Lecture 3: Pseudomonas aeruginosa Flashcards
What is pseudomonas aeruginosa?
An opportunistic pathogen that causes infections in immunocompromised individuals.
Describe the structure of Pseudomonas aeruginosa
Gram-negative rod-shaped bacterium (bacillus).
Which diseases are caused by Pseudomonas aeruginosa?
Pneumonia (especially in ventilated patients).
Sepsis and septic shock.
Urinary Tract Infections (UTIs).
Skin and soft tissue infections, including burn wound infections.
Is it true that P. aeruginosa is responsible for
one in ten hospital-acquired infections?
Yes
What are some sources of pathogen Pseudomonas aeruginosa?
Water sources: Public washrooms, rivers, and streams.
Animals: Domestic pets and farm animals.
Contaminated oil spills
is it true that Pseudomonas aeruginosa is a major cause of nosocomial infections (hospital-acquired infections) ?
Yes
Biotyping of gram negative bacteria
What groups can gram negative bacteria be split into?
coccobacilli, cocci, bacilli
Case Study: PA Superbug Infection
Patient presentation:
- 38-year-old male with symptoms of UTI (fever, chills, pain during urination, incomplete voiding).
- No history of recent UTIs or sexually transmitted infections.
- Progressed to septicemia (blood infection).
Diagnosis:
Phenotypic testing:
- Gram-negative bacillus identified through gram staining.
- Non-lactose fermenting (distinguishes PA from other gram-negative bacteria like E. coli).
- Oxidase-positive (indicating aerobic metabolism).
- Confirmed as Pseudomonas aeruginosa.
- Source investigation:
- Suspected sources:
- House water supply.
- Newly purchased hot tub (filled with stream water).
- Unrelated external source.
Genotypic analysis linked the strain in the patient’s blood to the contaminated hot tub.
What is RFLP (Restriction Fragment Length Polymorphism) ?
Detection and dentification of variations in homologous DNA sequences through the use of restriction enzymes, which cleave the DNA into fragments of varying lengths.”
What is normal electropheresis?
Separates DNA based on size
①A DNA sample is prepared
②The DNA is digested with one or more restriction enzymes that recognise and cut DNA at specific sites:
③A loading buffer with a blue dye is added and the samples are applied to the gel
④A current is applied, and the DNA fragments are separated on the basis of size
DNA is visualised by incorporation of a fluorescent dye – ethidium bromide – that intercalates between the bases of dsDNA, and is then viewed and photographed under uv illumination.
What is PFGE (Pulsed Field Gel Electrophoresis)?
- Standard gel electrophoresis cannot separate very large molecules of DNA.
- Pulsed Field Gel Electrophoresis (PFGE) is a variation that introduces an alternating voltage gradient to improve the resolution of larger molecules.
- Instead of constantly running the voltage in one direction, the voltage is periodically switched among three directions; one that runs through the central axis of the gel and two that run at an angle of 60 degrees either side.
- The pulse times are equal for each direction resulting in a net forward migration of the DNA.
- This procedure takes longer than normal gel electrophoresis due to the size of the fragments being resolved and the fact that the DNA does not move in a straight line through the gel
What are the advantages of PFGE?
✓ Good for analysing recent evolutions
e.g. Hospital outbreaks.
✓ Highly discriminative.
✓ Faster than other genotyping methods
What are the disadvantages of PGE?
X Requires many hours of work.
X Very technical & hard to reproduce.
X The results may be subjective.
Describe some phenotypic methods of identification
Gram Staining:
- Differentiates bacteria into gram-positive (purple) and gram-negative (pink).
- PA is gram-negative.
Lactose fermentation:
- Tests for the ability to metabolize lactose.
- PA is a non-lactose fermenter.
Oxidase test:
- Detects cytochrome oxidase enzyme in aerobic bacteria.
- PA is oxidase-positive.
Is it true that there has been a rapid and disturbing rise in carbepenem resistance in Pseudomonas aeruginosa?
Yes
What are Carbapenems?
A last-resort antibiotic (class of beta-lactams).
What are the known mechanisms of Carbapenem resistance in P. aeruginosa?
- Reduced permeability: Downregulation of porins (channels for antibiotic entry).
- Efflux pumps: Proteins actively pump antibiotics out of the bacterial cell.
- Carbapenemase production: Enzymes that break down carbapenem, rendering it ineffective.
Concern: Resistance genes are often carried on plasmids, allowing rapid spread across species.
Concern: Resistance genes are often carried on plasmids, allowing rapid spread across species.
Describe reistance to quinlones
Quinolones (e.g., ciprofloxacin) target DNA gyrase (enzyme essential for bacterial DNA replication).
Target modification: Mutations in DNA gyrase prevent quinolones from binding.
Efflux pumps: Expel quinolones from bacterial cells.
Why is the presence of carbapenemases in bacteria ,including in Pseudomonas aeruginosa strains, is a significant concern for public health?
due to the limited treatment options available for these resistant infections.
How can the resistance mechansims of carbapenemase production be tested?
- Spread a carbapenem-sensitive strain on an agar plate.
- Place a filter disk containing carbapenem in the centre.
- Streak test strains into the zone of inhibition.
- Growth of test strains in the inhibition zone indicates carbapenemase production (enzymes degrade carbapenem).
What do Quinolones (ciprofloxacin) inhibit ?
DNA gyrase (topoisomerase)
For the exams
Revise:
Phenotyping of Pseudomonas aeruginosa
RFLP & PFGE.
Carbapenem and quinolone resistance in Pseudomonas aeruginosa
Detection and Control of carbapenemase-producing bacteria (modified Hodge test).
Summary
- Nucleic acid typing techniques show superior strain discrimination.
- Rapid disturbing threat of carbapenem and quinolone resistance in Pseudomonas aeruginosa
- Carbapenem resistance: carbepenemases
- Quinoline resistance: mutations in GyrA, or aquision of effluc pumps
- These resistance phenotypes have spread to other Gram-negative pathogens
