Laboratory protocol for antimicrobial susceptibility testing (AST)

Introduction

Antimicrobial susceptibility testing (AST) is an important laboratory procedure used to determine the effectiveness of antimicrobial agents against isolated microorganisms. It plays an indispensable role in guiding both empirical and definitive therapy for infectious diseases. By identifying specific antimicrobial agents to which a microorganism is susceptible, AST aids clinicians in selecting appropriate treatments, optimizing patient outcomes, and preventing the emergence and spread of antimicrobial resistance (AMR).

AMR is a global health crisis that occurs when microorganisms evolve mechanisms to resist the effects of antimicrobial drugs. This phenomenon poses a significant challenge to public health, limiting therapeutic options and increasing morbidity and mortality rates. Consequently, understanding the mechanisms of antimicrobial resistance and implementing effective AST protocols are essential steps in combating this growing threat. The protocol detailed below is used in our lab and provided here for the convenience of our students to ensure consistency and accuracy in their results.

Principle of antimicrobial susceptibility testing

The principle underlying AST is to expose microorganisms to standardized concentrations of antimicrobial agents and assess their growth or inhibition under controlled conditions. Various methods are employed, but the core concept remains the same: if a microorganism is susceptible to an antimicrobial agent, its growth will be inhibited or killed, while resistant microorganisms will continue to grow despite the presence of the drug.

Mechanisms of antimicrobial action

Antimicrobial agents exert their effects through diverse mechanisms, targeting different cellular components or processes of microorganisms. These mechanisms include:

1. Inhibition of cell wall synthesis: This mode of action is characteristic of many antibiotics, such as penicillins and cephalosporins. They interfere with the synthesis of peptidoglycan, an essential component of the bacterial cell wall.

2. Disruption of cell membrane integrity: Agents like polymyxins target the bacterial cell membrane, leading to leakage of cellular contents and eventual cell death.

3. Inhibition of protein synthesis: Antibiotics such as aminoglycosides and tetracyclines bind to ribosomes and disrupt protein synthesis, hindering bacterial growth.

4. Inhibition of nucleic acid synthesis: Quinolones and fluoroquinolones inhibit DNA replication by targeting DNA gyrase, an essential enzyme for bacterial DNA replication.

5. Metabolic inhibition: Sulfonamides and trimethoprim interfere with folic acid metabolism, which is vital for bacterial growth.

Understanding these mechanisms is essential for accurately interpreting AST results and selecting appropriate therapeutic options.

Procedure for antimicrobial susceptibility testing (AST)

Disk diffusion method (Kirby-Bauer test)

This protocol details the disk diffusion method for antimicrobial susceptibility testing (AST) used in our lab, and is intended primarily for our students to ensure accuracy and consistency. The disk diffusion method, commonly known as the Kirby-Bauer test, is a standardized technique used to assess the susceptibility of bacteria to antimicrobial agents. This method involves inoculating a standardized bacterial suspension onto an agar plate and placing antibiotic-impregnated disks on the surface. After incubation, the diameter of the zone of inhibition surrounding each disk is measured and compared to established interpretive standards to determine susceptibility.

Materials:

• Mueller-Hinton agar plates

• Antimicrobial susceptibility disks (containing known concentrations of antibiotics)

• Sterile cotton swabs

• Inoculation loop

• Sterile saline solution (0.85%)

• Graduated cylinder

• Spectrophotometer or densitometer

• Non-selective culture medium

• Incubator

• Calipers or ruler

Procedure:

1. Preparation of bacterial suspension

   • Select a pure, well-isolated colony of the test organism from a non-selective culture medium.

   • Suspend the colony in sterile saline solution to match the turbidity of a 0.5 McFarland standard. This can be visually compared or measured using a spectrophotometer or densitometer.

2. Inoculation of agar plate

   • Dip a sterile cotton swab into the bacterial suspension and remove excess fluid by pressing the swab against the inside of the tube.

   • Inoculate the entire surface of the Mueller-Hinton agar plate by streaking the swab back and forth in three directions, rotating the plate 60 degrees after each pass.

3. Application of antibiotic disks

   • Using sterile forceps, place the desired antimicrobial disks onto the inoculated agar plate at predetermined distances.

   • Ensure that the disks are evenly spaced and do not overlap.

4. Incubation

   • Invert the agar plate and incubate at 35 ± 2°C for 16-18 hours.

5. Measurement of zone of inhibition

   • After incubation, measure the diameter of the zone of inhibition around each antibiotic disk in millimeters.

   • Use calipers or a ruler for accurate measurement.

6. Interpretation

   • Compare the measured zone diameters to established interpretive standards provided by clinical and laboratory standards institutes (e.g., CLSI, EUCAST).

   • Interpret the results as susceptible, intermediate, or resistant based on the zone diameter and the breakpoint for each antimicrobial agent:

     • Susceptible: The organism is inhibited by the standard therapeutic dose of the antimicrobial agent.

     • Intermediate: Susceptibility is uncertain; clinical efficacy may be variable.

     • Resistant: The organism is not inhibited by the standard therapeutic dose of the antimicrobial agent.

Caution

• Biosafety: Handle microorganisms following appropriate biosafety level precautions to minimize risks.

• Antibiotic resistance: Overuse of antibiotics can contribute to the development of antimicrobial resistance. Use antibiotics judiciously and in accordance with established guidelines.

• Interpretation: Proper interpretation of results requires expertise in microbiology and antimicrobial therapy. Consult with a clinical microbiologist when necessary.

• Quality control: Regular quality control is essential to ensure the accuracy and reliability of results.

Quality control

• Positive and negative controls: Include standard strains with known susceptibility patterns as positive and negative controls in each test run.

• Disk potency: Ensure that antibiotic disks are within their expiration date and stored according to manufacturer specifications.

• Media preparation: Prepare Mueller-Hinton agar according to manufacturer’s instructions and quality control specifications.

• Inoculum preparation: Verify the accuracy of the McFarland standard for inoculum preparation.

• Zone size interpretation: Use standardized guidelines (e.g., CLSI, EUCAST) for interpreting zone sizes.

Troubleshooting

1. Problem: No growth or poor growth

   • Possible cause: Incorrect inoculum preparation, improper incubation conditions, contaminated media

   • Solution: Prepare inoculum correctly, check incubator temperature and CO2 levels, use fresh media.

2. Problem: Excessive growth

   • Possible cause: Over-inoculation, contaminated media, improper incubation conditions

   • Solution: Adjust inoculum density, use fresh media, check incubator temperature and CO2 levels.

3. Problem: Inconsistent zone sizes

   • Possible cause: Variation in inoculum density, improper disk application, disk deterioration, media variation

   • Solution: Prepare inoculum consistently, apply disks correctly, use fresh disks, standardize media preparation.

4. Problem: No zones of inhibition

   • Possible cause: Resistant organism, disk placement error, media problems

   • Solution: Confirm organism identification, check disk placement, and use fresh media.

5. Problem: Discrepancies with reference laboratory

   • Possible cause: Technical errors, different interpretive criteria, different antimicrobial agents

   • Solution: Review test procedures, compare interpretive criteria, and check antimicrobial agent specifications.

Additional considerations

• Organism identification: Accurate identification of the microorganism is critical for appropriate antimicrobial selection.

• Patient factors: Consider the patient’s clinical condition, immune status, and potential allergies when selecting antimicrobial therapy.

• Combination therapy: In some cases, combination therapy with multiple antimicrobial agents may be necessary.

• Susceptibility testing methods: Other susceptibility testing methods, such as broth microdilution and E-test, may be used for specific purposes or organisms.

• Emerging resistance mechanisms: Stay updated on emerging antimicrobial resistance mechanisms to guide therapeutic decisions.

By following these guidelines and implementing appropriate quality control measures, laboratories can ensure the accuracy and reliability of antimicrobial susceptibility testing results, which are essential for effective antimicrobial therapy.

References

1. Ali F, Silvy TN, Hossain TJ, Uddin MK, Uddin MS. 2021. Prevalence and antimicrobial resistance phenotypes of Salmonella species recovered at various stages of broiler operations in Hathazari, Bangladesh. Int J One Health, 7, 158–164. (doi:10.14202/IJOH.2021)

2. Hossain TJ. 2024. Methods for screening and evaluation of antimicrobial activity: A review of protocols, advantages, and limitations. European Journal of Microbiology and Immunology, 14(2), 97–115. (doi:10.1556/1886.2024.00035)

3. Paul S, Hossain TJ, Ali F, Hossain ME, Chowdhury T, Faisal IK, Ferdouse J. 2024. Assessment of the in-vitro probiotic efficacy and safety of Pediococcus pentosaceus L1 and Streptococcus thermophilus L3 isolated from Laban, a popular fermented milk product. Arch Microbiol, 206, 82. (doi:10.1007/s00203-023-03812-5)