Laboratory Testing for Methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA)

Key points

  • There are many methods laboratorians can use to test for MRSA.
  • Four phenotypic methods are recommended for the detection of MRSA.
  • The oxacillin and cefoxitin breakpoints for Staphylococcus aureus (S. aureus) are different than those for other staphylococci.

Overview

Oxacillin/methicillin resistance

When an isolate carries an altered penicillin-binding protein (PBP2a) encoded by the mecA gene, the protein binds beta-lactams with lower avidity, resulting in resistance to this class of antimicrobial agents. Mechanisms other than mecA are rare.

All cells in a culture may carry the genetic information (mecA) for resistance, but only a small number may express the resistance in vitro (i.e., heteroresistance). Heteroresistance occurs in staphylococci resistant to penicillinase-stable penicillins, such as oxacillin.

MRSA

Methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA) is a type of bacteria that is resistant to most beta-lactam agents, including cephalosporins and carbapenems, with the exception of ceftaroline, a fifth-generation cephalosporin.

Healthcare-associated MRSA isolates are frequently resistant to other commonly used antimicrobial agents, including erythromycin, clindamycin, tetracycline and fluoroquinolones. Community-associated MRSA are typically more susceptible to these agents.

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, although these isolates remain rare.

Testing history

Although laboratorians used methicillin as the agent of choice for testing staphylococci in the early 1990s, it is no longer commercially available in the U.S. We now use oxacillin or cefoxtin. Oxacillin maintains its activity during storage better than methicillin and is more likely to detect heteroresistant strains. Cefoxitin is a better inducer of the mecA gene, gives clearer endpoints and is easier to read than tests with oxacillin. However, we still use the acronym MRSA to describe these isolates because of the historic role of methicillin.

Test methods

Susceptibility test methods

  • Cefoxitin or oxacillin broth microdilution and agar-based test.
  • The cefoxitin disk diffusion method. (For routine susceptibility testing or as a back-up method).
  • A plate containing 6 μg/ml of oxacillin in Mueller-Hinton agar supplemented with NaCl (4% w/v; 0.68 mol/L).1

For methods of inoculation and interpretive criteria, see most recent version of CLSI M100.

Additional methods

  • Nucleic acid amplification tests, such as the polymerase chain reaction (PCR), to detect the mecA gene, which mediates oxacillin resistance in staphylococci.
    • Note: mecA PCR tests will not detect novel resistance mechanisms or uncommon phenotypes (e.g., mecC or borderline-resistant oxacillin resistance).
  • Anti-PBP2a monoclonal antibodies available as latex agglutination or immunochromatographic membrane assays.1
  • Commercially available chromogenic agars.

Laboratory guidelines

Phenotypic methods recommended for the detection of MRSA include:

  • Cefoxitin broth microdilution.
  • Oxacillin broth microdilution.
  • Cefoxitin disk diffusion testing.

A screening agar containing 6 μg/ml of oxacillin in Mueller-Hinton agar supplemented with NaCl (4% w/v; 0.68 mol/L) is an alternative method of testing for MRSA. It should be noted that oxacillin disk diffusion testing is not a reliable method for S. aureus.

Accurate detection of oxacillin/methicillin resistance can be difficult due to the presence of two subpopulations (one susceptible and one resistant) that may coexist within a culture of staphylococci.2 CLSI M100 recommends incubation at 35°C +/- 2°C with the caveat that testing at temperatures above 35°C may not detect all methicillin-resistant staphylococci.

Interpreting results

Isolates that test positive for mecA or PBP2a or resistant by any of the recommended phenotypic methods should be reported as methicillin (oxacillin) resistant. It should be noted that the CLSI susceptibility breakpoints for S. aureus are different than those for other Staphylococcus species.1

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

CLSI Interpretive Criteria (in μg/ml) for Oxacillin MIC Tests
Pathogen Susceptible Intermediate Resistant
 S. aureus and S. lugdunensis  ≤ 2 μg/ml  N/A  ≥ 4 μg/ml
S. epidermidis  ≤ 0.5 μg/ml  N/A  ≥ 1 μg/ml
S. pseudintermedius and S. schleiferi  ≤ 0.5 μg/ml  N/A  ≥ 1 μg/ml
Staphylococcus spp. other  ≤ 0.5 μg/ml  N/A  ≥ 1 μg/ml

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

CLSI Interpretive Criteria (in μg/ml) for Cefoxitin MIC Tests
Pathogen Susceptible* Intermediate Resistant**
 S. aureus and S. lugdunensis  ≤ 4 μg/ml  N/A  ≥ 8 μg/ml
S. epidermidis  N/A  N/A  N/A
S. pseudintermedius and S. schleiferi  N/A  N/A  N/A
Staphylococcus spp. other  N/A  N/A  N/A

2 Bannerman, TL. 2003. Staphylococcus, Micrococcus and other catalase-positive cocci that grow aerobically. In P.R. Murray, E.J. Baron, J.H. Jorgensen, M.A. Pfaller, R.H. Yolken [eds.], Manual of Clinical Microbiology 8th ed. ASM Press, Washington, D.C.

CLSI Interpretive Criteria (in mm) for Oxacillin Disk Diffusion Test

CLSI Interpretive Criteria (in mm) for Oxacillin Disk Diffusion Test
Pathogen Susceptible Intermediate† Resistant
 S. aureus and S. lugdunensis  N/A  -  N/A
S. epidermidis  ≥ 18 ml  -  ≤ 17 ml
S. pseudintermedius and S. schleiferi  ≥ 18 ml  -  ≤ 17 ml
Staphylococcus spp. other N/A  -  N/A

CLSI Interpretive Criteria (in mm) for Cefoxitin Disk Diffusion Test

CLSI Interpretive Criteria (in mm) for Cefoxitin Disk Diffusion Test
Pathogen Susceptible* Intermediate† Resistant**
 S. aureus and S. lugdunensis  ≥ 22 ml  -  ≤ 21 ml
S. epidermidis  ≥ 25 ml  -  ≤ 24 ml
S. pseudintermedius and S. schleiferi  N/A  -  N/A
Staphylococcus spp. other ≥ 25 ml  -  ≤ 24 ml

N/A = not applicable
* Report as oxacillin/methicillin-susceptible
** Report as oxacillin/methicillin-resistant
†There is no intermediate category with the disk diffusion test

Print
Content Source:
National Center for Emerging and Zoonotic Infectious Diseases (NCEZID)
  1. CLSI. 2007. Performance standards for antimicrobial susceptibility testing. CLSI approved standard M100-S17. Clinical and Laboratory Standards Institute, Wayne, PA.
  2. Bannerman, TL. 2003. Staphylococcus, Micrococcus and other catalase-positive cocci that grow aerobically. In P.R. Murray, E.J. Baron, J.H. Jorgensen, M.A. Pfaller, R.H. Yolken [eds.], Manual of Clinical Microbiology 8th ed. ASM Press, Washington, D.C.