OVERVIEW OF ANTIBIOTICS SUSCEPTIBILITY TESTING

Introduction

Antibiotic susceptibility testing is a laboratory technique used to determine the effectiveness of antibiotics against specific bacterial or fungal strains. It helps in guiding appropriate antibiotic treatment for infectious diseases. The testing involves exposing the microorganism to different antibiotics and measuring its growth response or determining the minimum inhibitory concentration (MIC), which is the lowest concentration of the antibiotic that inhibits visible growth of the organism.

Here is a general overview of the process involved in antibiotic susceptibility testing:

1. Sample collection: A sample is collected from the infected site, such as blood, urine, sputum, or wound swab, depending on the suspected infection.
2. Isolation and identification of the organism: The collected sample is cultured on specific media to isolate and grow the microorganism. Once isolated, the organism is identified using various techniques like biochemical tests, DNA sequencing, or mass spectrometry.
3. Inoculation: The identified organism is then inoculated onto agar plates, such as Mueller-Hinton agar for bacteria or Sabouraud agar for fungi. The organism is spread evenly across the plate surface.
4. Antibiotic discs or dilution plates: Antibiotic susceptibility testing can be performed using two main methods: disk diffusion and broth dilution.
   • Disk diffusion method: Paper discs impregnated with specific concentrations of antibiotics are placed on the agar surface. The plate is then incubated under controlled conditions to allow the organism to grow.
   • Broth dilution method: Serial dilutions of antibiotics are prepared in liquid growth medium. Each dilution is inoculated with the organism and incubated. The growth is observed, usually by turbidity or by adding a dye that changes color when the organism grows.
5. Interpretation: After incubation, the results are interpreted based on the growth inhibition zones around the antibiotic discs (disk diffusion) or the lowest concentration of antibiotic that inhibits visible growth (broth dilution). Standard guidelines, such as those established by the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), are followed to interpret the results accurately.
6. Reporting: The results of the susceptibility testing are reported as sensitive, intermediate, or resistant, indicating the effectiveness of antibiotics against the tested organism. This information helps clinicians select the most appropriate antibiotic therapy for the patient.

It’s important to note that the specific techniques and protocols used may vary depending on the laboratory and the organism being tested. Moreover, new techniques and technologies may be developed over time to improve the accuracy and efficiency of antibiotic susceptibility testing.

 

AST Theory and Measurement

 

Kirby-Bauer disk diffusion testing

Kirby-Bauer disk diffusion testing, also known as the disk diffusion method or antibiogram, is a commonly performed test in clinical laboratories to determine the susceptibility of bacteria to various antimicrobial agents. It is a qualitative method used to assess the effectiveness of antibiotics or other antimicrobial drugs against specific bacterial strains.

Here’s a general overview of how the Kirby-Bauer disk diffusion test is conducted in a clinical laboratory:

1. Bacterial Isolation: The first step is to isolate the bacteria from the patient sample, such as blood, urine, or wound swab, on an agar plate. The bacteria are streaked evenly across the agar surface to obtain isolated colonies.
2. Preparation of Antimicrobial Disks: Small filter paper disks impregnated with specific antibiotics or antimicrobial agents are placed on the agar surface. These disks come pre-saturated with a standardized amount of the drug.
3. Incubation: The agar plate, with the antimicrobial disks in place, is then incubated at the optimal temperature for the specific bacteria being tested. Incubation allows the bacteria to grow and interact with the antibiotics.
4. Zone of Inhibition Measurement: After incubation, the plate is examined. The antimicrobial agent diffuses into the surrounding agar, creating a concentration gradient. If the bacteria are susceptible to the drug, it will not be able to grow near the disk, creating a clear zone of inhibition around the disk. The size of this zone is measured using a caliper or a ruler.
5. Interpretation: The diameter of the zone of inhibition is compared to established interpretive criteria, usually provided by organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). These guidelines categorize the bacterial susceptibility as susceptible, intermediate, or resistant based on the zone size.
6. Reporting: The results of the Kirby-Bauer test, including the name of the antimicrobial agent tested and the interpretation of susceptibility, are reported to the healthcare provider. This information helps guide the selection of appropriate antibiotics for treating the bacterial infection.

It’s important to note that the Kirby-Bauer test is just one method of antimicrobial susceptibility testing, and there are other more advanced techniques available in clinical laboratories. However, the disk diffusion method remains widely used due to its simplicity, cost-effectiveness, and ability to provide quick results for routine testing.