Top 8 Tips for Performing Antimicrobial Susceptibility Testing with Gram-Positive Organisms

Staphylococcus spp.

• mecA/mecC genes → confers methicillin resistance (MRSA):

Cefoxitin test is the recommended primary phenotypic screen, this can be performed either by disk diffusion or another susceptibility testing method available on the market.

MIC determination with oxacillin can also be used. Oxacillin disks are less reliable due to heterogeneous expression methicillin resistance mediated by the mecA or mecC gene.

• Inducible clindamycin resistance should be confirmed using the D-test, which involves testing erythromycin and clindamycin separately and together to detect inducible resistance.

• Vancomycin resistance in S. aureus includes VRSA (vancomycin resistant S. aureus), VISA (vancomycin intermediate S. aureus), and hVISA (heterogeneous vancomycin intermediate S. aureus). VRSA resistance is mediated by the vanA gene, whereas VISA and hVISA exhibit more complex resistance mechanisms involving various chromosomal mutations. Broth microdilution is the recommended method for accurate detection.

Enterococcus spp.

• vanA/vanB genes → confers vancomycin resistance (VRE): Clinically significant resistance is most often mediated by plasmid-encoded ligases. These enzymes replace the terminal D-Ala in the peptidoglycan precursor with D-Lac, thereby reducing glycopeptide binding and conferring vancomycin resistance.
  • VanA-producing strains: Show resistance to both vancomycin and teicoplanin.
  • VanB-producing strains: Typically remain susceptible to teicoplanin because the resistance operon is not induced by this agent. However, some heterogenous expression of teicoplanin resistance can be encountered.

It is important to confirm that the isolate is not E. gallinarum or E. casseliflavus, which intrinsically carry chromosomal enzymes that mediate low-level vancomycin resistance and are generally not considered significant from an infection-control perspective.

• High-level aminoglycoside resistance (HLAR) → limits the effectiveness of synergistic therapy with cell wall–active agents. Detection requires testing high concentrations of aminoglycosides, typically gentamicin or streptomycin, to determine whether synergistic activity is still achievable.

Avoid reporting antibiotics that are intrinsically ineffective for the organism. Examples include:

• Enterococcus spp.→ resistant to cephalosporins, trimethoprim–sulfamethoxazole, low-level aminoglycosides

• Staphylococcus spp. → naturally resistant to nalidixic acid due to poor drug affinity for the bacterial DNA gyrase. This is why nalidixic acid is not used clinically against staphylococci.

• Some Staphylococcus spp. possess intrinsic mutations or absent fosfomycin transporters, limiting drug entry. This contributes to variable intrinsic fosfomycin susceptibility across the genus.

Always consult the EUCAST or CLSI guidelines when establishing your reporting rules. Suppress results for agents that are intrinsically inactive - even if the testing system generates a zone or MIC value. Recognizing intrinsic resistance is essential to prevent inappropriate or ineffective therapy.