Biofilms

Question 1

How do biofilms form? P.aeruginosa, different shapes? (problems for liquid flow in medical device?)

Answer 1

Cell-cell and cell-surface contacts, quorum sensing signals, sticky EPS (extracellular polymeric substrate):

P.aeruginosa can form mushroom shapes in low flow

And form filamentous streamers in high flow (can block tubes, reduces flow rate)

Question 2

Biofilm heterogeneity induced by nutrient gradients? 3 ways?

Answer 2

Metabolic substrate gradient (like oxygen in aerobes) concentration lowers towards centre

Metabolic product gradient (e.g. methane in methanogens) increases concentration towards centre

Metabolic intermediate reaches high concentration part-way through biofilm then decreases (e.g. denitrifying bacteria use up waste nitrite?).

Question 3

3 different genetic mechanisms for creating heterogeneity in bacterial biofilms?

Answer 3

1) Adaptive gene expression (different genes, induced by different environments/gradient) (e.g. facultative anaerobes in oxygen concentration gradient)
2) Mutation/genetic variation and natural selection (e.g. non-mucoid mutants of Strep.pneumoniae –> better attachment, less protection)
3) Stochastic/random gene expression (can lead to persister cells in infection, more variety [independent of environment] –> more chance of resistance/resilience)

(“bistable switch” formation, e.g. EPS on or off in Bacillus subtilis, SinI anti-repressor sometimes expressed, de-represses Eps operon (from SinR repressor)

Question 4

Why are biofilm infections difficult to treat (and even diagnose)? examples?

Answer 4

Difficult to treat: Resistant to phagocytosis due to large size.

• *Resistant to many antibiotic drugs** (due to variability and slow growth)
• *Persister cells** (due to stochastic gene switching, and other genetic variation)

Diagnosis is difficult because they are localised infections.

Examples: P.aeruginosa in CF patients
Infective endocarditis (Strep. vegetations on damaged endothelium, layers of fibrin and platelets interspersed!)
Medical device infections

Question 5

4 diagnostic criteria for biofilm infection diagnosis?

Answer 5

1. Pathogenic bacteria surface associated (invasive investigation)
2. Direct examination reveals bacteria clustered encased in matrix
3. Localised infection
4. Abx resistant infection (when planktonic cells would be sensitive)

Question 6

Three mechanisms by which Biofilms could be resistant to ABx?

Answer 6

Slime matrix (EPS) forms barrier or dilutes Abx

Slow growing, slowly metabolising cells less susceptible (e.g. to beta lactams)

Genetic heterogeneity –> resistant persisters left behind

Question 7

3 Stages of staphylococcal biofilm lifecycle: key molecules in each stage?

Answer 7

1. Attachment (MSCRAMMS and autolysin AtlE)
2. Maturation (PIA, polysaccharide intercellular adhesion, deacetylated by icaB. –>positive charge, interacts with -ve [lipo]teichoic acids)
3. Dispersal: Agr quorum sensing promotes dispersal! Increased detachment factors PSM (phenol-soluble-modulins) and decreased attachment factors (EPS and autolysin AtlE)

Question 8

What is Disperin B?

Answer 8

PIA degrading enzyme found in wound dressings used to discourage biofilm formation.