Antimicrobial Susceptibility Testing (AST) Types and Limitations

In the management of infectious diseases, especially those caused by frequently drug-resistant pathogens, susceptibility (sensitivity) testing is employed to identify effective antimicrobial drugs. However, there are instances where susceptibility testing may not be necessary, as the likely susceptibility reactions of a pathogen can be anticipated. For example:

• Proteus species are generally resistant to nitrofurantoin and tetracyclines.
• Streptococcus pyogenes is typically susceptible to penicillin.
• Klebsiella pneumoniae is generally resistant to ampicillin.
• Anaerobes are susceptible to metronidazole.

It is crucial to avoid conducting susceptibility tests on commensal organisms or contaminants. Testing these organisms could mislead clinicians and potentially lead to patients receiving ineffective and unnecessary antimicrobial therapy. This not only risks causing side effects but also contributes to the development of resistance in other potentially pathogenic organisms. Thus, careful consideration and targeted testing are essential to guide appropriate and effective antimicrobial treatment.

Susceptibility Testing Techniques

Susceptibility testing is performed to determine the effectiveness of antimicrobial agents against specific pathogens. Various techniques are employed for susceptibility testing, and the choice of method depends on factors such as the type of microorganism, available resources, and the specific antimicrobial agents being tested.

1. Disk Diffusion (Kirby-Bauer) Method:

   • Principle: Paper disks containing standardized concentrations of antimicrobial agents are placed on an agar plate inoculated with the test microorganism. The zone of inhibition around each disk is measured and correlated with the organism’s susceptibility.
   • Application: Widely used for bacteria, including aerobes and facultative anaerobes.

2. Broth Dilution Method:

   • Principle: Serial dilutions of antimicrobial agents are prepared in liquid broth, and the test microorganism is added. The minimum inhibitory concentration (MIC) is determined as the lowest concentration that inhibits visible growth.
   • Application: Provides quantitative MIC data and is used for both bacteria and fungi.

3. Etest (Epsilometer Test):

   • Principle: Combines aspects of both disk diffusion and broth dilution. A plastic strip with a gradient of antimicrobial concentration is placed on an agar plate, and the intersection of the elliptical zone of inhibition with the strip indicates the MIC.
   • Application: Particularly useful for slow-growing or fastidious microorganisms.

4. Agar Dilution Method:

   • Principle: Similar to broth dilution but involves incorporating various concentrations of antimicrobial agents directly into the agar. MIC is determined by the lowest concentration inhibiting growth.
   • Application: Suitable for bacteria and fungi.

5. Automated Systems:

   • Principle: Utilizes automated instruments to perform susceptibility testing. These systems use predefined algorithms to interpret results based on growth patterns or changes in optical density.
   • Application: Provides efficient and standardized testing, often used in clinical laboratories.

6. VITEK Systems:

   • Principle: An automated system that uses cards containing dehydrated antimicrobials. The system measures microbial growth and provides susceptibility results.
   • Application: Commonly used for bacterial identification and susceptibility testing.

7. Molecular Methods:

   • Principle: Detects specific resistance genes or mutations associated with resistance using molecular techniques such as polymerase chain reaction (PCR).
   • Application: Rapid identification of resistance mechanisms, especially in situations where traditional methods may be time-consuming.

The choice of susceptibility testing method depends on factors such as the type of microorganism, the availability of resources, and the specific requirements of the clinical or research setting. Standardized guidelines, such as those from the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), help ensure the accuracy and reproducibility of susceptibility testing results.

Limitations of Antimicrobial Susceptibility Tests

While antimicrobial susceptibility tests are valuable tools for guiding treatment decisions, they have certain limitations that should be considered.

1. Standardization Issues:

   • Challenge: Lack of standardized methods across laboratories may lead to variations in testing procedures and interpretation criteria.
   • Implications: Inconsistencies in results may affect the comparability of data between different laboratories.

2. Time Constraints:

   • Challenge: Traditional susceptibility tests can take 24 to 48 hours to produce results.
   • Implications: Delayed results may impact timely initiation of appropriate antimicrobial therapy, especially in critical cases.

3. Detection of Heteroresistance:

   • Challenge: Some testing methods may not detect heteroresistance, where subpopulations of microorganisms within the same culture exhibit different susceptibility patterns.
   • Implications: Heteroresistance may lead to treatment failure if the resistant subpopulations are not identified.

4. Exposure to Antimicrobials:

   • Challenge: Prolonged exposure of microorganisms to suboptimal concentrations of antimicrobials during testing may influence susceptibility results.
   • Implications: False susceptibility results may occur, leading to inappropriate treatment decisions.

5. Limited Scope of Testing:

   • Challenge: Testing typically focuses on a predefined set of antimicrobials, and emerging resistance may not be captured.
   • Implications: The test may not provide a comprehensive view of the resistance profile, especially for newly developed antimicrobial agents.

6. Influence of Inoculum Size:

   • Challenge: The size of the inoculum used in testing can influence results, with larger inocula potentially masking the effects of antimicrobials.
   • Implications: Inaccurate results may be obtained if inoculum size is not standardized.

7. Biofilm Formation:

   • Challenge: Susceptibility tests may not accurately reflect the resistance of microorganisms within biofilms, which are common in certain infections.
   • Implications: Infections involving biofilm-forming bacteria may not be effectively treated based solely on susceptibility test results.

8. Lack of Clinical Correlation:

   • Challenge: In vitro susceptibility may not always correlate with clinical outcomes.
   • Implications: A microorganism may be susceptible in vitro, but clinical response can be influenced by factors such as host immune response, site of infection, and drug pharmacokinetics.

9. Limited Predictive Value for Some Pathogens:

   • Challenge: For certain pathogens, such as Mycobacterium tuberculosis, susceptibility tests may have limitations in predicting treatment outcomes.
   • Implications: Treatment decisions may require additional information beyond susceptibility test results.

• Emergence of New Resistance Mechanisms:

  • Challenge: Rapid evolution of resistance mechanisms may outpace the development of new testing methods.
  • Implications: Novel resistance mechanisms may not be detected, leading to delayed awareness and response.