Antimicrobial Susceptibility Testing (AST)

A. Procedures in Antimicrobial Susceptibility Testing

- Antimicrobial susceptibility testing is performed on bacteria isolated from clinical specimens
to determine which antimicrobial agents might be effective in treating infections caused by
bacteria.
- MIC ( Minimal Inhibitory Concentration ) or Disk Diffusion
- CLSI ( Clinical and Laboratory Standards Institute )
- ISO (International Standards Organization)

1. Reasons and Indications for Performing Antimicrobial Susceptibility Tests

- Antimicrobial susceptibility testing should be performed on a bacterial isolate from a clinical
specimen if the isolate is determined to be a probable cause of the patient’s infection and the
susceptibility of the isolate to particular antimicrobial agents cannot be reliable predicted
based on previous experience with the bacteria at health care facility.
- Susceptibility testing of isolates can also provide information on decreases in the
susceptibility of bacteria to antimicrobials.
- ECV (Epidemiologic cutoff values)

Factors to consider when determining whether testing is warranted:

1. Body site from which the bacterium was isolated
2. Presence of other bacteria and quality of the specimen from which the organism was
grown
3. Host’s Status

2. Selecting Antimicrobial Agents For Testing and Reporting

- FDA ( Food and Drug Administration)
- It is important that the antibiotics tested by the laboratory match the institutional formulary as
closely as possible. From the laboratory perspective, limiting factor for the number of
antibiotics tested is usually the number that can be practically tested with a particular method.
- The patient population must be considered in the choice of antimicrobial agents to be tested.

 Selection of Test Batteries

o A supplemental battery that contains antimicrobial agents with enhanced activity may
be included by laboratories that encounter a significant number of bacteria resistant
to the more commonly used antimicrobials

 Reporting of Susceptibility Test Results

o The final decision about the antimicrobials to
report is made once the identity of the isolate is
known, along with the overall susceptibility results
and specimen source.
o Reporting Protocols
3. Traditional Antimicrobial Susceptibility Test Methods
- Inoculum Preparation and Use Of Mcfarland Standards
o Inoculum Preparation
o Mcfarland Turbidity Standards
o Inoculum Standardization

- Dilution Susceptibility Testing Methods

o Principle: Dilution antimicrobial susceptibility test methods are used to determine the
MIC, or the lowest concentration of antimicrobial agent required to inhibit the growth
of the bacterium
o Once the MIC is determined, the organism is interpreted as nonsusceptible,
susceptible, intermediate, resistant

- Antimicrobial Stock Solutions

- Broth Macrodilution / tube dilution MIC tests.

- Broth Microdilution Test

o Breakpoint (Cutoff MIC panels)
o Trailing Growth and Skipped Wells
o Skipped Wells involve growth at higher concentrations and no growth at one or more
of the lower concentrations.

- Agar Dilution Tests

o DISK DIFFUSION TESTING

- Kirby-Bauer Test
- Establishing Zone Diameter Interpretive Breakpoints
- Test Performance
o Disk Storage
o Inoculation & Incubation
o Reading Plates and Test Interpretation
- Modified Methods of Testing Slow Growing or Fastidious Bacteria
o MH agar and additional required supplemental nutrients, modified incubation
conditions

 Streptococcus pneumoniae & Streptococcus species

o D-zone Test

 Haemophilus influenzae & Haemophilus parainfluenzae

 Neisseria gonorrhea & Neisseria meningitidis
 Helicobacter pylori
 Susceptibility testing Agents of Bioterrorism
 Anaerobes
 Infrequently encountered or Fastidious Bacteria (Aeromonas, Corynebacteria, Vibrio,
Pasteurella, Moraxella catarrhalis, HACEK,

- Additional Organism and Antimicrobial Agent Testing Concerns

 Detection of Oxacillin Resistance in Staphylococci
o Oxacillin Screen Plate

 Vancomycin Resistance or Diminished Susceptibility in Staphylococcus aureus

 Inducible Clindamycin Resistance in Staphylococci

 Enterococci
o Detection of High Level Aminoglycoside Resistance in Enterococci
o Detection of Vancomycin Resistance in Enterococci
 Extended Spectrum Beta Lactamases

 Carbapenemeses
o Klebsiella pneumoniae Carbapenemase

4. I . Automated Antimicrobial Susceptibility Test Methods

- Principles of Technologies Used

- Currently Available Automated Systems

- Readers Devices for Broth Microdilution Susceptibility Tests

- Automated Instruments Systems
1) BD Phoenix System

2) Microscan Walkaway SI

3) TREK sensititre

4) VITEK 1, VITEK 2, VITEK 2 Compact

II. Nanoautomated Antimicrobial Susceptibility Test Method: ETEST

- Etest ( AB biodisk, Solna, Sweden)

5. Interpretation of In Vitro Antimicrobial Susceptibility Test Results

- The inhibition zone size and MIC interpretive criteria published by CLSI and FDA
1. Microbiological data
2. Pharmacokinetic Data
3. Results of Clinical Studies obtained during the phase before FDA approval and
marketing of an antimicrobial agent and

6. Methods of Detecting Antimicrobial – Inactivating Enzymes

- Beta Lactamase Tests
- Quality Control of Antimicrobial Susceptibility Tests

Selecting an Antimicrobial Susceptibility Test Method

 Susceptibility Challenge

- Rapid Susceptibility Determination

- Susceptibility Testing Of Biofilms

B. Special Antimicrobial Susceptibility Tests

1. Antimicrobial Concentration Test Assay

2. Minimum Bactericidal Concentration Test

- Minimum inhibitory concentration Test
 Identify the amount of antimicrobial agent required to inhibit the growth or
multiplication of a bacterial isolate.

3. Controlling Test Variables

- MBC tests are subject to more technical
pitfalls than MIC tests, and several
variables must be rigidly controlled during
MBC testing.
4. Interpretation concerns
- Paradoxic (Eagle Effect)

5. Time-Kill Assays
- Measure of rate of killing of bacteria by an antimicrobial agent (as determined by examining
number of variable bacteria remaining at various intervals after exposure to agent.

6. Synergy Tests
- Measure of susceptibility of a bacterial isolate to a combination of two or more
antimicrobial agents
7. Serum Bactericidal Test
- Measure of the highest dilution or titer of a patient’s serum that is inhibitory to patient’s
own infecting bacterium and highest dilution or titer that is bactericidal.

8. Measurement of Antimicrobial Agents In Serum And Body Fluids

1. Biological Assays
2. Immunoassays
3. Chromatography Assays