Lecture 4: Key gram positive superbug Staphlococcus Auerus

Question 2

What is the classification of staphlococcus aureus?

Answer 2

Gram-positive cocci with a thick peptidoglycan layer in the cell wall.

Question 3

What is staphlococcus aureus found in under the microscope?

Answer 3

Clusters

Question 4

How can we distinguish between other gram posisitve cocci like Streptococcus and Enterococcus ?

Answer 4

• By carrying out specific enzymatic and biochemical tests

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Question 5

What does the catalase test do?

Answer 5

Differentiates Staphylococcus species. (catalase-positive) from Streptococcus species. (catalase-negative).

Question 6

How is the catalase test carried out?

Answer 6

• A drop of hydrogen peroxide is added to the bacterial sample
• Bubbles form due to breakdown of hydrogen peroxide into oxygen and water by the catalase enzyme if staphylococcus is present.

Question 7

What does the coagulase test do?

Answer 7

Differentiates S. aureus (coagulase-positive) from other Staphylococcus species.

Question 8

How is the coagulase test carried out?

Answer 8

• S. aureus produces coagulase, an enzyme that converts fibrinogen to fibrin, causing clot formation.
• Slide Test: A drop of plasma is mixed with the bacterial colony. Clotting/agglutination indicates a positive result. (for S. aureus)
• Tube Test: Bacterial sample is incubated with plasma, and clotting is observed after several hours.

Question 9

What is the purpose of hemolysis on blood agar?

Answer 9

• To observe the effects of S. aureus on red blood cells

Question 10

Describe hemolysis on blood agar

Answer 10

• Beta-Hemolysis: Complete lysis of red blood cells, forming clear zones (typical for S. aureus).
• Alpha-Hemolysis: Partial lysis with greenish discoloration.
• Gamma-Hemolysis: No lysis.

Question 11

What does MLST stand for?

Answer 11

• Multi-Locus Sequence Typing

Question 12

What is MLST?

Answer 12

• A genotyping method that is used for the typing of multiple genetic locci.
• It identifies bacterial strains and determines genetic relationships between isolates.

Question 13

Describe the method of MLST

Answer 13

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 (www.mlst.net)

Question 14

Who developed the Pseudomonas aeruginosa MLST Database?

Answer 14

Chris Dowson and Barry Curran at the University of Warwick, UK (Curran et al 2004 JCM )

Question 15

What are the applications of MLST?

Answer 15

• Tracking sources of infection
• Studying genetic diversity and resistance mechanisms

Question 16

What does Whole Genome Sequencing (WGS) do?

Answer 16

Provides complete genetic profiles, allowing high-resolution analysis of resistance genes and strain relationships.

Question 17

Describe how Restriction Fragment Length Polymorphism (RFLP) works

Answer 17

DNA is cut using restriction enzymes, producing unique fragment patterns for strain identification.

Question 18

Can S. aureus be acquired from being hospitalised?

Answer 18

Yes

Question 19

When were all staphylococci susceptible to penicillin?

Answer 19

In 1941

Question 20

By which year were 72% of nosocomial staphylococci resistant to penicillin?

Answer 20

By 1952. This means that susceptibility decreased, particularly in hospital settings.

Question 21

In which year was methicillin introduced?

Answer 21

1959

Question 22

Within 2 years of the introduction of methicillin, what happened?

Answer 22

• Methicillin-resistant staphylococci (MRSA) appeared.
• This resistance has continued since then, but is fortunately waning.

Question 24

Is being hospitalised a huge risk factor for contracting MRSA?

Answer 24

Yes

Question 26

Is colonisation of the nares a significant risk factor for subsequent S. aureus infection? explain...

Answer 26

- Yes - In >80% of S. aureus bacteraemias, the infecting strain is identical to a nasal colonising strain detected prior to onset of bacteraemia. - Hence, Improved hospital screening of patients: swabs may be taken the inside of the nose, throat, armpit, groin and any areas of damaged or open skin is required.

Question 27

What's the difference between methicillin and penicillin?

Answer 27

Methicillin is chemically very similar to penicillin but possess an acyl side chain that prevents hydrolysis of the β-lactam ring by β-lactamases

Question 28

When did Methicillin resistance first emerge in S. aureus?

Answer 28

In the 1960s

Question 29

What are the risk factors for community-acquired MRSA?

Answer 29

- Close-contact activities (e.g., sports). - Shared contaminated objects (fomites like towels, razors, and surfaces). - Immunocompromised individuals (e.g., HIV-positive). - Skin trauma (e.g., tattoos, cuts).

Question 30

What is a fomite?

Answer 30

Any surface/object that is contaminated with bacteria, and so poses as a source of infection

Question 31

How do bacteria survive for a long time on fomites?

Answer 31

Some bacteria can form biofilms, a protective matrix that helps them survive longer on surfaces, particularly in moist environments.

Question 32

Describe the evolution of penicillin-resistance in S. aureus

Answer 32

- 1940-1950s: use of penicillin to treat staphylococcus aureus infections - 1944: First case of penicillin-resistant S. aureus was detected - 1952: 72% of isolates of S. aureus were penicillin resistant

Question 33

By which mechanism did S. aureus gain resistance to penicillin?

Answer 33

acquisition of β-lactamases, which are enzymes that break the β-lactam ring of penicillin

Question 35

How did S. aureus develop low level resistance to Methicillin?

Answer 35

Developed by hyperproduction of β-lactamase

Question 36

How did S. aureus develop high level resistance to Methicllin?

Answer 36

- Mediated by the modification of the target for penicillin, the penicillin binding proteins PBPs (reduced affinity for methicillin), meaning the antibiotic can no longer effectively bind to them and disrupt the bacterial cell wall.

Question 37

Describe how bacteria use PBP2’ (which is encoded by the mecA gene) to acquire resistance to methicillin

Answer 37

- Encoded by the mecA gene, carried on the staphylococcal cassette chromosome (SCCmec). - This alternative PBP has low affinity for beta-lactams, enabling cell wall synthesis despite methicillin presence. - The likely mecA donor is S. sciuri

Question 38

Is Vancomycin the last choice glycopeptide antibiotic used for treating MRSA infections?

Answer 38

Yes

Question 39

Describe how vancomycin works

Answer 39

- Vancomycin binds the terminal ala-ala motif of the peptide chains of cell wall peptidoglycans, and prevents access of the PBP. - This results in no bacterial cell wall syntheisis

Question 40

How has resistance to vancomycin been acquired?

Answer 40

- D-alanine-D-alanine motif in peptidoglycan chains is altered, reducing vancomycin binding while maintaining structural integrity.

Question 41

Which phenotypic biotyping methods are discussed here?

Answer 41

Gram staining, catalase, coagulase, and hemolysis tests.

Question 42

What genotypic biotyping methods are discussed here?

Answer 42

MLST, RFLP, WGS.

Question 43

Which resitstance mechnisms are described here?

Answer 43

Beta-lactamase production, PBP2a, and peptidoglycan precursor modification.

Question 44

What preventative methods can be used against MRSA infections?

Answer 44

- Nosocomial infections: Screening, isolation, and improved hygiene. - Community infections: Public education and prudent antibiotic use.

Question 45

Summary

Answer 45

- Staphyloccus aureus has acquired resistance to methicillin (MRSA) - The incidence of MRSA in hospitals has been lowered properly worldwide - Extremely disturbing emergence of VRSA (Vancomycin-resistant S. aureus).

Question 46

For the exams

Answer 46

- Phenotyping of S. aureus - Principle of MLST - Risk factors of hospital acquired infections with S. aureus - Methicillin and vancomycin mode of action resistance in Pseudomonas aeruginosa - Resistance to Methicillin and vancomycin