Laboratory Testing

What are the antibiotic susceptibility patterns of clinical S. aureus isolates?

Approximately 10% of S. aureus isolates in the United States are susceptible to penicillin. However, many S. aureus strains, while resistant to penicillin, remain susceptible to penicillinase-stable penicillins, such as oxacillin and methicillin. Strains that are oxacillin and methicillin resistant, historically termed methicillin-resistant S. aureus (MRSA), are resistant to all ß-lactam agents, including cephalosporins and carbapenems, although they may be susceptible to the newest class of MRSA-active cephalosporins (e.g, ceftaroline). Strains of MRSA causing healthcare-associated infections are often resistant to other commonly used antimicrobial agents, including erythromycin, clindamycin and fluoroquinolones, while strains causing community-associated infections are often resistant only to ß-lactam agents, erythromycin and occasionally to fluoroquinolones. Since 1996, MRSA strains with decreased susceptibility to vancomycin (minimum inhibitory concentration [MIC], 4 – 8 μg/ml) and strains fully resistant to vancomycin (MIC ≥ 16 μg/ml) have been reported.

How should clinical laboratories test for MRSA?

In addition to broth microdilution testing, the Clinical and Laboratory Standards Institute (CLSI), recommends the cefoxitin disk diffusion test or a plate containing 6 μg/ml of oxacillin in Mueller-Hinton agar supplemented with 4% NaCl as alternative methods of testing for MRSA. For specific details on these methods, see CLSI Approved Standard M100. In addition, there are FDA-approved assays for molecular detection of the mecA gene and commercially available chromogenic agars that can be used for MRSA detection. Finally, latex agglutination or immunochromatographic membrane tests for PBP2a can be used to detect MRSA.

Is it difficult to detect oxacillin/methicillin resistance?

MRSA is resistant to all β-lactams because of the presence of mecA, a gene that produces a pencillin binding protein (PBP2a) with low affinity for β-lactam antibiotics. Mechanism of oxacillin resistance other than mecA are rare. 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 (see Bannerman, TL. 2003). All cells in a culture may carry the genetic information (mecA) 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 penicillins, such as oxacillin.

Cells expressing heteroresistance grow more slowly than the oxacillin-susceptible population and may be missed at temperatures above 35°C. This is why CLSI recommends incubating isolates being tested against oxacillin at 33-35°C (maximum of 35°C) for a full 24 hours before reading (see CLSI Approved Standard). Isolates tested against cefoxitin using either disk diffusion or broth microdilution should also be incubated at 33-35°C but can be read after 16-18 and 16-20 hours, respectively.

Can all susceptibility tests detect MRSA?

When used correctly, broth-based and agar-based tests usually can detect MRSA. The cefoxitin disk diffusion method can be used in addition to routine susceptibility test methods or as a back-up method.

Are there additional tests to detect oxacillin/methicillin resistance?

Nucleic acid amplification tests, such as the polymerase chain reaction (PCR), can be used for direct detection of mecA, the most common gene mediating oxacillin resistance in staphylococci. However, mecA PCR tests will not detect novel resistance mechanisms such as mecC or uncommon phenotypes such as borderline-resistant oxacillin resistance. An alternative method for detection of MRSA is the use of anti-PBP2a monoclonal antibodies available as latex agglutination or immunochromatographic membrane assays. Finally, there are commercially available chromogenic agars that can be used for MRSA detection.

How is the mecA gene involved in the mechanism of resistance?

Staphylococcal resistance to oxacillin/methicillin occurs when an isolate produces an altered penicillin-binding protein, PBP2a, which is encoded by the mecA gene. The variant penicillin-binding protein binds β-lactams with lower avidity, which results in resistance to this class of antibiotic agents.

What are the breakpoints for testing the susceptibility of staphylococci to oxacillin?

The CLSI clinical breakpoints for S. aureus are different than those for coagulase-negative staphylococci (CoNS) — see CLSI approved standard M100.

Oxacillin disk diffusion testing is not reliable for detecting oxacillin/ methicillin resistance. Cefoxitin should be used as a surrogate for disk diffusion testing.

Interpretive Criteria (in μg/ml) for Oxacillin MIC Tests

Level of resistance of 2 pathogens to the Oxacillin MIC tests.
Pathogen Susceptible Intermediate Resistant
 S. aureus and S. lugdunensis  ≤ 2 μg/ml  N/A  ≥ 4 μg/ml
 CoNS  ≤ 0.25 μg/ml  N/A  ≥ 0.5 μg/ml


Interpretive Criteria (in μg/ml) for Cefoxitin MIC Tests

Level of resistance of 2 pathogens to the  Cefoxitin MIC tests.
Pathogen Susceptible Intermediate Resistant
 S. aureus and S. lugdunensis  ≤ 4 μg/ml  N/A  ≥ 8 μg/ml
 CoNS  N/A  N/A  N/A


Interpretive Criteria (in mm) for Cefoxitin Disk Diffusion Test

Level of resistance of 2 pathogens to the Cefoxitin disk diffusion test.
Pathogen Susceptible Intermediate Resistant
 S. aureus and S. lugdunensis  ≥ 22 mm  N/A  ≤ 21 mm
 CoNS  ≥ 25 mm  N/A  ≤ 24 mm
Why are oxacillin and cefoxitin tested instead of methicillin?

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 mecA gene, and tests using cefoxitin give more reproducible and accurate results than tests with oxacillin.

If oxacillin and cefoxitin are tested, why are the isolates called “MRSA” instead of “ORSA”?

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, and this was modified to include cefoxitin later. The acronym MRSA is still used by many to describe these isolates because of its historic role.