1. Developed by JALAL SHEIKH, Ph.D.
Antimicrobial Susceptibility Testing (AST) in Clinical Microbiology Laboratories
Antimicrobial Susceptibility Testing (AST) in Clinical Microbiology Laboratories
This presentation will cover the fundamentals of AST, various methods of AST and QA/QC of AST including the importance of CLSI guidelines for efficient reporting of AST.
Developed by
JALAL SHEIKH, Ph.D.

2. Acknowledgements Dr. Robert Miller - Principal Investigator
The presenters would like to thank:
NIH/DAIDS -Daniella Livnat and Mike Ussery
This project has been funded in whole or in part with Federal funds from the Division of AIDS (DAIDS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract No. HHSN C, titled Patient Safety Monitoring in International Laboratories.
Johns Hopkins University
Dr. Robert Miller - Principal Investigator
Barbara Parsons - Operations Manager
Kurt Michael - Project Manager
SMILE Staff
This presenter would like to acknowledge:
Daniella Livnat and Mike Ussery with the Division of AIDS (DAIDS)
And the following people from Johns Hopkins University:
Dr. Robert Miller - Principal Investigator
Barbara Parsons - Operations Manager
Kurt Michael - Project Manager
And all SMILE members.

3. Basic components of this presentation
Basics of Antimicrobial Susceptibility Testing (AST)
Different classes of antibiotics and antimicrobial agents based on their mechanisms of actions
A practical guide: steps associated with AST methods
QA/QC on AST
This presentation will be covered by the following :
Basics of AST including the fundamentals of AST
Different classes of antibiotics and antimicrobial agents based on their mechanisms of actions
A practical guide: steps associated with AST methods
QA/QC on AST

4. Objectives of this presentation:
Decisions of performing AST
Different steps associated with AST
Effective reporting of antimicrobial agents/antibiotics
Why CLSI?
The objectives of this presentation are:
Decisions of performing AST – factors need to be considered before AST is being performed
Different steps associated with AST – technical key points should be covered in laboratory SOP
Effective reporting of antimicrobial agents – how to interpret AST results and effective reporting to physicians

5. Questions to ask before initiating AST
What is the organism?
What was the site of infection?
What antibiotics/Antimicrobial agents need to be
included/tested?
What method of AST will be implemented?
How to report the results?
How to ensure the accuracy of AST results (QA/QC)?
What are the questions you need to ask before initiating AST include:
What is the organism?
What was the site of infection?
What antibiotics/Antimicrobial agents need to be included/tested?
What method of AST will be implemented?
How to report the results?
How to ensure the accuracy of AST results (QA/QC)?

6. Microorganisms and choice of Antibiotics/antimicrobial agents
Single drug of choice: No AST required
Commensal or opportunistic pathogen: special request
from physician
Multiple choice of drugs: AST required
Antibiotics selection for testing and reporting: CLSI guidelines
A decision on AST can be based as follows:
If there is only a single drug of choice for a particular organism from a particular source: No AST is required
If the organism is a commensal or opportunistic pathogen: a special request should be obtained from the physician before doing AST
If multiple choices of drugs are the option, AST is required
Selection of antibiotics for testing and reporting is not done solely by the microbiology lab but rather by joint decisions made by the committee comprising members from infection control, pharmacy, physicians and microbiology lab based on CLSI and national committee’s recommendations.
Premade panels comprising wide range of antibiotics for automated instruments are also one form of selection but in that case lab should be careful for reporting recommended antibiotics only.

7. Difference between Antibiotics and antimicrobial agents
Antibiotics: Organic compounds produced by microorganisms that kills or inhibits the growth of other microorganisms.
Antimicrobial agents: Synthetic chemical compounds that kills or inhibits the growth of other microorganisms.
Although clear distinction exists between antibiotics and antimicrobial agents, both terms can be used interchangeably. The common property of both compounds is to kill or inhibit the growth of microorganisms.

8. Mechanisms of antimicrobial activity
Antimicrobial agents work against microorganisms through different mechanisms and categorized them accordingly which include:
Inhibiting cell-wall synthesis known as Cell-wall synthesis inhibitors
Inhibiting protein synthesis known as Protein synthesis inhibitors
Inhibiting nucleic acid or RNA/DNA synthesis known as Nucleic acid synthesis inhibitors
Inhibiting synthesis of essential metabolites known as Essential metabolites synthesis inhibitors
Inhibits cell-wall synthesis
Inhibits protein synthesis
Inhibits nucleic acid (RNA/DNA) synthesis
Inhibits synthesis of essential metabolites

9. Bacterial Structure: Targets of antimicrobial agents
These are diamgram representations of both Gram-positive and Gram-negative cellular compartments and their cell-walls. Antimicrobial agents have specific target to different cellular components.

10. Different classes of antibiotics based on mechanisms of actions (1)
Inhibitors of cell-wall synthesis:
- Penicillin (natural and semi-synthetic)
- Cephalosporins (1st/2nd/3rd/4th and 5th generation)
- Bacitracin
- Vancomycin
- Isoniazid (INH)
- Ethambutol
Examples of Antimicrobial agents those are broadly categorized into different classes based on their cellular targets. They are:
Inhibitors of cell-wall synthesis (e.g., penicillin, cephalosporin, bacitracin, vancomycin, isoniazid, ethambutol etc.)

11. Different classes of antibiotics based on mechanisms of actions (2)
Inhibitors of protein synthesis:
- Chloramphenicol
- Aminoglycosides (Streptomycin, neomycin, gentamicin
- Tetracycline
- Macrolides (Erythromycin for gram-positives)
2. Inhibitors of protein synthesis (e.g., tetracycline, chloramphenicol, gentamicin, erythromycin etc.)

12. Different classes of antibiotics based on mechanisms of actions (3)
Inhibitors of Nucleic Acid synthesis:
- Rifamycin
- Quinolones
- Fluoroquinolones
Inhibitor of folic acid synthesis:
- Sulfonamides (Sulfa drugs)
3. Inhibitors of nucleic acid synthesis (e.g., quinolones, fluroquinolones, rifamycin etc.)
4. Inhibitors of folic acids (e.g, sulfonamides)

13. Different methods of AST
Diffusion method: Kirby-Bauer Disk Diffusion
Dilution method: MIC (Minimal Inhibitory Concentration)
Combination of diffusion and Dilution method: E-test
AST can be performed by three different methods:
Qualitative method which is known as diffusion method often referred as Kirby-Bauer disk diffusion method;
Quantitative method often referred as MIC or Minimal Inhibitory Concentration; and
Combination of diffusion and dilution method known as E-test.

14. Kirby-Bauer Disk Diffusion method (1)
Preparation of inoculum
Standardize inoculum suspension
Inoculate Mueller Hinton Agar (MHA)
Dispense antibiotic disks
Incubate overnight at 37°C
Basic criteria to perform Kirby-Bauer disk diffusion method:
Prepare bacterial inoculum in a suitable broth medium (e.g., Trypticase Soy Broth) or normal saline (0.85% NaCl) from a fresh culture
Standardize inoculum suspension (adjust with turbidity standard)
Inoculate Mueller Hinton Agar medium uniformly by spreading the bacteria with an inoculated sterile cotton swab
Dispense antibiotic disks according to CLSI guidelines (M100-S21) or your local laboratory guidelines based on joint decisions made by the Infection Control, Pharmacy, physicians and microbiology lab. Premade commercial panels for automated instruments are also one form of selection.
Incubate the inoculated media with antibiotics disks for overnight at 37°C

15. Kirby-Bauer Disk Diffusion method (2)
Measure zone sizes
Interpret the zone sizes with established chart:
- Sensitive (S)
- intermediate Sensitive (I) and
- Resistant (R)
5. Measure and record zone sizes produced surrounding the discs;
6. Interpret zone sizes as sensitive (S), intermediate (I), and resistant (R) according to established chart
7. Reporting of AST based on CLSI and site laboratory guidelines

16. QC/QA: Kirby-Bauer Disk Diffusion: Factors affecting the zone of inhibition (1)
Thickness of medium: should be 4 mm
- Thicker medium – narrow zone
- Thin medium – larger zone
pH of medium: should be neutral pH
- Acidic pH: larger zone size for Tetracycline, Methicillin and Novobiocin
- Alkaline pH: larger zone size for Aminoglycosides and Erythromycin
To obtain the quality standard and maintain the quality (QC/QA) of Kirby-Bauer disk diffusion method, the following factors are crucial:
Thickness of medium (Mueller-Hinton Agar) should be 4 mm (thicker medium will yield narrow zone whereas thin medium will produce larger zone);
pH of the medium should be neutral (Acidic pH will yield larger zone sizes for tetracycline, methicillin and novobiocin whereas alkaline pH will yield larger zone sizes for aminoglycosides and erythromycin);

17. QC/QA: Kirby-Bauer Disk Diffusion: Factors affecting the zone of inhibition (2)
3. Potency of antibiotic disks: Right concentration
- Zone size narrower if antibiotic concentration not maintained
4. Inoculum density: based on McFarland standard
-0.5 (mostly non-fastidious gram-negatives)
-1.0 (mostly fastidious slow-growers)
- Light inoculum: Large zone size
- Heavy inoculum: Small zone size
3. Potency of antibiotics (zone sizes would be narrower if concentration not maintained)
4. Inoculum density: 0.5/1.0 McFarland standard
-0.5 (mostly non-fastidious gram-negatives)
-1.0 (mostly fastidious slow-growers)
Impact of inoculum:
- Light inoculum: Large zone size
- Heavy inoculum: Small zone size

18. QC/QA: Kirby-Bauer Disk Diffusion: Factors affecting the zone of inhibition (2)
5. Placing the disks: immediately
6. Spacing the disks: 2.5 cm
7. Incubation time: hours
8. Incubation temperature: 35°C-37°C
5. Placing the antibiotic disks immediately after inoculum spreading;
6. Spacing the disks 2.5 cm from each other;
7. Optimal incubation time is hours;
8. Optimal incubation temperature is 35°C-37°C.

19. Procedure for Dilution method: Broth / Agar dilution (1)
Quantitative method
Microbroth dilution: MIC
(Minimal Inhibitory Concentration)
Agar dilution method
MIC: The lowest concentration of any antibiotics that inhibits the growth of a microorganism.
MIC (which stands for Minimal Inhibitory Concentration) is the lowest concentration of any antibiotics that inhibit or kill the bacteria. It is a quantitative method and can be performed either by broth dilution or agar dilution method.
In broth dilution, different concentration (typically two fold dilutions) of antibiotics are added to suitable growth medium in a series of tubes or wells of microtiter dishes which is inoculated with a defined number of microorganisms. After overnight incubation, the MIC would be calculated based as the lowest concentration of antibiotics of growth where no visible growth or sediment is present.

20. Procedure for Dilution method: Broth dilution
Making dilutions of antibiotics in 96-well plate or tubes
Standardize inoculum preparation
Inoculation of MIC tray or tubes
Incubate at 35°C-37°C for overnight (16-18 h)
Read the turbidity under the light box or automated
reader
Steps needed to perform broth dilution method:
Making dilutions of antibiotics with Mueller-Hinton or appropriate broth in 96-well microtiter dishes or clear tubes
Prepare standardized inoculum of microorganism which needs to be tested
Inoculate the MIC tray or tubes
Incubate overnight at 35°C-37°C
Read the turbidity in the MIC tube or bacterial pellet at the bottom of the microtiter well under the light box or an automated reader.

21. Combining diffusion and dilution method: E-test (AB Biodisk, Solna, Sweden)
The E test, consisting of a continuous stable gradient of antimicrobial agent provides 15 twofold dilutions on a strip
The E-test strip is placed on the surface of an agar plate inoculated with test organisms
Incubate overnight at 35°C-37°C
MIC is measured on the test strip scale where the zone of inhibition intersect the strip
MIC is also obtained through a commercially available test strips known as E-test, which contains exponential gradients of antibiotics. It combines both diffusion and dilution of antibiotics. The antimicrobial agents are impregnated into test strips by providing 15 twofold dilutions. The recommended medium is inoculated with standardized inoculum and the strip is placed into inoculated agar medium, where the antibiotics diffuses into the medium.
After overnight incubation at 35°C-37°C, the MIC is read at the point of intersection where the inhibition zone has started.

22. Selecting and reporting of antimicrobial agents: Why CLSI ?
CLSI: Clinical Laboratory Standards Institute
To improve patient testing and health care services
International, interdisciplinary, nonprofit, standards-developing, educational organization
Recognized internationally worldwide
* CAP, Accutest and other EQA-providers follow CLSI guidelines.
CLSI guidelines are followed by all clinical laboratories throughout the United States and other countries worldwide.
Formerly known as National Committee for Clinical Laboratory Standards or NCCLS.
CLSI is a well-respected standards-developing, international, non-profit educational organization.
It publishes standard, guidelines and committee reports based on consensus guidelines, publications, epidemiological trends etc.
Both CAP, Accutest and other EQA-providers follow CLSI guidelines.

23. CLSI publications CLSI published three types of publications: Standard
Guideline
Report
CLSI publications are categorized as:
Standard: Document developed through consensus process which identifies specific, essential requirements for materials, methods, or practices.
Guideline: Document developed through consensus process describing criteria for a general practice, procedure or material for voluntary use.
Report: Document that has not been subjected to consensus review and is released by the Board of Directors.

24. CLSI Subcommittee on AST
Reviews data from a variety of sources and studies
To establish AST methods
To establish interpretive criteria
To establish QC parameters
This process is continually refined and updated annually.
Responsibilities of CLSI subcommittee on AST
Reviews and analyzes data collected from a variety of sources and studies to establish:
AST methods;
Interpretive criteria and
QC parameters.
This process is continually refined and updated annually because:
Over time, microorganisms acquire more resistance to certain antimicrobial agents
The field of medical science is ever changing and microbiological methods and QC parameters may be refined to ensure more accurate and better performance of susceptibility test methods

25. CLSI document: M100-S21
Performance Standards for Antimicrobial Susceptibility Testing
CLSI reference methods for AST:
Disk Diffusion method (Kirby-Bauer)
Dilution method: MIC Testing
CLSI M100-S21 document describes the Performance Standards for Antimicrobial Susceptibility Testing.
It is a 172-page document
It is continuously updated each year because over time microorganisms susceptibility to an antimicrobial agent may decrease, resulting in a lack clinical efficacy and/or safety.

26. A detailed presentation will be available on pSMILE resources.
How to use CLSI
M100-S21 Document ?
A detailed presentation will be available on pSMILE resources.
A detailed presentation on how to use CLSI M100-S21 document will be available on pSMILE resources. Please use that presentation in conjunction with this AST presentation which might be more beneficial for your lab. To select and report antibiotics and antimicrobial agents,

27. If you have any questions please contact SMILE
Thanks for your attention.
SMILE strongly encourage your feedback for further improvement of this presentation. If you have any question or concern please contact your SMILE primary coordinator or contact directly to Kurt Michael at