Oxacillin Information and Courses from MediaLab, Inc.

Most S. pneumoniae strains gain penicillin resistance by altering the penicillin-binding proteins in their cell wall. Penicillin molecules that cannot find a penicillin binding site cannot interfere with cell wall synthesis. Several different types of penicillin binding proteins may be involved, explaining the various levels of intermediate resistance that may be encountered with different strains of S. pneumoniae. Because different penicillin binding proteins may be involved, the level of penicillin resistance cannot be predicted by the oxacillin screening test. Infections caused by isolates of S. pneumoniae showing penicillin resistance in the intermediate range may be successfully treated by administering high doses of antibiotic. For this reason, the level of resistance with an accurate minimum inhibitory concentration (MIC) test must be determined for all clinically significant isolates of S. pneumoniae.

Antibiotics inhibit bacterial growth by interfering with one or more cellular processes. Beta lactams are a large group of cell wall active antibiotics used to treat a wide variety of infections. S. aureus cell wall synthesis is dependent on the proper functioning of a number of enzymes. The beta-lactam antibiotics exert their effect by binding with one specific type of enzyme, transpeptidase, thus interfering with its ability to catalyze the final stage of peptidoglycan synthesis, resulting in defective cell wall formation. The beta-lactams comprise four main groups of antibiotics; all have the beta-lactam ring as their basic chemical structure: Penicillins (penicillin, oxacillin/methicillin, ampicillin and piperacillin) Cephalosporins Carbapenems Monobactams The spectrum of antimicrobial activity is dependent upon the particular structural modification of the beta-lactam ring. The transpeptidases are commonly referred to as penicillin-binding proteins (PBPs). Different bacterial species have distinct PBPs, resulting in very specific drug interactions.

When a clinical isolate is presumptively identified as S. aureus, susceptibility testing will be performed by either the Standardized Disk Diffusion (Kirby-Bauer) or Broth Dilution (MIC) methods, using the following testing conditions as recommended by the Clinical and Laboratory Standards Institute (CLSI): Medium: MHA for disk diffusion; CAMHB + 2% NaCL for oxacillin, methicillin, and nafcillin; CAMHB supplemented up to 50 ug/ml calcium for daptomycin Inoculum: Direct colony suspension (0.5 McFarland Standard) Incubation: 35° C (Testing at temperatures above 35° C may not detect MRSA); ambient air; disk diffusion; 16to 18 hours; dilution methods; 16 to 20 hours. All methods: 24 hrs for oxacillin, methicillin, nafcillin, and vancomycin.

Resistance to oxacillin is most accurately determined by testing for mecA or for the protein expressed by mecA, the penicillin-binding protein 2a (PBP 2a, which is also referred to as PBP 2'). Isolates of staphylococci that carry the mecA gene, or that produce PBP 2a should be reported as oxacillin-resistant according to CLSI guidelines. If MIC tests are performed in addition to disk diffusion, isolates for which oxacillin MICs are > 4µg/mL, are mecA-negative, or PBP 2a negative should be reported as oxacillin-resistant. Such isolates may have a rare resistance mechanism other than mecA, and may also test susceptible to cefoxitin by disc diffusion. In these scenarios, oxacillin resistance should be reported in accordance with the MIC value.

As previously discussed, multiplexing is assaying several different genes in the same reaction. Multiplexing has become possible due the high specificity of probe design. Development of multiplexing PCR reactions can be complicated because it has to be ensured that the probes do not interfere or react with each other. The process can also be time-consuming because each reaction may require different conditions. Figuring out the ideal mix for all the probes can take time. When developing a new multiplex test, it is important to weigh the time involved and costs against running each PCR reaction individually. There are many pre-established multiplex tests that can be purchased and used in laboratories today. One example is a multiplex PCR test that can distinguish between Staphylococcus aureus and coagulase-negative Staphylococcus and determine the presence of the gene that is resistant to oxacillin. Another example is a multiplex PCR test that can detect all four species of Plasmodium, the cause of malaria.