Lecture 6/7: Microbiology of Cystic Fibrosis

Question 1

What is cystic fibrosis?

Answer 1

A genetic disorder that leads to chronic lung infection

Question 2

What is cystic fibrosis caused by?

Answer 2

Autosomal recessive mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Gene

Question 3

What is the CFTR gene responsible for?

Answer 3

An ATP-dependent chloride channel

Makes exocrine secretions too thick, sticky and salty

Question 4

What proportion of people are carriers for CF?

Answer 4

1/25 are carriers

Question 5

What proportion of people are born with CF?

Answer 5

1/3000 born with CF

Question 6

What is the most common CFTR mutation in Europeans?

Answer 6

Phe508

Question 7

What does the Phe508 mutation cuase?

Answer 7

Misfolded protein tagged for protease destruction

Question 8

Where does expression of the CFTR gene cause thickened exocrine secretions?

Answer 8

Thickened gastric mucus - meconium ileus

Thickened mucus in pancreatic ducts - totally blocked

Lack of digestive enzymes and poor nutrient absorption

Question 9

When was the CF gene identified?

Answer 9

1989

Question 10

When was the sweat chloride test developed?

Answer 10

1959

Question 11

What did thick, sticky mucus in the lungs and sinuses lead to?

Answer 11

Chronic colonisation by pathogens

Question 12

What effects do the sticky mucus in the lungs lead to?

Answer 12

Pancreatitis/ diabetes, liver damage due to bile backlog

Reduced fertility due to thick cervical mucus or blocked vas deferens

Question 13

When did a massive breakthrough occur due to the understanding on the molecular biology of CF?

Answer 13

2010s

Question 14

What percentage of people die from respiratory failure?

Answer 14

90%

Question 15

What growth conditions come associated of those with CF?

Answer 15

Nutrient availability
Oxygen tension
Temperature
pH
Concentration of inflammatory cells
Activation of inflammatory cells
Local microbial competition
Host epithelial cell interactions

Question 16

What is the effect of CF lung infection on the patient?

Answer 16

Bronchiectasis: bronchi widen and fill with mucus

Decrease in FEV - down to 20% normal function by age 20-30 not uncommon

Question 17

What is bronchiectasis?

Answer 17

When bronchi widen and fill with mucus

Question 18

What are the microbial niches of someone with CF lung infection?

Answer 18

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

Question 19

What makes biofilms so peristent?

Answer 19

Genetic adaptation or changes in expression

Quiescence/ persistence allow bacteria to hide from antibiotics that target

Efflux pumps

Antibiotic breakdown/ modification

Target modification/ bypass

Question 20

What are the key pathogens surrounding CF lung infection?

Answer 20

Staphylococcus aureus

Pseudomonas aeruginosa

Burkholderia cepacia complex

Aspergillus fumigatus (fungus)

Question 21

What is the microbiology of staphylococcus aureus?

Answer 21

Gram positive

Thick peptidoglycan layer (protects from desiccation)

Persistence on surfaces and be transmitted indirectly

Facultative anaerobe

Question 22

What antibiotic resistance has been associated with Staphylococcus aureus?

Answer 22

Beta-lactamases, efflux pumps and mecA gene

Question 23

What are the virulent factors associated with Staphylococcus aureus?

Answer 23

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

Question 24

What is the treatment of Staphylococcus aureus?

Answer 24

Flucloxacilin, fusidic acid, vancomycin, linezolid

Question 25

What is the ultimate CF pathogen?

Answer 25

Pseudomonas aeruginosa

Question 26

What is the microbiology of pseudomonas aeruginosa?

Answer 26

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

Question 27

What is the treatment of pseudomonas aeruginosa?

Answer 27

A range of antibiotics used Tobramycin binds bacterial ribosomes, blocking protein synthesis

Question 28

What kind of bacterial cells won't be affected by tobramycin?

Answer 28

Cells with no protein synthesis Biofilm in quiescent stage Only targets metabolically active cells - no translation happening

Question 29

What do biofilms grown on polycarbonate membranes contain?

Answer 29

Inner zones of low oxygen and metabolically inactive cells

Question 30

What is the microbiology of the burkholderia cenocepacia complex?

Answer 30

Gram negative Intrinsic resistance to many antibiotics Motile Can be transmissible

Question 31

What percentage of colonisation is by the Burkholderia cepacia complex?

Answer 31

2-8%

Question 32

What is Cepacia syndrome?

Answer 32

Aggressive pneumonia Bacteraemia

Question 33

What niche does B. cenocepacia occupy?

Answer 33

Invades phagocytes and epithelial cells Rather than existing in extracellular biofilm Moves into alveoli causing pneumonia

Question 34

What is the treatment of Burkholderia cenocapacia complex?

Answer 34

Range of oral/ IV antibiotics

Question 35

What is the fungus associated with cystic fibrosis infection?

Answer 35

Aspergillus fumigatus

Question 36

What are the characteristics of Aspergillus fumigatus?

Answer 36

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

Question 37

What is the treatment of Aspergillus fumigatus?

Answer 37

Steroid to dampen immune response and antifungal agent

Question 38

What are potentiators?

Answer 38

Open and close mutations

Question 39

When was Ivacaftor approved and how was it discovered?

Answer 39

Available on the NHS in late 2016 High throughput screen for CFTR potentiators

Question 40

What are the 3 correctors used for misfolding in CF>?

Answer 40

Elexacaftor Tezacftor Lumacaftor

Question 41

What is Ivacaftor used to treat with CF mutations?

Answer 41

Potentiator for gating

Question 42

What is Orkambi? (combination of, treatment for, year made available on NHS)

Answer 42

Lumacaftor and Ivacaftor For people with 2 copies of F508 2019

Question 43

What is Symkevi? (combination of, treatment for, year made available on NHS)

Answer 43

Tezacaftor and Ivacaftor For people with 2 copies F508 or F508 + another residual function mutation 2019

Question 44

What is Trikafta/Kaftrio? (combination of, treatment for, year made available on NHS)

Answer 44

Elexacaftor, Tezacaftor, Lumacaftor 2020 For people with 2 copies F508 or F508+ another residual function mutation 2020

Question 45

What pathogen dominates the CF infected lung over time?

Answer 45

Pseudomonas aeurginosa

Question 46

What are the 4 types of bacterial interaction?

Answer 46

Niche construction Cross-protection Competition Signalling

Question 47

What is the bacterial interaction niche interaction?

Answer 47

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

Question 48

How does niche construction occur?

Answer 48

Fermentation of gram-positive anaerobes

Question 49

What is the bacterial interaction - signalling?

Answer 49

Many bacteria use diffusible small molecules to co-regulate the expression of virulence factors - quorum sensing Increases virulence factor production

Question 50

What are some examples of cross-species quorum sensing with P. aeruginosa?

Answer 50

Signals from oropharyngeal flora have shown to upregulate P. aeruginosa virulence P. aeruginosa signals can trigger biofilm formation by S. aureus

Question 51

What is the bacterial interaction - cross-protection?

Answer 51

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

Question 52

What factors affect whether cystic fibrosis communities are relevant?

Answer 52

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

Question 53

Why do evolutionary changes occur of the community in the lung?

Answer 53

Adaptation to the host To chronic lifestyle Clinical intervention Rest of the community

Question 54

Why are chronic infections complex, polymicrobial ecosystems?

Answer 54

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

Question 55

How is a pig lung a good match for modelling chronic CF lung infection?

Answer 55

Good match for human lung structure and chemistry Ethical Realistic, cheap and high throughput

Question 56

How much of a colistin dose can enter the biofilm matrix?

Answer 56

10-20%