Microbiota Flashcards
Human is microbial ecosystem
Humans = eukaryotes + bacteria + archaeabacteria + viruses + parasites
Cell: 1:1
Genome: mainly prokaryotic
Weight: mainly human
microbiota vs. microbiome
Microbiota: cells
Microbiome: genes
Interaction between human and microbes
Symbiosis
3 types of symbiosis
Mutualism : both benefit
Parasitism: one benefits, one harmed
Commensalism: one benefits, one unaffected
Under some situation the relationship chan shift from mutualism to parasitism
Holobiome
human genome and microbiome
Why study Microbiome
Metabolize >50 xenobiotics, altering their activity, toxicity, pharmacokinetics, bioavailability, etc.
Synthesize essential vitamins and nutrients, metabolize polysaccharides, and modulate the immune system
Change animal behavior and mating preferences
Make you UNIQUE: humans have 99.9% identical genomes, but differ a LOT in their microbiome
How to study microbiome
Traditionally: culture-based methods
Gut bacteria:
Known temperature, pH, anaerobic environment
But most cells need other members of the community (bacterial consortium)
Culture-independent methods
16S rDNA-based sequencing studies
•Who is there?
Deep genomic sequencing studies
“metagenomics”
What do they have the capacity to do?
mRNA sequencing: “metatranscriptomics”
•What are they actually doing?
Metabolomics
•What are they actually doing?
16S rDNA gene analyses
Phylogenetic marker found in all species
16S rDNA gene contains regions that are identical (slow-evolving) for all bacteria, and some that are variable (fast-evolving) and have unique sequences in individual bacterial species
Specific primers allow us to target and amplify regions of interest found in most of the microorganisms present in the environment.
After PCR amplification, it is possible to sequence the region and make comparisons with other members of the microbial community or members of its own species (large pre-existing databases)
90% of the study only focus on V4 region
Metagenomics
Advanced methods for sequencing all genomic DNA present in a sample
• Human sequences removed with bioinformatics tools
• Tell what genes are present
• Metabolic activities can be inferred
Microbial communities are site specific
Human microbiota is dominated by 3-5 phyla
Skin
Actinobacteria
Staphylococci, Streptococci, Diphtheroids (e.g. Propionobacterium acnes), …
Resist to UV and other strong stimuli
Some of the metabolic by-products of the skin bacteria are volatile fatty acids (body odor)
Nose
Staphylococci, Streptococci
Mouth/Oral cavity
- Streptococcus
- No teeth: aerobes
- Teeth: predominantly anaerobes (anaerobic environment between teeth and in gums)
- Involved in tooth decay
- Linked to the gastrointestinal microbiota
Urogenital tract
Vagina: main genus is Lactobacillus
– Produces lactic acid to maintain low pH
– Inhibits growth of other microorganisms ex. Fungi and yeast
– Vaginal microbiome varies greatly between individuals and varies with menstrual月经 cycle
Penis
-Pseudomonadaceae and Oxalobactericeae most abundant
- Penis and urethra support distinct bacterial communities
- Circumcision reduces putative anaerobic bacterial families (Clostridia and Prevotella)
- Sexual activity can alter microbial diversity
GI tract
~70% of total microbiome is in the colon
- Mostly anaerobes, some facultative (300:1)
- Predominantly 2 phyla: Bacteroidetes, Firmicutes
- Many different species (est. ~1000)
Foetus, human placenta
Should have low number of microbe
Microbe is a negative sign for baby development
How do we acquire a microbiome?
Large amounts of microbes are acquired at birth and colonization varies with Delivery mode (Vaginal vs. C-section)
How do we acquire a microbiome
Large amounts of microbes are acquired at birth and colonization varies with Delivery mode (Vaginal
vs. C-section)
Establishment of facultative anaerobes (Enterobacteriaceae) and Bifidobacteria first. After six months, obligate anaerobes predominate.
At 3 years, adult-like microbiota
Does the microbiome change over time
Phyla remain stable over the course of months
Species and strains are much more variable
Mouse model
mammalian model with controlled conditions and interventions (genetics, diet, disease, therapeutic drugs)
Germ-free = axenic = sterile / no microbiota
• Can nearly be obtained with broad-range antibiotic treatment. But side-effects can be seen and not 100% effective.
• Real axenic mice are generally obtained by hysterectomy rederivation and maintained in isolators
Gnotobiotic
all microbes are known
Axenic and gnotobiotic mice
Mouse eat each other’s poop, in some cage share the microbe
Microbiota-associated mice models provide us with means to determine causality and mechanisms of interactions
Diet
Major driver of the diversity of the gut microbiota
The obesity epidemic
-Obese individuals have a distinct gut microbiome from lean individuals
-Several confounder exist: diet, genetics, environment, etc
-Twin: obese and lean; monozygotic have different gut microbial communities; dizygotic twin have even more different gut microbial communities
-use animal models to determine the role of gut microbiome
Mouse model: control genetic and diet
Leptin mutant+normal lean mouse
Leptin
Hormone made of fat cells that regulates the amount of fat stored in the body, “satiety” hormone
Animal models of Type 2 diatbetes
Obese mice have a different gut microbiota
Obese mice and WT mice fed the same diet
Amplicon sequencing (variable region of 16S rDNA)
Obese mice have significantly higher proportions of
Firmicutes : Bacteroidet
Cause & effect of gut microbiota and obesity
Transfer gut microbial community from the obese individual to lean individual. The mice putting on more weight despite controlling for the diet
After confirmation of microbiome transfer, ratios of Fimicutes:Bacteriodetes were increased in the recipient mice
Mice receiving feces from obese mice had more body fat after 2 weeks DESPITE no differences in the amount of food eaten per group
How to find the causal relationship between gut microbiota and obesity
Shotgun sequencing of gut microbiome form lean and obese littermate mice
Genes used to harvest energy from carbohydrates higher in obese mice
Fecal calorimetry measurements showed that obese mice had less energy left in their feces than lean mice
Translation the animal experiment result to human
Germ-free mice were colonized with feces form twins discordant不一致 for obesity. Mice receive obese twin microbial community would put on body weight
Diet remains key: Additional data showed that a “lean” microbiota cannot colonize well when mice are fed high fat/low fiber diets
Subsequent studies show the altered F:B ratio is not always present and that housing conditions remain important
Antibiotics and the gut microbiota study
Took 7 day clindamycin
For nine months after exposure, only one resistance strain detected
Even after 24 months, gut microbiota diversity remained low
Antibiotics alter the gut microbiota
Antibiotics decrease gut bacterial diversity
Particularly disruptive during early childhood
At sub-therapeutic doses, antibiotics can promote weight gain. Some people give livestock sub-therapeutic doses to increase their weight
Unintended effects of antibiotics
Antibiotics can promote ”pathobiont” expansion
• Pathobionts: members of the microbiota in healthy hosts, normally don’t cause disease
Most common adverse effect of clindamycin is Clostridioides difficile-associated diarrhea
C. difficile infection: antibiotic associated diarrhea
~2% of the population are “healthy carriers”
Present in the environment, spore formers
• Healthy microbiota: colonization resistance
• Highest risk factor: broad spectrum, antibiotic treatment
Colonization resistance
Other bacteria already take the niche to prevent C. difficile growth
Already have C. difficile but other bacteria produce some molecules will limit the expansion of C. difficile
Vicious cycle of C. difficile
Treatment can’t be to use another antibiotics to against it
1st line treatment: discontinuation of antibiotic usage, metronidazole or vancomycin course
FMT
Fecal microbiota transplant
FMT as treatment of C. difficile
FMT: Possible last-resort treatment for patients suffering from recurring C. difficile infections
Infusion of fecal bacteria from a healthy individual into a recipient
-get stool sample from healthy patient
-screen the sample
-no other pathogen in it
-transplant the sample to patient by colonoscopy or infusion
Even a better treatment
Data of the disease cause of C. difficile
Up to 25% some patients have treatment resistant or recurrent C. difficile.
Multiple relapses in the same patient are common, and up to 10 or more bouts of relapsing colitis have occurred in some patients
C. difficile infections are linked to 14,000 deaths in the U.S. yearly.
Current concerns about FMT
Total community transplant
other possible pathobionts and/or pathogens
- how to choose the correct “healthy” donor?
- what is a “healthy” microbiome?
- more data comes out correlating certain microbiome constituents with more and more diseases…
- Vertical transmission of microbiome (mother to child): considerations for future generations?
Society buy-in (taboo of fecal matter)
Defined bacterial mixtures will be easier from a
regulatory standpoint
Also considered for many diseases: inflammatory bowel diseases (ulcerative colitis and Crohn’s disease), autoimmune disorders, metabolic syndrome, obesity, diabetes, multiple sclerosis, Parkinson’s disease, …
• More challenging (chronic conditions)
• More studies needed to define the best indications, optimal timing, frequency, mode of delivery, and optimal donor for each patient
• Several clinical trials in progress
• Other ways to manipulate microbiome: probiotics, prebiotics, diet, bacteriophages, …
The Gut Brian axis: Bi-directional communication
between gut and brain
Short chain fatty acids (SCFAs) are the fermentation end products of dietary carbohydrates.
SCFA have immunoregulatory properties (pro-, anti-inflammatory).
Butyrate acts as an energy source for enterocytes.
Bacterial metabolites, such as SCFAs and neurotransmitters, can cross the blood-brain barrier (BBB) and regulate neurological functions
Blood-brain barrier: highly selective semipermeable barrier of cells restricting access of molecules from blood to brain
Bacteria and neurotransmitter produced
Bacillus: Dopamine, norepinephrine
Bifidobacterium: GABA (gamma-aminobutyric acid)
Enterococcus: Serotonin
Escherichia: Norepinephrine, serotonin
Lactobacillus: Acetylcholine, GABA
Streptococcus: Serotonin
Immune cells and cytokines cross the BBB and can regulate neurological functions, including the hypothalamic-pituitary-adrenal axis (HPA)
All these molecules are active in both the brain and the gut
Body-wide microbial interactions
Many diseases have co-occurring pathologies:
– Up to 50% of adults with IBD and 33% of patients with IBS have pulmonary inflammation or impaired lung function
– Individuals with asthma have functional and structural changes in their gut microbiota
– Changes in the oral microbiome are associated with preterm birth and abortions
– Patients with rheumatoid arthritis have a higher prevalence of periodontal disease
– 7-11% of patients with IBD also have psoriasis, and the prevalence of IBD in individuals with psoriasis is 4 times
Probiotics: definition
live microorganisms which when administered in adequate amounts confer a health benefit to the host
Current issues with probiotics
Inadequate studies: low sample size, quality of study design, lack of in vivo data, self-reported diagnostics and measurements, lack of details about probiotic strains used, … 还有安慰剂效应
• Limited mechanistic understanding of effects of current probiotics
• Probiotic industry largely unregulated
• Publication of negative results required
• Need for a standardized and validated measure of “health benefit to the host”
• No adequate tests to predict functionality of probiotics in the human body
Information still required: strain(s)of probiotics, dose, duration,
frequency, timing, indications, and side-effects
• Rare cases of infections associated with probiotics
• Transferrable microbial properties (antibiotic resistance genes, virulence genes, …)
• Effectiveness of probiotic for altering a disease state is not provided currently
Probiotics vs. FMT test
Antibiotics: ciprofloxacin and metronidazole
7-day treatment in humans, 14 days in mice
Resulted in significant decrease in gut bacterial diversity
Bi-daily probiotics administration (same 11-strain cocktail) to 21 human volunteers
3 test group
Probiotics, auto FMT, spontaneous recovery
Result of the test of Probiotics vs. FMT
• Mice with a native gut microbiota are not colonized by probiotics. Antibiotics mildly enhance probiotic colonization in mucosal layers.
• In humans, probiotic colonization is site- and person-specific. Antibiotics significantly enhance probiotic colonization in mucosal layers
• After antibiotics exposure, probiotics delay gut microbiome reconstitution and its metabolism.
• In contrast, FMT from the same person (autologous FMT) restores mucosal microbial diversity and metabolism
Helminth infection
Worm
Free-living organisms and parasites of most animals (almost universal feature of vertebrate animals) and plants
Three major assemblages of parasitic helminths recognized: nematodes, cestodes, and trematodes
Helminth infection concerns 25% of the world population
Symptom and disease causing of helminth infection
• Symptoms of helminth infections: diarrhoea, abdominal pain, weakness, reduced nutritional input, chronic intestinal bleeding (causing anemia), intestinal obstruction and rectal prolapse, death
• Co-infection common
• Modulate host immune response (induce Th2 response), allowing for decade-long infections.
• Conflicting reports that helminths protect from malaria (Plasmodium sp.)
The hygiene hypothesis
Autoimmune disorders Vs. Helminths infestation
Gut helminth
Intestinal helminths possess immunomodulatory capacities and can alleviate bleeding in IBD.
Their interactions with the gut bacteria can alleviate asthma symptoms.
Have been shown to decrease the number of Bacteroides species involved in intestinal inflammation
Worm therapy concerns
Frequency, duration, which worm(s)?
Is the whole/live worm needed?
What are the direct effects of the helminths on the (human) host?
Interaction with other chronic or acute diseases?
Bacteriophages
Viruses infecting bacterial, exist everywhere bacteria are present
Can be recognize by immune system
Self-replicating obligatory parasites without inherent metabolism
High morphological diversity
Typically outnumber bacteria 10:1
Several molecular tools used in the lab come from phage research
DNA polymerase
restriction enzymes
CRISPR
Phage diversity
DNA and RNA phages (circular, linear, ss and ds)
Phages with capsid or envelope
Highly mosaic structure
Most phages remain unknown
No common genetic marker (no 16S rDNA equivalent), mutate so fast
Limited, incomplete databases
Replication cycle of phages
Lyric cycle
Lysogenic cycle
2 variations of lysogenic cycle
-Chronic cycle
-pseudolysogeny cycle
phage in human
Majority of DNA phages
RNA phages mostly link to diet
Most phages are integrated in bacterial cells (prophages)
Unique communities, temporally stable, high similarity between relatives and household members (“core phageome” – Manrique et
al., 2016, PNAS)
Can directly/indirectly modulate immune response
Phage to bacteria ratio lower than other systems (between 0.1 and 3)
Caudovirales (Myoviridae, Podoviridae, Siphoviridae), Microviridae, crAssphage, Lak phages
Phage populations are dynamic
Healthy Infant: Majority lytic phage highly abundant but not diversified
Healthy adult: Mostly prophages, integrated less diversified, higher abundance of microbial community
Inflamed gut: more extracellular viruses, less diversified bacteria
Disease-specific changes in virome diversity, increased phage richness, increased abundance of free phages
Phage regulate bacterial
Phage has limit host range
Phage might encode antibiotic gene
Type 2, 3, maybe 4 secretion system encoded by phages
Current questions about bacteria phage
• Can phages be used as biomarkers?
• Can we use phages to manipulate gut bacterial communities?
• Are phages shaping the microbiota-immune cross-talk?
Stunting
Height-for-age > 2 STDEV below the WHO Child Growth Standards median
Stunting affects 22% of children <5 years old
Irreversible after the first 2-3 years of life
poor maternal health and nutrition, inadequate feeding practices, and repeated infections.
Phage and child stunting
Healthy and stunted phages change gut bacterial
communities in an age-specific manner
Stunted phages allow for Proteobacteria to develop in an age-specific manner
Results suggest an intervention time window, under the age of 23 months, for microbiome manipulation
bacterial consortium
bacteria need to grow with other members in the community
