Antimicrobial susceptibility testing (AST) measures an antimicrobial agent’s ability to inhibit the growth of a microorganism. Several AST methods are available to determine bacterial susceptibility to antimicrobials. The disk diffusion method is an inexpensive and very common method.
AST is carried out using standardized laboratory methods and is interpreted according to guidelines established by organizations such as the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST).
Breakpoints are MIC values or zone diameters that classify an organism as susceptible (S), intermediate (I), or resistant (R) to a specific antimicrobial agent. Breakpoints vary not only according to the antimicrobial but also to the organism. This document summarizes the updated zone diameter breakpoints and interpretation criteria for Staphylococcus aureus in the disk diffusion method as published in the CLSI M100, Performance Standards for Antimicrobial Susceptibility Testing, 35th Edition (2025).
CLSI 2025 Breakpoints for Staphylococcus aureus
The following table lists the CLSI 2025 zone diameter breakpoints for Staphylococcus aureus. All values represent zones of inhibition (ZOI) measured in millimeters (mm).
| Tier | Antimicrobial agent | Sensitive | Intermediate | Resistant | Remarks |
|---|---|---|---|---|---|
| 1 | Azithromycin (15 µg) | ≥18 | 14–17 | ≤13 | |
| 1 | Clindamycin (2 µg) | ≥21 | 15–20 | ≤14 | |
| 1 | Cefoxitin (30 µg) | ≥22 | ≤21 | Surrogate for Oxacillin | |
| 1 | Erythromycin (15 µg) | ≥23 | 14–22 | ≤13 | |
| 1 | Nitrofurantoin (300 µg) | ≥17 | 15–16 | ≤14 | Urine only |
| 1 | Oxacillin | (MIC) | |||
| 1 | Tetracycline (30 µg) | ≥19 | 15–18 | ≤14 | |
| 1 | Vancomycin | (MIC) | |||
| 1 | Trimethoprim–Sulfamethoxazole (1.25/23.75 µg) | ≥16 | 11–15 | ≤10 | |
| 2 | Linezolid (30 µg) | ≥26 | 23–25 | ≤22 | |
| 2 | Penicillin (10 U) | ≥29 | ≤28 | ||
| 3 | Ceftaroline (30 µg) | ≥25 | 20-24 (SDD) | ≤19 | |
| 4 | Ciprofloxacin (5 µg) | ≥21 | 16–20 | ≤15 | |
| 4 | Dalbavancin | (MIC) | |||
| 4 | Gentamicin (10 µg) | ≥15 | 13–14 | ≤12 | |
| 4 | Levofloxacin (5 µg) | ≥19 | 16–18 | ≤15 | |
| 4 | Oritavancin | (MIC) | |||
| 4 | Telavancin | (MIC) | |||
| Chloramphenicol (30 µg) | ≥18 | 13–17 | ≤12 | ||
Selection of Antibiotics for AST of Staphylococcus aureus
- Tier 1: Appropriate for routine, primary testing and reporting.
- Tier 2: Appropriate for routine, primary testing. Reporting may follow institution-specific cascading rules.
- Tier 3: Introduced for institutions with high levels of multidrug-resistant organisms. Appropriate for routine, primary testing and reporting but must follow institution-specific cascade rules.
- Tier 4: Reserved for testing/reporting on request. Used if other antimicrobial agents in other tiers are not optimal because of various factors.
Testing conditions
- Medium: Muller Hinton Agar (MHA)
- Inoculum: Colony suspension, equivalent to a 0.5 McFarland standard
- Number of antibiotic discs: Maximum 12 discs on 150 mm plate, no more than 6 discs on 100 mm plate
- Distance between discs: Discs should be placed no less than 24 mm apart (center to center).
- Incubation: 35C±2°C, ambient air
Intrinsic Resistance
Staphylococcus aureus has intrinsic resistance to certain antibiotics due to natural genetic features. Intrinsic resistance is so common that susceptibility testing is not required. A small percentage (1-3%) may appear susceptible due to method variation, mutation, or low levels of resistance expression.
Staphylococcus aureus (and all other gram-positive bacteria) are intrinsically resistant to the following antibiotics:
- Aztreonam
- Colistin/Polymyxin B
- Nalidixic acid
Methicillin-resistant Staphylococcus aureus (MRSA), determined through cefoxitin or oxacillin testing, is considered resistant to:
- Other β-lactam agents
- β-lactam/β-lactamase inhibitor combinations
- Cephalosporins except Ceftaroline, and
- All carbapenems
References
- CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 35th ed. CLSI supplement M100. Clinical and Laboratory Standards Institute; 2025.
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