Methicillin-Resistant Staphylococcus aureus (MRSA) Detection and Identification Methods
- Gram +ve coccus
- Grape like clusters of cells
- Resitant to methicillin and other penicillins
- May also be referred to as ORSA [oxacillin resistant]
Methicillin-resistant Staphylococcus aureus (MRSA) were first reported in the early 1960's and are now regarded as a major hospital acquired pathogen worldwide. The term methicillin resistant is historically used to describe resistance to any of this class of antimicrobials. Today in the USA approx. 35% of hospital strains of S. aureus are resistant to methicillin (or other penicillin antibiotics), and in recent years the emergence of vancomycin-resistant S. aureus (VRSA) has caused additional concern.
Resistance occurs when the organism has a mecA gene producing an altered penicillin binding protein, PBP2a (also known as PBP2') and either an oxacillin MIC of 2mg/l or a methicillin MIC of 4mg/l.
Infected and colonised patients are the reservoir of MRSA both in hospitals and the community with transmission generally being via contact with health workers.
Effective, rapid laboratory diagnosis and susceptibility testing is critical in treating, managing and preventing MRSA infections.
Laboratory screening for MRSA is a complex balance between speed of result, sensitivity, specificity and cost.
Currently the majority of screening is carried out using plate based methods. Surveys suggest that this methodology group accounts for >90% of the screening tests performed.
However a number of alternative methods including broth based methods, chromogenic media, rapid screening kits, molecular assays and automated systems are increasingly being used. Isolation from screening swabs can be a lengthy procedure, due to the number of 'contaminating' organisms that are present in swabs from non-sterile sites.
Broth based enrichment media are commonly employed to increase sensitivity. However this is at the expense of speed of result. NaCl is generally added to the base broth along with either methicillin, oxacillin, [and more recently] cefoxitin. Indicator compounds can also be used to give an early indication of the presence of MRSA.
Solid Agar Media: There are no universal standardised methods for screening and isolation of MRSA using solid agar media. Many selective media are available, and these rely on inhibitors such as NaCl and/or antibiotics to aid selection, together with a pH indicator to highlight presumptives. Examples are Mannitol Salt Agar containing 7% NaCl with either 4mg/L methicillin or 2mg/L oxacillin; Oxacillin Resistant Screening Agar with 5.5% NaCl and 2mg/L oxacillin; Baird Parker Medium with 8mg/L ciprofloxacin; Mueller Hinton Agar with 4% NaCl and 6mg/L oxacillin. Sensitivity at 24hrs incubation is variable with 48hrs incubation often required for an acceptable result.
Recently developed chromogenic media combine primary growth and selectivity with differentiation from coagulase negative staphylococci. These media show improved specificity when compared with traditional media. Sensitivity is also improved but requires 48hrs incubation to achieve >85%.
The majority of molecular methods used for the detection of MRSA are in-house, relying on multiplexed PCR primers detecting genes specific for S. aureus (nuc,fem) and mecA detecting methicillin resistance. Most are only suitable for use with pure cultures and not screening of swabs due to the presence of coagulase negative staphylococci carrying the methicillin resistant gene mecA. Newer commercially available amplification assays targeting mecA in combination with other specific markers such as coagulase and have shown encouraging results
There have been a number of developments with bioluminescence in particular the use of adenylate kinase (AK), an enzyme found in all cells that produce ATP from ADP. AK measurement is more sensitive than ATP-based systems and allows routine detection of 50 organisms or more in a sample. Early performance data shows results equivalent to conventional plate culture methods whilst providing results within 5 hours.
Traditionally confirmation of S. aureus is performed using the slide coagulase test (clumping factor) and the tube coagulase test (free coagulase). Positives on the slide coagulase test should be confirmed with the tube coagulase test. DNase media plates can also be used but positives require additional confirmation.
Agglutination kits are widely available and can be used to confirm S. aureus by detecting protein A and clumping factor, although some strains of MRSA have low levels of these proteins. Newer kits now work by also detecting surface antigen. Other latex kits detect PBP2a which occurs within the cell membrane and requires lysis of the cells for detection.
A wide range of commercial biochemical kits are available, both manual and automated. These are based on an array of biochemical tests giving a profile assessed against databases/tables. Many automated systems combine biochemical identification of S. aureus with antibiotic sensitivity panels for the confirmation of MRSA.
Antibiotic Sensitivity Methods
Methods for methicillin and oxacillin susceptibility testing are extensive and published data is contradictory with regard to recommendations.
There is no single method that is suitable for all MRSA strains. Standard methods are published by the British Society for Antimicrobial Chemotherapy (BSAC) and in the USA, by the Clinical Laboratory Standards Institute (CLSI), previously known as NCCLS.
Minimum Inhibitory Concentration [MIC] by the dilution method has traditionally been the reference method.
BSAC recommend the use of Mueller Hinton or Columbia agar with 2% NaCl and 104 cfu/ml inoculum incubated at 30°C. CLSI recommend Mueller Hinton Agar with 2% NaCl and 104 cfu/ml inoculum incubated at 33-35°C.
Molecular methods which detect the mecA gene are replacing MIC as the reference method.
Antibiotic sensitivity testing using disc diffusion methods remain the most widely used but results are influenced by a range of factors including medium, NaCl concentration, temperature, inoculum and test agent.
A number of recent studies using cefoxitin disc diffusion method suggest greater reliability than with oxacillin. No special medium or incubation temperature is required and the test is less affected by hyper-producers of penicillinase.
The most recent CLSI supplement (M100-S14) suggests the use of 30µg cefoxitin discs using a breakpoint of <= 19mm as indicative of resistance of S. aureus to oxacillin. Other media based methods include agar, broth based breakpoint methods (2mg/L oxacillin, 4mg/L methicillin) and agar screening methods recommended by CLSI (Approved standard M7-A6).
MRSA is no longer only an infection that is acquired in hospitals [HA-MRSA], although this remains a primary source of transmission.
Increasingly MRSA can be acquired in the community [CA-MRSA] and indeed from pets [PA-MRSA?]. This perhaps is the more worrying trend due to the potentially large host population and emphasises the need for significantly increased controls of how and when antibiotics are used.
Widespread delivery of antibiotics outside clinically significant uses can only lead to further selection of organisms resitant to higher levels of antibiotics.
Definitions: What are ESKAPE pathogens?
Often referred to in the media as 'superbugs', the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) are considered to be the leading cause of hospital-acquired infections globally.
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