Antimicrobial Susceptibility Testing (AST)- Types and Limitations

Antimicrobial susceptibility testing (AST) is a laboratory procedure that determines how effective an antimicrobial agent is against a bacterial or fungal isolate. AST is important for guiding the appropriate choice and dosage of antimicrobial therapy for infections, preventing the emergence and spread of antimicrobial resistance, and monitoring the epidemiology of resistant organisms.

AST involves exposing a standardized inoculum of the test organism to different concentrations or amounts of an antimicrobial agent, either in liquid or solid media. The growth or survival of the test organism is then measured and compared to that of a control organism or a reference value. The results of AST are usually reported as one of the following categories:

• Susceptible (S): The test organism is inhibited by a concentration or amount of the antimicrobial agent that is achievable in the body with standard dosing. The antimicrobial agent is likely to be effective for treating the infection caused by the test organism.
• Intermediate (I): The test organism is inhibited by a concentration or amount of the antimicrobial agent that is higher than the susceptible breakpoint but lower than the resistant breakpoint. The antimicrobial agent may be effective for treating the infection caused by the test organism, depending on the site and severity of the infection, the pharmacokinetics and pharmacodynamics of the drug, and the immune status of the patient.
• Resistant (R): The test organism is not inhibited by a concentration or amount of the antimicrobial agent that is achievable in the body with standard dosing. The antimicrobial agent is unlikely to be effective for treating the infection caused by the test organism.

There are different methods and techniques for performing AST, each with its own advantages and limitations. In this article, we will discuss two major types of AST techniques: dilution susceptibility tests and disc diffusion susceptibility tests. We will also describe two common variants of disc diffusion susceptibility tests: Kirby-Bauer NCCLS modified disc diffusion technique and Stokes disc diffusion technique. We will explain how these techniques work, what are their benefits and drawbacks, and how to interpret their results.

Antimicrobial susceptibility testing (AST) is the process of determining the ability of an antimicrobial agent to inhibit or kill a microorganism. AST is essential for guiding appropriate and effective treatment of infections, preventing the emergence and spread of antimicrobial resistance, and monitoring the epidemiology of resistant strains.

There are two main types of AST techniques: dilution and disc diffusion. Both techniques rely on measuring the growth or survival of the test organism in the presence of different concentrations or amounts of an antimicrobial agent. The results of AST are usually expressed as susceptible, intermediate, or resistant, based on predefined criteria that correlate with clinical outcomes.

Dilution techniques measure the minimum inhibitory concentration (MIC) or the minimum bactericidal concentration (MBC) of an antimicrobial agent. MIC is the lowest concentration of an antimicrobial agent that prevents visible growth of the test organism, while MBC is the lowest concentration that kills the test organism. Dilution techniques can be performed in broth or agar media, manually or semi-automatically. Dilution techniques are more accurate and precise than disc diffusion techniques, but they are also more labor-intensive, time-consuming, and expensive.

Disc diffusion techniques measure the zone of inhibition around a disc or a tablet impregnated with a known amount and concentration of an antimicrobial agent. The zone size reflects the diffusion rate of the antimicrobial agent in the agar medium and the susceptibility of the test organism. Disc diffusion techniques are simpler, faster, and cheaper than dilution techniques, but they are also less sensitive and specific, and more prone to variability due to factors such as inoculum size, disc content, agar quality, and incubation conditions.

There are different methods of performing disc diffusion techniques, such as the Kirby-Bauer NCCLS modified technique and the Stokes technique. The Kirby-Bauer NCCLS modified technique is a standardized method that follows strict guidelines for disc content, inoculum density, agar type, incubation time and temperature, and zone interpretation. The Kirby-Bauer NCCLS modified technique is recommended by WHO for clinical and surveillance purposes and for promoting reproducibility and comparability of results between laboratories. The Stokes technique is a less standardized method that compares the zone sizes of the test organism with that of a control organism on the same plate. The Stokes technique is used in laboratories that have difficulties in obtaining or storing discs correctly or meeting the other conditions required for the Kirby-Bauer NCCLS modified technique.

Both dilution and disc diffusion techniques have advantages and limitations for AST. The choice of technique depends on several factors such as the availability of resources, the type and number of organisms to be tested, the clinical relevance and urgency of the results, and the quality assurance and quality control measures in place.

Dilution susceptibility tests are used to measure the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of an antimicrobial agent against a bacterial isolate. The MIC is the lowest concentration of the antimicrobial that prevents visible growth of the bacteria, while the MBC is the lowest concentration that kills the bacteria.

Dilution susceptibility tests can be performed in broth or agar media, using manual or semi-automated methods. The broth dilution method involves adding serial dilutions of the antimicrobial to tubes or wells containing a standardized inoculum of the test organism. The agar dilution method involves preparing plates with different concentrations of the antimicrobial in the agar and inoculating them with a standardized amount of the test organism. In both methods, after overnight incubation, the MIC is determined by observing the lowest concentration that shows no growth.

The MBC can be determined by subculturing from the tubes or wells that show no growth at the MIC onto antibiotic-free media and observing for growth after another overnight incubation. The MBC is the lowest concentration that shows no growth on the subculture.

Dilution susceptibility tests are useful for epidemiological purposes or when a patient does not respond to treatment, relapses, or has immunosuppression. They can also provide information on the pharmacokinetics and pharmacodynamics of the antimicrobial agent, such as its serum levels, tissue penetration, and bactericidal activity. However, dilution susceptibility tests are more labor-intensive, time-consuming, and expensive than disc diffusion tests. They also require more standardized conditions and quality control measures to ensure accuracy and reproducibility. Dilution susceptibility tests are not suitable for fastidious or slow-growing organisms that require special media or incubation conditions.

Disc diffusion techniques are used by most laboratories to test routinely for antimicrobial susceptibility. A disc of blotting paper is impregnated with a known volume and appropriate concentration of an antimicrobial, and this is placed on a plate of susceptibility testing agar uniformly inoculated with the test organism. The antimicrobial diffuses from the disc into the medium and the growth of the test organism is inhibited at a distance from the disc that is related (among other factors) to the susceptibility of the organism. Strains susceptible to the antimicrobial are inhibited at a distance from the disc whereas resistant strains have smaller zones of inhibition or grow up to edge of the disc.

Disc diffusion techniques are simple, inexpensive and easy to perform. They can be used to test a large number of organisms and antimicrobials simultaneously. They also provide a visual representation of the susceptibility pattern of the test organism. However, disc diffusion techniques have some limitations as well. They are not suitable for bacteria that are slow-growing, need special nutrients, or require CO2 or anaerobic incubation. They are also affected by various factors such as inoculum size, disc content, agar composition, pH, temperature and incubation time. Therefore, disc diffusion techniques need to be standardized and controlled carefully to ensure reproducibility and comparability of results between laboratories.

There are two main types of disc diffusion techniques: the Kirby-Bauer NCCLS modified disc diffusion technique and the Stokes disc diffusion technique. These techniques differ in their degree of standardization, quality control and interpretation criteria. The Kirby-Bauer NCCLS modified disc diffusion technique is recommended by WHO for clinical and surveillance purposes as it is more reliable and consistent than the Stokes disc diffusion technique. The details of these techniques will be discussed in the next points.

The Kirby-Bauer NCCLS modified disc diffusion technique is a widely used method for antimicrobial susceptibility testing. It is based on the principle that the growth of a bacterial strain is inhibited by an antimicrobial agent at a certain concentration and distance from the source of the agent. The technique involves the following steps:

• A standardized inoculum of the test organism is prepared by suspending a pure culture in a saline solution and adjusting the turbidity to match a 0.5 McFarland standard. This ensures that the inoculum contains approximately 1.5 x 10^8 colony forming units (CFU) per ml.
• A sterile cotton swab is dipped into the inoculum and used to streak the entire surface of a Mueller-Hinton agar plate in three directions, forming a lawn of bacteria. The plate is allowed to dry for 3 to 5 minutes before applying the antimicrobial discs.
• Antimicrobial discs containing known concentrations of different agents are placed on the surface of the agar using sterile forceps. The discs should be spaced at least 24 mm apart from center to center and at least 15 mm from the edge of the plate. The discs should be gently pressed onto the agar to ensure contact.
• The plate is incubated at 35°C for 16 to 18 hours in ambient air. During this time, the antimicrobial agents diffuse from the discs into the agar and create concentration gradients around them. The bacteria grow on the agar except where they are inhibited by the antimicrobial agents.
• After incubation, the diameter of the zone of inhibition around each disc is measured in millimeters using a ruler or a caliper. The zone size reflects the susceptibility of the test organism to the antimicrobial agent. The larger the zone, the more susceptible the organism is.
• The zone sizes are interpreted by comparing them with standard tables provided by the National Committee for Clinical Laboratory Standards (NCCLS). The tables assign breakpoints for each antimicrobial agent and bacterial species combination, which classify the test organism as susceptible (S), intermediate (I), or resistant (R) to the agent. The breakpoints are based on the correlation between zone sizes and minimum inhibitory concentrations (MICs), as well as clinical and pharmacological data.
• The results are reported as S, I, or R for each antimicrobial agent tested. If an organism is susceptible to an agent, it means that it can be effectively treated with that agent at usual dosages. If an organism is intermediate to an agent, it means that it may be treated with that agent at higher dosages or in combination with other agents. If an organism is resistant to an agent, it means that it cannot be treated with that agent.

The Kirby-Bauer NCCLS modified disc diffusion technique has several advantages over other methods of antimicrobial susceptibility testing. It is simple, inexpensive, and reproducible. It can test multiple agents on a single plate and provide results within 24 hours. It can also detect some mechanisms of resistance, such as beta-lactamase production, by observing changes in zone sizes or shapes.

However, the technique also has some limitations and challenges. It requires strict adherence to standardized procedures and quality control measures to ensure accuracy and reliability. It can only be used for well-evaluated bacterial species that grow well on Mueller-Hinton agar and do not require special nutrients or atmospheric conditions. It cannot measure MICs or MBCs directly, which may be useful for some clinical situations or research purposes. It may also be affected by factors such as inoculum size, disc potency, agar thickness, diffusion rate, and interpretation criteria.

Therefore, the Kirby-Bauer NCCLS modified disc diffusion technique is a valuable tool for antimicrobial susceptibility testing, but it should be performed and interpreted with caution and expertise.

The Stokes disc diffusion technique is a simple and inexpensive method for testing the susceptibility of bacteria to antimicrobials. It is based on the principle that the diameter of the zone of inhibition around a disc impregnated with an antimicrobial is proportional to the susceptibility of the bacteria to that antimicrobial. Unlike the Kirby-Bauer NCCLS modified disc diffusion technique, which uses standardized discs, inoculum, agar, and interpretation criteria, the Stokes technique uses a control strain of known susceptibility to compare with the test strain on the same plate. This allows for some variation in the factors that affect the diffusion of the antimicrobial, such as the thickness and composition of the agar, the amount and potency of the antimicrobial in the disc, and the incubation conditions.

The Stokes technique involves the following steps:

• Prepare a suspension of the test organism and a control organism (usually Staphylococcus aureus) in sterile saline or broth, adjusting the turbidity to match a 0.5 McFarland standard. This corresponds to approximately 1.5 x 10^8^ colony forming units (CFU) per ml.
• Using a sterile cotton swab, inoculate a plate of Mueller-Hinton agar with the test organism by streaking it over the entire surface in three directions, rotating the plate by 60 degrees each time. Repeat the same procedure with another plate using the control organism.
• Place discs containing different antimicrobials on each plate, ensuring that they are well spaced and do not overlap. Press them gently to ensure good contact with the agar. Alternatively, use Rosco Diagnostica antibiotic tablets (Neo-Sensitabs) instead of paper discs for better stability and accuracy.
• Incubate the plates at 35°C for 16-18 hours in ambient air.
• Measure the diameter of the zone of inhibition around each disc or tablet in millimeters, including the diameter of the disc or tablet itself. Record the results for both the test and control organisms.
• Compare the zone sizes of the test organism with those of the control organism. If they are within ±3 mm of each other, then the test organism is considered to have the same susceptibility as the control organism for that antimicrobial. If they differ by more than 3 mm, then there may be an error in the test or a difference in susceptibility between the two organisms.
• Interpret the results using tables or charts that correlate zone sizes with susceptibility categories (such as susceptible, intermediate, or resistant) for each antimicrobial and bacterial species. These tables or charts are usually based on clinical and epidemiological data and may vary depending on local guidelines and policies.

The Stokes technique has some advantages and disadvantages compared to other disc diffusion techniques. Some of the advantages are:

• It is simple, cheap, and easy to perform with minimal equipment and training.
• It can be used for bacteria that are not well evaluated by standardized techniques, such as fastidious or anaerobic bacteria.
• It can compensate for some variations in disc content, agar quality, and incubation conditions by using a control strain.

Some of the disadvantages are:

• It is less reproducible and comparable than standardized techniques, as it depends on subjective measurements and comparisons.
• It may not detect subtle differences in susceptibility between strains or resistance mechanisms that affect MICs but not zone sizes.
• It may give misleading results if there is cross-contamination between discs or plates, if there is poor quality or storage of discs or tablets, or if there is inappropriate selection of control strain or interpretation criteria.

The Stokes technique is a useful method for testing antimicrobial susceptibility in resource-limited settings or when standardized techniques are not available or applicable. However, it should be performed with care and quality control to ensure reliable and accurate results. Whenever possible, it should be supplemented or replaced by more standardized and validated techniques such as the Kirby-Bauer NCCLS modified disc diffusion technique.

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