Interphase förel 1-6 Flashcards
NCD
Non-communicable disease, medical condition/disease not caused by infectious agents. Can refer to chronic diseases.
Main types are cardiovascular diseases, cancer, chronic respiratory diseases and diabetes. Also allergic disorders such as food allergy and allergic rhinitis among others.
The probability of dying of a NCD is the highest in developing regions.
We’re expecting an increased cost of NCDs in upcoming years.
NCD triggers
Stress
vit D deficiency
global mobility
air pollution
alcohol & smoking
consumption of antibiotics and other pharma-products
increased sanitation and hygiene
C-section as birth mode
formula feeding
Direct health effects of human microbial residents (local)
- Further digestion; provides 10-15% of host energy
- Nutrients for the gut epithelium
- Colonization resistance against pathogens
- Formation of health-promoting metabolites
- Formation of (geno-)toxic compounds
Indirect health effects of human microbial residents (systemic)
- Interaction with gut-associated lymphoid tissue (GALT) = 60-70% of our immune system
- Gut microbiome is largest endocrine organ, producing >30 hormone-like compounds
- Interaction with brain through gut-brain axis.
Humoral response of gut-brain axis
Gut-brain:
- Gastrointestinal hormones
- Inflammatory mediators
Brain-gut:
- HPA axis
Neural response of gut-brain axis
- Autonomic nervous system
Enteric nervous system
Issues related to microbiota-gut-brain-axis
- Stress
- Metabolic disorder
- Obesity
- Diabetes
- Functional Gastrointestinal Disorders
- IBS
- Neurodegenerative Disorders
- Alzheimer’s
- Parkinson’s
- Neurodevelopmental disorders
- Autism
- Schizophrenia
- Addiction
- Alcohol dependence
Microbiology
monocultures in test tubes
Microbiome
totality of microbes, their genes and their interactions with the environment. An ecosystem inside the human, highly organized, interactive and dynamic ecological union.
Resident microbiome
symbiotic interactions with the host
Transient microbiome
passing through stimulating resident microbiome; supply metabolites for cross-feeding and growth factors etc., reducing/inhibiting pathogens, indirect impact through interaction with host epithelium
Two types of selective pressure
- Bottom up : due to competition between microbes, stimulating the developement fo new and specialized niches
- Top down: by the host resulting in functional redundancy to maintain itself
Multiple species that represent a variety of taxonomic groups can share similar, or even identical roles in the ecosystem
Functional niche
Not a physical location in the ecosystem. Has functional role of the microbe in the ecosystem.
Positive interactions between microbes create new niches. Occupation of all niches; all nutrients in the ecosystem are used.
Negative interaction: negative feedback to community. Balance of + and - results in a ‘climax community’. Dynamic stability.
Colonization resistance
Pre-emptive colonization. Fewer chances for pathogens. Comes from interaction processes between and with all microbial groups; not from a single organism. Every organism in the climax community has a specific role.
Quorum sensing
Regulating bacterial gene expression in function of the population density by production of signaling molecules, ‘auto-inducers’
Bacteria sense the population density. There is a critical cell population. Two/three types of auto-inducers. They are important for biofilms and microcolonies among others
Types of Quorum sensing
Type-1 or AI-1: Species-specific, intraspecies communication = AHL ring with carbon chain specific for the species (G-)
Type-2 or AI-2: Not specific, international language for interspecies communication and communication with other microorganisms (fungi, protozoa) = Furanosyl borate diester
Type-3 or AI-3: Found in EHEC, bacterium-human host communication (cross talk AI-3 and hormone epinephrine)
Quorum sensing gene regulation for:
Biofilm formation
Virulence and toxin production
Conjugation; exchange genetic material, e.g antibiotic resistance
Enzyme production and uptake of micronutrients
Bacteriocin production
Therapeutic potential of Quorum sensing (3)
Development of pharmaceuticals specifically inhibiting the QS system → avoiding virulence/toxicity
Quorum quenching = induce QS system at low cell density to stimulate a host response long before the pathogen population is large enough to sustain infection
Signals as a biosensor marker; e.g recognizing cancer microenvironments
Abilities of top-down host microbiome
Outgrow chemical and physical stresses such as pH, bile acids and peristalsis etc..
Fend off immune response from host
Tissue tropism
Tissue tropism
Choice of a specific species to adhere to a specific place
Different types of host cells; extracellular matrix, other microbes, excretion molecules
Mechanistic explanation: receptor on host tissue recognizes complementary adhesin from microbe.
Microbiome homeostasis
property of a microbial community to maintain their stability while adjusting to conditions optimal for survival. Stability attained is actually in a dynamic equilibrium, in which continuous change of the conditions occurs between pre-set limits (homeostatic range).
Dysbiosis
Term for microbial imbalance, when microbiome homeostasis on or inside the host is not successful. Tipping points; depending on resilience.
Rivet hypothesis
Removal of either type of species does not affect the structure of the community much and it is likely that the function of the community will be preserved. Only removing many species will give a functional change
Walker’s “drivers and passengers hypothesis”
Removal of a low-abundance species does not alter the structure significantly, but removal of a keystone species gives a major structural change, which is likely associated with loss of community function and major reorganization for the community.
Alpha diversity
mean species diversity in sites or habitats at local scale
Beta diversity
differentiation among habitats/sites, absolute species turnover
Gamma diversity
total species diversity in a landscape. Gamma = alpha * beta
Nutritional and physicochemical laws concerning bacterial colonization
Liebig’s law: law of the minimum.
Bacteria dependent on many parameters. One specific parameter determines growth of the bacterial cell. Growth of bacterium follows rate limiting component in the ecosystem.
Shelford’s law: law of the tolerance.
Has to do with non-nutritional factors such as pH and temperature. Every factor has boundaries within which the organism is able to grow. Organism will only grow if environmental parameter values are within proper range (homeostatic range)
3 general steps of metabolism of carbon sources in the large intestine
1st step: Hydrolysis to oligomers and monomers
2nd step: Fermentation of monomers to fatty acids
3rd step: Is situation dependent. Further conversion to methane (colon)
Sequential steps by different microbial groups
Physicochemical aspects affecting bacterial colonization
- Temp
- pH
- Oxygen
- Redox potential
- Atmospheric composition
- Water activity
- Salinity
- Light
- Shear forces and microgravity
Nutritional aspects affecting bacterial colonization
- Building blocks of biomass
- Co-factors of enzymes
- Other micronutrients
- Vitamins
Sources; host, other bacteria, nutrition (only for GI tract)
Carbon source: Skin - lipids, respiratory tract - Mucins and proteins, large intestine - non-digested food
Which chemicals does epithelial skin membrane produce that destroy MO?
- Skin acidity (pH 3-5) - inhibit bacterial growth
- Sebum - toxic to bacteria
- Stomach mucosae secrete conc HCl and protein-digesting enzymes
- Saliva and lacrimal fluid contain lysozyme
- Mucus - trap MO entering respiratory and digestive system
Mucus
Components made by Goblet and epithelial cells
Host defense system vs nutrient source
- Has receptors for some bacterial adhesins (lectins): bacteria are ‘trapped’
- Effector molecules from innate and adaptive immune system: antimicrobial components
- Continuous secretion
- Protection against physical and chemical processes, e.g. enzymes and acid
- Humidifies and lubricates: transport of components, passage of digested food through GIT
Difference mucus and mucin
Mucus contains >90% H2O, inorganic ions, organic compounds. 2-10% mucin. Mucin is 70-80% carbohydrates.
Mucin
Can be gel forming or non gel forming
Serves as a nutrient substrate
Complex structure to give bacterial collaboration for breaksdown
keratinisation
Occurs on epithelium skin layer. Making it dry, chemcially and physically reistant surface inhospitable to microbes.
Desquamination
Process that removes any microbes that have succeeded in colonizing the epithelial surface.
Apical and basolateral side of epithelium
Apical - Contact with “the other world”
Basolateral - contact with basal lamina and underlying tissue
MAMP
Microbe associated molecular patterns
- LPS
- Peptidoglycan
- Lipoprotein
- Protein
Are recognized by PRR; pattern recognition receptors
TLR
Toll like receptor
10 different types
Specific for different modulins
Interaction of TLR with ligand activated signalling pathway
Immune control by endogenous microbiota
- Pathogens induce pro-inflammatory response
- Immune suppressive effects from microbiota
- Induction of pro-inflammatory cytokines
- Induction of TGF-beta by Lactobacillus sp
- Antimicrobial peptides
Biofilm
A way of spatial organization of a microbial community. Adhesion, growth and reproduction when nutritional needs are fulfilled. Hydrodynamic and mechanic forces counteract the adhesion process. Daughter cells can adhere somewhere else when mobile. Shedding of epithelial cells; desquamation.
Biofilms are the most natural form for microbes to occur in nature
Microcolonies
adherent microbial aggregates in extracellular polymeric substances (EPS)
How microcolonies form biofilm (5 steps)
- Reversible adhesion
- Irreversible adhesion
- Maturation I
- Maturation II
- Dispersion
The biofilm is formed on body tissue when it has proper anatomical conditions and on particles in colon. Has a high hydration. Only 10-50% of the biofilm is bacterial.
Colon mucosal biofilm
Atypical biofilm. Growth within a dynamic mucus layer; bacterial and host compounds, host inflammatory responses, continuous mucus renewal. Low densities of around 10^5-10^6 CFU/mL mucus. Always “immature”
Bioavailibility
- Gastrointestinal digestibility and solubility
- Endothelial absorption
- Distribution
- Utilisation
Can be studied in vivo with animal studies and human studies
Bioaccessibility
- Degree of digestive enzymatic hydrolysis
- Release of components from food
Can be studied in vitro with solubility assays, dialysability assays, simualted gastrointenstinal model
Bioactivity
- Endothelial absorption
- Transportation
- Transformation
- Tissue uptake
- Physiological response
Can be studied in vitro using Caco2 cell line or HT-29 cell line or in vivo using animal studies or human studies
Redox tower of microbial metabolism
MO can use a wide range of e- donors (edibles) and e- acceptors (breathables) to generate energy for metabolism. Can couple any oxi reaction to nearly any red reaction as longer as the e- donor is higher on the tower than the e- acceptor.
Host microbe metabolic interactions at 3 levels, which?
- Macronutrients
a) Carbohydrates e.g starch, poly/oligosaccharides, lactose…
b)Proteins eg peptides, albumin, hemoglobin, insulin…
c) Lipids eg triglycerides, fatty acids, cholesterol, phospholipids…
d) Combinations of the above
- Glycoprotein (=protein + oligosacc. side chain) e.g mucin
- Lipoprotein (=protein + lipid) e.g HDL and LDL - Micronutrient level
- Special focus on bioactive “extranutritrional” compounds like antioxidants, antiinflammatory, anticarcinogenic etc.
- Phytoestrogens - plant hormones that resemble natural human hormones. Can be used to prevent hormone-related diaseases as well as to fix distrubed hormone balance during menopause. - Xenobiotics
- Pollutants, “foreign to biota”
- Carcinogens, drugs, environmental pollutants, food additives, hydrocarbons, pesticides.
- Host metabolism - focus on excretion
The pan-genome/supra-genome
= the full complement of genes in a clade (species for Bacteria and Archaea)
a) Core pan-genome
b) Shell pan-genome
c) Cloud pan-genome
a) Core pan-genome: genes present in all individuals
b) Shell pan-genome: shell genes present in two or more strains
c) Cloud pan-genome: unique cloud genes specific to single strains Also referred to as accessory or dispensable genome
a) Closed pan genome
b) Open pan genome
a) Closed: very few genes added per sequenced genome after sequencing many strains, size can be predicted
b) Open: so many genes added per additional sequenced genome that predicting the size of the full pan-genome is not possible
Pan genome size affected by..
…population size and niche versatility.
What is meant with “Old friends” concerning hygiene and microbes?
Drastic decrease to MO because of use of antibiotics, antimicrobials and disinfectants. Giving lack of immune training, causing immune system to be overly sensitive → exaggerated responses. There are diseases in the industrialized western world that do not occur in rural areas.
Due to lack of exposure of MO in western world today we may actually need specific microbial compounds to train our immune system, so called “old-friends” such as B fragilis and H pylori or Helicobacter hepaticus. H. hepaticus uses a type 6 secretion system common to 25% of all bacteria which is a defense mechanism of MO against other MO. This can calm the immune system in the body. We rely on the system to upregulate Treg. However, at certain conditions it becomes a renegade and behaves as a pathogen which stresses the relationship. This means that certain alleged pathogens can have an important symbiotic relationship with the host.
Factors affecting microbiome developement (6)
- Pregnancy: uterine microbiome, third trimester, placental microbiome. In the third trimester the intestinal barrier is still very thin, giving a higher risk of obtaining pathogens.
- Time of birth: preterm versus term. Premature babies are more sensitive, less immune development.
- Mode of birth: vaginal delivery versus C-section
- Diet: breastfeeding versus formula
- Exposure to antibiotics
- Others: place of birth (hospitalization versus at home), presence of siblings, daycare attendance…
Birthmode contribution microbiome development
Study performed ten years ago saw that babies born vaginally had a microbiome resembling the mother’s vagianl microbiome whereas those delivered with C-section developed a microbiome similar to the mother’s skin.
It was also shown that birth mode had no big effect on microbiome development and that possible initial differences fade away one a feeding pattern is established.
However, higher levels of pro-inflammatory cytokines of vaginally born babies suggests that children born with C-section might have a higher risk of developing asthma and allergies since their immune system is less alert.
Rubbing of swabs from vagina and anal to the skin and mouth of the child could be performed to partially restore the vaginal microbe to children born with C-section. Studies performed in this currently.
Aging, factors affecting the microbiome (3)
- Elderly people experience dietary changes - less supportive of microbiome fitness.
- Medication and drug use typically increases, so does degree in hygiene.
These contributions give a higher vulnerability to and frequency of developing diseases.
Onset of physiological changes depend on the individual and is affected by lifestyle.
- From studies it has been seen that depending on residence location, different microbiota. Long stay residential care gives higher vulnerability.
Exposome
Assembly of exposures over a lifetime; Ab, drugs, environmental pollutants. Combination and accumulated effects over time may give changes in immune and microbiome status that can negatively affect our health status.
Other antimicrobial agents that are not antibodies
- Organic acids: Carboxylic acids able to alter physiology of bacteria, causing metabolic disorders preventing proliferation and causing death. Can increase bodyweight, improve feed conversion ratio, reduce colonization of pathogen in the intestine
- Antimicrobial peptides: Novel therapeutic agents, small molecules, broad antimicrobial spectrum
- Phytogenics: Essential oils etc
Antibody perturbation of human gut microbiome, timeline + risks
At day 4 of Ab treatment you’re able to see a significant drop in species richness. After 180 days, the value has gone up but is still low. Might also be new composition of microbiome. The difference depends on the individual.
If the microbiome does not recover, blooming can occur.
Every single use of antibiotics will increase the prevalence of resistance
Recovery associated bacteria (RABs)
20 bacterial species associated with post antibiotic recovery. Primary colonizing species. Able to use both host and diet-derived energy sources. Break down complex carbohydrates to support growth of other bacteria.
Primary colonizers - take the first steps in colonization
Secondary and tertiary and further making the range broader.
Restoration of metabolic interaction can be evaluated by seeing cross-feeding.
Positive feedback loop.
Blooming
Overgrowth of ab-resistant MO that benefit from empty space both physically and functionally. Certain of these species are harmless. May become life-threatening, such as Cl. difficile infection with diarrhea. Colonization becomes possible because it has an “open” field to play on
Developement of Antimicrobial resistance, mechanisms (3)
- Selective pressure; resistant mutant will have large fitness advantage → rapid spread.
- Horizontal gene transfer: resistance transferred from non-pathogen to pathogen. Exchange between individuals but also species.
- Resistome: Collection of AMR genes and their precursors in both pathogenic and nonpathogenic bacteria. Consists of four types of genes.
Resistome gene types (4)
Resistance genes found on pathogenic bacteria
Resistance genes found on AB producers
Cryptic resistance genes: Embedded in chromosome, can confer AMR but have not been selected in response to recent exposure of AB
Precursor genes: Do not confer AMR, encode proteins with other metabolic functions but with a basal level activity against the antibiotic molecule or affinity to the molecule. May evolve to a full resistance gene under appropriate selective pressure.
Antimicrobial strategies that spare the microbiome (3)
Use of compounds more benign to the microbiome flora.
Bacteriophage therapy
Disarming pathogens instead of killing them. Block virulence factors and regulate virulence to control the pathogens.
Plant based diet vs animal based diet
Plant based diets give increased fiber and nutrient intake and reduced fat and protein intake whereas an animal diet had the opposite effect. This also has a large effect on the microbiome.
- With animal based diets the lipid consumption is higher, giving a higher amount of bile salts to digest the lipids. Bile salts acts as a natural detergent but also AM causing stress to the microbiome.
- Bacteria specialized in fiber degradation also decreases with animal based diet, which is negative since these also produce butyrate. Butyrate gives energy to the cells in our intestines and also acts anti-inflammatory and anti-carcinogenic.
- AA fermentation products increase which is related to production of compounds that are toxic and able to damage the gut epithelium giving a reduced gut barrier integrity. AB diet may also give a higher tolerance to certain MO.
Importance of human milk given to babies, constituents in milk
Human milk contains IgA, lysozymes which cow milk lack.
Human milk also contains other things than nutrients. Human milk oligosaccharides (HMO) stimulate bifidobacterium infantis which is an efficient HMO degrade and acts as a prebiotic for the child.
> 150 HMOs have been identified. Composition differs between mothers and throughout the breastfeeding period. Four distinct milk groups. Have been found also in amniotic fluid → continuous exposure to them from conception onwards.
HMOs cannot be digested, but is meant for the MO. Normally pathogens are able to bind mucin structures in the gut, but with HMOs the pathogen will bind to those instead of the epithelium. HMOs also have an effect in brain development. HMOs have many different important aspects.
HMOs are lacking in formula milk. Even Though the other differences are small, the impact can be huge.
Human milk offers the neonate a vital protection against infections and harmful substances. Breastfeeding is the most important contributing factor to the gut microbiome development in the first years of life.
Prebiotics
“A prebiotic is a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host wellbeing and health”
Required properties of prebiotics
- Resist hydrolysis and absorption in the upper GIT
- Fermentable by only one or a limited number of potentially beneficial bacteria
- Induce an alteration in the microbial composition towards more healthy one
- Induce beneficial effects towards host
Dietary fiber
Dietary fiber means carbohydrate polymers with ≥10 monomeric units, which are not hydrolyzed by the endogenous enzymes in the small intestine of humans
Recommended amounts of dietary fiber is far above actual consumption for most people. Vegetarians and people living in rural Africa has higher consumption. Sources of dietary fiber is from whole grain foods; wheat bran, arabinoxylans, arabinogalactans…
A proven health benefit with dietary fibers is fecal bulking and improved bowel transit. Other aspects are yet not totally proven.
Fecal transplant
Fecal transplant from healthy to diseased individual could be a strategy to give better health to individual. However, there is no thing as a “superdonor” and it is hard to understand how this transplant could be used, you need to know mode of action of the MO you transplant.
Infection transmission is a large issue. Patient acceptance is fine if the patient is suffering with their disease.
Maybe there could be something in the fecal matter of healthy individuals that can also in itself cause disease..
The idea of instead selecting specific microbes for a certain effect could be a better idea. Recolonization of microbiotics.
Probiotics
So far no probiotics with proven health benefits on the market, apart from helping persons with lactose intolerance.
A probiotic is only such if you can define it at strain level with a strain determined effect. The characterization of the strain is important.
Difference pro and prebiotics
Probiotics are live microorganisms that are similar to the beneficial microorganisms found in the human gut. They are often called “good” or “helpful” bacteria because they help keep the gut healthy. Probiotics are available in supplements and foods, such as yogurt.
Prebiotics are non-digestible carbohydrates that act as food for the beneficial microorganisms in the gut. They help to stimulate the growth and activity of these microorganisms, and are found in a variety of foods, including bananas, onions, garlic, and whole grains.
In general, probiotics are taken to help restore the balance of beneficial microorganisms in the gut, while prebiotics are taken to help support the growth and activity of these beneficial microorganisms. Both probiotics and prebiotics can be important for maintaining a healthy digestive system.
Compartments of UPPER GIT
- Mouth
- Pharynx
- Esophagus
- Stomach
- Duodenum (small intenstine)
Compartments of LOWER GIT
Small intestine
- Jejunum
- Ileum
Large intestine
- Cecum and appendix
- Ascending colon
- Transverse colon
- Descending colon
- Sigmoid colon
- Rectum
- Anal canal
Food breakdown through the GIT
a) Mouth
b) Stomach
c) Small intestine
d) Colon
a) Mouth - Chewing and mixing with saliva. Transit time 10s - 2 min
b) Stomach - Mechanical and enzymatic processing of ingesting bolus. Transit time 15 min - 3 hours.
c) Small intestine - Breaking down of macromolecules and absorption of nutrients. Transit time 2-5 hours.
d) Colon - Microbial fermentation of undigested food and water re-absorbation. Transit time 12- 24 hours.
Which role od these have in digestive system?
a) Mucosa
b) Submucosa
c) Muscularis
d) Serosa
a) Mucosa - innermost layer of digestive system, in contact with lumen. Responsible for secreting mucus, also contains villi and microvilli that hep absorb nutrients from food.
b) Submuscosa - lies underneath mucosa, help support mucosa and give it the nutrients it needs
c) Muscularis - Smooth muscle tissue. Contracts and relaxes to move food.
d) Serosa - Outermost layer of digestive system. Cover outside of digestive tract and help protect it.
How does O2 and SCFA level vary through the GIT
O2: High in beginning, become lower and lower
SCFA: Low in beginning, become higher and higher
Parts of epithelium in oral cavity
- Masticatory: moderate thickness, subjected to mechanical stress, keratinized (hard pallate)
- Lining: 500 µm thick, more flexible, non-keratinized (soft pallate, cheeck, inner side lips)
- Specialized: mix of keratinised and non-keratinized (tongue with lingual papillae)
Teeth - covered of…
Covered with enamel - hardest substance and highest mineral content in the human body.
Enamel restores itself via remineralisation. saliva: rich in Ca and PO4 for remineralisation
96-97% mineral: hydroxylapatite and fluorapatite [Ca10(PO4)6F2]
Saliva functions
lubrication, digestion, buffering capacity, antibacterial defense, clearance, rinsing, solubilization, swallowing
Composition saliva
- 99.5% water
- Electrolytes
- High MW components: Ig (IgA is most important), enzymes, mucin
- N-components: ureum, uric acid
- Amylase, lipase: aid digestion during chewing and during first 30 minutes in the stomach (not yet inactivated by gastric pH)
Gingival crevicular fluid,
Gingival crevicular fluid (GCF)
Fluid from depression between tooth and gums
Nutrient rich:
- Host cells
- Proteins, carbohydrates, inorganic ion
- Desquamated cells, leukocytes
- IgA, IgG (most important), IgM, albumin, transferrin, complement, fibrinogen, antitrypsin, macroglobulin and lipoprotein
- Vit K, hemin: essential components for certain species
Has a lot of inflammatory properties.
Nutritional factors for microbial colonization in oral cavity
- Food matrix
- Saliva mucin
- Secretion products from mucusa
- Gingival crevicular fluid
- Metabolites from adhered bacteria
Physicochemical factors for microbial colonization in oral cavity
- Temperature
- pH saliva
- O2 level
Redox potential
Physical removal of microbials in oral cavity
- Remove bacteria; chewing, movement, saliva flow
- Extra co-aggregation of bacteria: Mucin and statherin keep bacterial cells in saliva
- Swallowing : Transfer to acidic stomach environment
Innate immune system in oral cavity
- Saliva: lysozyme (0.11 mg/mL), lactoperoxidase, secretory leukocyte protease inhibitor, lactoferrin, transferrin
- Mucosa: antimicrobial peptides with specific spectrum
- Cathelicidine (LL37): Active against several G+ and G-, Neutralizes effects from LPS, Requires appropriate conditions: inactive at high ionic strength
- Human beta defensin hBD-1: constitutive expression (continuous)
- hBD-2: expression upregulated by LPS and IL-1
- hBD-3: active at µg/L level
* Expression upregulated by TNF and bacteria
* Interferes with peptidoglycan synthesis - IgA: primarily from saliva
- IgG
*Primarily in GCF
*Opsonisation, neutralisation microbial enzymes and toxins, interfere with microbial adhesion
Adaptive immune system in oral cavity
- Intra-epithelial lymfocytes (IEL): repair
- Langerhans cells: antigen-presenting
- GCF/Gingival crevical fluid: delivers lymphocytes, monocytes, and neutrophil recruitment - most important polymorphonuclear leukocytes (PMNs)
Different factors affecting oral microbiota and their variables (4)
A) Age - Changes in host and habits, horizontal transfer of MO, microevolution, diversity changes
B) Host & Environment - Genetic factors, immune system, environment, diet and habits, changes in host defenses
C) Habitat - Oxygen, surface, nutrition, pH, oral hygiene, salivary flow and GCF, shedding or not
D) Biofilm maturation - Density, environment, microbial interactions, immune response of host
Streptococcus spp
G+, facultative anaerobes, catalase negative
Cocci that tend to form pairs or bent or twisted chains
order Lactobacillales (lactic acid bacteria, Firmicutes)
> 50 species
Classified based on hemolytic properties
- α-hemolytic: oxidize Fe in hemoglobin
- β-hemolytic: complete rupture of red blood cells => serotypes
- γ-hemolytic: no hemolysis
Many are commensals: Mouth, skin, intestine and upper respiratory tract
Fermented food: yoghurt, Emmentaler cheese…
Some are pathogenic:
- Meningitis, flesh-eating bacteria, bacterial pneumonia…
- AB resistance, E.g. S. pneumoniae, S. pyogenes
Veillonella spp
G-, anaerobic, diplococci
order Lactobacillales (lactic acid bacteria, Firmicutes)
Harmless, beneficial
Lactate fermentation
Methylmalonyl-CoA pathway
Dental plaque formation (4 steps)
A) Formation of acquired enamel pellicle’
Thin layer (0.1-1 µm thickness) on tooth surface: sorption proteins, glycolipids
B) Proline-rich proteins, histatin (outside), lysozyme, MUC5B, amylase, IgA, IgG, bacterial glucosyltransferases and glucan
C) Adherence of organisms with appropriate adhesins for these components
Streptococcus species (and other G+): 12-32% surface, critical cell concentration 5x10^5 cells/mm2, quorum sensing related
D) Formation of dental plaque:
Fusobacterium nucleatum and other G- attach to first adherence organisms
Diseases of oral cavity with microbial factor
- Caries -tooth decay
- Periodontal disease: Gingivitis; inflammation of gums and periodontitis - more servere form of gingivitis, non reversible
- Oral muscostitis from chemo-/radiotherapy
Stomach compartments;
a) Cardia
b) Body/corpus
c) Fundus
d) Pylorus
e) Goblet cells
f) Parietal cells
g) Zymogen cells
h) Enteroendocrine cells
i) Pepsinogen
a) Cardia - entrance
b) Body/corpus - food mixing
c) Fundus - cupola, gather swalloed gases
d) Pylorus -outlet
e) Goblet cells - mucus
f) Parietal cells - gastic acid
g) Zymogen cells - pepsinogen
h) Enteroendocrine cells - ghrelin, the hunger hormone
i) Pepsinogen - pepsin; pH optimum 1-3
Antimicrobial defense mechanisms of the stomach
Innate immunity: IgA, pepsin, lysozyme, peptides
Gastric microbiota
Very low numbers: low pH, peristalsis
Yet, highly diverse
5 major phyla
Dominant genera: Prevotella, Streptococcus, Veillonella, Rothia, Haemophilus
Common bacterial gastric infections
- Helicobacter pylori infection
- < Proteobacteria
- Virulence
- 50% global population
- 1-3% results in gastric cancer
Parts of small intestine
- Duodenum
-Jejenum
-Ileum
Small intestine epithelium ; cell types and absorption
Epithelial cells:
- Enterocytes – absorption
- Goblet cells – mucus production
- Enteroendocrine cells – secretion
Absorption:
- Selective membrane against xenobiotics, enzymes, bacteria
- Muscularis mucosa, lamina propria (supports epithelium, lymph ducts, nerves, macrophages, lymphocytes)
- Epithelium in direct contact with chyme (= intestinal fluid)
- 150 -200 m2
Antimicrobial defense mechanisms of small intestine - physical
Since the small intestine has a less thick mucin layer it needs more defense mechanisms.
-Physical removal
Desquamation
Complete renewal every 2 days
2x10^11 cells/d
Antimicrobial defense mechanisms of small intestine - innate
- Antibacterial peptides
- α-defensins
*Human Defensin 5 HD5, HD6: Paneth cells - β-defensins: epithelial cells
- hBD-1: constitutive
- hBD-2: responds to IL1, bacterial infection…
- Secretory phospholipases A2
- Histone H1:
- Displays activity against Salmonella spp.,
- Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, and E. coli
- Released upon apoptosis of villi: hence active against invasive bacteria
Antimicrobial defense mechanisms of small intestine - adaptive
Intestinal epithelium: sIgA
- Blocks bacterial adhesion to epithelium
- Microbial aggregation
Production of immune response:
- Presentation of antigen by antigen-presenting cell (APC, e.g. B-cells, dendritic cells, macrophages)
- Uptake of antigen by M cells of Peyer’s patches
Bacteroides fragilis
- Commensal, found in all mammals
- Became famous for its production of zwitterionic polysaccharides (ZPSs), which have a unique structure and function
- Polysaccharide A (PSA)
- ZPSs interact with the immune system
- = non-peptide antigens
- PSA suppresses IL-17 (pro-inflammatory)
*PSA induces CD4+ T-cell activation
- E.g. Bacteroides fragilis relieves mice of Helicobacter hepaticus induced colitis by producing PSA. According to a study.
- B. fragilis as a trigger for the host immune system
When PSA enters the cell, it is degraded by an oxidation reaction which requires presence of NOS2 and the generation of NO. PSA fragments are loaded onto the MHC II molecule HLA-DR present in the endosome. PSA induces upregulation of expression of MHC II molecules and co-stimulatory molecules. - B. fragilis correcting immune defects in GF mice
B. fragilis produces the immunomodulatory PSA that seems to be sampled by dendritic cells. DC migrate to initiate T-cell responses. The immune system of GF mice is highly skewed towards a Th2 phenotype and colonization with B fragilis corrects this
Enterobacteriaceae
- 30 genera, 150 species
- Non-sporulating, facultative anaerobe, G-, mobile
- Contains (opportunistic) pathogens
- Ferment glucose to organic acids
- NO3- to NO2-
- Antigen polysaccharide: “enterobacterial common antigen”
- Most dominant: Escherichia, Proteus, Citrobacter, Enterobacter
- Classification E. coli: type O antigen (LPS), K antigen (capside), H antigen (flagella)
Diseases of the small intestine with a microbial factor
Celiac disease (CeD) = long-term autoimmune disorder that occurs in genetically predisposed individuals upon ingestion of gluten
- Microbial alterations cause or consequence?
- Bacterial infections might stimulate or protect against CeD development
Small intestinal infectious diseases
- Viral, bacterial or parasitic infections that cause gastro-enteritis (inflammation)
- Immuno-compromised individuals, baby’s & infants, the elderly…!
Small intestinal inflammation disease
- Crohn’s disease
- Insulin resistance, metabolic syndrome (central obesity) and prediabetes (type 2 diabetes
Cancer
Microorganisms that cause intestinal infections
Bacterial contamination of food
Infection (microbial agent) or intoxication (toxins)
Symptoms:
- Small intestine: vomiting, watery diarrhea
- Large intestine: diarrhea, possibly leading to dysentery
Escherichia coli:
Vast majority are helpful commensals
Pathogenic strains:
- ETEC: Enterotoxigenic E. coli (traveler’s diarrhea)
- EIEC: Enteroinvasive E. coli
- EPEC: Enteropathogenic E. coli
- EHEC: Enterohemorrhagic E. coli (epidemics)
Salmonella enterica:
- Salmonella enterica Enteritidis
- Salmonella enterica Typhi/Paratyphi (typhoid fever)
Large intestine, microbial fermentation
Microbial fermentation:
- Bacterial community: 10^12/g dry weight
- Sugars (saccharolysis), proteins (proteolysis)
- SCFA production 400-600 mmol/d ~ 10% daily energy requirement
- N-source: proteolysis (50%), amino acids (35%) and ammonium (15%)
- Production of vitamines: vit K, vit B12
Antimicrobial defense mechanisms of large intestine -innate
Continuous, thick mucus layer
Cells: macrophages, neutrophils, eosinophils, mast cells
Pathogen recognition by TLR and NOD receptors
Effector molecules:
* Cathelicidine (LL37) = peptide with 37 amino acids
- Crypts colon (macrophages, neutrophils)
- Binds and neutralizes LPS
- Requires active form of vitamin D (sensitivity TBC patients)
* α/β-defensins
* RELM-b:
- Produced by Goblet cells
- Expulsion of the nematode parasite Trichuris muris
Antimicrobial defense mechanisms of large intestine - adaptive
IgA and AMP
Colonization resistance in large intestine
Direct CR - secreted compounds.
Indirect (immune-mediated) CR: commensals activate immune responses that target pathogenic bacteria, E.g. Bacteroides thetaiotaomicron enhances expression of REGIIIγ, an antimicrobial peptide that targets G+ bacteria. Remove pathogenic bacteria.
Endogenous colon microbiota
10^12 bacteria/g: most densely colonized niche on earth
Interindividual variability
> 1000 species distributed among individuals
60 common species
Phylogenetically diverse, but functionally similar (15% of bacterial genes are unique)
Intraindividual variability
Microbial composition evolves throughout life
Microbial composition varies with mode of birth, diet, lifestyle, AB consumption…
Frequent bacterial divisions in GI tract (2)
- Bacteroidetes (Bacteroides, Prevotella)
- Firmicutes (Clostridium, Lactobacillus, Enterococcus, Eubacterium, Staphylococcus
Enterotype
An enterotype is a classification of living organisms based on the bacteriological composition of their gut microbiota
Associated with long term diet
Bacteroides thetaiotaomicron
- Anaerobe, G-, non-sporulating, non-mobile
- very broad metabolic potency toward carbohydrates
- Mucin degrading and proteolytic
- Most require hemin and vit B12
- Genome size 6.26 Mb
- Has no organelles for host adhesion
- When food is scarce - able to change polysaccharide binding proteins
Prevotella spp
- Anaerobe, G-, non-spore-forming, non-motile, rod shape
- Colonize by binding of attaching to other bacteria or epithelial cells
- Create a larger infection in previously infected areas
- Natural antibiotic resistance
- More common in non-westernized populations consuing plant-rich diet.
Butyrate producers
Formerly categorized as Clostridium clusters IV and Clostridium clusters XIVa.
Now, family- and genera-based nomenclature:
- Family Ruminococcaceae (formerly Clostridium clusters IV)
Ex. Faecalibacterium prausnitzii (5-14% of total fecal gut microbiota)
G+, anaerobic (extremely oxygen-sensitive under lab conditions), rod
Health-promotive properties:- One of the main butyrate producers in human colon
- Anti-inflammatory properties (MAM production
- Enhances intestinal barrier functioning
Depleted in gut diseases: inflammatory bowel disease (IBD), colorectal cancer (CRC), obesity, asthma, major depressive disorder
Uses low concentrations of oxygen as a competitive growth advantage
Potential diagnostic biomarker?
* Health versus disease: IBD, CRC
* IBD subtype: UC, CD
* Disease progression: IBD flare-ups
- Family Lachnospiraceae (formerly Clostridium clusters XIVa)
Ex. Agathobacter rectalis (Eubacterium rectale: up to 13% of total fecal gut microbiota)
G+, obligatory anaerobic, non-spore-forming rods
Saccharolytic, chemo-organotrophic
Main fermentation products on peptone yeast glucose (PYG) medium were butyrate, acetate, hydrogen and lactate - Others: Eubacterium hallii, Butyricicoccus pullicaecorum, Anaerostipes spp.
Can be used by epithelial cells as energy source to get a better barrier function.
Butyrate producers stimulate T-reg.
Bifidobacteria
Actinobacteria, high GC content
Anaerobe, non-sporulating, non-mobile, G+, rods
32 species
First colonizers after birth
Unique carbohydrate metabolism: “Bifid” shunt
Acetate:lactate production in ratio 2:3
Acid tolerant, pH optimum 6.5
Broad range glycosidases: starch, xylan, pectine, inuline, dextran…
Mucus degrading enzymes : sialidase, fucosidase, galactosidase, glucosidase…
NH4+ as N-source
Vitamin production: thiamin, folic acid, nicotinic acid, pyridoxine, cyanocobalamin and biotin
Interesting as probiotic
Health promoting activities of bifidobacteria
Interference with pathogen adhesion
Bacteriostatic through acetate and lactate production
Antimicrobial component against Bacteroides fragilis, Clostridium perfringens, Vibrio cholerae, Listeria monocytogenes, Salmonella and Escherichia coli
3500 Da lipophilic component against Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa and E. coli
Bacteriocin: bifidocin B against Listeria, Enterococcus, Bacillus, Lactobacillus and Pediococcus
Enterococcus spp
Facultative anaerobe, non-sporulating, G+, catalase neg.
17 species
Order Lactobacillales (lactic acid bacteria, Firmicutes)
E. faecalis, E. faecium most dominant
Tolerant organisms
10-45°C, bile salts, acids, base, hypotonic, hypertonic
Most are AB resistent: specific E. faecalis strain as hospital strain
Cytolysin: against bacteria, erythrocytes…
Proteases: against connective tissue, fibrinogen
Polysaccharide coat: protects against phagocytosis
H2O2: kills off bacterial cells
Sulphate reducing bacteria (SRB)
Inverse relation with methanogens:
Competition for H2
Can not both be dominant
Archaea
A methanogen
Is non mobile, anaerobe, G+. Most frequently Methanobrevibacter smithii. Performs methanogenesis. Mimics host glycans.
Microbial habitats in the colon count
High bacterial count in lumen, becomes lower when closer to epithelial cells, which is good.
Colon mucoscal layers
Outer:
- 3-4x volume expansion
- Loose polymeric network
- Removable by suction
- Colonized by microbes using flagella, mucus-binding proteins, …
- Mostly long curved rod-shaped bacteria
Inner:
- Several 100 um thick
- Lamellar, highly organized layer of stacked mucins
- Firmly attached
- Largely devoid/ very low numbers of microbes
- Mostly short coccoid rods
Difference in fluid shear of the colon
High:
- Turbulent mixing of nutrients
- Physical perturbations
- Higher survival percentage of Salmonella infected mice over a longer time
Low:
- Diffusion of nutrients
- Much less physical perturbations
- Reprograms gene expression, physiology and stress resistance of (opportunisitc) pathogens
Oxygen gradients in colon
Low oxygen:
- Growth advantage for F. prausnitzii
- Growth advantage for “nanaerobes” e.g, Bacteroides spp.
- Enhance infective properties of pathogens
Colon host defence molecules
Secretion of antimicrobial peptides and sIgA much lower than in small intestine
BUT
remain trapped in mucus and only marginally leach to lumen
–> Comparable / higher antimicrobial activity than in small intestine?
Colon crypt niche
- Low mucus viscosity –> selecting for short coccoid rods
- High oxygen partial pressure –> selecting for aerotolerant species
- Specific types & concentrations of host glycans
–> Glycan mediated crypt occupancy
Colon mucoscal biofilm keypoints
- The colon mucosal environment is a highly structured environment
- Across the mucus layer, a variety of microenvironments are found impacting mucosal biofilm composition and functionality
- Microbes actively participate in shaping the mucosal environment
→ Towards a better insight into the mucosal biofilm for improved monitoring and modulation of human health
Diseases of large intestine with microbial factor
- Large intestinal infectious diseases
- Viral, bacterial or parasitic infections that cause gastro-enteritis (inflammation)- Immuno-compromised individuals, baby’s & infants, the elderly…!
- Large intestinal inflammation diseases
- IBD: Crohn’s disease, Ulcerative Colitis
- Cancer
Difference between interaction between bile salt and bacteria in the GIT between healthy and IBD subject
Healthy:
- Normal microbiota enzymatic activity
- Desulfation
- Small production of primary BA and sulfated BA and a lot of secondary BA.
IBD:
- Lower microbiota enzymatic activity
- Dysbiosis giving higher amount of primary BA.
- Lower desulfation
- Higher amount of sulfated BA and lower amount of secondary BA. Giving rise to a inflammation loop
Virulence factors
- Flagella; Long filamentous structures, Propellors → motility, Major role in host colonization
- Extracellular polysaccharides: Slime layer surrounding cells, Functions: adhesion (sticky) and protection ↔ host defenses & AB. It is very rare for bacteria to be in suspension, it wants to adhere to an area. With the slime layer the bacteria can become isolated from the host environment. It offers protection.
- Lytic enzymes and toxins: Damage the host and there are many different types. For example hemolysin that breaks down blood cells.
- Secretion systems: Transport of virulence factors out of virulent cell. There are different types.
Helicobacter pylori
- Microaerophilic, mobile, non-sporulating, G-
- Catalase, urease and oxidase positive (identification)
- Urease = 20% total protein !
- Produced ammonium functions as protective layer that allows migration across mucus layer to epithelium
- Adhesins: BabA, LPS
- Virulence factors: cag (cytotoxin associated gene) pathogenicity island (31 genes)
- Interindividual variability
- Creates a pH neutral environment; able to survive in the stomach.
Clostridial toxins
The genus Clostridium
G+, anaerobe, sporulating, some are motile
Can be health-promotive: e.g. clusters IV and XIVa (old taxonomy)
Can be virulent: toxin production!
E.g. Clostridium perfringens → gas gangrene, enteritis necroticans, food poisoning
E.g. Clostridium difficile → life-threatening AAD
Chron’s disease
CD is a chronic relapsing inflammatory condition usually with flare-ups alternating with periods of remission, and an increasing disease severity and incidence of complications as time goes on. Triggering factor is unknown.
It can affect any part of the gastrointestinal tract from the mouth to the anus. 30-60% of patients have procititis where they experience rectal bleeding, tenesmus, urgency
Complications of Chron’s disease
- Fistula formation - abnormal channels of communication between loops of bowl, bladder and skin. If CD in the colon, fistula can form between colon and vagina or between colon and perineum. Skapar nya vägar i magen
- Obstruction - Typically from structures of adhesions which narrow the lumen, blocking passage of intestinal contents.
Difference in tarm between Normal, chron’s and Ulcerative colitis
- Normal: Wide with little fat
- CD: Fissures, thickened walls, fat wrapping around
- UC: Ulceration of tisssue, surviving mucosa, loss of naustra, crypt distortion
Treatment of IBD
Never cured, goal is to achieve and maintain remission
Flare-ups and periods of remission can be controlled with:
- Antibiotics
- Local anti-inflammatory drugs: 5 aminosalicylates (5-ASA), e.g. sulfasalazine, mesalamine. Not every patient responds to 5-ASA.
- Strong anti-inflammatory drugs: corticosteroids (short-term, control inflammation)
- Immunomodulators (long-term, maintain remission) e.g. azothioprine, 6-mercaptopurine
- Biologicals: antibodies against TNF (anti-TNF) and other chemokines (IL12, ICAM, α4integrins)
- Ab - Used but have a lot of side effects.
- Pig parasites - can help train the immune system and make it able to cope better with pro-inflammatory diseases
- Probiotics
- Nutritional support: identify and avoid trigger foods
Cause of IBD
- Not one clear cause
- Likely to be an outcome of interactions between genetic predisposition, environmental factors and subsequent reaction of host immune system
- Genetic susceptibility can be from mutation.
- NF-kb is essential in the proinflammatory pathway, will trigger TNF-alpha. The protein structure is important in immune response. For patients with CD it was found that 50% of patients have 1 or 2 mutations.
*NOD2 mutation - Like a TLR, appears to be an intracellular pattern-recognition receptor for bacterial endotoxin. Lead to defective function of macrophages, defective epithelial cell responses, defective conditioning of APCs
- NF-kb is essential in the proinflammatory pathway, will trigger TNF-alpha. The protein structure is important in immune response. For patients with CD it was found that 50% of patients have 1 or 2 mutations.
Microbial factors in IBD
- (Colitogenic) microbial dysbiosis
- Reduced diversity, which renders the microbiome more susceptible to pathogens or pathobionts
- Increase in invasive species:
- Enterobacteriaceae (adhesive species such as AIEC; Proteus)
- Other gamma-proteobacteria
- Decrease in protective species:
- Butyrate producers: Faecalibacterium prausnitzii, Roseburia spp.)
IBD and systemic inflammation
- Susceptibility for local inflammation is related with susceptibility for systemic inflammation
- Significant overlap in genetic susceptibility loci for IBD with a variety of inflammatory diseases involving extraintestinal tissues
- Frequent extraintestinal disease manifestations of joints, skin and eyes:
- Musculosketelal diseases
- Uveitis
Note: local inflammation in rheumatic disease can also become systemic and cause IBD
Chronic low-grade and systemic inflammation in metabolic syndrome (obesity)
Metabolic syndrome is a clustering of at least three of the five following medical conditions: central obesity, high blood pressure, high blood sugar (insulin resistance), high serum triglycerides, and low serum high-density lipoprotein (HDL)
Adipocytes in adipose tissue produce cytokine-like hormones: adipokines, e.g. leptin, adiponectin, visfatin, resistin, …
Macrophages in adipose tissue also produce TNF-α, IL-6, …
Metabolic syndrome → elevated inflammatory state
- Pro-inflammatory C-reactive protein, TNF-α, plasma resistin, IL- 6 and IL-18 are up
- Anti-inflammatory adiponectin is down
Energy homeostasis
Affected by hormones. Hormonal imbalance will give you hunger feelings when not needed.
Long-term regulators in adipose tissue:
food intake-inhibiting hormones
leptin and adiponectin
Hormones produced in the GI tract and pancreas - short-term
food intake-inhibiting: peptide tyrosine-tyrosine PYY, glucagon-like peptide 1 GLP-1, oxyntomodulin OXM, cholecystokinin CCK, pancreatic polypeptide PP, amylin
Food intake-stimulating: ghrelin
Akkermansia muciniphilia
might be able to be used for obesity and type 2 diabetes. Inverse correlation for humans between body weight and presence of A. muciniphila. Is associated with a healthier metabolic status and better clinical outcomes after caloric restriction. The pathway is not totally understood yet, but it seems to be able to make the epithelial barrier stranger and decrease inflammation.
Neurological disorders and the gut-brain axis
Correlations between diseases and microbiome shifts
E.g. Antibiotic use and schizophrenia
E.g. Bacteroidetes and autism severity
E.g. Lower Bifidobacterium and Lactobacillus in depression
Intervention studies with probiotics showing improved symptoms of e.g. IBS, autism, depression, anxiety…
Production of neurotransmitters by gut microbiota
E.g. Dopamine
Correlations between early-life microbiome stressors and disease
E.g. ADHD and delivery mode, type of feeding, gestational age…
IBS
Diagnostic criteria (Rome IV)
Recurrent abdominal pain, on average, at least 1 day per week in the last 3 months, associated with 2 or more of the following criteria:
- Related to defecation
- Associated with a change in frequency of stool
- Associated with a change in appearance (form) of stool
Criteria fulfilled for the last 3 months with symptom onset at least 6 months before diagnosis.
Gut microbiota neurotransmitters and neuroactive compounds
- SCFA; Affects microglial maturation.
- Serotonin; Happiness hormone. Produced in the colon (90%) and brain(10%). Dysregulation can lead to IBS, cardiovascular disease, osteoporosis.
- γ-aminobutyric acid (GABA)
- Dopamine
Respiratory tree (7)
- Trachea
- Mainstem bronchus
- Lobar bronchus
- Segmental bronchus
- Bronchiole
- Alveolar duct
- Alveolus
Airway surface liquid
5-100 µm thick
Three components:
- Layer of periciliary fluid
- Surfactant (Clara cells)
- Superficial gel or mucous layer (Goblet and serous cells)
Microvilli around cilia determine the thickness of ASL
Respiratory tract mucus
- Extends from the intermediate airway to the upper airway and is approximately 2-10 μm thick in the trachea
- Secreted by Goblet cells and the submucosal glands
- At least 8 MUC genes expressed in the respiratory tract
- Main gel-forming: MUC5AC and MUC5B
Mucociliary clearance
The most important antimicrobial defense mechanism for airways
Trapping of particles in the mucus layer. Transport from the lower respiratory tract to the pharynx. 200 cilia per cell. 30 nm long hooks from the tip. The cilia vibrates rhythmically. Waves 5-20 nm/min.
If the periciliary fluid is too long - the cilia do not reach the mucus. If too short - mucus hinders cilia movement.
Disruption impairs the pulmonary function and lung defense and increases risk of infection → cough is neede for airway hygiene.
Interferance of mucociliary movement
Upon adhesion, bacteria is able to hinder mucociliary movement. This can damage cilia, inhibit ciliary activity, change mucus viscosity and modulate the amount of mucus.
Bacteria adhesives to ASl first. The mucus has a ligand/receptor binding and hydrophobic interactions. There is a balance between matrix that acts to remove bacteria or intermediary stage for bacteria to migrate to epithelium. The periciliary layer is less renewed due to lack of activity of the cilia, there is a larger chance for colonization.
Epithelium - more permanent place for colonization. Only few bacteria able to adhere to ciliated cels. Upon damage there is a release of other receptors for further bacterial adhesion.
Innate immune system components in nasal fluid
- Lysozym (10-30% of total protein)
- Lactoferrin (2-4% total protein)
- igA (15% total protein), igG (4% total protein) → agglutination of bacteria, better removal, avoid adhesion.
- Statherin - broad spectrum activity
- Elafin: Neutralizes pro-inflammatory effects from LPS.
- H2O2 and peroxidase: reactive oxygen components, also broad-spectrum
Alveolar Lining Fluid
Lysozym, free fatty acids, immunoglobulins (IgG !), Fe-binding proteins and surfactant proteins
IgG: serves as opsonin enhancing phagocytosis
Surfactant protein A (SP-A) binds macrophages and bacteria (also opsonin function) and stimulates phagocytosis by PMN, macrophages
- Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenza
- Recognizes di-mannose bonds and lipide A domain of LPS
SP-D: binds to other types of bacteria
- E. coli, Klebsiella pneumoniae, Mycobacterium tuberculosis
- Recognizes core oligosaccharides of LPS
Keystone species of airway microbiota
Keystone species with exceptionally large benefit, e.g. Corynebacterium spp. and Dolosigranulum spp., exclusion of Streptococcus pneumonia
Chronic rhinosinusitis
Chronic (>12 weeks) inflammation of lining of the sinuses creating extra mucus
Affects ~ 10.9 % of adult population in Europe
Sinusitis is number one indication for antibiotic prescription in outpatients
Aetiology unknown:
- Infection? Pathogen colonization and microbiota imbalance might be initial causes of chronic immune response and inflammation.
- Inflammation? Dysfunctional immune barrier, inflamed mucosal epithelium and obstructed sinuses resulting from allergic or non-allergic inflammation may promote secondary bacterial overgrowth and dysbiosis, setting the stage for CRS.
Role of microbiota in disease aetiology, persistence and modification
Cystic fibrosis
Most common lethal genetic disorder amongst caucasians. Life expectancy ca 40 years. Single primary cause = dysfunctional CF transmembrane conductance regulatory (CFTR) chloride channel. Affects every secretory organ in the body.
- Normal lungs have no osmotic gradient and hydration of the mucus layer.
- CF lungs where the CFTR does not work we have an increased uptake of sodium, leading to mucus dehydration and the mucus becoming very viscous make cilia movement inpossible. Microbes are therefore not moved away and can stay resident and use their flagella to move towards epithelial cells to start infect process.
Opportunistic bacteria exploit the static CF mucus and this gives chronic infection characterized by intense neutrophilic inflammation
Male urogenital system
One combined system
Externally: foreskin and urethral opening, penis, scrotum → skin microbiota
Internally: Testicles, epididymis, sperm duct, accessory glands, bladder, urethra = exit for urine and semen, the only space for specific urogenital bacterial colonization. Male urethra is 5x longer than the feminine counterparts, which leads to risk fo bladder/kidney infection being much lower.
Female urogenital system
2 separate systems
Urinary tract
Reproductive system: Ovaries, fallopian tubes, uterus, cervix, vagina and vulva. Most important microbial colonization: Cervix, vagina and vulva.
Labia majora: Adipose tissue, sebaceous and suduoriferous glands.
Labia minora: Especially sebaceous
Antimicrobial defense mechanisms of urogenital system
Desquamation epithelial cells, urinary flow.
Urine
Antimicrobial: Urea, peptides, antibodies.
Antiadhesion: TH protein, sigA
Sperm
Contains Zn from prostate which hinders growth of certain MO
Prostate liquid: Phospholipase A2 is active against G+
Spermidine, spermine, lactoferine etc etc.
IgA and IgG
Urethra: Covered with IgA/G/M → barrier.
Urinary microbiota, thoughts about it previously vs today
Previously:
Sterile, only microbes during urinary tract infections
Now: Urinary microbiome
Difference between healthy control and urogenital diseases
Significant differences between sexes
Variety too high to speak of comparable communities between individuals of the same age and gender
Vaginal mucosa
- 3 layers: basal, intermediary layer (glycogen production), surficial layer (microfilaments, rigidity)
- Lymphocytes, Langerhans cells
- Constant remodeling during menstruation cycle:
- Estrogens: Proliferation, maturation, desquamation
- Progesterone: Inhibits maturation
- Before menarch, after menopause: Thin epithelium
- All cells are mucus secreting: 10mg/day
- 700 mg mucus/day during ovulation
Antimicrobial defense mechanisms of vagina -innate
MUCUS
Receptor for bacterial adhesins
Desquamation (hormonal regulation)
Contains antimicrobial components
- Lysozyme: lowest during ovulation
- Lactoferrin: cycle dependent (only 10 µg/mg protein upon menses), contraceptives lower lactoferrin
- Secretory leukocyte protease inhibitor: highest during ovulation
hBD
Natural antibiotics, 3-4 kDa cationic peptides, affect bacterial cell wall permeability: amphiphilic property
hBD-1: constitutively
hBD-2: induced upon infection
Defensin HD-5
LL37, calprotectine
Group D Streptococci - death cause of newborns, how prevent?
During conception: cervical mucus hardens at the cervix and forms a plug which blocks the uterus → Physical barrier + antimicrobial components against Streptococcus agalacticae (group D streptococci = most important cause of death with newborns). 2-3 weeks before giving birth, swab test is performed to detect eventual group D streptococci. if positive, get treated with prophylactic when giving birth.
Antimicrobial defense mechanisms of vagina - adaptive
Vaginal mucosa differs from mucosa elsewhere
No lymphoid follicles:
- Primary mucosal immune response
- Dendritic cells: antigen presenting
- Immature B and T cells
Antibodies locally produced, but also circulatory
Hormonal control:
- IgA and IgG are higher before ovulation
- IgA first half of menstrual cycle (high estrogen)
Number of Ig-producing and -containing cells in cervix is 4x that from vagina
Hinder microbial adhesion, neutralize toxins, induce phagocytosis by neutrophils
Vaginal microbiota changes over time
a) Neonate
b) Premenarchal
c) Postmenarchal
d) Pregnant
e) Postmenopausal
Neonate:
- Conditions in newborn baby girl = adult woman
- After 3 weeks, normalization to child-like environment
Premenarchal - before first menustral period of woman
- Peptostreptococcus, Bacterioides, Clostridium, Eubacterium as anaerobes
- Staphylococcus epidermis, coryneforms as facultative anaerobes
Postmenarchal / premenopausal - having period
- mostly lactobaciullus dominated
- During menses
* Stability decreases and correlates with estrogen levels
* Increased iron levels –> Accelerated growth of Gardnerella and Lactobacillus iners, other lactobacillus decrease in abundance.
Pregnancy
- Overall increased stability (lowering susceptibility to infection)
- Less diverse, more Lactobacillus-dominated
- Accumulation of glycogen (< estrogen) 🡪 lactate
- Increasing vascularisation: higher O2 partial pressure, redox
- Bacterial vaginosis and preterm delivery? To be elucidated…
- Postpartum vaginal microbiome up to 1 year: less lactobacilli, more vaginosis-associated bacteria, higher resemblance to gut microbiome
Postmenopausal
- Less lactobacilli, because of the environment changing
- More anaerobic species: Gardnerella vaginalis, Mobiluncus, Bacteroides
- More viridans streptococci
- Lower glycogen concentration
- Thinner epithelium
- Absence receptors for bacterial adhesins
- Changes in immune defense
- Changes in production of vaginal fluid
Health status of vaginal microbiome is associated with..
…low community diversity. Many community state types of healthy reproductive-aged women are dominated by Lactobacillus species and remain fairly stable, but not permanent. Glycogen deposits in the vaginal epithelium are being used by the species in anaerobic glycolysis, giving lactic acid production.
In disease states, the communities observed are more diverse and less stable.
Lactobacillus spp in vaginal microbiome
G+, facultative anaerobe or microaerophilic, rod-shaped, non-sporulating, catalase negative.
Order Lactobacillales (lactic acid bacteria, Firmicutes).
10^7-10^8 cells/g vaginal fluid
Affinity for vaginal epithelial cell adhesion
Sugar fermentation
Homofermentative: glucose → lactate
Heterofermentative: glucose → formate, acetate, lactate, ethanol
Lactate and acetate are bacteriostatic
Many adhesins
Lipoteichoic acids, fimbriae, glycoproteins…
Produce bacteriocins and H202
Lactic acid, reuterin, antimicrobial proteins!
Note: H202 minor contribution in vagina: requires oxygen, anaerobic lactobacilli do not produce peroxides. H2O2 is inactivated by the reducing capabilities of cervicovaginal fluid and semen. Production of H2O2 by Lactobacillus is very low, compared to state of Bacterial Vaginosis.
4 most common strains: Jensenii (41%), Crispatus (38%), Gasseri, Iners
Adhesion to vaginal endothelial cells (VEC).
Gardnerella vaginalis
Only species of Gardnerella genus (Order Bifidobacteriales)
G-variable: age of the culture may influence staining results (Cfr. Mycoplasma)
Non-sporulating, facultative anaerobic, nonmotile coccobacilli
Virulence: pilli, LPS
EPS
Acetate, lactate
Sialidase
Siderofores
Bind Fe-containing groups (haemoglobin, lactoferrin…, not transferrin)
Haemolysin: lyses neutrophils and endothelial cells
Bacterial vaginosis, BV
- Microbial community out of balance
- Proteolytic enzymes: production polyamines (smell) that in turn induce secretion
- Not to be confused with Candida albicans infection
- “Clue cells”: Epithelial cells covered with bacterial cells
- Unclear what drives the process. A drop in lactobacillus species, which gives increased pH.
- Lactobacillus LBP could be used as a novel therapeutic strategy in BV. They believe it is the production of reuterin by lactobacillus reuteri that can help.
Semen microbiota
A new research area. Perhaps correlation between semen microbes and fertility
Differential abundance of specific bacterial genera correlated with sperm parameters
- Prevotella - abnormal spermiogram (at least one deficiency, total motility)
- Staphylococcus - normal spermiogram (total motility)
- Lactobacillus - normal morphology
Intra-uterine infection (when pregnant)
- Accounts for 25-40% of preterm births
- Most common species are genital Mycoplasma
Several potential routes of intra-uterine colonization
Microbial evolution during pregnancy - 3rd trimester
In 3rd trimester maternal gut microbiome changes.
Increased fat mass, higher glucose values and lower insulin sensitivity.
Maybe because if child is able to gain energy quicker then, it has higher potential for survival, therefore energy intake has to the as efficient as possible.
