Lecture 12. Key Gram Negative Superbug

Question 1

What nosocomial infections can Pseudomonas aeruginosa cause?

Answer 1

Pneumonia
Septic shock
Urinary tract infection
Gastrointestinal infection
Skin and soft tissue infections

Question 2

How many hospital-acquired infections is Pseudomonas aeruginosa responsible for?

Answer 2

One in ten

Question 3

What are examples of reservoirs of Pseudomonas aeruginosa?

Answer 3

Water sources
Paws and hair samples
Contaminated oil spills

Question 4

What does PFGE stand for?

Answer 4

Pulsed Field Gel Electrophoresis

Question 5

What charge does DNA have?

Answer 5

Negative

Question 6

If placed in an agarose gel in an electric field, where will the DNA travel?

Answer 6

Towards the positive electrode

Question 7

How does gel electrophoresis split the DNA?

Answer 7

The gel matrix is difficult for large DNA fragments to move through so large fragments lag behind while small fragments move through the gel relatively rapidly

Question 8

What fluorescent dye is used to visualise DNA in gel electrophoresis?

Answer 8

Ethidium bromide

Question 9

What are the limitations of standard gel electrophoresis?

Answer 9

Cannot separate very large molecules of DNA

Question 10

What is PFGE?

Answer 10

A variation of standard gel electrophoresis that introduces an alternating voltage gradient to improve the resolution of larger molecules

Question 11

What is the main difference between standard gel electrophoresis and PFGE?

Answer 11

Instead of constantly running the voltage in one direction, in PFGE 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

Question 12

What takes longer, standard gel electrophoresis or PFGE?

Answer 12

PFGE

Question 13

Advantages of PFGE

Answer 13

Good for analysing recent evolutions (e.g Hospital outbreaks)
Highly discriminative
Faster than MLST

Question 14

Disadvantages of PFGE

Answer 14

Requires many hours of work
Very technical & hard to reproduce
The results may be subjective

Question 15

Where can P. aeruginosa grow easily?

Answer 15

Hot tub

Question 16

What is Multilocus sequence typing (MLST)?

Answer 16

A technique in molecular biology for the typing of multiple genetic loci

Question 17

How does Multilocus sequence typing (MLST) work?

Answer 17

Isolates of microbial species are characterised by deriving DNA sequences of internal fragments of multiple genes
Relatively small fragments (approximately 450-500 bp) are amplified using specific PCR primers as these can be accurately sequenced on both strands using an automated DNA sequencer

Question 18

What are the steps in MLST?

Answer 18

1. Amplify ~450 bp fragments of seven house-keeping genes
2. Sequence the seven gene fragments
3. Assign each different sequence at a locus a different allele number
4. These allele numbers give an allelic profile of the isolate
5. Compare the allelic profile of the isolate to those of all other isolates in a central database

Question 19

What is the method of high throughput 16S rDNA sequencing?

Answer 19

Universal primers are used to amplify (by PCR) the rDNA from an isolate
The amplified DNA is sequenced
Sequences are compared

Question 20

What are carbapenemases?

Answer 20

β-lactam antibiotics with a broad spectrum of activity. Their structures are generally resistant to β-lactamases

Question 21

What has there been a rapid and disturbing spread of in Gram-negative bacteria?

Answer 21

Extended-spectrum β-lactamases (ESBL) and multiple carbepenemases

Question 22

What causes carbapenem resistance?

Answer 22

Down-regulation of porins
Acquisition of carbapenemases
Up-regulation of efflux pumps

Question 23

What causes quinolone resistance?

Answer 23

GyrA: the mutated DNA gyrase resists quinolone binding
NfxB: globally disregulated physiology that upregulates the efflux pumps encoded by OprN and OprJ