- Q) What is MRSA (Methicillin Resistant Staphylococcus aureus)?
- A) MRSA is a Staphylococcus aureus isolate which is resistant to Oxacillin and other ß-lactamase stable penicillins (methicillin, nafcillin, cloxacillin, flucloxacillin, and dicloxacillin).
- Q) What is the mechanism of resistance in Staphylococcus aureus?
- A) Methicillin resistance is associated with acquisition of mec A gene, which transcribe alteration in PBP (penicillin binding protein). The typical or intrinsically (methicillin) resistant Staphylococcus aureus isolates are associated with high Oxacillin MIC (>16 ug/ml).
- Q) What are borderline methicillin resistance Staphylococcus aureus?
- A) Borderline methicillin resistance Staphylococcus aureus usually lack mec A gene but express resistance to oxacillin by other resistance mechanism. The MIC for Oxacillin in such strains is at or just above the susceptible breakpoint (>2 ug/ml).
- Q) What is the procedure for inoculum preparation?
- A) Prepare inoculum from four or five isolated colonies of similar colony morphology grown overnight (18-24 hours) on nonselective media (sheep blood agar or chocolate agar plate).
- Q) How the inoculated plates should be examined?
- A) Examine plates closely with transmitted light. In case of disc susceptibility methods look for sensitive and resistant zone sizes. For screening agar method, observe for the presence or the absence of growth.
- Q) What is the usual sensitivity pattern in MRSA isolates?
- A) Most of the MRSA isolates also demonstrate resistance to multiple classes of antimicrobial agent, including clindamycin and erythromycin and sometimes quinolones, chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole and the aminoglycosides.
- Q) What are the quality control measures that should be adopted for sensitivity testing?
- A) Following recommendations should be taken into consideration:
Depth of Muller- Hinton agar should not be less or more than 20 ml in 90 mm Petri plate.
Inoculum density should be adjusted to McFarland 0.5 turbidity standards.
Incubation time and temperature should be according to the recommended protocol of each method.
- aureus ATCC 29213 —– oxacillin susceptible.
- aureus ATCC 43300 —– oxacillin resistant.
These QC strains should be run parallel when the test is performed.
- Q) Why are MRSA important?
A)Pathogenicity: MRSA have many virulence factors that enable them to cause disease in normal hosts. For example, MRSA are frequent causes of healthcare-associated bloodstream and catheter-related infections. MRSA are also an emerging cause of community-associated infections, especially skin and soft tissue infections and necrotizing pneumonia.
Limited treatment options: Vancomycin and two newer antimicrobial agents, linezolid and daptomycin, are among the drugs that are used for treatment of severe healthcare-associated MRSA infections. Although some strains remain susceptible to trimethoprim-sulfamethoxazole, gentamicin, or rifampin, these drugs are not typically used as first-line agents. Because of the rapid emergence of resistance to rifampin, this drug should never be used as a single agent to treat MRSA infections.
MRSA are transmissible: A MRSA outbreak can occur when one strain is transmitted to other patients or close contacts of the infected persons in the community. Often this occurs when a patient or health-care worker is colonized with aa MRSA strain (i.e., carries the organism but shows no clinical signs or symptoms of infection) and, through contact, spreads the strain to another person. Hand washing and screening patients for MRSA should be performed to decrease transmission and reduce the number of patients infected with MRSA.
- Q) Is it difficult to detect Oxacillin/Methicillin resistance?
- A) Accurate detection of Oxacillin/Methicillin resistance can be difficult due to the presence of two subpopulations (one susceptible and the other resistant) that may coexist within a culture of staphylococci. All cells in a culture may carry the genetic information for resistance, but only a small number may express the resistance in vitro. This phenomenon is termed heteroresistance and occurs in staphylococci resistant to penicillinase-stable penicillin, such as oxacillin. Cells expressing heteroresistance grow more slowly than the oxacillin-susceptible population and may be missed at temperatures above 35oC. This is why CLSI recommends incubating isolates being tested against oxacillin, methicillin, or nafcillin at 33-35oC (maximum of 35oC) for a full 24 hours before reading.
- Q) Why are oxacillin and cefoxitin tested instead of methicillin?
- A) First, methicillin is no longer commercially available in the United States. Second, oxacillin maintains its activity during storage better than methicillin and is more likely to detect heteroresistant strains. However, cefoxitin is an even better inducer of the mec A gene and disk diffusion tests using cefoxitin give clearer endpoints and are easier to read than tests with oxacillin.
- Q) If oxacillin is tested, why are the isolates called “MRSA” instead of “ORSA”?
- A) When resistance was first described in 1961, methicillin was used to test and treat infections caused by S. aureus. However, oxacillin, which is in the same class of drugs as methicillin, was chosen as the agent of choice for testing staphylococci in the early 1990s. The acronym MRSA is still used by many to describe these isolates because of its historic role.
- Q) How should clinical laboratory screen for MRSA
METHODS FOR DETECTING METHICILLIN ARE AS FOLLOWS
RESISTANCE IN STAPHYLOCOCCUS AUREUS
|Methods||Antibiotic concentration||Inoculum Density||Media||Incubation temperature||Incubation time||Result interpretation|
|Oxacillin disc||1ug||Adjust inoculum to 0.5% McFarland standard in 0.9% saline||Make lawn on Muller Hinton agar (20 ml in 90 mm Petri plate)||30-35oC||24 hrs||Zone size
>13mm = sensitive
< 13mm = Resistant
Salt agar screening
|6ug/ml||Adjust to inoculum to 0.5% McFarland standard in 0.9% saline||Streak small area (10-15mm) on Muller Hinton Agar.
Several organism can be tested on each plate by spot inoculation on surface of media)
|30-35oC||24hrs||Growth present= resistant
|Cefoxitin disc||30ug||Adjust inoculum to 0.5% McFarland in 0.9% saline||Make lawn on Muller Hinton agar||35oC||18 hrs||Zone diameter
< 21mm =resistant
* If intermediate results are obtained when testing Oxacillin 1ug disc for S.aureus then test for cefoxitin disk (mecA or PBP2a), or Oxacillin MIC test, or Oxacillin salt agar-screening test. Report the result of alternate test result rather than the intermediate result.
CLINICAL MICROBIOLOGY PROCEDURES HANDBOOK — ASM (American Society Manual 3rd edition)
CLSI (Clinical and laboratory standard instituted) Jan 2014 Performance standards for antimicrobial susceptibility testing. CLSI approved standard M100-S24. Clinical and Laboratory Standards Institute, Wayne, PA.
Updated July 2018
Year 1 Resident
Department of pathology and laboratory medicine
The Aga Khan University Hospital, Karachi