Laboratory Detection of: Oxacillin/Methicillin-resistant Staphylococcus aureus

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. Hospital-associated MRSA isolates often are multiply resistant to other commonly used antimicrobial agents, including erythromycin, clindamycin, and tetracycline, while community-associated MRSA isolates are often resistant only to ß-lactam agents and erythromycin. Since 1996, MRSA strains with decreased susceptibility to vancomycin (minimum inhibitory concentration [MIC], 8-16 μg/ml) and strains fully resistant to vancomycin (MIC ≥ 32 μg/ml) have been reported.

1. 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.
2. 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 resistance to rifampin, this drug should never be used as a single agent to treat MRSA infections.
3. MRSA are transmissible.
   An 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 Healthcare worker is colonized with an 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. Handwashing and screening patients for MRSA should be performed to decrease transmission and reduce the number of patients infected with MRSA.

The National Committee for Clinical Laboratory Standards (NCCLS), now called the Clinical and Laboratory Standards Institute (CLSI), recommends the cefoxitin disk screen test, the latex agglutination test for PBP2a, or a plate containing 6 μg/ml of oxacillin in Mueller-Hinton agar supplemented with NaCl (4% w/v; 0.68 mol/L) as alternative methods of testing for MRSA. For methods of inoculation, see CLSI Approved Standard M100-S15 (1).

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 (2). 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 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, methicillin, or nafcillin at 33-35° C (maximum of 35°C) for a full 24 hours before reading (1).

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.

Nucleic acid amplification tests, such as the polymerase chain reaction (PCR), can be used to detect the mecA gene, which mediates oxacillin resistance in staphylococci.

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

The CLSI breakpoints for S. aureus are different than those for coagulase-negative staphylococci (CoNS) (1)

N/A = not applicable

* Report as oxacillin susceptible
** Report as oxacillin resistant
†There is no intermediate category with the cefoxitin disk diffusion test

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 disk diffusion tests using cefoxitin give clearer endpoints and are easier to read than tests with oxacillin.

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.

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 8 th ed. ASM Press, Washington, D.C.