Lecture 6/7: Microbiology of Cystic Fibrosis Flashcards
What is cystic fibrosis?
A genetic disorder that leads to chronic lung infection
What is cystic fibrosis caused by?
Autosomal recessive mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Gene
What is the CFTR gene responsible for?
An ATP-dependent chloride channel
Makes exocrine secretions too thick, sticky and salty
What proportion of people are carriers for CF?
1/25 are carriers
What proportion of people are born with CF?
1/3000 born with CF
What is the most common CFTR mutation in Europeans?
Phe508
What does the Phe508 mutation cuase?
Misfolded protein tagged for protease destruction
Where does expression of the CFTR gene cause thickened exocrine secretions?
Thickened gastric mucus - meconium ileus
Thickened mucus in pancreatic ducts - totally blocked
Lack of digestive enzymes and poor nutrient absorption
When was the CF gene identified?
1989
When was the sweat chloride test developed?
1959
What did thick, sticky mucus in the lungs and sinuses lead to?
Chronic colonisation by pathogens
What effects do the sticky mucus in the lungs lead to?
Pancreatitis/ diabetes, liver damage due to bile backlog
Reduced fertility due to thick cervical mucus or blocked vas deferens
When did a massive breakthrough occur due to the understanding on the molecular biology of CF?
2010s
What percentage of people die from respiratory failure?
90%
What growth conditions come associated of those with CF?
Nutrient availability
Oxygen tension
Temperature
pH
Concentration of inflammatory cells
Activation of inflammatory cells
Local microbial competition
Host epithelial cell interactions
What is the effect of CF lung infection on the patient?
Bronchiectasis: bronchi widen and fill with mucus
Decrease in FEV - down to 20% normal function by age 20-30 not uncommon
What is bronchiectasis?
When bronchi widen and fill with mucus
What are the microbial niches of someone with CF lung infection?
Mucus gives some protection from immune cells
Decreased oxygen (get anaerobes can live in the lung)
Amino acids from damaged tissues as carbon source
Tissue damage - increased iron
Hyperinflammatory response
Biofilm
What makes biofilms so peristent?
Genetic adaptation or changes in expression
Quiescence/ persistence allow bacteria to hide from antibiotics that target
Efflux pumps
Antibiotic breakdown/ modification
Target modification/ bypass
What are the key pathogens surrounding CF lung infection?
Staphylococcus aureus
Pseudomonas aeruginosa
Burkholderia cepacia complex
Aspergillus fumigatus (fungus)
What is the microbiology of staphylococcus aureus?
Gram positive
Thick peptidoglycan layer (protects from desiccation)
Persistence on surfaces and be transmitted indirectly
Facultative anaerobe
What antibiotic resistance has been associated with Staphylococcus aureus?
Beta-lactamases, efflux pumps and mecA gene
What are the virulent factors associated with Staphylococcus aureus?
Toxins that damage cell membranes
Toxins that cause tissue damage
Invasins that promote spread in tissues
Molecules that protect bacteria from immune-produced oxygen radicals
Small colony variants - very persistent, resistant to host antimicrobial peptides
Hypermutable or mutator strains
What is the treatment of Staphylococcus aureus?
Flucloxacilin, fusidic acid, vancomycin, linezolid
What is the ultimate CF pathogen?
Pseudomonas aeruginosa
What is the microbiology of pseudomonas aeruginosa?
Gram negative
Outer membrane gives intrinsic resistance to many antibiotics
Huge genome (large availability to modify environment)
Hypermutable clones arise
Forms biofilms using range of polymers
Biofilms aids persistence - hard to break down and clear
What is the treatment of pseudomonas aeruginosa?
A range of antibiotics used
Tobramycin binds bacterial ribosomes, blocking protein synthesis
What kind of bacterial cells won’t be affected by tobramycin?
Cells with no protein synthesis
Biofilm in quiescent stage
Only targets metabolically active cells - no translation happening
What do biofilms grown on polycarbonate membranes contain?
Inner zones of low oxygen and metabolically inactive cells
What is the microbiology of the burkholderia cenocepacia complex?
Gram negative
Intrinsic resistance to many antibiotics
Motile
Can be transmissible
What percentage of colonisation is by the Burkholderia cepacia complex?
2-8%
What is Cepacia syndrome?
Aggressive pneumonia
Bacteraemia
What niche does B. cenocepacia occupy?
Invades phagocytes and epithelial cells
Rather than existing in extracellular biofilm
Moves into alveoli causing pneumonia
What is the treatment of Burkholderia cenocapacia complex?
Range of oral/ IV antibiotics
What is the fungus associated with cystic fibrosis infection?
Aspergillus fumigatus
What are the characteristics of Aspergillus fumigatus?
Forms long-lived spores (inhaled and penetrate deeply into alveoli)
Hypersensitive immune response to spores dur to existing infection in CF
Proteases can damage and evade immune cells produce toxins
What is the treatment of Aspergillus fumigatus?
Steroid to dampen immune response and antifungal agent
What are potentiators?
Open and close mutations
When was Ivacaftor approved and how was it discovered?
Available on the NHS in late 2016
High throughput screen for CFTR potentiators
What are the 3 correctors used for misfolding in CF>?
Elexacaftor
Tezacftor
Lumacaftor
What is Ivacaftor used to treat with CF mutations?
Potentiator for gating
What is Orkambi?
(combination of, treatment for, year made available on NHS)
Lumacaftor and Ivacaftor
For people with 2 copies of F508
2019
What is Symkevi?
(combination of, treatment for, year made available on NHS)
Tezacaftor and Ivacaftor
For people with 2 copies F508 or F508 + another residual function mutation
2019
What is Trikafta/Kaftrio?
(combination of, treatment for, year made available on NHS)
Elexacaftor, Tezacaftor, Lumacaftor
2020
For people with 2 copies F508 or F508+ another residual function mutation
2020
What pathogen dominates the CF infected lung over time?
Pseudomonas aeurginosa
What are the 4 types of bacterial interaction?
Niche construction
Cross-protection
Competition
Signalling
What is the bacterial interaction niche interaction?
Amino acids and short-chain fatty acids are used a primary carbon sources
Produced from mucins by anaerobic bacteria that metabolise the mucins by fermentation
How does niche construction occur?
Fermentation of gram-positive anaerobes
What is the bacterial interaction - signalling?
Many bacteria use diffusible small molecules to co-regulate the expression of virulence factors - quorum sensing
Increases virulence factor production
What are some examples of cross-species quorum sensing with P. aeruginosa?
Signals from oropharyngeal flora have shown to upregulate P. aeruginosa virulence
P. aeruginosa signals can trigger biofilm formation by S. aureus
What is the bacterial interaction - cross-protection?
Mixed-species biofilm can have higher antibiotic tolerance than single -species biofilm
Similar effect of within-species diversity
Diversification of a founding clone - lots of micro-niches in the lung
What factors affect whether cystic fibrosis communities are relevant?
How close/ far apart the species are in the lung
Environment or community in different individual patients
Genotype of infecting strains
Evolution within the lung over time
Why do evolutionary changes occur of the community in the lung?
Adaptation to the host
To chronic lifestyle
Clinical intervention
Rest of the community
Why are chronic infections complex, polymicrobial ecosystems?
The environment and community bacteria inhabit is crucial in determining their gene expression and physiology
Influences their virulence and susceptibility to antibiotics
Hard to predict which antibiotics will work against these infections
How is a pig lung a good match for modelling chronic CF lung infection?
Good match for human lung structure and chemistry
Ethical
Realistic, cheap and high throughput
How much of a colistin dose can enter the biofilm matrix?
10-20%
