Microbiome

What is the composition of microbes in humans?

--The human body hosts ~1013-1014 bacterial cells (1013 human cells) --Microbes make up 1-3% of human body mass --Microbes inhabit all surfaces exposed to the environment --The majority of microbes inhabit the large intestine, with ~1,000 different species present

Why is the microbiome beneficial?

-The human microbiome is the collection of microbes that reside on and within humans -Most species of the microbiome are not harmful, but actually assist in maintaining human health

Where does the microbiome come from? What influences it?

1) Birth; Babies acquire mother's microbiome at birth, and from breast milk 2)Diet is another key way in which the human microbiome is altered. It has been shown that changes in diet can rapidly alter the proportion of different species found in the microbiome. 3)Environment can also influence the microbiome, including different temperatures, humidities, and other environmental factors, including the different microbes that you may be exposed to in your home, or other environments.

How is rRNA-based species identification done?

1)An environmental sample is taken, and gDNA is extracted from all the species. 2)Each species will contain a 16S rRNA gene, containing both a conserved genetic sequence, which is conserved across (essentially) all species, as well as a variable region, unique to that particular species. 3)Universal 16S PCR primers are then used (in the highly conserved region) to amplify all of the 16S genes in the gDNA collection. These amplified DNA sequences are converted into a sequencing library. 4)This sequencing library can then be sequenced, using different sequencing techniques (which we won't go into here), which will read out the genetic sequence of both the conserved and variable regions of the different sequences present in your sample. 5)If we put all this sequencing data into a computer, we can use reference databases, and other tools to figure out which species the different 16S sequences originated from. 6)We can then turn this into information that tells us which species are present in our sample, at what relative proportions, and how they are genetically related to each other.

How is C.difficile diagnosed?

1)Colonoscopy, identifying pseudomembranes 2)ELISA or PCR assays to detect toxins A & B 3)Cell cytotoxicity assays (looks for the effects of C. diff toxins on human cells in culture)

How does metagenomic analysis work?

1)First, genomic DNA is isolated directly from the environment being analyzed (in other words, without culturing). The sample may be collected from swabs, or fecal samples, and gDNA is isolated directly from the sample. 2)The gDNA is then sequenced 3) the data is analyzed

What are microbiome therapeutics? (What is done to restore it?)

1)Probiotics are live microbial species used to confer a health benefit ---Typically refers to microbes ingested with goal of modifying or restoring the microbiome (ex. for C. diff infection) 2)Prebiotics are fiber compounds said to stimulate growth of microbiome species

What is the microbiome?

A microbiome is a community of microorganisms that comprise a given environment -Includes bacteria, archaea, fungi, viruses, etc. -Microbes of the microbiome may include pathogens, commensals, and symbionts

What is another important technique for microbiome analysis?

Another important technique for microbiome analysis is using animal models to assess the phenotypic impact of a microbiome. This is often done by microbiome transplants, when a microbiome is transplanted into a germ-free animal (which has no microbiome at all), we may gain insight into the functionality of that particular microbiome

How is C.difficile transmitted?

Bacteria may be endogenous to microbiome, or spread by fecal-oral transmission within hospitals (frequently a hospital-acquired infection) C. diff infections are on the rise

How does C.difficile cause infection?

C. diff produces spores which are robust to harsh environments. Live on surfaces for months (survive alcohol-based disinfectants), survive stomach acid, many antibiotics C. diff produces several toxins (A&B), and harmful enzymes Toxins cause epithelial cell apoptosis Inflammation leads to pseudomembrane formation and watery diarrhea.

What are the symptoms of C.difficile infection?

C. difficile causes an infection of the colon: pseudomembranous colitis, resulting in diarrhea, colon inflammation Symptoms: flu-like, watery diarrhea, abdominal pain, fever CDI can progress to a life-threatening infection

How does one get C.difficile?

C. difficile causes disease when competing bacteria are reduce with antibiotic treatment (especially broad-spectrum antibiotics)

What happens when the gut microbiome is disrupted?

Dysbiosis in the gut microbiome can influence auto-immune inflammation of the GI tract and Inflammatory Bowel Disease (IBD) - Crohn's disease, ulcerative colitis

What is dysbiosis?

Dysbiosis occurs when microbiome species composition is altered May occur from changes in diet, environmental changes, antibiotics, etc.

What are other forms of species identification?

Eukaryotic species (fungi, parasites) can be identified from 18S rRNA or the internal transcribed spacer (ITS) region of rRNA Viral metagenomics is difficult, as they lack a unique rRNA-like region, and may also be incorporated into the bacterial genome Viral DNA can be analyzed by isolating viruses, ex. through filtration methods, and sequencing their entire genome

What is a new form used to restore patients microbiome?

Fecal microbiota transplantation is a new technique that aims at restoring a patients microbiome, and treating C diff infections. Transplantation of fecal microbiome species from a healthy individual, to a recipient via enema, colonoscope, nasogastric tube For patients suffering with severe C. difficile infections that are otherwise difficult to treat with antibiotics

What C. difficile?

Gram-positive, motile, anaerobic, bacillus Present in the colon of 2-5% of people

Where can microbes be found in the human body?

Microbes inhabit many human niches, especially: Skin Nose Mouth (saliva and oral mucosa) Respiratory tract Gastro-intestinal tract Urogenital tract conjunctiva *Even within a niche, like the skin, different parts of the skin harbour very different organisms

How is the microbiome beneficial to human metabolism?

Microbes of the microbiome also aid in human metabolism, by 1) chemically breaking down carbohydrates, 2)helping the absorbing of nutrients, 3) and synthesizing specific vitamins for their human hosts.

What is the effect of dysbiosis in the immune system?

Microbiome interacts with immune system, regulates its development Microbiome can also influence auto-immune disease, in which an abnormal immune response is mounted against one's own tissues Allergies, asthma, rheumatoid arthritis, MS, type I diabetes, lupus...

What happens if microbiome is disrupted?

Microbiome is critical for health, disruption of the microbiome has serious consequences for disease

What is an example of significant microbiome alterations?

Obesity is correlated with significant microbiome alterations This shift is correlated with a shift in microbial genes related to carbohydrates metabolism

Why important role in human health does the microbiome serve?

One important role of the microbiome is to fight off other pathogenic microbes. They can do so by physically blocking pathogen entry, competing with pathogens for nutrients and space, and actively inhibiting pathogens through secreted toxins this way, when the microbiome is depleted (say due to antibiotic treatment), the patient may be more susceptible to some types of pathogenic infections, by both endogenous and exogenous pathogens (ie. VRE, C. difficile, Salmonella).

How is C.difficile prevented?

Prevention: bleach-based disinfectants, limiting hospital spread, appropriate antibiotic prescriptions

How was microbial species identification done?

Previously, microbial species has based on culturing them, and then assessing their phenotypes, or sequencing them. BUT Inaccurate, slow, incomplete

How is C.difficile treated?

Resistance to most: Beta-lactams (penicillins and cephalosporins), aminoglycosides, tetracyclines, erythromycin, and recently, fluoroquinolones Discontinuation of antibiotics may resolve, oral rehydration Treatment with vancomycin, metronidazole (doesn't kill spores, ~25% relapse)

Are prebiotics and probiotics actually help?

Scientific evidence remains insufficient to prove a cause and effect relationship between consumption of probiotic products and any health benefits (European Food Safety Authority)

How is the microbiome beneficial for the immune system?

The microbiome also promotes human health by helping to "train" the immune system. During early stages of human development, microbes (particularly those of the GI system), interact with the host immune system and help it develop prenatal and postnatal immune systems are very different, and that postnatally, thanks to colonization by microbiome bacteria, the immune system develops and matures rapidly, making it ready for potential infections by pathogenic microbes.

Is the microbiome an important reservoir for opportunistic infections?

The microbiome can be an important reservoir for opportunistic infections. Remember...microbiome protects host from pathogens When microbiome is disrupted (often from antibiotics), pathogens are allowed to take over and cause infection (ex. Candida, C. difficile, VRE) Some antibiotics are not very specific, and will also kill off the microbiome

What is the most common way to identify the species makeup of a metagenomic sample?

The most common way to identify the species makeup of a metagenomic sample is through rRNA sequencing. 16S rRNA in prokaryotes, and 18S rRNA in eukaryotes are components of the small ribosome subunit, which contain both highly conserved regions, across all species and hyper-variable regions, unique to a species/genus. T

What is the most common, non-cultured based method of doing microbiome analysis?

The most common, non-culture-based method of doing microbiome analysis is through metagenomics. Metagenomes are the entire collection of genetic material (genomes) from a diverse group of organisms, such as a microbiome.

What happens to germ-free animals that are born and raised in sterility with no microbiome?

These animals have weak immune systems, high susceptibility to infections and are prone to inflammatory bowel disease, asthma, and other autoimmune disorders

What happens when microbiomes from matches lean and obese human twins are transplanted into germ-free mice?

Transfer of microbiota from matched lean and obese human twins, into germ-free mice can also alter weight gain in mice

What happens when transplating the microbiata of obsese mice into germ-free mouse?

Transplanting the microbiota of obese mice into a germ-free mouse is sufficient to produce obese mice

Why is the microbiome important?

Understanding the microbiome is important, as microbial species are a critical part of our lives and our environment. -Microbial species make up ~60% of Earth's biomass -Fewer than ~1% of species have been identified -Humans are home to at least as many bacterial cells as human cells. These microbiomes play an important role in human physiology