Disc Test for Detecting Staphylococcus aureus Strains Producing Type A and Type C β-Lactamases

Microbiol Spectr. 2023 Aug 17;11(4):e0022023. doi: 10.1128/spectrum.00220-23. Epub 2023 Jul 6.


Staphylococcus aureus can produce β-lactamases capable of hydrolyzing penicillins and first-generation cephalosporins. The propensity of type A and type C β-lactamase-producing S. aureus (TAPSA and TCPSA) to hydrolyze cefazolin at a high inoculum is termed the cefazolin inoculum effect (CIE). Strains with a CIE have a theoretical risk of causing treatment failure and are unable to be detected routinely by most laboratories. We developed a high-performing yet straightforward β-lactamase disc test that identifies and differentiates both TAPSA and TCPSA and is suitable for routine diagnostic laboratory workflows. Clinical isolates of S. aureus resistant to penicillin were identified, and their blaZ genes were sequenced. MICs were determined at low and high inocula (5 × 105 CFU/mL and 5 × 107 CFU/mL), and isolates demonstrating a CIE were characterized. A semimechanistic model was established to describe differential hydrolysis patterns, and candidate models were iteratively assessed using area-under-the-curve analysis from competitor receiver operating characteristic (ROC) curves. Biomarker thresholds were derived from Youdon index-derived optimal cutoff values. Genetic analysis of 99 isolates identified 26 TAPSA isolates and 45 TCPSA isolates. The model best differentiating TAPSA from non-TAPSA utilized cefazolin-to-cephalothin ratio analysis (sensitivity, 96.2%; specificity, 98.6%). The model best differentiating TCPSA from non-TCPSA incorporated cefazolin, cephalothin, and oxacillin (sensitivity, 88.6%; specificity, 96.6%). TAPSA and TCPSA can be differentiated using three antibiotic discs on a single agar plate. The test has potential value in typing the β-lactamase type from isolates from patients that are candidates for or have failed cefazolin therapy. IMPORTANCE The key significance of this article is that it details a straightforward method of performing a disc test that can differentiate Staphylococcus aureus isolates that are likely to be associated with a cefazolin inoculum effect and theoretical risk of cefazolin treatment failure from isolates that are less likely to be associated with a cefazolin inoculum effect.

Keywords: Staphylococcus aureus; cefazolin inoculum effect; disc test.

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Anti-Bacterial Agents / therapeutic use
  • Cefazolin* / therapeutic use
  • Cephalothin / therapeutic use
  • Humans
  • Microbial Sensitivity Tests
  • Oxacillin
  • Staphylococcal Infections* / diagnosis
  • Staphylococcal Infections* / drug therapy
  • Staphylococcus aureus / genetics
  • beta-Lactamases / genetics


  • Cefazolin
  • beta-Lactamases
  • Cephalothin
  • Anti-Bacterial Agents
  • Oxacillin