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 Enterobacterales excluding Salmonella and Shigella spp. species in the disk diffusion method as published in the CLSI M100, Performance Standards for Antimicrobial Susceptibility Testing, 35th Edition (2025).
These organisms include various important human pathogens, such as:
- Citrobacter freundii, Citrobacter koseri
- Enterobacter cloacae
- Escherichia coli, Escherichia hermannii
- Hafnia alvei
- Klebsiella aerogenes (formerly Enterobacter aerogenes)
- Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella variicola
- Morganella morganii
- Proteus mirabilis, Proteus vulgaris
- Providencia stuartii, Providencia rettgeri
- Serratia marcescens
- Yersenia enterocolitica
CLSI 2025 Breakpoints for Enterobacterales
The following table lists the CLSI 2025 zone diameter breakpoints for Enterobacterales other than Salmonella and Shigella. All values represent zones of inhibition (ZOI) measured in millimeters (mm).
| Tier | Antimicrobial agent | Sensitive | Intermediate | Resistant | Remarks |
|---|---|---|---|---|---|
| 1 | Ampicillin (10 µg) | ≥17 | 14-16 | ≤13 | |
| 1 | Amoxycillin-clavulanate (20/10 µg) | ≥18 | 14-17 | ≤13 | |
| 1 | Ampicillin-sulbactam (10/10 µg) | ≥15 | 12-14 | ≤11 | |
| 1 | Cefotaxime (30 µg) | ≥26 | 23-25 | ≤22 | |
| 1 | Ceftriaxone (30 µg) | ≥23 | 20-22 | ≤19 | |
| 1 | Gentamicin (10 µg) | ≥18 | 15-17 | ≤14 | |
| 1 | Ciprofloxacin (5 µg) | ≥26 | 22-25 | ≤21 | |
| 1 | Levofloxacin (5 µg) | ≥21 | 17-20 | ≤16 | |
| 1 | Nitrofurantoin (300 µg) | ≥17 | 15-16 | ≤14 | Urine only |
| 1 | Piperacillin-Tazobactam (100/10 µg) | ≥25 | – | ≤20 | |
| 1 | Trimethoprim/Sulfamethoxazole (1.25/23.75 µg) | ≥16 | 11-15 | ≤10 | |
| 2 | Amikacin (30 µg) | ≥20 | 17-19 | ≤16 | |
| 2 | Cefepime (30 µg) | ≥25 | 19-24 | ≤18 | |
| 2 | Cefuroxime (30 µg) | ≥18 | 15-17 | ≤14 | |
| 2 | Imipenem (10 µg) | ≥23 | 20-22 | ≤19 | |
| 2 | Meropenem (10 µg) | ≥23 | 20-22 | ≤19 | |
| 2 | Tetracycline (30 µg) | ≥15 | 12-14 | ≤11 | |
| 2 | Tobramycin (10 µg) | ≥17 | 13-16 | ≤12 | |
| 3 | Ceftazidime-avibactam (30/20 µg) | ≥21 | – | ≤20 | |
| 3 | Fosfomycin (200 µg) | ≥26 | 13-15 | ≤12 | Urine only (E. coli) |
| 3 | Imipenem-relebactam (10/25 µg) | ≥25 | 21-24 | ≤23 | |
| 3 | Meropenem-vaborbactam (21/10 µg) | ≥18 | 15-17 | ≤14 | |
| 4 | Aztreonam (30 µg) | ≥21 | 18-20 | ≤17 | |
| 4 | Ceftazidime (30 µg) | ≥21 | 18-20 | ≤17 | |
| Cefixime (5 µg) | ≥19 | 16-18 | ≤15 | ||
| Chloramphenicol (30 µg) | ≥18 | 13-17 | ≤12 | ||
| Colistin/Polymyxin B | (MIC) | ||||
| Ofloxacin (5 µg) | ≥16 | 13-15 | ≤12 | ||
Selection of Antibiotics for AST of Enterobacterales
- 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 for Enterobacterales
- 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, 16-18 hours
Intrinsic Resistance of Enterobacterales
The table below provides an overview of intrinsic resistance characteristics among common Enterobacterales species:
| Organism | Intrinsic Resistance To |
|---|---|
| Enterobacteriaceae (General) | Clindamycin, Daptomycin, Fusidic acid, Glycopeptides (Vancomycin), Lipoglycopeptides (Oritavancin, Telavancin, Dalbavancin), Linezolid, Quinupristin-Dalfopristin, Rifampin, Macrolides (Erythromycin, Clarithromycin, Azithromycin) |
| Klebsiella pneumoniae | Same as Enterobacteriaceae plus: Ampicillin, Ticarcillin |
| Citrobacter koseri | Same as Enterobacteriaceae plus: Ampicillin, Ticarcillin |
| Citrobacter freundii | Same as Enterobacteriaceae plus: Ampicillin, First- and second-generation cephalosporins, Amoxicillin-clavulanate, Ampicillin-sulbactam |
| Enterobacter spp. | Same as Enterobacteriaceae plus: Ampicillin, First- and second-generation cephalosporins, Amoxicillin-clavulanate, Ampicillin-sulbactam |
| Proteus vulgaris | Same as Enterobacteriaceae plus: Ampicillin, First- and second-generation cephalosporins, Tetracyclines, Tigecycline, Nitrofurantoin, Polymyxins (Colistin, Polymyxin B) |
| Proteus mirabilis | Same as Enterobacteriaceae plus: Tetracyclines, Tigecycline, Nitrofurantoin, Polymyxins (Colistin, Polymyxin B) |
References
- CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 35th ed. CLSI supplement M100. Clinical and Laboratory Standards Institute; 2025.
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