Lecture 4: Key gram positive superbug Staphlococcus Auerus Flashcards
What is the classification of staphlococcus aureus?
Gram-positive cocci with a thick peptidoglycan layer in the cell wall.
What is staphlococcus aureus found in under the microscope?
Clusters
How can we distinguish between other gram posisitve cocci like Streptococcus and Enterococcus ?
- By carrying out specific enzymatic and biochemical tests
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What does the catalase test do?
Differentiates Staphylococcus species. (catalase-positive) from Streptococcus species. (catalase-negative).
How is the catalase test carried out?
- 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.
What does the coagulase test do?
Differentiates S. aureus (coagulase-positive) from other Staphylococcus species.
How is the coagulase test carried out?
- 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.
What is the purpose of hemolysis on blood agar?
- To observe the effects of S. aureus on red blood cells
Describe hemolysis on blood agar
- 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.
What does MLST stand for?
- Multi-Locus Sequence Typing
What is MLST?
A genotyping method that is used for the typing of multiple genetic locci. It identifies bacterial strains and determines genetic relationships between isolates.
Describe the method of MLST
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)
Who developed the Pseudomonas aeruginosa MLST Database?
Chris Dowson and Barry Curran at the University of Warwick, UK (Curran et al 2004 JCM )
What are the applications of MLST?
- Tracking sources of infection
- Studying genetic diversity and resistance mechanisms
What does Whole Genome Sequencing (WGS) do?
Provides complete genetic profiles, allowing high-resolution analysis of resistance genes and strain relationships.
Describe how Restriction Fragment Length Polymorphism (RFLP) works
DNA is cut using restriction enzymes, producing unique fragment patterns for strain identification.
Can S.aureus be acquired from being hospitalised?
Yes
When were all staphylococci susceptible to penicillin?
In 1941
By which year were 72% of nosocomial staphylococci resistant to penicillin?
By 1952. This means that susceptibility decreased, particularly in hospital settings.
In which year was methicillin introduced?
1959
Within 2 years of the introduction of methicillin, what happened?
methicillin-resistant staphylococci (MRSA) appeared. This resistance has continued since then, but is fortunately waning.
Is being hospitalised a huge risk factor for contracting MRSA?
Yes
Is colonisation of the nares a significant risk factor for subsequent S. aureus infection? explain…
- 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.
What’s the difference between methicillin and penicillin?
Methicillin is chemically very similar to penicillin but possess an acyl side chain that prevents hydrolysis of the β-lactam ring by β-lactamases
When did Methicillin resistance first emerge in S.aureus?
In the 1960s
What are the risk factors for community-acquired MRSA?
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).
What is a fomite?
Any surface/object that is contaminated with bacteria, and so poses as a source of infection
Describe the evolution of penicillin-resistance in S. aureus
- 1940-1950s: use of penicillin to treat staphylococcus aureus infections
- 1944: First case of S. aureus was detected
- 1952: 72% of isolates of S. aureus were penicillin resistant
By which mechanism did S.aureus gain resistance to penicillin?
acquisition of β-lactamases, which are enzymes that break the β-lactam ring of penicillin
How did S.aureus develop low level resistance to Methicillin?
Developed by hyperproduction of β-lactamase
How did S.aureus develop high level resistance to Methicllin?
Mediated by 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.
Describe how bacteria use PBP2’ (which is encoded by the mecA gene) to acquire resistance to methicillin
- 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
Is Vancomycin the last choice glycopeptide antibiotic used for treating MRSA infections?
Yes
Describe how vancomycin works
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
How has resistance to vancomycin been acquired?
- D-alanine-D-alanine motif in peptidoglycan chains is altered, reducing vancomycin binding while maintaining structural integrity.
Which phenotypic biotyping methods are discussed here?
Gram staining, catalase, coagulase, and hemolysis tests.
What genotypic biotyping methods are discussed here?
MLST, RFLP, WGS.
Which resitstance mechnisms are described here?
Beta-lactamase, PBP2a, and peptidoglycan precursor modification.
What preventative methods can be used against MRSA infections?
- Nosocomial infections: Screening, isolation, and improved hygiene.
- Community infections: Public education and prudent antibiotic use.
Summary
Staphyloccus aureus has acquired resistance to methicillin (MRSA)
The incidence of MRSA in hospitals has been lowered properly worldwide
Extremely disturbing emergence of VRSA
For the exams
- 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
