Antibiogram Kirby Bauer Disc Diffusion Method Dilution Tests Etest Automation in antimicrobial susceptibility testing

1. Antimicrobial Susceptibility Testing
Prepared By Lilian K. Nyarieko
M.SC-Medical Lab Sciences, JKUAT
Medical Laboratory Scientist- Afya International Hospital
2025

2. OBJECTIVES
• Antibiogram
• Kirby Bauer Disk Diffusion testing.
• Significance of the MIC and the MBC in Susceptibility testing.
• Automation

3. Introduction
• Testing the effectiveness of antimicrobial drugs against specific
organisms is important in:
 Identifying their spectrum of activity
 Determining the therapeutic dosage

4. Empirical diagnosis: Antibiogram
• An antibiogram is a vital tool in clinical microbiology and antimicrobial
stewardship.
– Compiles data on the susceptibility of bacterial pathogens to various antibiotics.
– Aids healthcare providers in selecting effective empiric therapies and monitoring
resistance trends.
• What is it? a cumulative report -
– Summarizes the results of antimicrobial susceptibility testing (AST) for bacterial
isolates collected over a specific period, typically one year.
• Generated by clinical microbiology laboratories
• Reflects local resistance patterns within a healthcare facility or region.
• Reference for clinicians to guide empiric antibiotic therapy before specific culture
results are available.

5. Developing an Antibiogram: steps
1. Data Collection: Bacterial isolates are obtained from patient
specimens
2. Susceptibility Testing: Each isolate undergoes AST using
standardized methods
3. Data Aggregation: Results are compiled over a defined time
frame, ensuring a sufficient number of isolates (typically at
least 30 per organism) to provide statistically reliable data.
4. Report Generation: data is formatted into a report (as
Percentages susceptible to given drug)

6. Types of antibiograms
• Depending on clinical need, they are:
1. Routine (Cumulative) Antibiogram: Aggregates susceptibility data for common
pathogens over a set period.
2. Syndromic Antibiogram: Focuses on pathogens associated with specific clinical
syndromes, such as urinary tract infections.
3. Unit-Specific Antibiogram: Tailored to specific hospital units like the ICU,
reflecting unique resistance patterns.
4. Combination Antibiogram: Evaluates the efficacy of antibiotic combinations against
particular pathogens.
5. Rolling or Real-Time Antibiogram: Continuously updated to reflect the most
current susceptibility data.

7. Clinical Applications
1. Guiding Empiric Therapy
2. Monitoring Resistance Trends: identify emerging resistance patterns.
3. Informing Stewardship Programs: promoting the judicious use of
antibiotics.
To maximize the impact of antibiograms:
4. Education and Training
5. Integration with Clinical Decision Support Systems
(CDSS):incorporation into health records/HIMS
6. Regular Updates

8. Limitations
• In resource-limited environments
Developing antibiograms can be challenging due to
constraints like limited laboratory capacity and lack of
standardized protocols.
• However, there is demonstrated feasibility and benefits where
implementation has occured like Gambia and Ghana.

9. Kirby Bauer Disk Diffusion
• Most commonly used/starting point/been long in use.
• Uses:
– Muller Hinton Agar
• A confluent lawn (uniform, continuous layer) of a pure bacterial
isolate.
– Filter paper disks impregnated with target antibacterial drugs.
• Disks have known amounts of drug.

10. Kirby Bauer Disk Diffusion...
• Principle:
– Antibiotic diffuses into agar as bacteria grows.
– Antimicrobial activity- observed as a clear circular zone around the
disk (zone of inhibition)
– The diameter of the zone of inhibition, measured in millimeters, and
– Compared to a standardized chart.
– Determines the susceptibility/ resistance.

11. Factors that determine size of zone of inhibition
• Drug solubility
• Rate of drug diffusion through agar
• The thickness of the agar medium
• The drug concentration impregnated into the disk
• Limitations
Lack of standardization of these factors-limited information/wrong
interpretation
Inability to distinguish between bacteriostatic and bactericidal
activities
Inability to compare drug potencies or efficacies

12. Dilution Tests
• To counter the inability of disc
diffusion method to determine
doses/assess drug potencies.
• Determines a drug’s MIC and MBC.
The tests include:
 Macrobroth Dilution assay
 96-well microdilution trays
 E-test
Definitions
 Minimal inhibitory concentration
(MIC)- the lowest concentration of
drug that inhibits visible bacterial
growth.
 Minimal bactericidal concentration
(MBC)- the lowest drug concentration
that kills ≥99.9% of the starting
inoculum.

13. Dilution Tests...
• Macrobroth dilution assay
• A dilution series of the drug in broth is made in test tubes and equal
amounts of bacterial cells added.
The MIC is determined by examining for turbidity (cloudiness).
• Tubes with no visible growth are then inoculated onto agar media
without antibiotic to determine the MBC.
Generally, serum levels of an antibacterial should be at least three to
five times above the MIC for effective treatment.

14. Dilution Tests...
• 96-well microdilution trays
• Uses small volumes automated dispensing devices, as well as
the testing of multiple antimicrobials and/or microorganisms in
one tray.
• MICs are interpreted as the lowest concentration that
inhibits visible growth. Growth may also be interpreted
visually or by using a spectrophotometer or similar device to
detect turbidity or a color change .

15. Etest
• A combination of the Kirby-Bauer disk diffusion test and
dilution methods.
• Procedure:
• A confluent lawn of a bacterial isolate is inoculated onto the
surface of Muller Hinton agar .
• In place of circular discs, plastic strips that contain a
gradient of an antibacterial are used.
• An elliptical zone of inhibition is observed.

16. Etest
• To interpret the results, the intersection of the elliptical zone
with the gradient on the drug-containing strip indicates the
MIC.
• Multiple strips containing different antimicrobials can be
placed on the same plate, the MIC of multiple antimicrobials
can be determined concurrently and directly compared.
Limitation
• MBC cannot be determined with the Etest

17. Automation in AST
• Advantages
• Standardization-less
errors/minimal
variability.
• Rapid: Reduced TAT
• Data Integration with
LIMS/HIMS
Consideration for implementation
Cost: High Initial investment and
maintenance costs: cost-benefit analyses
are essential.
Training: to operate and maintain
automated systems effectively.
Infrastructure: Adequate laboratory
infrastructure,; space and information
technology support

18. Leading Automated AST Systems: Examples
• BD Phoenix™ System
– Utilizes broth microdilution
with redox indicators to
detect bacterial growth.
– Up to 99 panels
simultaneously, (MIC) results
in 4–16 hours
– Applications: wide range of
aerobic and facultative
anaerobic Gram-positive and
Gram-negative bacteria.
• VITEK® 2 System (bioMérieux)
• Photometric and fluorometric readings to
assess bacterial growth in AST cards
containing dehydrated antibiotics.
• Up to 240 cards simultaneously, 4–8
hours for rapidly growing organisms.
• Applications: Widely used for bacterial
identification and susceptibility testing in
clinical laboratories.

19. Leading Automated AST Systems: Examples...
• MicroScan WalkAway® System
(Beckman Coulter)
– Based on broth microdilution
with colorimetric detection using
fluorogenic substrates.
– 40 to 96-panel modules, MIC
results in 4.5–18 hours,
depending on organism growth
rates.
– Applications: medium to high-
volume laboratories requiring
comprehensive AST capabilities.
• ASTar® System (Q-linea)
• Performs fully automated
microdilution AST directly from
positive blood cultures using time-lapse
imaging and proprietary algorithms.
• MIC results in approximately 6 hours,
enhancing rapid decision-making in
bloodstream infections.
• For rapid phenotypic AST of Gram-
negative rods directly from blood
cultures.

20. Leading Automated AST Systems: Examples...
• Accelerate Pheno® System
• Combines rapid
identification with
phenotypic AST using
morphokinetic cellular
analysis.
• MIC results- 7 hours
directly from positive blood
cultures.
• Supports timely
antimicrobial therapy
decisions in critical care
settings.
• Sysmex Astrego System
• Employs proprietary microfluidic
technology for rapid AST.
• Susceptibility results in
approximately 30 minutes, suitable
for point-of-care settings.
• Applications: Aids in the appropriate
use of antimicrobials during initial
patient visits in primary care.

21. Leading Automated AST Systems: Examples...
• RASP (Robotic Antimicrobial Susceptibility
Platform)
– Automates broth microdilution AST
using robotic systems.
– Enhances throughput and
standardization in AST, particularly
useful in surveillance of antimicrobial
resistance.
– Facilitates One Health surveillance by
enabling high-throughput testing
across human and animal health
sectors.
Summary:
 Different automation Systems
utilize different
methodologies,
 Have varying TAT
 Can be employed in different
settings to meet different needs
like surveilance, point of care
testing, primary health care
and critical care

22. References
• Adu-Sarkodie, Y., Amponsah, J. A., Owusu-Ofori, A., Labi, A. K., &
Opare-Addo, M. (2023). Developing hospital antibiograms to support
antimicrobial stewardship in Ghana. PLOS Global Public Health, 3(4),
e0001480. https://doi.org/10.1371/journal.pgph.0001480
• Saidu, Y., Jarju, S., Ceesay, F., Bah, A. A., & Tapgun, M. (2023). Using
antibiogram data to support antimicrobial stewardship in The Gambia.
IJID Regional, 12, 98–103. https://doi.org/10.1016/j.ijregi.2023.04.006
• Clinical and Laboratory Standards Institute. (2023). Analysis and
presentation of cumulative antimicrobial susceptibility test data (4th ed.)
(CLSI standard M39-A4).
https://clsi.org/standards/products/microbiology/documents/m39/

23. References
Buehler, S. S., Madison, B., Snyder, S. R., Derzon, J. H., Cornish, N. E., Saubolle, M.
A., ... & Weissfeld, A. S. (2016). Effectiveness of practices to increase timeliness of
providing targeted therapy for inpatients with bloodstream infections: A laboratory
medicine best practices systematic review and meta-analysis. Clinical Microbiology
Reviews, 29(1), 59–103. https://doi.org/10.1128/CMR.00053-14
Hombach, M., Zbinden, R., & Böttger, E. C. (2012). Standardisation of disk diffusion
results for antibiotic susceptibility testing using the BD Phoenix, VITEK 2 and disk
diffusion system. European Journal of Clinical Microbiology & Infectious Diseases,
31(4), 627–633. https://doi.org/10.1007/s10096-011-1353-4

24. References
• Charnot-Katsikas, A., & Jacobs, M. R. (2021). Advances in
automated antimicrobial susceptibility testing systems. Clinical
Microbiology Newsletter, 43(18), 141–147.
https://doi.org/10.1016/j.clinmicnews.2021.08.001
• OpenStax. (2016). Microbiology: Chapter 14 - Antimicrobial
Drugs. OpenStax CNX.
https://openstax.org/books/microbiology/pages/14-introduction

25. ABBREVIATIONS
• AST-antimicrobial Susceptibility Testing
• TAT-Turn Around Time
• LIMS- Laboratory Information Management System
• HIMS- Hospital Information Management System
• ICU- Intensive Care Unit

26. The end.