Antimicrobial Susceptibility Testing (AST) Notes

Introduction to Antimicrobial Susceptibility Testing (AST)

• AST is essential in microbiology labs to assess isolated disease-causing organisms for their susceptibility to antimicrobial agents, including bacteria and fungi.
• The laboratory report indicating susceptibility or resistance to antibiotics is crucial for determining effective treatment.
• Accuracy and reproducibility in testing are paramount.

Considerations in Antibiotic Selection

• Activity Against Pathogen: Antibiotic must exhibit maximum activity against the targeted pathogen.
• Toxicity to Host: Take into account the drug's tolerance level in the host organism; some antibiotics can be harsh on the body.
• Impact on Normal Flora: The antibiotic should minimally disrupt the native microbiome.
• Pharmacologic Considerations: Assess potential interactions with other medications; factors such as patient age and pregnancy can influence choices.
• Cost-Effectiveness: The more resistant the bacteria, the higher the treatment costs; no single antibiotic is effective against all bacteria.

Understanding Susceptibility and Resistance

• Susceptible (S): Indicates that the patient will likely respond favorably to the treatment with the specified antimicrobial agent.
• Resistant (R): Indicates that treatment with the antimicrobial agent is expected to fail.
• Intermediate (I): Means treatment with the agent may or may not work; continuance of dosage is critical.
• Resistance patterns are dynamic, with Multi-Drug Resistant Organisms (MDROs) posing significant treatment challenges.

McFarland Standards in Testing

• Consistency in results requires the same bacterial concentration in ASTs.
• The McFarland Standard refers to glass tubes with varying bacterial concentrations, serving as a reference to yield turbidity consistent across organisms (commonly Staphylococcus aureus and E. coli).
• The most commonly used standard is the 0.5 McFarland Standard, often created using barium sulfate.
• Densitometers might also be utilized for reading standards.

Disk Diffusion Testing (Kirby-Bauer Method)

• Method involves paper disks impregnated with antibiotics, placed on an agar culture inoculated with the organism.
• A standardized inoculum (0.5 McFarland) is swabbed over the agar plate, followed by disk placement and incubation for 16-18 hours.
• The diffusion of antibiotics generates a zone of inhibition where bacterial growth is prevented, measured in millimeters (mm).

Evaluating Zone of Inhibition

• Larger zones indicate higher antimicrobial effectiveness, while zero zones indicate resistance.
• Various antimicrobial disks can be tested, ensuring they are properly spaced on agar to avoid interference.
• Measurement is performed using a ruler in millimeters, referring to specific CLSI tables for interpretation of results (sensitive, resistant, intermediate).

CLSI and Zone Size Interpretation

• Testing adheres to CLSI standards regarding disk concentrations, media standardization (agar formula, pH, depth), inoculum density, and temperature.
• Mueller-Hinton agar is the preferred medium for disk diffusion testing.
• Results can be directly referenced from CLSI tables for accurate antimicrobial susceptibility reporting.

Summary of Key Points

• Mueller-Hinton agar is the primary medium used for ASTs; testing is standardized and reproducible.
• Standardized density for organisms is generally measured at 0.5 McFarland for accurate results in testing.
• Zone sizes read in mm are pivotal in determining antimicrobial susceptibility categories: Sensitive (S), Resistant (R), and Intermediate (I).