand for? What is it used for? How does it work?
Denaturing Gradient Gel Electrophoresis (profiling microbe community). You take your sample and do PCR amplification of the 16S rRNA gene - one of the primers has a 40-GC-repeat (more hydrogen bonds) clamp. You then put them through gel electrophoresis with a concentration gradient of a denaturing agent - as the 16S genes will have differences, they will (mostly) break apart at different times (held together by clamp), and this differential break-up will affect their movement speed through the gel. In this way, you can identify differences in microbe composition, and potentially choose what to sequence.
