Ceftazidime-avibactam susceptibility testing evaluates the effectiveness of the combination of ceftazidime, a third-generation cephalosporin, and avibactam, a beta-lactamase inhibitor, against bacterial isolates. This combination is used to treat infections caused by Gram-negative bacteria, including those with beta-lactamase-mediated resistance.

Testing Procedures

Preparation:

• Bacterial Isolate: Use a pure culture of the organism to be tested.
• Inoculum Preparation: Adjust the bacterial suspension to a 0.5 McFarland turbidity standard for consistency.

Broth Microdilution Method:

• Preparation: Prepare a series of ceftazidime-avibactam concentrations in a microtiter plate. This involves creating a range of dilutions of ceftazidime-avibactam in a broth medium. Typically, a fixed ratio of ceftazidime to avibactam is used (e.g., 4:1).
• Inoculation: Add the standardized bacterial suspension to each well of the microtiter plate containing ceftazidime-avibactam.
• Incubation: Incubate at 35-37°C for 16-24 hours.
• Reading Results: Determine the minimum inhibitory concentration (MIC), which is the lowest concentration of ceftazidime-avibactam that prevents visible bacterial growth.
• Interpretation: Compare MIC values to established breakpoints to classify the isolate as:
  • Susceptible: MIC ≤ 8/4 µg/mL
  • Intermediate: MIC = 16/8 µg/mL
  • Resistant: MIC ≥ 32/16 µg/mL The exact breakpoints can vary based on guidelines from organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST).

Disk Diffusion Method:

• Agar Plate: Plate Mueller-Hinton agar with the standardized bacterial suspension.
• Ceftazidime-Avibactam Disk: Place a disk impregnated with ceftazidime-avibactam (e.g., ceftazidime 30 µg/avibactam 10 µg) on the agar surface.
• Incubation: Incubate at 35-37°C for 16-18 hours.
• Measurement: Measure the diameter of the zone of inhibition around the disk.
• Interpretation: Compare the zone diameter to standardized breakpoints provided by CLSI or EUCAST. For ceftazidime-avibactam, specific disk diffusion breakpoints might be provided in the future as the methodology is less commonly standardized than MIC testing.

Automated Systems (e.g., VITEK 2, Phoenix):

• Procedure: Automated systems can perform susceptibility testing for ceftazidime-avibactam using specific panels designed for combination therapies.
• Incubation and Reading: The system performs automatic incubation and growth measurement, providing susceptibility results based on pre-set algorithms and breakpoint criteria.