Antimicrobial Susceptibility Testing (AST): Introduction, Principle, Test Methods, Test Requirements, Procedure, and Result Interpretation, Application, and Keynotes

Introduction

Antimicrobial Susceptibility Testing (AST) is a laboratory technique used to assess the effectiveness of antimicrobial agents, such as antibiotics, against specific microorganisms. The aim of AST is to determine the susceptibility or resistance of a microorganism to different antimicrobial drugs. This information is crucial for guiding healthcare professionals in choosing appropriate antimicrobial therapy for treating bacterial or fungal infections.

The emergence and spread of antimicrobial resistance pose a significant challenge in the field of healthcare. AST plays a vital role in combating this problem by providing information on the susceptibility patterns of microorganisms to various antimicrobial agents. It helps healthcare providers make informed decisions about the selection, dosage, and duration of antimicrobial treatment, leading to improved patient outcomes and reduced antimicrobial resistance.

The choice of antimicrobial agents for treatment is not arbitrary and should be based on the specific characteristics of the infecting microorganism. AST helps in identifying the most effective drugs by assessing the growth inhibition or killing ability of microorganisms in the presence of different antimicrobial agents. It provides valuable information about the sensitivity or resistance of the microorganism to specific drugs and assists in tailoring treatment regimens to maximize therapeutic efficacy.

AST is performed using standardized laboratory methods and follows guidelines established by organizations such as the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). These guidelines ensure uniformity and consistency in testing procedures, interpretation criteria, and reporting of results.

Different methods are employed for AST, including the disk diffusion method (Kirby-Bauer method), broth dilution method, Etest method, and automated systems. Each method has its advantages, limitations, and specific applications, but they all aim to provide accurate and reliable information about antimicrobial susceptibility.

Principle

The principle of Antimicrobial Susceptibility Testing (AST) is based on the evaluation of the growth inhibition or killing effect of antimicrobial agents on specific microorganisms. The testing is performed to determine the susceptibility or resistance of a particular microorganism to different antimicrobial drugs.

The principle of AST can be summarized as follows:

1. Isolation of the microorganism: The first step in AST is the isolation and identification of the microorganism from the clinical sample. This involves culturing the organism on appropriate growth media and performing biochemical or molecular tests to identify the species.
2. Inoculation: A standardized suspension of the isolated microorganism is prepared and inoculated onto the surface of an agar plate or added to a liquid broth. This ensures a consistent number of microorganisms for testing.
3. Exposure to antimicrobial agents: Different antimicrobial agents, such as antibiotics or antifungal drugs, are applied to the culture medium or broth. The antimicrobial agents can be in the form of disks impregnated with specific concentrations of drugs (disk diffusion method), different concentrations of drugs in a liquid broth (broth dilution method), or plastic strips with a gradient of drug concentrations (Etest method).
4. Incubation: The culture plates or broth cultures containing the microorganisms and antimicrobial agents are incubated under optimal conditions for the growth of the microorganism. Incubation time and temperature may vary depending on the organism being tested.
5. Measurement of growth inhibition: After incubation, the growth of microorganisms in the presence of antimicrobial agents is evaluated. In the disk diffusion method, the diameter of the zones of inhibition around the drug-containing disks is measured. In the broth dilution method, the Minimum Inhibitory Concentration (MIC) is determined, which is the lowest concentration of the drug that inhibits the visible growth of the microorganism. In the Etest method, the intersection point of the elliptical zone of inhibition with the drug gradient strip indicates the MIC value.
6. Result interpretation: The results of AST are interpreted based on established guidelines provided by organizations like the CLSI or EUCAST. The interpretation categorizes the microorganism as susceptible, intermediate, or resistant to the tested antimicrobial agents. This information helps clinicians in selecting the most appropriate antimicrobial therapy for treating the infection.

Test Methods

Antimicrobial Susceptibility Testing (AST) employs various test methods to determine the susceptibility or resistance of microorganisms to antimicrobial agents. The commonly used AST methods include:

1. Disk Diffusion Method (Kirby-Bauer Method):
   • A standardized suspension of the microorganism is spread on an agar plate.
   • Disks impregnated with specific concentrations of antimicrobial agents are placed on the agar surface.
   • After incubation, the zones of inhibition around the disks are measured and compared to interpret the susceptibility of the microorganism. The zone sizes are influenced by the diffusion of the drug through the agar and the susceptibility of the organism.
2. Broth Dilution Method:
   • Different concentrations of antimicrobial agents are prepared in liquid broth.
   • The microorganism is inoculated into the broth containing antimicrobial agents.
   • After incubation, the lowest concentration of the drug that inhibits visible growth, known as the Minimum Inhibitory Concentration (MIC), is determined.
   • The MIC values are compared to interpret the susceptibility of the microorganism.
3. Etest Method:
   • A plastic strip with a gradient of antimicrobial agent concentrations is placed on an agar plate inoculated with the microorganism.
   • After incubation, the intersection point of the elliptical zone of inhibition with the strip indicates the MIC value.
   • The MIC values obtained from the strip are compared to interpret the susceptibility of the microorganism.
4. Automated Systems:
   • Automated systems, such as the VITEK system or the Phoenix system, use specialized panels containing antimicrobial agents.
   • The panels are inoculated with the microorganism, and the system automatically measures growth or turbidity.
   • The system interprets the growth patterns and provides MIC values or categorical interpretations of susceptibility.

Each method has its advantages, limitations, and specific applications. Factors such as ease of use, turnaround time, cost, and availability of resources influence the choice of method in a particular laboratory or clinical setting. It’s important to follow standardized protocols and interpret the results based on established guidelines provided by organizations like the CLSI or EUCAST to ensure accurate and reliable AST results.

Test Requirements

Antimicrobial Susceptibility Testing (AST) requires specific test requirements to ensure accurate and reliable results. The following are the key requirements for performing AST:

1. Pure culture of the microorganism: A pure culture of the microorganism is essential for AST. It involves isolating and identifying the microorganism from the clinical sample using appropriate laboratory techniques, such as culture media, biochemical tests, or molecular methods. A pure culture ensures that the results obtained are specific to the target microorganism.
2. Standardized inoculum: AST requires a standardized inoculum of the microorganism. The inoculum density should be adjusted to a specific McFarland standard, which ensures consistent and reproducible growth across different tests. McFarland standards provide a turbidity standard equivalent to a specific concentration of microbial cells.
3. Quality control strains: Quality control strains are used to validate the performance and accuracy of the AST method. These strains are well-characterized reference strains with known susceptibility patterns. They should be included in each batch of AST testing to verify the accuracy and precision of the test results.
4. Antimicrobial agents: A panel of antimicrobial agents is needed for testing. The selection of antimicrobial agents depends on the type of microorganism being tested and the infection site. The chosen agents should represent the commonly used drugs for treating the specific type of infection.
5. Culture media: Appropriate culture media are required to support the growth of the microorganism and facilitate AST. Different media may be used depending on the microorganism being tested, such as Mueller-Hinton agar for bacteria or Sabouraud agar for fungi. The media should meet the quality standards and provide optimal growth conditions for the microorganism.
6. Sterile discs, plates, or strips: For methods like the disk diffusion method or Etest, sterile discs or strips impregnated with specific concentrations of antimicrobial agents are required. These discs or strips should be stored properly and handled aseptically to prevent contamination and ensure accurate results.
7. Incubator: An incubator is necessary to provide controlled temperature and humidity conditions for the growth of microorganisms during the incubation period. The temperature and duration of incubation may vary depending on the microorganism and testing method.
8. Standardized interpretation criteria: Established guidelines and interpretive criteria provided by organizations like the CLSI or EUCAST should be followed to interpret the results accurately. These criteria define the breakpoints for categorizing microorganisms as susceptible, intermediate, or resistant to antimicrobial agents.

It’s crucial to follow standardized protocols, maintain quality control measures, and ensure the use of appropriate materials and equipment to obtain reliable and reproducible AST results. Adhering to these requirements helps in guiding appropriate antimicrobial therapy and combating antimicrobial resistance effectively.

Procedure

The procedure for Antimicrobial Susceptibility Testing (AST) typically involves the following steps:

1. Isolation and identification of the microorganism: Obtain a pure culture of the microorganism from the clinical sample using appropriate laboratory techniques. Identify the microorganism at the species level using biochemical tests or molecular methods.
2. Preparation of standardized inoculum: Prepare a standardized inoculum of the microorganism. Adjust the density of the inoculum to a specific McFarland standard, which provides a turbidity equivalent to a specific concentration of microbial cells.
3. Inoculation of culture media: Inoculate the standardized inoculum onto appropriate culture media. For bacteria, Mueller-Hinton agar is commonly used, while Sabouraud agar is used for fungi. Streak the agar surface or spread the inoculum evenly using a sterile loop or swab.
4. Application of antimicrobial agents: a. Disk Diffusion Method (Kirby-Bauer Method):
   • Place sterile paper disks impregnated with specific concentrations of antimicrobial agents onto the agar surface.
   • Gently press the disks to ensure good contact with the agar.
   • Use a disk dispenser or forceps to place the disks in a standardized manner, maintaining appropriate distances between them.
   b. Broth Dilution Method:
   • Prepare a range of antimicrobial agent concentrations in liquid broth.
   • Add the standardized inoculum to each broth tube, ensuring the appropriate drug concentrations are achieved.
   • Include positive growth and negative control tubes without antimicrobial agents.
   c. Etest Method:
   • Place a plastic strip with a gradient of antimicrobial agent concentrations onto the agar surface.
   • Ensure proper alignment of the strip and press it gently onto the agar.
   • Incubate the plates or broth cultures under appropriate conditions (temperature, duration) suitable for the growth of the microorganism.
5. Incubation: Incubate the culture plates or broth cultures under optimal conditions for the growth of the microorganism. The temperature and duration of incubation may vary depending on the microorganism and testing method used. It is important to maintain proper temperature and humidity control during incubation.
6. Measurement and interpretation of results: a. Disk Diffusion Method:
   • After incubation, measure the diameter of the zones of inhibition around each disk.
   • Use a ruler or a calibrated measuring device to obtain accurate measurements.
   • Compare the zone sizes with interpretive criteria provided by organizations like the CLSI or EUCAST to determine the susceptibility or resistance of the microorganism to the antimicrobial agents.
   b. Broth Dilution Method:
   • Examine the broth tubes for visible growth after incubation.
   • The lowest concentration of the drug that inhibits visible growth is considered the Minimum Inhibitory Concentration (MIC).
   • Compare the MIC values with interpretive criteria to determine the susceptibility or resistance of the microorganism.
   c. Etest Method:
   • After incubation, observe the elliptical zones of inhibition and locate the intersection point with the strip.
   • The MIC value is read from the scale on the strip at the intersection point.
   • Compare the MIC values with interpretive criteria to determine the susceptibility or resistance of the microorganism.
7. Reporting of results: Record and report the AST results, indicating the susceptibility or resistance of the microorganism to each tested antimicrobial agent. Follow the established guidelines for result reporting, including the interpretation categories (susceptible, intermediate, resistant).

It is important to follow standardized protocols, maintain quality control measures, and adhere to interpretive criteria to ensure accurate and reliable AST results.

Result-Interpretation

The interpretation of Antimicrobial Susceptibility Testing (AST) results involves categorizing the susceptibility or resistance of a microorganism to different antimicrobial agents. The interpretation is based on established guidelines and breakpoints provided by organizations such as the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The following are the general categories used for result interpretation:

1. Susceptible (S): The microorganism is inhibited by the antimicrobial agent at a concentration that can be achieved in the body, indicating that the drug is likely to be effective for treatment.
2. Intermediate (I) or Indeterminate: The microorganism shows an intermediate response to the antimicrobial agent, indicating that the drug’s effectiveness may be limited or variable. Clinical decision-making should consider factors such as the site of infection, host factors, and alternative treatment options.
3. Resistant (R): The microorganism is not inhibited by the antimicrobial agent at concentrations achievable in the body, suggesting that the drug is unlikely to be effective for treatment.

It’s important to note that interpretation categories may vary depending on the specific organism, antimicrobial agent, and clinical situation. The breakpoints and interpretive criteria provided by relevant guidelines should be consulted for accurate result interpretation.

Additionally, some guidelines provide additional categories such as “N” (non-susceptible) or “F” (full resistance) to indicate certain situations where further testing or expert consultation is required. These categories are used in specific circumstances and may not be universally applicable.

When reporting AST results, it is essential to provide clear and concise information, including the microorganism’s name, the antimicrobial agent tested, and the interpretation category (S, I, or R). This information helps guide healthcare professionals in selecting appropriate antimicrobial therapy for the treatment of infectious diseases and in combating antimicrobial resistance.

Application

Antimicrobial Susceptibility Testing (AST) has several important applications in clinical practice and public health. The main applications of AST include:

1. Guiding antimicrobial therapy: AST results play a crucial role in guiding healthcare professionals in selecting appropriate antimicrobial therapy for the treatment of bacterial or fungal infections. By determining the susceptibility or resistance of the microorganism to specific antimicrobial agents, AST helps in tailoring treatment regimens to maximize therapeutic efficacy and improve patient outcomes.
2. Optimizing drug selection and dosing: AST provides information about the effectiveness of different antimicrobial agents against specific microorganisms. This helps in optimizing drug selection by identifying the most effective drug for treating a particular infection. Additionally, AST can assist in determining the appropriate dosage of antimicrobial agents to ensure adequate drug exposure and minimize the development of resistance.
3. Surveillance of antimicrobial resistance: AST plays a vital role in monitoring and surveillance of antimicrobial resistance patterns. By collecting and analyzing data on susceptibility profiles, AST helps in identifying emerging resistance trends, monitoring the spread of resistance, and informing public health strategies to combat antimicrobial resistance on a local, national, and global scale.
4. Infection control and prevention: AST aids in infection control and prevention strategies by identifying multidrug-resistant organisms (MDROs). The information obtained from AST helps in implementing appropriate infection control measures, such as isolation precautions and targeted antibiotic stewardship interventions, to prevent the spread of resistant microorganisms within healthcare settings.
5. Epidemiological studies and research: AST results contribute to epidemiological studies and research by providing valuable data on antimicrobial resistance patterns, the prevalence of specific resistance mechanisms, and the efficacy of new antimicrobial agents. This information helps in understanding the dynamics of antimicrobial resistance, developing new treatment strategies, and informing public health policies.
6. Antibiotic stewardship: AST is a cornerstone of antibiotic stewardship programs aimed at promoting the rational and appropriate use of antibiotics. By providing information on antimicrobial susceptibility, AST supports efforts to optimize antibiotic prescribing, reduce unnecessary antibiotic use, and minimize the selection and spread of resistant microorganisms.

Keynotes

Here are some keynotes on Antimicrobial Susceptibility Testing (AST):

1. Purpose: AST is performed to determine the susceptibility or resistance of microorganisms to specific antimicrobial agents. It helps guide appropriate antimicrobial therapy and contributes to the management of infectious diseases.
2. Methods: Various methods are available for AST, including the disk diffusion method, broth dilution method, Etest method, and automated systems. Each method has its advantages, limitations, and specific applications.
3. Test Requirements: AST requires a pure culture of the microorganism, standardized inoculum, quality control strains, antimicrobial agents, culture media, sterile discs/plates/strips, an incubator, and adherence to standardized protocols.
4. Result Interpretation: The interpretation of AST results involves categorizing microorganisms as susceptible (S), intermediate (I), or resistant (R) based on established breakpoints and interpretive criteria provided by organizations like the CLSI or EUCAST.
5. Applications: AST is used to guide antimicrobial therapy, optimize drug selection and dosing, surveil antimicrobial resistance, implement infection control measures, support epidemiological studies and research, and promote antibiotic stewardship.
6. Importance in Public Health: AST plays a crucial role in combating antimicrobial resistance, informing public health policies, and preserving the effectiveness of antimicrobial agents.
7. Standardization: Following standardized protocols, quality control measures, and interpretive criteria is crucial to ensure accurate and reliable AST results.
8. Collaborative Efforts: Collaboration between clinical microbiologists, healthcare professionals, and public health authorities is essential for effective AST implementation, data sharing, and monitoring of antimicrobial resistance patterns.
9. Continued Monitoring: Regular monitoring of local and global resistance patterns through AST is necessary to adapt treatment guidelines, inform empirical therapy choices, and develop strategies to combat emerging resistance.
10. Integration with Antibiotic Stewardship: AST is an integral part of antibiotic stewardship programs, promoting the rational and appropriate use of antibiotics to minimize resistance development.

Further Readings

1. Clinical and Laboratory Standards Institute (CLSI): The CLSI publishes guidelines and standards for AST. Their documents, such as the CLSI M100 document series, provide detailed information on AST methods, interpretive criteria, and quality control procedures. You can access their publications at: https://clsi.org/standards/
2. European Committee on Antimicrobial Susceptibility Testing (EUCAST): EUCAST provides guidelines and breakpoints for AST in Europe. Their documents offer updated recommendations on AST methods and interpretive criteria. You can find their resources at: http://www.eucast.org/
3. Infectious Diseases Society of America (IDSA): The IDSA offers guidelines and expert recommendations on various aspects of infectious diseases, including antimicrobial susceptibility testing. Their guidelines provide practical guidance on AST interpretation and management of specific infections. Visit: https://www.idsociety.org/
4. Journal of Clinical Microbiology (JCM): JCM is a reputable journal that publishes research articles and reviews on clinical microbiology, including AST. Reading articles in JCM can provide insights into new developments, methodologies, and research findings related to AST. You can access their articles at: https://jcm.asm.org/
5. UpToDate: UpToDate is an online clinical resource that offers comprehensive and evidence-based information on various medical topics, including infectious diseases and antimicrobial therapy. Their articles on AST provide practical guidance for clinicians. Visit: https://www.uptodate.com/
6. Antimicrobial Agents and Chemotherapy (AAC): AAC is a scientific journal that publishes original research articles and reviews on antimicrobial agents and their susceptibility testing. Reading articles in AAC can provide you with the latest advancements and research in the field of AST. Access their articles at: https://aac.asm.org/
7. World Health Organization (WHO): The WHO provides resources and guidance on antimicrobial resistance, including AST. Their reports and guidelines offer global perspectives and strategies to combat antimicrobial resistance. Visit their website at: https://www.who.int/