Antimicrobial Susceptibility Testing (AST)

Antimicrobial Susceptibility Testing (AST)

  • Antibiotic susceptibility testing, also known as AST is a commonly used method for evaluating resistance to antibiotics and determining the treatment plan for patients in clinical environments.
  • There are several different ways to conduct AST like the dilution of broth, agar and disc diffusion tests.
  • The disc diffusion method, or ‘KirbyBauer procedure involves spreading bacteria onto an agar plate , and then placing discs of paper impregnated with antibiotics onto the plate. After incubation the growth of bacteria is monitored.
  • The areas around the discs where there is no growth of bacteria can be observed are known as zones of inhibition. These zones prove that the antibiotic is effective in stopping the growth of bacterial or killing bacteria. By measuring the size of the zones we are able to assess the effectiveness of antibiotics as well as assess resistance to antimicrobials.
  • For the treatment and management of infections, especially when they are caused by pathogens which are typically resistant to drugs susceptibility (sensitivity) tests are used to identify effective antimicrobials.
  • Testing for susceptibility is typically not recommended when the susceptibility reaction of a pathogen could be identified, for instance:
    • Proteus species are generally intolerant to nitrofurantoin, tetracyclines and nitrofurant
    • S. Pyogenes is generally susceptible to penicillin.
    • K. pneumoniae is generally ampicillin-resistant,
    • Anaerobes are at risk of metronidazole.
  • Tests for susceptibility should never be conducted on a variety of commensal organisms or contaminants as it could confuse the physician and lead to the patient receiving uneffective and ineffective antimicrobial therapy which could cause adverse side reactions and resistance to other pathogenic organisms.

Techniques of Antimicrobial Susceptibility test

Antimicrobial susceptibility tests in the laboratory can be done by using:

  • A dilution technique
  • A disc diffusion technique.

Dilution susceptibility tests

  • Semi-automated or manual dilution susceptibility tests can be performed at Microbiology Reference Laboratories for epidemiological reasons or when a patient fails to respond to treatment that is thought to be sufficient, or is relapsed while receiving treatment or if there is an immunosuppression condition.
  • Dilution methods determine the concentration of inhibitory minimum (MIC).
  • They may also be used to determine the minimum concentration of bactericidal (MBC) that is, in essence, the smallest amount of antimicrobial that is required to eliminate bacteria.
  • A Dilution test is conducted by adding dilutions from an antimicrobial to agar or broth medium. A standardized inoculum from the test organism is added. After incubation over a period of time and incubation for several days, the MIC is defined as the lowest level of antimicrobial needed to stop visible growth. When comparing the MIC value against the known concentrations of the drug in blood as well as other fluids in the body the likelihood of a clinical response can be determined.

Disc diffusion susceptibility tests

  • Disk diffusion techniques are utilized by many laboratories to determine routinely susceptibility to antibiotics.
  • Blotting paper is coated with a specified volume and the right amount of an antimicrobial and then placed on a dish of susceptibility-testing agar that is uniformly inoculated with the organism of test.
  • The antimicrobial disperses through the disc medium, and the development of the organism is impeded when it is located away from the disc. This distance is linked (among other things) with the degree of susceptibility to infection.
  • Strains that are susceptible to antimicrobials are restricted in a certain distance from the disc, while resistant strains have smaller inhibition zones or can grow to the edges that of disc.
  • To be used for surveillance and clinical purposes in order to improve reproducibility and comparability among labs, WHO recommends the (NCCLS) modified Kirby-Bauer disc diffusion method.

Kirby-Bauer NCCLS modified disc diffusion technique

  • The reliability of this standardized method is dependent on, for each species, the use of discs with the correct antimicrobial composition and an inoculum that gives confluent growth, as well as a trustworthy Mueller Hinton Agar.
  • The test procedure must be adhered to in all aspects. After the incubation period at 35deg C over 16-18 hours zones are measured and then interpreted by using NCCLS standards. They are derived from the relationship that exists between the size of zones and MICs.
  • The NCCLS Kirby-Bauer method should only be used to evaluate bacteria. It’s not suitable for bacteria that grow slowly or require special nutrients or require anaerobic or CO2 incubation.

Stokes disc diffusion technique

  • In this technique both control and test organisms will be inoculated together on the same surface.
  • The sizes of the zones of the test organism are directly compared with the control.
  • This technique isn’t as well-defined as Kirby-Bauer’s method and is employed in labs especially when the precise amount of antimicrobial contained in discs isn’t confirmed due to the difficulty of finding discs and storage in a proper manner or when the other requirements to use the Kirby-Bauer technique can’t be fulfilled.
  • One way that labs using the Stokes method could switch to a method that is similar to the WHO Kirby-Bauer recommendation is to use extremely solid Rosco Diagnostica antibiotic tablets (Neo-Sensitabs) instead of less durable paper discs.
A disk diffusion test with an isolate of Escherichia coli from a urine culture. The diameters of all zones of inhibition are measured and those values translated to categories of susceptible, intermediate, or resistant using the latest tables published by the CLSI.
A disk diffusion test with an isolate of Escherichia coli from a urine culture. The diameters of all zones of inhibition are measured and those values translated to categories of susceptible, intermediate, or resistant using the latest tables published by the CLSI. | Source:

Disadvantages of antimicrobial susceptibility tests

Susceptibility tests determine the antimicrobial effect against bacteria in conditions of laboratory (in in vitro activities) and not in the human body (in in vivo activity). Therefore, it is not possible to conclude that an antimicrobial agent that blocks or kills the growth of an organism in vitro will prove to be effective in treating.


Making the right choice for antimicrobial treatment requires a thorough examination of the patient’s medical health, any other underlying conditions (e.g. kidney disease or liver) as well as the type and location of infection the history of hypersensitivity to medications, the health status of the patient and whether the woman is expecting. It is also important to be aware of the actions of different medications, such as their absorption rates and diffusion into tissues metabolism, excretion, and the possibility of toxicity and adverse effects on the normal microbiome. Cost and availability of a medication should also be taken into consideration.



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