Amoxicillin-Clavulanic Susceptibility Testing evaluates the effectiveness of the amoxicillin-clavulanic acid combination, a β-lactam antibiotic paired with a β-lactamase inhibitor, against bacterial pathogens. This combination enhances the spectrum of activity of amoxicillin by inhibiting β-lactamase enzymes that could otherwise degrade it. Here’s a detailed overview of the testing process:

Testing Methods

• Disk Diffusion Method:
  • Preparation: An agar plate is inoculated with a bacterial suspension to create a uniform lawn of growth.
  • Application: Discs impregnated with amoxicillin-clavulanic acid are placed on the agar surface. Often, separate discs for amoxicillin alone and amoxicillin-clavulanic acid are used to detect the effect of clavulanic acid.
  • Incubation: The plate is incubated at 35-37°C for 18-24 hours.
  • Measurement: The diameter of the zone of inhibition around each disc is measured. A larger zone around the amoxicillin-clavulanic disc compared to the amoxicillin-only disc indicates that the clavulanic acid is effective in overcoming β-lactamase-mediated resistance.
• Broth Microdilution Method:
  • Preparation: Bacterial suspensions are diluted in a microtiter plate containing broth with varying concentrations of amoxicillin-clavulanic acid.
  • Incubation: The plate is incubated at 35-37°C for 18-24 hours.
  • Evaluation: The minimum inhibitory concentration (MIC) of amoxicillin-clavulanic acid is determined by identifying the lowest concentration that inhibits visible bacterial growth. Comparing the MIC of the combination to that of amoxicillin alone helps to assess the effect of clavulanic acid.
• Etest Method:
  • Preparation: An agar plate is inoculated with the bacterial strain.
  • Application: A strip containing a gradient of amoxicillin-clavulanic acid concentrations is placed on the agar surface.
  • Incubation: The plate is incubated at 35-37°C for 18-24 hours.
  • Measurement: The MIC is determined by reading the point where bacterial growth intersects with the strip’s gradient. Comparing this with the MIC of amoxicillin alone, if tested separately, helps to assess the clavulanic acid’s effect.

Technical Specifications

• Antimicrobial Concentration: The concentration of amoxicillin and clavulanic acid in the disc or dilution ranges is standardized according to established guidelines to ensure accurate susceptibility testing.
• Quality Control: Testing procedures follow protocols from organizations such as CLSI (Clinical and Laboratory Standards Institute) to ensure reliability and reproducibility.

Applications

• Clinical Diagnostics: This testing is crucial for determining the effectiveness of the amoxicillin-clavulanic acid combination in treating infections caused by β-lactamase-producing bacteria. It helps in selecting appropriate antibiotics and ensuring effective treatment.
• Antibiotic Stewardship: The test supports the management of antibiotic use and helps prevent the development of resistance by confirming the effectiveness of combination therapies.