Microbial Ecology Final Exam Study Guide Questions

Probiotics

- "live microbes that when given an adequate amount have a beneficial effect on host" - often in research use in vitro or animal models and animal strains of probiotics different from human probiotics (changes in colonization efficacy) - if we are just talking about Antibiotic Associated Diarrhea, then there is some evidence that probiotics (such as eating yogurt) can decrease changing of diarrhea - in terms of C. diff, not enough evidence to officially use as treatment

How does circulating levels of LPS potentially tie into diabetes? What about the exact structure of LPS?

- Finland vs Russia - B. dorei made LPS that suppressed immune responses to it and to other LPS types like E.coli - immune system never learned tolerance = microbial disturbance on impact of development for reason for having T1D - for T2D, higher than normal circulating amount of LPS - T2D: decrease in butyrate producers = could increase circulating LPS and allow for increased inflammation - metabolic endotoxemia

Be able to explain the experiments and results from the 2013 study by Ridaura et al.

- Gut microbiota from twins discordant for obesity modulate metabolism in mice - transplanted human microbiota into mice - started with 1500 people (750 twin pairs), adults, discordant for obesity (one obese and one lean), consistent trend over couple of years - starting with that, ended up with FOUR twin pairs that fit criteria - took fecal sample from lean twin, put it in mouse, stayed lean - took obese fecal sample, put it in mouse, became obese - microbiota appears to be the sole factor influencing obesity - same diet, environment, age, how they were raised; only changed was microbiota - stability? diet? - series of cohousing experiments: cage of lean mice and a cage of obese mice (two of each) - control: dropped new lean mouse in cage of lean mice and dropped new obese mouse in cage of obese mice (no changed) - put obese mouse in cage of lean mice? - put lean mouse in cage of obese mice? - the obese mice become leaner but nothing happens to the lean mice (do not become obese) - arguing that the lean microbiota appears to be more stable, obese microbiota might be changeable - saw "invasion" of lean microbiota into obese animals - only saw this when using a "lean" diet (non-Western diet; low fat-high plant polysaccharide diet) - gave people the idea that fecal transplants might work for obesity because lean microbiota could invade but will also need to be close attention to EF to get it to work (like diet)

Describe the study from Kim et al. What is the effect of the maternal microbiota on the MIA offspring? What data support this?

- Is there something about the microbiota of females that makes their infection increase the baby's risk for autism? - Role of maternal microbiome? - MIA model again - use same immunostimulant - Asking about the effect of maternal microbiota? - Still looking at behavioral differences in the babies - Maternal bacteria promote abnormal behaviors associated with neurodevelopmental disorders in MIA offspring - Looking at behaviors in baby mice - USV (ultrasonic vocalizations) - high pitched noises seen in autistic mice - Repetitive behaviors seen in autistic mice - Time that mouse interacts with other mouse vs rubber stopper - Expectation is that autistic mouse would interact more with rubber stopper (not sure how this really relates to human autistic behavior) - Also looking at markers of inflammation - Treatment given to mothers: control or vancomycin - Saline +/- antibiotic and immunostimulant +/- antibiotic - If you don't change mother's microbiota, the immunostimulant increases autistic behaviors in babies - If you add vancomycin, you don't see (in many cases) a difference in the offspring - Changing the mother's microbiota changed the impact of the immunostimulant - Conclusion: Only certain pregnant females that if they get an infection, there is an increase in autism (based on their microbiota) Infection during pregnancy? - Some suggestion of 2nd or 3rd trimester (not the first) - Severity of infection - Higher risk with significant fever or hospitalization Mice - Because a difference is seen in IL17, what microbes are known to stimulate IL17? - C57BI/6 mice - Two different vendors - mice from different vendors had different levels of SFB - Taconic- Lots of SFB (segmented filamentous bacteria) → expect to see higher rates of IL17 - Inducer of IL-17 production - Jackson labs - No detectable SFB → not expect to see high rates of IL17 SFB in the pregnant mothers promotes abnormal behaviors in MIA offspring - Looking at the mothers on the x-axis - Mice from taconic vs jackson lab (jax) - Cohoused and gavaged have SFB - Only case where immunostimulant doesn't matter is in the jax only mice with no SFB - "Certain pregnant females" = those that have a microbiota that promotes IL17- Get infection during pregnancy, there is an increased risk of autism for the babies Where are we? - Heterogeneity and diagnosis- How to group individuals with autism? - Animal and human studies - Fecal transplants - Study to Explore Early Development (SEED) - Trying to recruit huge numbers of kids and track many aspects of behavior - CDC

Type 1 diabetes

- Juvenile diabetes, but onset anytime - More commonly diagnosed in children - Autoimmune disorder - Attacks insulin-producing cells in pancreas (destroys them) --- By the time of diagnosis, usually a decent portion of the pancreas has been destroyed - Big problem: lack of insulin production

What is the evidence for microbial involvement in Type 1? Be able to explain several points

- Largely circumstantial evidence a. concordance rate in monozygotic twins (genetics) - Concordance rates - between 30-60% ... room for EF b. migrant studies (EF) - Migrant - when people move to Sweden...within 1-2 gens...the T1 incidence matches that of individuals that were born and raised in Sweden c. viruses -- recently diagnosed -- animal models -- epidemic outbreak? -> Viruses - viral involvement -> Being a trigger in susceptible individuals -> Recently diagnosed...more often than not have suffered from (a variety of) viral infection -> Animal models - can trigger onset with viral infection -> Epidemic outbreak...1993...mumps/measles epidemic...2 years later, that state had a bloom in T1D d. general role in inflammatory disorders --- hygiene hypothesis ---autoimmunity: -> Increase in allergies and asthma : see increase in T1D -> Leads to immune system imbalance that can lead to many inflammatory disorders but also autoimmune disorders > increase in allergies and asthma = see increase in T1D > leads to immune system imbalance that can lead to many inflammatory disorders but also autoimmune disorders e. rodent studies - microbial changes and impacts ---- increased Bacteriodetes --- reduced Firmicutes - rodent models ---- antibiotics ---- hormones f. human studies - diabetes prediction and prevention study (Finland) - children with T1D: increased Bacteroidetes to Firmicutes - TEDDY study -> recruited over 12,000 participants when study started based on idea that there some chance, they would develop T1D...only 800 of them had (less than 10% of population ended up with what they were looking for - Multifactorial studies - Finland study: T1 prevalence in Finland vs Russia; have 6x higher prevalence than Russia...suggestion of EF Microbiota - altered bacteroidetes/firmicutes ratio - low mucin, low butyrate, low microbial diversity Hypothesis for Involvement - Two Phases 1. Birth to first T1D-associated antibodies 2. Antibodies to T1D - Have a kid that is born, track them until they have first specific antibodies to insulin producing cells...period of time after this until they have T1D - Think a microbial disturbance has to happen here that leads to improper development of immune system - To cause process to continue/speed up - environmental trigger - Maybe a viral infection or gastrointestinal infection - lead to reduced microbial diversity or might lead to unusual inflammation for period of time - immune system development happens early on - evidence that microbial metabolites might matter when pregnant - this would be the time period where microbiota might be important

Describe the study from Hsaio et al. What defects were observed in the MIA offspring? What effect did B. fragilis have on the MIA offspring?

- Maternal immune activation (MIA) model - If pregnant women get some type of infection during pregnancy, there is a higher likelihood that the baby will get autism - Inject pregnant mice with an immunostimulant (polyinosinic:polycytidylic acid) - Could also inject mice with a virus - Look at the offspring for autistic related behaviors - Gave pregnant mice saline as control or the immunostimulant → give birth → measure behavior in babies, look at barrier function or inflammation - Babies in this study got the Bacteroides fragilis treatment - Everything in this study was measured in the offspring - Questions being asked in this study: Does MIA model really lead to babies with autistic behaviors and can microbes be used to treat these behaviors? - Alternation of gut permeability - changes in barrier function (leakier gut) - Serum metabolites - 4-ethylphenylsulfate (4EPS)and p-cresol - Indolepyruvate - Increases in these serum metabolites in the babies when the mother had been given the immunostimulant - In autistic humans, we also see increases in these serum metabolites (maybe these act as biomarkers for autism) - Treatment with Bacteroides fragilis - Gut permeability and serum metabolites were fixed - Did not work with an enterococcus → suggests some type of specificity - DSS → increase in leakiness and inflammation - Mothers for S got saline, mothers for P got immunostimulant - Not much gut leakiness if mothers get saline - If mothers receive immunostimulant, you see gut leakiness in babies - Mothers given saline → babies have better barrier function (more proteins involved in barrier function) - More inflammation in the babies if mothers are given immunostimulant - Same experiment as before, but babies are now given B. fragilis... can we change the defects with microbes? - Treating babies with B. fragilis returns things to normal - Conclusion: if a pregnant female has some type of infection during pregnancy, it leads to an increased risk in the offspring of autism. But you may be able to correct that by changing the microbiota of the offspring early enough

Why was the hygiene hypothesis originally proposed (What trend was seen? What data was analyzed?)

- Strachan - hay fever, hygiene, and household size - longitudinal study of 17, 414 British children, all born in one week in March that year (1989) and followed them for 20 years - 16 factors (perinatal, social, and environmental): socioeconomic status, family size, EFs: rural vs urban, pets vs no pets, etc. - looked for correlations with the factors and hay fever, eczema, and allergic rhinitis - for the last few decades prior to this, noticed overall increase in allergies and asthma; did not have an understanding of what drove that, so looked for things that changed in the population - found that there was a good correlation between family size, birth order, and allergies, most specifically hay fever - the more children in the family, the less likely you were to see hay fever - the oldest children in these families had the highest risks of allergies (hay fever) and the younger ones in order have the lowest risks initial proposal - infection in early childhood may prevent allergic diseases - recent declines in family size and increases in sanitation, and hygiene have led to decreased childhood infections, particularly within families - in larger families they probably shared more infectious diseases - increase in allergies and asthma is because the family sizes have been getting smaller so kids are having less overall childhood diseases - decrease family size and increase in hygiene led to decrease in overall infectious diseases in children - original hypothesis = decreases rate of diseases is leading to higher numbers of allergies/people with more allergies

Type 2 diabetes

- associated with metabolic syndrome - associated with obesity but can get it with any shape or size - cells in body become resistant to insulin; even though they are being produced, see less of an impact - body tries to increase insulin production and over time, pancreas get worn out - tied to a variety of other metabolic issues...insulin production is fine, just cells are not responding to it

Explain epithelial cell proliferation and migration (in the general way that was discussed in class)

- at base of crypts, have intestinal stem cells where all new cells form from - proliferation: most stem cells after making new cells - the new ones travel up villi and differntiate into enterocytes, goblet cells, enteroendocrine cells - serve their function - after a period of time, go to top of villi and are either sloughed off or apoptosis happens and they die - small percentage of remaining cells will migrate down to crypts and become Paneth cells goblet cells = make mucus enteroendocrine cells = produce hormones (serotonin) paneth cells = produce antimicrobial proteins (defensins) epithelial cells = barrier function

What were the initial data that suggested that the microbiota might be associated with obesity?

- back to GF animals - obesity was probably first disorder they tried to tie to microbiota - based on difference between GF and conventionalized mice - noted early on, if you take GF animals and conventionalize them, they eat less (20-30% less) but they weigh more (in terms of % of body fat; 40% more fat than GF animals) - first suggestion: difference in metabolism (slower metabolism, absorb more nutrients, eat less and weigh more) - but actually, the GF animals had slower metabolism - has to be connection between gut microbe and mice

Thuricin CD

- bacteriocin, CD stands for C. diff - produced by Bacillus thuringiensis - very narrow spectrum of activity; appears to only attack C. diff - used to prevent disruption - using this narrow spectrum works better than other ones because it targets C. diff which is not a normal member of the microbiota so not included in any other microbe's food web so able to wipe it out without harming other microbes - with normal treatment (Vancomyocin or Metronidazole) and even Lacticin 3147 (broad spectrum antibiotic), see total disruption of the microbiota even though Lac 3147 is a bacteriocin like Turicin CD; all show increase in proteobacteria (phylum level) - at family level, treatment with TCD see some changes like increase in Enterobacteriaceae, but no dramatic changes like those seen with V, M, and Lac - important because it is a natural product (from C. diff)

What is the gut-brain axis?

- communication - bidirectional communication between gut and brain - things that happen in brain influence what happens in gut and vice versa - driven in many ways: hormonal signaling, immunological signaling, neurotransmitters, other signaling (vagus nerve) - functions: a. gut - changes in barrier function that is influenced by things in the brain, changes in mucus levels, motility, immune system presence, hormone levels b. brain - changes in feelings like anxiety, ties with depression, and other things stress and depression: - by themselves can alter gut microbiome (change community) - anything that influences gut microbiome can be influenced by this - depression = higher circulating levels of pro-inflammatory cytokines

What are the proposed functions for bile salt hydrolase activity in terms of how it benefits bacteria?

- deconjugation which is removal of the Taurine and Glycine - extremely variable - many different types of organisms - one or more - inside or outside cell - exponential vs stationary - specificity in recognizing certain (de)conjugated bile acids with specific amino acids or none at all importance (deonjugation) a. colonization b. detoxification c. virulence factor d. nutrient acquisition

Are probiotics a possible treatment for certain disorders of the central nervous system? Why or why not?

- effects seem to be extremely strain specific - some evidence of impacts on depression, anxiety, and perhaps stress-associated problems - some evidence of impacts on pain perception: > particularly GI pain perceptions > people with IBS not only have functional issues but some report gastrointestinal pain > question about whether it is actual pain or perception of pain > are they getting increases in pain signals from what others would perceive as small amounts > if it is pain perception, maybe probiotics can help with that > measure GI pain perception? > same for animals and people > balloon, more sensitive pain perception = the less they can blow it up Nothing definitive so far!

SER-109

- fecal transplant alternative - most promising - poop in a pill - purified spore preparation (bacteria that form endospores) DATA Phase 1 clinical trial showed good promise: 87% cure rate for recurrent C. diff; no risks because purified and know what is in the pill Phase 2 - FAILED - no significant reduction in recurrence - even broken down by age group (less than 65 and greater than 65) - based on screening, the samples did not act like they expected - under 65 had lower reoccurrence than expected for placebo - over 65 had higher rates than expected for placebo - in over 65 pop without recurrence, found better establishment from microbes from treatment that those with recurrence - if organisms were able to establish themselves, they prevented recurrence but the presence and absence of those microbes varied - phase 1: good establishment with high and low dose - phase 2: lower establishment with variable levels Phase 3? - changes: increase dose and screen for toxins - screening not just for presence/absence of C. diff, but for toxins produced by C. diff - preliminary results? - at 8, 12, and 24 weeks with higher dose and toxin screening, found that this did substantially prevented recurrence across age groups - finishing analysis and asking for FDA approval in mid 2022 - first more purified form of fecal transplant with FDA approval

What are the Conjugated bile acids?

- have amino acids added to them (Taurine and Glycine) - this happens in the liver - made by the body not microbial activity - TCA - GCA - TCDCA - GCDCA

Explain the data involving Salmonella enterica serovar Typhimurium infection that supports the concept of colonization resistance

- healthy microbiota can prevent colonization and overgrowth of invading microbes - first investigated in 1950s by Bohnoff et al - effect of antibiotic treatment on infectious dose of Salmonella enterica serovar Typhimurium - animal model - wild type mice with healthy microbiota - difficulty to consistently colonize them with salmonella - if treated with antibiotics first, could use dose of salmonella that was 100,000 times lower and consistently colonize gut microbiota with salmonella - damage gut microbes first in order to get it to colonize How successful is the pathogen? - first, conventionalized mice, normal microbiota - see lots of variation - colonizes between high levels and none at all - difference is due to mice being from multiple different studies - "house" microbiota was different from study to study and has colonization resistant efficiency at different levels (some better than others) - antibiotic treatment - every mouse gets high levels of colonizations - GF mice - every mouse gets high levels of colonization - LCM = low complexity microbiota > group of 8 different microbes, based on knowledge at the time, 8 (types) representative microbes required as bare minimum to have good gut microbiome > from CR aspect just as bad as antibiotic and GF > every mouse got high level of colonization now, to have CR, need diverse microbiome, and specific microbiome in terms of host association (human microbiome will not protect mouse as well as mouse microbiome will) - in absence of microbes, can get high levels of pathogen colonization - shown with many pathogens - take advantage if gut microbiota is damaged - therapeutics - put in normal microbiota - should decrease pathogen if not wipe it out completely

Explain the results from the Wheeler et al study (fungal dysbiosis).

- immunological consequences of intestinal fungal dysbiosis - mice given antifungal (Flucanazole) - decrease in fungi leads to decrease in allergic responses (expecting this) - treated mice had increased inflammation and increased antibodies (increased allergic response when changed fungal community) - tested two other antifungals and got the same results - antifungals increase inflammation as measured by antibody levels and cell types - antifungals increased airway allergic response - decreased Penicillium brevicompactum and Candida tropicalis - increased Aspergillus amstelodami, Wallemia sebi, and Epicoccum migrum - those those caused the increase in inflammation

In general, what have animal studies suggested in terms of the compositional changes in obese vs lean animals? Human studies?

- increased ratio of Firmicutes to Bacteroidetes a. in ob/ob mice b. in diet-induced obesity ("Western diet") c. in "humanized" mice - decreased diversity - Original studies said there is a signature for obesity and in animals it does not matter the cause - Signature of a phenotype - They had an increased level of Firmicutes and a decreased level of Bacteroidetes - This was true whether the obesity was triggered genetically (ob/ob), by diet, or by humanizing the mice (with fecal samples from obese person) - Does not matter, see this in obese mice regardless of mode of obesity F:B ratio - Decreased diversity Specifically richness - less species

What types of microbial changes are potentially associated with allergic diseases? (either cause or effect)

- microbial changes associated with allergic disease - very among studies - decreased diversity - different studies look at different phyla to different fungi to different viruses to everything - cannot say if you have this, you baby will have allergy - same type of trend seen for IBD and obesity - decrease in diversity = negative thing - do not know which microbes are involved at all possible mechanisms - altered immune system development or response a. Treg cells b. dendritic cells c. regulation of IgE d. Th1/Th2 and Th17 - how a. early microbial exposure b. microbial metabolites

Describe IBS in general (symptoms, cause, etc)

- most widespread GI disorder - chronic disorder - unknown cause - 15% of the population suffers from IBS - various onset ages - heterogeneous symptoms/presentation 1. inflammation: in GI tract but mild relative to IBD 2. abnormal motility: chronic diarrhea, chronically constipated, alternative between the two, no normal bowl movement 3. abnormal pain/sensitivity: in GI tract 4. many individuals also suffer from anxiety and/or depression - abnormal % of people with IBS suffer - Gut-Brain Axis and in which direction the abnormalities might be traveling - gut affects brain or brain affects gut - antidepressants used to treat this are not first line treatments for depression - do not understand how it helps but helps a subset of people

What is the evidence for involvement of environmental factors in the development of allergic diseases?

- rapid increase - difference in western vs not western communities - increase primarily in westernized or developed communities - antibiotic usage (first started this in WWII) - everything that happens in first year of life: a. pregnancy b. delivery c. feeding style d. family size e. antibiotic / infection history f. vaccinations g. pets

What impacts do microbes have on epithelial proliferation, migration, and differentiation? Gut motility? Mucus? Barrier Function? GALT? Blood vessel formation? Basic structures in GI tract (crypts and villi)?

- reduced proliferation - reduced migration - reduced differentiation - reduced gut motility - reduced amount of mucus and its composition - alters barrier function = leakier gut - GALT is underdeveloped so lower immune system presence - BV formation is decreased - thinner and taller villi - shallower crypts MORE DETAIL IN FIRST CARD

Over the last 20 years (including recent data): How has the definition of the hygiene hypothesis shifted?

- revamped definition of the hygiene hypothesis - reduction in antigen exposure - imbalance in immune responses - favors chronic inflammatory conditions - allergic disease, IBD, autoimmune diseases - increased in immune related disorders (vague) driven by decreased antigen exposure (environmental antigens, animal antigens, antigens to different types of pollen, lives in cities, spend time in inside a lot) - mechanism: immune system imbalance - favors diseases where inflammation is not well controlled like asthma, allergies, IBD, other autoimmune disorders - in microbial world, "Old Friends" hypothesis and the "Disappearing Microbiota" hypothesis - in terms of decreased antigens is actually a decrease in microbial diversity Rise in asthma and allergies in this time period, all infectious diseases decreased, correlates with increase in autoimmune disorders

Non-toxigenic C. difficile

- some strains of C. diff do not produce toxins thus do not cause disease or damage to microbiota; but those that do are the ones that cause damage - they have found that is you colonize someone with non-toxic C. diff before they are infected with toxic C. diff, then it outcompetes the toxic ones and causes no damage - however, how do you know know who to colonize since it has to be done ahead of time? - another issue is HGF; temperate phages can sometimes carry toxic C. diff, so how do you know it will not evolve/develop into the bad C. diff once inside? - we do not know exactly how everyone will respond to this - 80% of the people given the non-toxic C. diff will never be a risk for developing the toxic kind so it can be an unnecessary intervention

How is the brain thought to influence the microbiota?

- stress causes compositional changes (phylum or genus level) - decrease in Bacteroides and increase in Clostridium - mechanisms are largely unknown - potential alterations to intestinal environment: a. motility - driven by hormone levels b. mucus - amounts in gut c. barrier function - in gut

What are the Deconjugated bile acids?

- the amino acids (Taurine and Glycine) are removed - CA - CDCA - this happens in the colon through microbial activity - these are the ones converted to the secondary bile acids

What makes a "good" fecal transplant donor?

1. A healthy microbiome - do not know what this looks like though - but do know it should contain High Diversity and Absence of Pathogens 2. Phenomenon of "Super-Donors" - people whose donations have had ridiculously high success rates > see recipient microbiome change to look like donors - do not know what identifies someone as this - found to have high diversity but other than that, do not know what makes them super - questions about particular species or functions (large number of butyrate producers?), overall we don't know 3. What about recipients? - what is necessary of person receiving donation in order for donor community to establish itself - looking at immune response to transplant and what role it plays 4. Is there a combination of donors and recipients that are ideal?

Describe the ways in which pathogens might be able to infect despite having a healthy commensal microbiota (how do they subvert colonization resistance?)

1. Antibiotic treatment - for some pathogen, this is biggest risk factor, like C. diff - C. diff is bad competitor - antibiotics disrupt gut microbiota that opens room for C. diff 2. Specific characteristics / mechanisms of the pathogen - pathogen has adapted to get around mechanisms - bibliocholera - cholera toxin -> diarrhea 3. Help from the normal microbiota (?!) - in few cases, pathogens take advantage of healthy microbiota - most literature based off of viruses so far - shown but not understand if: --- poliovirus --- norovirus - mice with damaged gut microbiome or none at all, difficult to infect with (above); can infect easier with healthy microbiome - virus ability to attach to LPS 4. Pathogen mechanisms - virulence gene expression (toxins) - successful competition of niches and nutrients (?) --- colonize unusual areas --- use unusual nutrients - some pathogen can use entirely different sugars than non-pathogenic - ability to thrive in the inflammatory environment (?) (some can) - localization to specific areas --- successful colonization

What are the functions of the normal microbiota?

1. Bile acid modification 2. Fermentation of complex carbohydrates into volatile fatty acids (VFAs) (butyrate) 3. Vitamin synthesis: Thiamine, Riboflavin, Pyridoxine, B12, and K 4. Proper development and functioning of GI tract 5. Proper development of the immune system (including recognition of the microbiota) 6. Protection against pathogens (invasion and overgrowth) (colonization resistance) 7. Gut-brain axis - microbes in gut can influence behavior, personality, mental/neurological medical conditions

What are some of the defects found in germ-free rodents (in terms of gastrointestinal tract development)?

1. Cecum - larger than normal and full of mucus 2. Surface Area: - decreased because of changes to the crypts and villi 3. Crypts and Villi: - villi are TALLER but NARROWER - crypts are more SHALLOW 4. Epithelial Cells: - reduced turn over, proliferation, and migration - crypts and villi units can be formed in the absence of microbes, but particular steps are modified - Paneth cells (slower lysozyme production); slower maturation and turn over (less of these in GF mice) - decreased # of goblet cells and enteroendocrine cells - slower repair if damage occurs - leakier gut and more prone to inflammation - slower gastric emptying and movement through intestine because less enteroendocrine cells, less Serotonin in gut, peristalsis is slower barrier function - leakier gut in GF mice - epithelial lining not replace at proper rate - less mucus protecting epithelial cells - tight junctions affects because influenced by certain microbes - decreased tight junctions in GF mice 5. Mucus: - less mucus and different thickness/composition - different types and quantities of Mucins (specialized carbs in mucus) - if Muc2 is disrupted in mice, they are more prone to intestinal inflammation and more prone to bacterial overgrowth 6. Lymphoid Tissue: - GALT (gut-associated lymphoid tissue) is underdeveloped - large areas surrounding cut = Peyer's Patches (where GALT is concentrated) - very immature, less present, when present, do not contain many nodes - lowered immune presence in GF animals; lower lymphocytes 7. Blood Vessels: - based on what occurs once GF mice are colonized microbes - increased oxygen demand by gastrointestinal epithelial cells - blood vessels under epithelial cells - GF animals have reduced BV formation around GI tract - could be driven by less surface area, decrease oxygen demand, not a huge need for BV formation - when conventionalized, crypts and villi correct, surface area increases, oxygen demand increases, BV formation increases

What are some of the mechanisms through which an undistributed gastrointestinal microbiota might be able to prevent C. difficile infection?

1. Competition - C. diff is not a good competitor - compete in terms of nutrients - if it eats same thing as other microbes, those other microbes eat it faster and more efficiently and outcompete C. diff 2. Bile Metabolism - disruption of ^ - some bile acids that appear to stimulate C. diff (in terms of germination) (primary) - or appear to be able to kill vegetative C. diff (secondary) - based on ratios of bile acids - balance is important in terms of infection 3. SCFA - normal (make them) - ferment - influence immune system - play role in nutrients, inflammation, gene expression, could directly damage C. diff - 4. Direct Antagonism - some microbes produce antimicrobial things - bacteriocins made by Bacillus

What is the evidence for microbial involvement in human studies?

1. Differences in Gut Microbiota a. cannot differentiate between cause and effect b. microbial changes could be causing inflammation or vice versa 2. Antibiotic Exposure a. increase antibiotic use and increased rates of allergies/asthma = positive correlation when track antibiotic use in first year of life 3. Microbial Exposure a. correlation b. rise in allergies goes along with increasing C-sections, increasing use of formula, decrease in family size, decrease in microbial transfer (pandemic) 4. Probiotics a. can influence allergic risks of babies 5. C. albicans a. normal to find in mouth, GI tract, etc. b. too much of it becomes a problem c. can take over immunosuppressed person d. associated with # of problems when it appears to be the dominant member

What is the evidence that suggests a link between the microbiota and the gut-brain axis? (what evidence is there that it should perhaps be called the microbiota-gut-brain axis)?

1. Encephalopathy associated with late stage liver disease - decrease in brain function - memory, end up in come - liver directly impacts function of brain - for period of time, if given patients antibiotics (do not impact liver), can reverse the disease for a period of time - by altering gut microbes, changing impact of disease on brain 2. Co-occurrence of certain GI conditions and psychiatric disorders (along with changes in the microbiota) - autism - large % of them has some type of GI dysfunction, range in severity, range in inflammation - IBD (Chron's/UC) - large number of them have anxiety or depression - IBS - most tightly tied to brain and gut axis - about 50% of people with this have anxiety, depression, or both; common treatment is antidepressants (help alleviate gut symptoms in addition to helping with associated anxiety and depression) - co-occurance of Gut and Brain dysfunctions is interesting idea in terms of communication 3. Side effects of antibiotic treatment - especially in the elderly, when on antibiotics, see many behavioral changes - includes things like some changes in terms of sleep patterns, aggression, etc.

What are the potential mechanisms through which the microbiota may impact obesity? Be able to explain the ideas/data behind each of these.

1. Energy extraction - fermentation of non-digestible carbohydrates-production of SCFAs > some absorb more nutrients from food than others > most likely influenced by microbial degradation of complex carbs that are broken down into sugars and fermented into SCFA that we absorb - regulation of host genes (Fiaf - fasting-induced adipocyte protein) > Fiaf is an off switch for fat storage > when expressed, less energy is stored as fat > certain microbes can turn this off, so more energy is stored as fat 2. SCFAs - conflicting data > energy source (absorb 90% of SCFAs) > anti-inflammatory potential (barrier function) ("leaky gut") > signals (to suppress some things involved with obesity) 3. Signaling - hormones (satiety) > Leptin can influence microbes; microbes can influence Leptin - bile acids as a ligand for a variety of important receptors (directly involved in absorption of fats, but also can function as ligands) > TGR5 > Farnesoid X (FXR) => a FXR -/- GF mouse when conventionalized will not change in weight; those with FXR did see weight gain when conventionalized => FXR -/- has less Firmicutes than WT and more Bacteroidetes than WT 4. Circulating levels of LPS - low grade inflammation - metabolic endotoxemia - LPS is an endotoxin (too much can be lethal) - people with obesity, T2D, and metabolic syndrome have higher circulating levels of LPS; this is metabolic endotoxemia - higher circulating levels of LPS cause a constant low level of inflammation which puts stress on the body and causes changes that tie in these disease (obesity, T2D, and metabolic syndrome) - mice with more LPS have an (a) increase in fat storage/weight and (b) increase fasting glucose/higher overall blood glucose

Over the last 20 years (including recent data): What epidemiological data has been reported?

1. Household size and structure - family size: larger size, tend to see less inflammatory disorders - birth order: most likely the older one has the disorder - gender: does not hold up, suggests boys (older brothers) are better at bringing home antigens for younger siblings - genetics: caveat, the hypothesis does not allow for role of genetics but we know there are genetic risk factors for conditions 2. Day care - no agreement in literature between more or less infectious diseases - timing difference between this and public school 3. Variations in socioeconomic status - affluent - urban poor? - the more well-off people were, the more likely they were to have allergies and asthma - more recent literature suggests it has more to do with where they live than overall status - if you look at wealthy people in cities, do have higher risks, but so do the poor people in the cities 4. Animal Exposure - farmers - have overall lower risk of allergies than average person (increase antigen exposure) - pets - variable/cats not super protective but dogs maybe (most family with dogs do not have people with allergies but does not hold up well) - prenatal - interesting studies that look at rates of pregnant women encounters with animals; more animals she has contact with while pregnant, there is a correlation with lower allergies in the child

What are the possible mechanisms of communication between the microbiota, the brain, and the gut?

1. Neural Pathways: Vagus Nervs - Lactobacillus rhamnosus administration promoted exploration in mice - only when the vagus nerve was fully functional - vagus nerve runs from brain to gut and controls a large number of anatomic functions in the body - if you give the Lac probiotic, the mice were less anxious and depressed but only is the Vagus Nerve was intact 2. Immunological and endocrine mechanisms - circulating levels of cytokines - systemic effect; cytokines are high in people with depression - influence on epithelial cell migration and proliferation impacts enteroendocrine cells which directly impacts serotonin levels and movement of hormones 3. Bacterial metabolites a. Tryptophan metabolites - kynurenic acid correlation with Schizophrenia b. Neurotransmitters - GABA, dopamine, noradrenaline - microbes can alter metabolites but some can directly produce them - some Lac. species can produce them - directly influence parts of the nervous system c. Fermentation end products a. lactic acid b. SCFA - acetate, propionate, and butyrate - barrier function - alteration of immune response - gene expression of particular microbes - wide ranging effects - glucose metabolism - can they get into brain? these listed can potentially get to brain but not in large amount - a lot of studies say they are acting through other mechanisms to affect the brain

What are the causes of antibiotic-associated diarrhea?

1. Pathogen-independent a. drug - speeding up gastrointestinal times - pulling water into gastrointestinal tract b. loss of commensals - loss of normal microbes - get more complex carbs sitting in colon and not being broken down and fermented - different osmotic balance in colon - lack of SCFA 2. Pathogen-associated a. not always identified b. most common is C. difficile - causative agent in about 25% of AAD

Over the last 20 years (including recent data): How have the proposed immune mechanisms changed?

1. Th1/Th2 a. very popular in the 1980s b. suggests that Th1 responses are not stimulated correctly, so Th2 cells expand instead c. support has weakened for several reasons - see rise in both Th1 and Th2 mediated diseases - when looking at microbes that are considered to be Old Friends, would want them to increase Th1 responses to balance it out, but some of them much more strongly increase Th2 levels - make the issue worse with Old Friends hypothesis 2. T Regulatory Cells a. important for homeostasis - player that helps immune system recognize what it should and should not react to b. proposed that recognition of microbes leads to activation of dendritic cells which stimulate T reg cells - "Old Friends" - if exposed to variety of microbes, can activate T reg cells that will develop tolerance to that variety of microbes - develop tolerance to majority of antigens exposed to - if you do that, immune system will be balanced - missing them, do not develop tolerance immune system overacts to everything 3. iNKT Cells a. potential mechanism - invariant natural killer T cells - 2012 study - GF mice: more prone to allergies and colitis; due to expansion of iNKT cells? - find huge number of specialized T cells in both areas in GF mice - if given microbes, do not see increased amount of iNTK cells and not prone to allergies and colitis - can fake them out with antibody that prevents expansion of iNTK (also not prone to allergies and colitis) 4. LPS Variations a. gram-negative cells have LPS - can act as endotoxin - cause immune responses, inflammation, etc. - immune system recognizes it - different gram negatives have a different structure to their LPS's - number of them have differences in their lipid A portion - kids in Russia vs Finland, EF differences but not huge genetic differences, but rate of diabetes is very different between the two populations (T1D) - Finland = 6x higher risk of developing T1D than kids in Russia - high amount of B. dorei in kids in Finland while kids in Russia had more E. coli - different LPS structures between the two E. coli (Russia) - immunostimulatory LPS - activated the immune system like LPS is supposed to do B. dorei - suppressed inflammation/suppressed immune response - if mixed together, suppresses immune response that immune response that immune system can no longer recognize E. coli - in Russia, kids exposed to E. coli early on, lots of immunostimulant early on, immune system learns what it should and should not react to - in Finland, exposed to B. dorei suppressing Immune Response over and over again, immune system has not seen anything it knows, can start attacking anything >>> autoimmune disorders where it attacks body like T1D

What are some common characteristics for diseases in which the microbiota is considered to have a role?

3 main characteristics 1. Unknown Etiology (do not know the cause) - bad at predicting who will have the disease - problems with preventing it and problems with curing it - at best, see symptom management 2. Noticeable increase of the past several decades - faster than changes in genetics - environmental (something external is triggering this increase) 3. Wide range of presentations - severity of allergies and asthma or IBD/IBS (range of disorders covered under ASD also) - difference in severity, triggers, and age of onset (females more so after puberty than males)

What are the potential problems with Deconjugation activity?

A. changes chemical properties of bile acids B. allows for recovery and increases toxic effects C. potential problem for host? - # of derivatives that can lead to cancer formation - levels of free Taurine in gut (has some silver in it), evidence of microbial activity produces hydrogen sulfide D. a potential benefit for the host? - if deconjugated bile acid, means it made it to the colon, some will be excreted (good) - the more that are excreted in fecal material, the more cholesterol the body has to pull to make new bile acids - probiotics (with bile salt hydrolase activity) might be a way to lower cholesterol levels i. demonstrated in mice ii. from medical standpoint, variety of medications are used to treat high cholesterol, but one of the categories is Bile Acid Sequesters that bind up bile acids so you excrete more but has bad side effects iii. do not want to lower cholesterol levels just for that to increase risk of cancer

In terms of C. difficile colonization, what is known about asymptomatic carriers? Infants?

Asymptomatic - rate? - community acquired C. difficile - no symptoms, but carrying and shedding C. diff - population based surveys - found in 1-3% of healthy people (not high) - in terms of these people, we do not know if we have long term carriers, transient carriers - do not know role of these people - do not know a low about the strains they carry or how long they carry - symptoms and damage come from toxins and some strains do not produce toxins Infants -Esp. in first week - really likely to have C. diff in GI tract - 50% carriage -Higher rates than expected in adult population until age of 2 -Almost never see disease in infants -50% colonization --> 0% symptomatic disease -Theories: (both wrong) -1: maybe did not have toxic producing strains -2: something in GI tract did not allow toxins to be expressed -time they are born to age 2 have unstable and simple microbiota - niches where C. diff can colonize - not good enough to keep it out

How does age matter in terms of microbial exposure and immune system development/responsiveness? Does it always matter? Be able to explain ONE specific example discussed where age matters.

Age-independent influences - window of time does not matter, can fix the issues at any point in time Age-dependent influences - there is a window, specific time, at which you have to colonize the mice by and if you do not, you cannot fix the defect EVER Does not always mater Example: Exposure to a microbiota during early life regulates IgE levels in serum of adult mice and their sensitivity to orally induced anaphylaxis - IgE levels - in GF animals, make them prone to having anaphylactic responses - skewed to Th2 - some are GF, some given standard microbiota, others given low diversity - time window = 4 weeks - do nothing --> prone to anaphylactic responses - after 4 weeks - excessive levels of IgE and still susceptible - before 4 weeks - can prevent expansion of IgE and not prone but only if use fully complex microbiota (not with low diversity one) - specific microbes at specific time

What are some of the difficulties with investigating possible microbial involvement in autism spectrum disorders?

Autism - wide spectrum of disorders - mild to severe - some similarities in that we see some level on improper social interactions - can also see repetitive behaviors, food aversions, etc. - 1 in 54 children in the US; higher than in other parts of the world Risk Factors a. Genetics: one of the primary focuses - genes - potential specific genes - genetic disorders (ex: fragile X syndrome) - family - autism can be seen going through families - currently thinking is that genetics accounts for 50% of autism risk b. Gender - diagnosed more frequently in boys than in girls - keep in mind that it probably presents differently in boys vs girls c. Maternal Infection - if a mother gets a viral infection, there is a possibility of autism in the baby - relatively high fever in the pregnant mother could even cause autism d. Drug use during pregnancy - some prescription medications - opioid use may lead to autism in babies - thalidomide (no longer prescribe this to pregnant women) e. Parental age - older the parents, higher the risk f. Premature birth? Some studies suggest there may be a connection, some have not

What types of GI disorders are thought to be associated with autism? What unanswered questions remain in terms of GI disorders and autism?

Autism and the GI tract - GI issues can range from mild to severe (may be absent in some people) > across the literature, 9-91% of people with autism have GI disorders > most recent studies think it's 90% - also see differences in the types of GI disorders...some people have chronic inflammation, some have IBD, and some have functional disorders (IBS, constipation, diarrhea) - histological/pathological/immunological changes - functional disorders are common; correlation with autism severity - unanswered questions: 1. is there a real connection? 2. how might behaviors associated with autism impact GI function and inflammation? - recent paper looked at food restrictions for autistic individuals - argued that if you eat a low diversity diet it will decrease diversity in the gut microbiome that leads to increased functional disorders in the gut (primarily diarrhea) 3. are GI issues causes by some of the behaviors associated with autism or are autism and GI issues directly connected?

What is the evidence for microbial involvement in Type 2? Be able to explain several points

Based on connections with obesity 1. Conventionalized (previously) GF mice - increase in body fat AND triglycerides and blood glucose levels (lost control) - become obese and start to see metabolic problems associated with T2D 2. Metabolic endotoxemia - higher than normal circulating amount of LPS - chronic low level of inflammation seems to cause problems 3. Human studies - increase in Bacteroidetes to Firmicutes a. but, the more we looked at it, it fell apart b. decrease in butyrate producers - lead to decreased barrier function which could increase circulating LPS and allow for increased inflammation c. fecal transplant study - not significant impacts on obesity - 18 men with metabolic syndrome

Explain the characteristics of Crohn's disease vs ulcerative colitis.

Crohn's - can happen anywhere in GI tract - inflammation is patchy (cobblestone appearance; not continuous) - severe...multiple layers of tissue - deep inflammation UC - limited to colon/large intestine - inflammation is continuous - no healthy tissue mixed in with diseased area - surgery seems to be more successful because limited to just one area - surface level inflammation

What types of compositional and functional changes are proposed for dysbiosis as the possible cause/trigger/exacerbating factor for IBD?

Dysbiosis 1) lower overall diversity levels 2) absence of certain commensal/beneficial organisms? a. butyrate producers b. Clostridia (normal gut members) c. Faecalibacterium 3) increase of certain detrimental organisms? a. Proteobacteria b. microbes that produce metabolites that cause oxidative stress/or other negative metabolites - not consistent microbial changes - part is due to whether they have Crohn's or UC - has to do with severity or inflammation and treatments being use, key treatment is use of immunosuppressants - inflammation decrease diversity but low diversity might can cause inflammation - study: all forms of IBD were different from healthy controls, but ileal Crohn's was most distinct 4) role of metabolites - decrease in butyrate? - butyrate helps decrease inflammation and maintain barrier function - issues of oxidative stress, nitric oxide in gut, etc.

What is the evidence for microbial involvement in IBS? Include the specific information discussed in class, along with an explanation of the how the genetic risk factors, dietary factors, and post-infection IBS also support this idea.

Evidence for Microbial Involvement - intestinal permeability: decreased barrier function, increased permeability, leakier gut - fermentation profiles: various gases in breath and fecal material, a lot of those products in those are from microbes, can see different levels of things like short chain fatty acids and hydrogen - immune response a. antibodies b. toll-like receptors c. defensins - probiotics and antibiotics: some claim symptom relief and other do not with probiotics; antibiotics in some cases could be triggering or could alleviate symptoms for short period of time - microbial composition: depends on the study - GF mice: fecal samples from someone with IBS, put in GI mice, the mice have increased pain perception in GI tracts and had leakier guts = suggest microbial involvement Dysbiosis - imbalance between the host and the microbiota - imbalance is maintained in IBS patients - something (like listed below or stress, antibiotics) disrupted microbiome and it didn't bounce back - long term imbalance - less stability - more changing - lack of stability - in some people, based on genetics and mild inflammation, could argue immune response is involved (not in all people) Post-infection IBS - gastroenteritis is a trigger for some individuals - 6x to 7x increased risk - additional risk factors - differences in terms of timing of onset - following gastrointestinal infection get some type of viral or bacterial infection that causes intestinal inflammation and that causes some type of dysbiosis in terms of gut microbiota - the intestinal inflammation persists for at least 3 months following initial infection even after symptoms stop - cause dysbiosis to persistent; does not return to normal state (never); trigger that sets them on this course for rest of life Genetic Risk Factors - concordance rates - monozygotic twins - 22.4% - dizygotic twins - 9.1% - differentially expressed genes - response or recognition to bacterial antigens or invasion - EF's play a big role - genes are involved Dietary Impact - majority of patients report food intolerance; rates of food allergies? - importance of carbs - 65-80% of them could name a food they couldn't eat because of IBS but food allergies are no more common than those without IBS - food intolerances but not tru food allergies - low FODMAP - short term diet to get things to calm down - number of carbs like lactose, fructose, etc. that people with IBS had issues absorbing it - from microbial perspective, these are things that should be absorbed in small intestine, but if not, because of some issue with absorption, this means they make it to large intestine/colon and there they could be easily fermented by microbes - excess gas production, differences in osmotic balance in GI tract, changes in SCFA concentrations

What is the evidence for microbial involvement in autism? Be able to describe the evidence involving environmental factors, the gut-brain axis, fermentation profiles, and microbial dysbiosis.

Evidence for environmental factors - Rapid increase (?) - somewhat controversial - Maybe the increase in numbers is coming from more diagnoses (we are better at diagnosing autism) - Concordance rates - actual rates are all over the place in the literature - Monozygotic twins tend to have a higher rate than dizygotic twins - Diet - certain dietary changes can lead to behavioral changes - Looking at avoiding particular carbohydrates - In the US, rates of autism rapidly increased in a 12 year period (no changes in genetics...suggests environmental factors, but that's even fast for environmental factors) - Gut-brain axis - we know microbes are involved (brain development + function) - Germ-free mice = changes in their brain and behavior - Leaky gut - Microbes are important for barrier function - Leaky gut seen in animal models → suggests microbial involvement - Changes in fermentation profiles - Problems with carbohydrate absorption and transport - primarily certain types of disaccharides (dietary sugars) - Changes in ability to break down carbohydrates → changes what "food" the microbes are getting - Altered SCFA levels - Increase in propionate - Inject animals with propionate → mimic some of the behaviors associated with autism - But what does autism really look like in a mouse? - Can lead to GI problems, behavioral changes (increases in repetitive behaviors in animals) - Can also argue for a decrease in butyrate because it would lead to a decrease in barrier function and leaky gut - Altered fecal ammonia levels - Also observed in humans with autism - Changes in microbial composition - no consistent change throughout the literature - Impact of antibiotics and probiotics? - Some case studies that show antibiotics have a remarkable impact on autism related behaviors - Kid with ear infection → antibiotics for infection → behaviors changed (more outgoing, less repetitive behaviors, eat wider variety of food) - After antibiotics, the kid's behavior went back to before - Increased Firmicutes to Bacteroidetes - Presence of Sutterella genus - Decrease in Akkermansia muciniphila - Very little consistency. What are the complicating factors? microbial changes could be a cause, could be impacted by autism, could go along with the GI disorders, etc.

Are fecal transplants potentially going to be successful in treating or preventing diabetes? Why or why not? (think a bit beyond the particular study mentioned in class)

Fecal Transplant Study - 18 men with metabolic syndrome - two groups - everyone got fecal transplant except 9 got it from their own feces and 9 got it from lean donors - impacts lasted a year - they were in early stage T2D...it was stopped and/or reversed in number of them - microbial communities changed - at 12 month-mark everyone was back at where they started in terms of insulin intolerance, microbial community, and glucose levels - multiple transplants - require substantial changes in diet (low fat, low animal protein, high plant polysaccharide) - study not clear if asked men to make lifestyle changes/changed EF's - animal studies...many of the transplants only work long term if given a particular diet - lean can only invade obese microbiota if given high plant polysaccharide diet from another student: -to an extent, human studies showed that when prediabetic individuals got a fecal transplant from lan individuals, they were able to improve their insulin sensitivity and become no longer prediabetic -BUT if there are no diet and lifestyle changes their microbes revert back to prediabetic

What is the proposed mechanism for how Clostridium scidens may be able to prevent C. difficile infection?

First Figure a. adoptive transfer of resistance-associated intestinal bacteria after antibiotic exposure increases resistance to C. difficile infection b. when given suspension with PBS, a mixture of 4 "essential" microbes, and C. scidens (studies the most in terms of 7a/B), the mixture suppressed levels of C. diff the most but C. scidens did good as well c. when looking at toxins, the 4 mix and C. scidens had similar low levels d. when looking at body weight of the mice, all similar findings e. in terms of survival, the 4 mix did best then C. scindens f. correlation: C. scindens has strongest correlation g. biodiversity: pre-antibiotic had good diversity, with all treatments, none restored diversity not even the mix or C. scindens * appears to require a specific interaction* Second Figure a. C. scindens-mediated C. difficile inhibition is associated with secondary bile acid synthesis and is dependent on bile endogenous to intestinal content b. mice resistant to C. diff mostly have secondary bile acids (in more abundance than those not resistant) c. the bai operon is responsible for this activity: primary bile acids --> multi-step 7-dehydroxylation --> secondary bile acids d. DCA is most abundant pre-antibiotic, but also at same level with the 4 microbe mix and C. scindens post-antibiotic treatment e. correlation between CS abundance and DCA abundance f. Cholestyramine is a bile acid sequester - when it is present, there are no free bile acids - (-)CS and (-)Chol., bile acids present and mostly primary ones, so HIGH levels of C. difficile - (+)CS and (-)Chol., bile present, able to convert primary to secondary, see DROP in C. difficile levels - (+)CS and (+)Chol., no bile is present, CS is present but cannot act on anything because no bile acids, so the level of C. difficile INCREASES

Why might the fungal, viral, and bacterial communities all be involved?

Fungal - dysbiosis - could predict if FT worked based on yeast present Viral - disease-specific alterations in the enteric virome in inflammatory bowl disease - under explored at this point...hard to tie to a disease Bacterial - study showed similarity within households but a couple cases with IBD patients different form others in house Phage - DNA-based bacteriophages - in CD patients, had expansion of particular type of phage and rarefaction curves - study virome more and pay more attention to phage communities Overall, consider that fungi, viruses, bacteria, archaea, and protists are all present and potentially interacting.

How does the microbiota impact immune system development and function?

GF animals there are defects in immune development 1. GALT development - less peyer's patches and the ones present are immature + less mesenteric lymph nodes and those present are immature 2. antibody production - IgA, IgE, etc. are impacted in terms of levels of function 3. T cell subsets - T regulatory cells (regulatory inflammaotry responses), GF animals do not have maturation in terms of T reg cells -- skewed levels of T helper cells - Th1, Th2 (have more of this than should = more prone to allergies and inflammation), Th17 4. Epithelial cell proliferation, migration, and differentiation - less paneth cells, less antimicrobial compounds made in gut like defensins, problems with barrier function, innate part of the immune system host-specific microbes are necessary - colonized GF mice with either a mouse microbiota or a human gut microbiome - specific interactions required - mice colonized with human gut microbiota had some of the same immune defects found in GF mice microbial control - lymphoid structure and epithelial function - antimicrobial protein production; defensins - T cell subsets - balance of Th1, Th2, Th17, and Treg cells - production of particular cyotkines - systemic immunity

What general compositional changes are associated with IBS? What are some of the possible mechanisms behind IBS? (focus on those with possible microbial involvement)

General Compositional Changes - temporal instability - decreased diversity - otherwise: a. increase in Firmicutes; decrease in Bacteroidetes b. enrichment in Bacteroidetes c. increase in Lactobacillus spp.? d. compromised Lactobacillus spp.? Possible Mechanisms a. SCFA production imbalance - most studies suggest acetate and propionate are increased - butyrate decreased b. barrier function c. inflammation (subsets) d. enteroendocrine systemic alteration (motility) e. CNS involvement (gut-brain axis) 1. Gut microbiota - absorption of carbs 2. Immune activity 3. Intestinal permeability 4. Enteroendocrine activity - levels of serotonin, ability to impact GI motility

Fecal Transplant

History: - earliest record is in the 4th century BCE in China used to treat gastrointestinal illness - when first used for C. diff, mostly just for severe complications like pseudomembranous colitis, but has been used since 1960s, so overall long history of use Efficacy: - for recurrent C. diff, efficacy of preventing it is 90% efficient - better than antibiotic treatments - we do not know how they work though, just that recurrent C. diff is caused by damage to microbiome and transplant appears to be able to fix damage when given someone's healthy microbiome sample - do not know why they work either, and we do not know the most important members of the microbiome - so far, has been small studies like with 5 people and before they did the transplant, they filtered it through a 0.22-micron filter which filters out everything (like bacteria) but viruses, however, all 5 people were cured from C. diff - potential therapies: expedited approval only for recurrent C. diff, all else requires lots of paperwork; using transplants for anything microbiome could be involved with Dangers? - infection (screening; two people died in 2020 from fecal transplants because had multidrug resistant E. coli in donor samples and missed in screening - colonoscopy: anesthesia (deaths) - long-term impacts: > diseases -> transplant with healthy -> return to diseased microbiome > not big problem for C. diff cases but happens frequently when using it for other things > obese mouse --> transfer microbiome into lean mouse --> that mouse becomes obese - majority of side effects are mild and self-resolving - over 90% of those where received a transplant said they would do it again The "Brown Cross" - third, Open Biome is a nonprofit that advertises itself as a good place to get fecal sample for transplant from - first we look for samples from family members because 1. shared environment so similar microbes make them a good match (before infection of C. diff at least), and 2. they love you and are most willing - second, maybe storing our own samples prior to developing C. diff infection, but how do we know if we need to store it? The dirty aspect of fecal microbiota transplantation: - minor adverse events (common): abdominal discomfort, bloating, cramping, diarrhea/constipation, nausea/vomiting, low-grade fever - serious adverse effects (rare): high-grade fever, infection/sepsis, pneumonia, endoscopy complications - theoretical adverse events: unrecognized infection, inducing chronic diseases like colon cancer later in life

What are the subtypes of IBS? How might these complicate the understanding of microbial involvement?

IBS-D - diarrhea predominant - when having a flare-up, have lots of diarrhea IBS-C - constipation predominant Mixed IBS - alternate between D and C Un-subtyped IBS - no motility problems but have abnormal pain - pain can onset with bowl movement OR pain can be relieved by bowl movement Differences in inflammation, motility, brain involvement, and severity. No single signature confirmed in regard to microbes.

What are the new possible treatments for C. difficile infection?

If a disruption in colonization resistance is a major risk for C. diff infection, can we somehow either, 1. prevent the disruption or 2. restore colonization? Possible treatments: - Thuricin CD - Probiotic - Non-toxin producing C. difficile - Fecal transplants - SERP-109

What is the evidence for microbial involvement in animal studies?

Mice a. GF animals - observation that GF mice are prone to airway inflammation and GI tract inflammation suggesting microbes are involved in suppressing that b. antibiotic treatment - the more you give them, the more their risk for allergic diseases increases especially in pups - study: young age or adult mice, each given streptomycin or vancomycin - strep had not detectable impact on microbiome from global perspective; did not increase risk of allergies in young or adult mice - vancomycin caused decrease in diversity and major compositional changes and increased likelihood of allergies but only in the very young mice c. probiotics and other microbes - given to mother mice during pregnancy, or given to young babies, can suppress some of that allergic disease risk but depends on probiotic d. Candida albicans - yeast - normal to find this in variety of places including people - excess amount of this seems to be involved in mice in promoting inflammation - some components of yeast cell walls are known to be pro-inflammaotry - overgrowth of this can cause bacterial dysbiosis

Explain the proposed mechanisms behind colonization resistance (both microbe-microbe and host-microbe)

Microbe-Microbe interactions 1. Competition for niches and nutrients - niches = areas to live - carrying capacity = easy to use nutrients taken and all of mucous layer inhabited (no space for pathogen) - competition between different strains of a species - mouse colonizer with commensal E. coli (generic) in some cases that will prevent some pathogenic E. coli from being established because of overlap for niches and nutrients (EHEC and EPEC) 2. SCFA production - number of microbes in gut break down carbs in sugars and ferment those into SCFA - pathogen gene expression...some SCFAs cause pathogens to down regulate virulence factors (things it uses to establish disease, like secretion systems) 3. Direct attack - when microbes produce antimicrobials - Bacteriocins (short peptides that cells produce to attack closely related cells); bacteriocins work in different ways - some mess with metabolism, some cause target cells to form wholes in cell envelope, etc. 4. Bacteriophages - most common viruses in gut - recognize pathogen and can attack = effective - phage therapy ... to modify gut microbiota in targeted way - phages sit in mucous with legs out for when microbes go by, they have a better change to recognize and bind to them Microbe-Host interactions (complex) 1. Stimulation (or Suppression) of innate and adaptive immune responses - toll-like receptors - balance of T cells - number of commensals that can change it in either direction (suppress or stimulate) 2. SCFAs - butyrate and epithelial barrier function - primary energy source for lining of epithelial cells (typically butyrate) - for good barrier to keep microbes in gut and not elsewhere, butyrate is vital 3. Antimicrobial production - in small intestine, lots of Paneth cells, primary function is to produce defensins that act as antimicrobial agents in mucous layer and sometimes lumen - direct evidence they can influence microbial populations 4. Mucus layers - mucus formation - composition of mucus is impact by microbes as well as thickness of mucus - in terms of barriers and keeping it from getting right by epithelial cells - western diet, starves gut microbiome, not enough diet carbs, some cells will switch to get carbs in mucus, allows cells to get closer to epithelial cells...gut infection 5. Bile acid modification - body makes primary bile acid - will undergo # of changes as they travel from liver throughout body most will end up back in liver - the modifications that occurred in GI tract were done by gut microbes - including those only formed from microbial activity, see # of pathogens that respond differently to bile acids that gut microbes make as opposed to the ones that host makes

How was the hygiene hypothesis initially received by the scientific community? Why?

Skepticism - not popular in the late 80s - only a correlation then: as family size went down, we have seen decline in infectious diseases and that correlates with increase in allergies/asthma - at the time, the understanding of allergies and asthma was that they were triggered by infections Acceptance? - potential mechanism...he did not have one only this observation - still not universally accepted - what started the movement towards acceptance was the fact that they learned that not all T cells were the same and that you can look at two generalized groups (Th1 and Th2) Their thinking: - Th1 - respond to infectious agents - Th2 - responses were associated with allergies - balance between the two = everything is fine - not enough Th1, Th2 have major response, which they associated with allergies NOT CORRECT but helped people accept it more

Explain the evidence for microbial involvement in the development or exacerbation of IBD (include genetic risk, environmental factors, plus any other supporting evidence).

Microbial Involvement - Basic Evidence a. genetic risk factors -- how immune system recognizes microbes b. success of antibiotic treatment -- not a long-term solution -- but can be the factor that pushes someone in remission (out of an active flare up) -- can suppress inflammation c. fecal diversion therapy -- surgery; portion of intestine that is significantly inflamed, can divert fecal flow around that to give it time to calm down -- removed microbial exposure and the problem is "fixed" -- can relapse d. fecal bacteriotherapy -- fecal transplants -- huge amount of interest in this for IBD -- concern for possible dangers -- large number of microbes into someone who has damaged intestinal tracts, risk of holes, so do not want the fecal transplant to get into the bloodstream e. animal models -- immunocomprimised mice -- IL 10-/- mouse > anti-inflammatory cytokine - suppress inflammation > as long as they are GF, do not develop intestinal inflammation > if colonized with microbes, within 2 weeks, develop severe intestinal inflammation, and more prone to colon cancer later one > humans with IBD are more prone to colon cancer -- NOD2 -/- > mutation in this is the most common human genetic defect; recognition of peptidoglycan -- give mouse DSS; did not always cause intestinal inflammation; not used commonly now; interesting ties between microbial populations in mice and with certain immunodeficient mice Genetic Risk a. bacterial recognition by the immune system - TLR4, TLR3, TLR9 - NOD2 b. bacterial killing and degradation by the immune system - NOD2: single biggest genetic relationship with IBD (mutation in it is mostly commonly found in people with IBD) - ATG16L1 c. has to do with how immune system recognizes and deals with microbes; can affect barrier function Environmental Factors a. 50% concordance rate between monozygotic twins (10% in dizygotic twins) - stronger genetic component but drops for fraternal twins b. migrant studies - IBD is more common in Western countries -> NON-WESTERN = low rate of IBD -> move to U.S. -> in 1-2 generations, the rates of IBD skyrocket in that population c. rapid increase - increasing faster than genetics can take place

How is the microbiota impacted by C. difficile infection? What about recurrent C. difficile infection?

Microbiome - most have Bacteroidetes and Firmicutes - after on infection, slight changes but still mostly B. and F.s - with recurrent infection, none look normal - in the study, one person has only F.s, one has mostly Proteobacteria, and another has mostly Verrucomicrobia - the overall richness decreases after each infection Recurrent - after first infection, 20% chance to get another infection - after second infection, 40% chance to get third infection - after third infection, 60% chance for more infections - risk increases with every subsequent infection because treating with risk factor - damage to microbiome allows C. diff to enter that is mostly due to antibiotics then treating it with antibiotics

Are probiotics thought the help with the prevention of allergic diseases? The treatment?

Prevention - given to pregnant women and/or infants - Lactobacillus rhammosus GG (decrease in rates of dermatitis and eczema) - shown to decrease rates of skin allergies (for up to 4 years when they stopped - overall, possible protective effect against skin allergies in particular Treatment - L. rhammosus GG - Bifidobacterium species - L. reuteri - 50% chance of treatment beneficial effect (does not look like they help with treatment)

Over the last 20 years (including recent data): What microbiological data has been reported?

Role of Microbiota 1. Differences in allergic vs non-allergic individuals a. Question: are the differences driving the disease or is the disease driving the differences? 2. Exposure to Hep A, Toxoplasma gondii, and Helicobacter pylori (hookworms) a. In non-developed countries, the infection rate of H pylori is the majority of the population b. Have higher rates of parasitic infections, but lower rates of autoimmune disorders 3. Antibiotics a. Correlation between # of times a child is given antibiotics before age of 1 and their risks of allergies and asthma b. Changing the levels of microbial diversity 4. Immunomodulatory capabilities a. Bacteroides fragilis - production of capsular polysaccharide A (PSA) - probiotics - decrease in diversity, decrease in antigen exposure, overall increase in allergies/asthma

What is the evidence in support of a possible single causative agent for IBD? Is this the predominant theory behind this chronic disease?

Specific pathogens? - single vent? chronic infection? - NOT the predominant theory but there is literature about it - 3 commonly discussed pathogens: Mycobacterium avium subspecies paratuberculosis - causes disease similar to IBD in cows...intestinal inflammation, diarrhea, weight lose - IBD more common in cultures that drink cow's milk (argument, not fact) - big studies, do not find it more frequently in people with IBD E. coli; Adherent and Invasive (AIEC) - particularly in pediatric IBD - attach to epithelial cells, invade and survive within them (not all E. coli can do this) - in certain IBD pediatric populations, huge numbers (90%) of them have abnormal amounts of this - if you take IL10-/- mouse and mono-associate it with a single strain of AIEC, it has intestinal inflammation - not commonly found in adults Clostridium difficile - not necessarily saying it causes it - if someone is in remission, C. diff seems to be able to cause a relapse

What are the primary bile acids?

a. Cholate (CA) b. Chenodeoxycholate (CDCA) - made by the body, specifically in the liver from cholesterol - they become conjugated in the liver which means amino acids are added to them - the only two amino acids that can be added are Taurine and Glycine - TCA/TCDCA and GCA/GCDCA - these are the forms that leave the liver - go to small intestine where most will get reabsorbed and end up back in the liver - a small % of them go to the colon

Importance/Purpose of Deconjugation

a. Colonization - want to have gut microbe like Lactobacillus, maybe need BSH to be able to colonize - true for some gut microbes in literature b. Detoxification - based purely on culture-based studies, different conditions than in gut - typically, deconjugated forms are more toxic than conjugated forms - argument: have to have deconjugated form to be able to make secondary forms; secondary forms are the least toxic C. Virulence Factor - not sure why, used by some pathogens - Lis. Monocytogenes ...if BSH removed, does not cause infection D. Nutrient Acquistion - # of gut microbes have lost ability to make own amino acids, commonly found hose associated (get them from host) - theory: free Taurine and Glycine to use them as amino acids or use them as nitrogen source or other source of nutrients - evidence that this might be the case for Bacteroidetes, not widespread use

What are the secondary bile acids?

a. Deoxycholate (DCA) - converted from CA b. Lithocholate (LCA) - converted from CDCA - in the colon - made by microbes - the T and G are removed so that is how we get CD and CDCA - through microbial activity, CA is converted to DCA and CDCA is converted to LCA

How is the microbiota thought to influence brain development? The already developed brain?

a. GF animals - different neurotransmitter levels, myelination levels, development, blood brain barrier function but they do not have gut microbes b. Defects in responses to stress; altered behavior - different behavior than conventionalize mice - exaggerated stress response - exhibit more curiosity - early colonization = normalization; if colonize mice before 6 weeks old, can fix those issues - later colonization = incomplete normalization; wait until 14 weeks, cannot fully fix them c. Brain Chemistry is different - differences in neurotransmitters for example d. Behavior is altered - dietary changes; 50% lean ground beef diet > study: one group had normal diet, other on the lean diet, 3 weeks, in lean diet group, have more diverse microbial population and better memory capabilities and less anxious - antibiotic administration - transfer studies; behavioral traits were passed on with the microbiota transfers; BALB/c mice (anxious) vs Swiss mice (calm) > study: different strains of mice have different personalities, BALB/c tend to be anxious and Swiss tend to be calm; took GF BALB/c mice and put in Swiss transplant; that mice became calmer than normal BALB/c counterparts; did the reverse and the Swiss mice became more anxious

How are studies involving the microbiota-gut-brain axis conducted? What are some limitations to this research?

a. animal studies - light-dark preference (normally light dark and familiar places but will explore periodically) - step-down: innate curiosity, see how long it takes mouse to step down and explore - tail suspension: how long does it take mice to curl itself up to free its tail (depressed=long time) - forced swim: how long will the mice swim to the side (depressed=give up) - mazes and learning: memory and learning; how fast they can learn to complete the maze b. limitations on research - GF animals are widely used (not normal at all) > too big of a difference between GF and conventionalize mice and not just presence/absence of microbes for example > differences in GI tracts, structure, function, immune system responses, hormones, and brain chemistry - possible mechanisms for studying influence of alterations in the microbiota > driven by different factors > diet, change of diet, change microbiota but also changing behavior depending on how you change the diet > careful in terms of some alterations because can change other things like behavior independent of that > people are not mice - mice vs humans

What is the connection between bile and C. diff?

a. bile acids are thought to be involved in spore germination and outgrowth b. differing impacts for various bile acids, but evidence suggests that i. primary bile acids are used as signals for C. diff spore germination; signal that it is in the small intestine ii. secondary bile acids may directly inhibit vegetative cell growth; in healthy gut, should have plenty of these secondary bile acids to do this iii. but, if took antibiotics, can cause decreases levels of secondary bile acids resulting in lots of C. diff germination and nothing to stop the vegetative cells from growing

What are the potential problems with 7alpha/beta dehydroxylation?

a. changes chemical properties of bile acids b. allows for recovery and increases toxic effects c. potential problem for the host? - particularly deoxycholate d. correlation of particularly deoxycholate (DCA) with colon cancer risk e. too much of this activity may play role in gallstone formation f. literature now is looking at colon cancer g. do not want too much of this activity but still want in it, just in balance This been found in VERY FEW microbes (mostly Clostridium) while Deconjugation is found in VERY MANY microbes!

What are the functions of bile?

a. helps us absorb fats/fat soluble vitamins primarily in small intestine (in terms of our major digestion) b. certain bile acids (main component of bile) also get into bloodstream where they can interact with receptors so they can have systemic effects on the body - can be directly involved in glucose metabolism, cholesterol levels, fat storage, energy balance c. within small intestine, plays role in helping to control microbial growth - we want this! we do not want large numbers of microbes in SI because where microbes compete with us for nutrients so want small amount - antimicrobial capabilities, keep number of microbes low in SI How does bile impact so many function? a. in part, through acting as a ligand for a variety of important receptors: - TGR5 - Farnesoid X (FXR) Bile is amphipathic meaning a portion of it his hydrophobic and the other portion is hydrophilic a. it is good for interacting with fats and breaking them down further for absorption b. capable of getting into plasma membrane of cells and causing damage to bacteria

Why is microbial colonization problematic for the immune system?

a. immune system has to recognize this really diverse, heavily colonized area, this group of microbes - different groups, different antigens, huge numbers - but need immune system to not react to that - need it to recognize the microbes and see it as self b. but there are 10^13 cells per gram of intestinal content, how does it sort through that to identify a few Salmonella microbes, for example c. how can it get along with regular microbes but still defend against pathogens d. how does the immune system get trained?

What are the future directions in trying to understand the connection between the GI microbiota and IBS?

a. multifactorial approach - beyond gut microbiota - look at diet, infection history, stress, severity, age of onset b. closer investigation of the microbial populations - luminal vs mucosal populations - larger studies (dividing subjects into subtypes) c. fecal transplants - 7 trials - healthy sample but in person with IBS - 3 studies show no improvement but 4 did - DOSE is very important, minimum amount under which it will not work - DONOR seems to be very important, super donors - DELIVERY mechanism - DURATION is one enough to fit it for a lifetime or need multiple transplants? - no negative effects from fecal transplants

What disorders are associated with microbial involvement in the gut-brain axis?

a. multiple sclerosis...GF animals resistant to MS b. late-onset autism spectrum disorders - suggest microbes involved - autism might start in utero in terms of early brain development - late-onset: some cases where behavioral changes are not noted until after 18 months - argument that autism was always there just late-onset - some changes might be triggers by changes in microbiome - impact of antibiotics in terms of behavior for autism c. depression - depressed people have differences in gut microbiomes compared to non-depressed people - differences in immune system d. schizophrenia - differences in tryptophan levels

How does the immune system control the microbiota? (think containment, including stratification and compartmentalization, and composition)

a. must control contact with epithelial cells and invasion of tissues - mucus -- colon -- small intestine ----- antimicrobial peptides - secreted IgA (immunoglobulin A) b. what about when a breach occurs? - huge immune presence under inner layer of colon - dendritic cells, phagocytic cells, et.c - if it manages to get through, it should quickly be destroyed - if deemed to be dangerous enough, could be prevented - leading to systemic respond - it should be shut down right away if it is working right c. homeostasis between microbiome and immune system are necessary d. composition of microbiota can change in STRATIFICATION and COMPARTMENTALIZATION - stratification (layers): ability of immune system to keep some things in lumen, in outer mucus layer, out of inner mucus layer, away from epithelial cells - compartmentalization: keeping what should be in the gut in the gut and what should not be in there not in there - have to keep microbes away from epithelial cells because it too close can lead to intestinal inflammation - in colon, two layers of mucus (inner and outer) with lumen outside of outer layer: microbes can be in lumen and maybe outer layer, but do not think they should be in inner layer - in small intestine, only one layer of mucus -- crypts and villi ---- at bottom of crypts: paneth cells, produce defensins, particular cells have particular sensitivities to defensins ---- by changing defensions, can change microbial composition - secreted IgA: known to be able to bind to certain microbes and prevent them from being able to cross intestinal lining e. control of COMPOSITION - antimicrobial proteins are perhaps most well studied at this time (defensins produced by paneth cells) -- alpha-defensins -- regIII-gamma - some found in lumen (alpha) but RegIII stays in mucus - different targets - controls it differently - secretory IgA binding to cell - RegIII attacks cells in mucus layer - dendritic cells attacking cells - all being attacked because shouldn't be there - Alpha-defensins attacking microbes - systemic immunity ready to respond if anything crosses over

What is 7alpha/beta dehydroxylation activity?

a. occurs after deconjugation b. this is how we get the secondary bile acids

What are the risk factors for C. difficile infection? Explain the problem with antibiotics (think of risk factors and treatment options)

a. older individuals (>65) b. receiving medical care (hospitals) c. possible connection with some chronic conditions: IBD and obesity - decrease in diversity in both d. people you would expect to have a disrupted microbiota e. Antibiotics in the last few months - single biggest risk factor - single most common treatment as well - different antibiotics induce distinct changes to C. diff infection resistance and intestinal microbiota composition - each drug caused different types of changes in microbiome Antibiotic Treatment - stop antibiotics (which is normally the cause of C. diff) to allow microbiome to restore itself - then use antibiotics to treat C. diff infection a. vancomycin b. metronidazole - works in 25% of cases

What is enterohepatic circulation?

a. primary bile acids are synthesized and conjugated in the liver b. they are secreted into the gallbladder (concentrated in gallbladder) c. they are released into the small intestine when we eat because the hormone cholecystokinin causes the gallbladder to constrict which pushes the bile into the small intestine d. here it aids in digestion (fats and fat soluble vitamins) e. majority absorbed in the distal ileum f. carried back to the liver (recycled) - very efficient in recycling the bile acids - 95% of the bile that enters the small intestine is recycled - only 5% goes to the colon - in the colon, they are modified and either recycled to excreted in feces a. the ones reabsorbed in the small intestine are typically conjugated primary bile acids b. the ones reabsorbed in the colon are typically secondary bile acids

What is Deconjugation activity?

a. removal of taurine or glycine b. bile salt hydrolase enzymes - extremely variable in terms of: (1) organisms can have 1 or multiple enzymes (2) some enzymes work (intracellularly) inside cell and some work extracellularly (3) some expressed at different time points (exponential phase vs stationary phase) (4) recognize different things/specificity: some recognize any conjugated base, some recognize only ones that are attached to a specific amino acid, some recognize only deconjugated cholate or (CDCA) - many different types of organisms > found in huge numbers > major phyla of bacteria like Firmicutes, Bacteroidetes, and Actinobacteria but also Archaea c. importance/purpose OWN CARD d. when the amino acids, Taurine and Glycine, are removed e. "gate-way" reaction: nothing else can happen to the bile acids in terms of microbial standpoint until the amino acids are removed f. TCA/GCA --> CA (still primary bile acids)

What is the cecum?

a. small fermentation chamber b. at the end of the small intestine and at the beginning of the large intestine c. in conventionalized animals, it is full microbes d. in GF animals, it is much larger in size; this is a consistent finding when looking at GF animals in research e. full of mucus in GF

Lecture 34 - Obesity

compare mechanism of obesity and T2D development

What is sequenced when analyzing fungal communities?

qPCR - 18S rRNA gene & ITS regions

Which of the bile acids are produced directly through bacterial activity?

the secondary bile acids DCA and LCA a. deoxycholate b. lithocholate