Lecture 21: Gut and Oral Microbiome

What are the main metrics for characterizing microbial communities?

- diversity - taxonomy

What is 16s rRNA sequencing? It classifies up to the ____ . What does it reveal? What is metagenomics? It gives greater resolution in _____ . What does it reveal? What is transcriptomics? What does it reveal? What is metaproteomics and metabolomics? What does it tell us?

- "traditional microbiome sequencing"; conserved gene in bacteria, polymorphisms allow rapid, unbiased, and high-throughput identification of bacterial taxa. - family level (usually not genus, species, sub-species) - reveals who they are - fully sequence all microbial genomes to reveal physiological potentials. - taxonomic level. - reveals community capabilities based upon genes present - measures all RNA, usually by RNA sequencing - reveals what the microbes are trying to do (gene expression) - measures all proteins or all metabolites - tells us what physiologic activities are occurring.

What culture-independent technology can we use to assess our microbiota?

- 16s rRNA genomic sequencing (Traditional microbiome sequencing - Who is there?) - metagenomics (What are they capable of doing?) - transcriptomics (What are they doing) - metabolomics (How are they modifying their habitat?)

The surface area of the GI tract is ____ . The GI tract contains mostly ____ . Most of the stem cells located at the bottom of the crypts in the intestines turn into ____ .

- HUGE (like a tennis court) - anaerobes - absorptive enterocytes (replacing the intestinal epithelium)

Dr. D said that we just need to know that ___ and ____ are 2 examples of oral bacteria that cause systemic disease/disease in other parts of the body.

- S. sanguinis - fusobacterium nucleatum

The intestinal microbiota is ______ . It contains _____ different species. In the whole body, microbial cells roughly ___ the humber of human cells. The intestinal microbiome is _______ . It may contain ____ the number of genes in the human genome. The ____ is what you actually sequence.

- a complex community of bacteria, archaea, and eukarya that colonize the entire GI tract - 300-1000 (used to estimate 7000, but that was before genetic sequencing was done) - equal (about 3 x 10^13 cells!) - the cumulative genomes of the intestinal microbiota. - 100 times - microbiome

What is the definition of an organ? Is the microbiota considered an organ?

- a part of an organism that is typically self-contained and has a specific vital fxn, such as the heart or liver in humans. - nope.

In healthy people, the microbiota stimulate production of ___ and educated ____ . If host defenses are compromised, they can cause _____ or _____ . The Good News: antibodies elicited by normal flora bacteria can provide _____ . The Bad News: If normal flora bacteria share antigenic determinants with ____ , antibodies elicited by normal flora bacteria may ____ with tissues, leading to ____ . What is an example of one of these types of conditions?

- antibodies - T cells - opportunistic infections - excessive immune responses - protection against infection - normal tissue components - cross-react - autoimmune disease - inflammatory bowel disease (IBD) ma be an aberrant response to antigens of the normal GI flora.

Current research involving oral and gut microbes suggests that what is good for the mouth may ____ in other body sites. In other words, ____ . There is only a ____ overlap between oral and gut microbiomes.

- cause disease - habitat matters! Bacteria in one place may be good and in another place in body they may be bad. - tiny overlap

Microbial dysbiosis may contribute to ____ . These include ___ , ____ , _____ , and _____ .

- chronic diseases (dysbiosis = altered microbiome thought to drive disease) - inflammatory bowel diseases and colorectal cancer associated with expansion of mucosally-adherent E. coli, as well as other pro-inflammatory and pro-carcinogenic microbes (these are more pathobiotic, not opportunistic microbes) - diet-induced obesity (altered microbiota associated with increased fat deposition) - malnutrition and growth (microbiota transfer from humans with Kwshiokor (type of malnutrition) induced Kwshiokor in mice) - early life exposures influence host phenotypes (early antibiotic usage causes increased adiposity and sterile birth associated with immune diseases later in life)

The host and its microbiota have _____ on an ____ scale, and within an ___ over its lifetime. The composite of human and microbial cells, genes, and traits constitutes a _____. The human microbiota is a ____ and ____ microbial ____ . The microbiota impacts upon many different aspects of host _____ . ____ occur between the host and the microbiota. In general, our microbiota is more similar ____ over time (intra-individual) vs. between _____ (inter-individual).

- co-evolved - evolutionary - individual - superorganism - dynamic - complex - ecosystem - physiology (i.e. gut microbiota don't only impact the gut) - reciprocal interactions - within us - 2 individuals

What are the functions of the microbiota?

- competitive exclusion - digestion of food - intestinal physiology - immune system development

What are the traditional methods for assessing microbial communities?

- cultivation, microscopy, and molecular biology.

When oral commensal microbes colonize other sites, they can cause ___ in _____ individuals. What can the Strep. sanguinis that is found in the mouth cause in the heart? Why don't we try and eliminate S. sanguinis from the body? There are known links between ___ and ____ disease, like ____ , ___, and ____ . In terms of which bugs cause which diseases, we must distinguish ____ from causation, and consider other host factors like ___ and ___ .

- disease - susceptible** - can cause infectious endocarditis (really dangerous!) - because it competes for niche with S. mutans and thus may decrease caries and periodontitis. - periodontal - cardiovascular - atherosclerosis - coronary heart disease - stroke - association - comorbidities - gene variants

The human gut microbiota develops _____ and _____ . There are different microbiota between babies delivered via ___ and those delivered via ___ . C-section babies are colonized primarily by ___ microbes, while vaginal birth babies are colonized by ___ microbes. The long-term health effects of either birth route are ___ . After birth, ___ and ___ are the first bacteria to colonize. ____ persist until weaning. This is followed by _____ . By ____ , microbiota looks more adult-like. What is the significance of this age? By ____ , microbiota looks mostly like adult microbiome.

- during birth - after birth - c-section - vaginal birth - skin - vaginal - unknown - proteobacteria - other facultative aerobes - actinobacterua (bifidobacterium) - anaerobes (like bacteroidetes and firmicutes) - about 12 months of age - 12 months is about when solid foods are introduced - 3 years old

What is a germ-free organism? What is a gnotobiotic organism?

- germ-free: an organism that is free of microorgansisms including the normal flora - an environment for rearing organisms in which all colonizing microorganisms are known (ex: introduce E. coli to mouse -> becomes a gnotobiotic mouse because you know exactly which bacteria are present)

What was the goal of the Human Microbiome Project (HMP1)? The ____ of the microbiomes between subjects can differ significantly. Our microbiota are more similar by ___ than by ____ . Microbiomes of healthy subjects in the HMP1 share ____ .

- goal was to define a "core healthy microbiome" - taxonomic composition - function - taxonomy (analogy is that basketball players are all tall, can dribble, etc. -> all have different names and personalities, though) - similarities in their metabolic pathways (i.e. their potential functions)

The presence of normal flora of the GI tract affects the development of the ___ : Germ-free animals have fewer ____ , smaller _____ , and low levels of ____ . When germ-free animals are colonized, they exhibit a transient _____ because their immune cells have not been ____ to the normal flora. Lack of microbial exposure in early life may lead to an increased likelihood of allergic diseases, a phenomenon known as the _____ . There are correlations between ____ and _____ , as well as altered GI flora composition.

- immune system - T cells - Peyer's patches - secretory IgA - hyper-inflammatory response - tolerized (remember tolerance of T cells in the thymus!!) - the "hygiene hypothesis" - allergic airway disease - antibiotic use early in life

Cultivation provides _____ for further characterization. Cultivation is an ___ and ___ community representation. This is because it selects for clones that ____ on artificial media. Abundant clones grown in vitro are ___ those that dominate the in vivo community. Unfortunately, most bacteria cannot be cultivated ____ . Microscopy allows for ____ analysis of the community. The major downside is that it is difficult to _____ . Molecular biology utilizes _____ to do _____ .

- isolates - incomplete - biased - grow quickly - rarely - in vitro - spatial - distinguish between similar species - targeted PCR - microbiota sequencing

What is the significance of fusobacterium nucleatum?

- it is an oral microbe with pro-carcinogenic effects on the gut (has many mechanisms by which it promotes cancer, like containing factors that induce uncontrolled proliferation of epithelial cells, interference with chemotherapy drugs for colorectal cancer for some currently unknown reason, and may play some role in metastasis of colorectal cancer).

Metabolic activity of gut bacteria yields ___ that are used by the host. _____ is made by bacteria in the GI tract. Dietary polysaccharides are metabolized by _____ to be used by host and microbes. Gut microbes facilitate more ____ use of nutrients: Germ-free rats require ____ more calories to maintain body mass than normal rats require. There is evidence that the GI flora of obese people are more ____ at breaking down indigestible compounds into usable nutrients, compared to the bacteria found in non-obese people. Intestinal bacteria metabolize __ into the short-chain fatty acids ___, ____ , and _____. These microbial metabolites are taken up by the ______ .

- nutrients - Vitamin K - Bacterioides species - efficient - 30% - efficient - fiber - acetate - propionate - butyrate - colon epithelial cells

What are some beneficial effects of the microbiota on human health?

- promotes normal healthy development - barrier function (against pathogens) - essential for differentiation and maturation of the immune system. - nutrition and energy

What are the different functional classifications of microbes in the microbiota?

- resident, commensal, symbiont, pathobiont, and pathogen.

What are resident microbes? What are commensal microbes? Which bacteria were formerly considered commensal? What are symbionts? What are pathobionts? Pathobionts are not the same thing as ____ . What are pathogens?

- the microbe resides in your body, but we aren't implying anything about its function. - one partner benefits while the other is unaffected. - "normal flora," until we knew the extent of health benefits they provide us as hosts. - both the host and the microbe benefit from their association. - this resident microbe is normally harmless under most conditions. However, under certain conditions (genetic susceptibility, environmental factors, etc.) this microbe induces inflammation and/or disease. (always have carried the microbe, but something changed in the conditions to make them pathogenic/bad) - opportunistic infections - not a resident microbe, but invades the ecosystem, may out-compete the normal flora, and cause disease.

What is competitive exclusion? What is the evidence for this? What is an example of evidence for competitive exclusion? Commensal flora (not hurting or helping) physically occupy sites on epithelial cells, thus _______ . Some commensal bacteria produce ____ or ____ that inhibit the growth of other pathogens. _____ is effective for treating recurrent C. diff infection.

- the normal flora protect against invading pathogens. - evidence for such protection is that antibiotic treatment increases susceptibility to new infection with pathogens. - C. diff (when the immune system becomes suppressed, opportunistic pathogens like C. diff can cause infection) - blocking attachment by new pathogens - bacteriocins - antibiotics - fecal microbiota transplant (FMT) (but you have to make sure the fecal transplant is from someone with a super healthy gut microbiome, because not all microbiota are created equal)

Many factors impact the microbiome, but yours is still ____ . This is determined by ____ .

- unique to you (you can have slight shifts in gut microbiota -> same bacteria, but may express different genes) - birth mode, housemates and pets, disease, diet, antibiotics, etc.

What is butyrate used for? Acetate and propionate reach the liver by the _____ . Together, this gut/liver/brain axis modulates ____.

- used as fuel for enterocytes - portal vein - gluconeogenesis (IGN)

What is diversity? Community ecologists typically describe microbial diversity by: ____ What is taxonomy?

- variation in members of an ecosystem. - number of taxa and diversity (commonality) between samples. - assign high-throughput sequencing reads to taxonomic identities using established databases. (ex: high abundance of Fusobacterium)

Commensal bacteria of the gut stimulate ____ , _____ , and _____ . Commensal bacteria of the gut protect from infection: ______ and _____ .

- vascularization and development of intestinal villi - protective mucus production from epithelial cells - support cardiac output (germ-free rats have about ~30% less CO) - maintain skin and mucosal barrier function - pathogen exclusion (prevent invasion and growth of pathogens, like C. diff)