1. Antimicrobial Susceptibility Testing (AST)

2. Reasons and Indications for Antimicrobial Susceptibility Testing (AST)
Offer guidance to physician in selecting effective antibacterial therapy for a pathogen
Performed on bacteria isolated from clinical specimens if the bacteria’s susceptibility to particular antimicrobial agents is uncertain
Susceptibilities NOT performed on bacteria that are predictably susceptible to antimicrobials
Ex. Group A Strep

3. Why Need continues for testing for Antibiotic Sensitivity
Bacteria have the ability to develop resistance following repeated or subclinical (insufficient) doses, so more advanced antibiotics and synthetic antimicrobials are continually required to overcome them.

4. Selection of Test Batteries
Generally, labs choose antibiotics to test susceptibility for Gram Positive organisms and another for Gram Negative organisms
Too many choices can confuse physicians and be too expensive
Primary objective
Use the least toxic, most cost-effective, and most clinically appropriate agents
Refrain from more costly, broader-spectrum agents

5. Antibiotic sensitivity test:
the in vitro testing of bacterial cultures with antibiotics to determine susceptibility of bacteria to antibiotic therapy.

6. Important terms Minimum inhibitory concentration(MIC)
Lowest concentration of an antimicrobial agent that visibly inhibits the growth of the organism.
Minimum bactericidal concentration (MBC)
Lowest concentration of the antimicrobial agent that require to kill the organism.

8. Susceptible ”S”
Interpretive category that indicates an organism is inhibited by the recommended dose, at the infection site, of an antimicrobial agent
Intermediate “I”
Interpretive category that represents an organism that may require a higher dose of antibiotic for a longer period of time to be inhibited
Resistant “R”
Interpretive category that indicates an organism is not inhibited by the recommended dose, at the infection site, of an antimicrobial agent.

9. Methods of Performing AST
Disk diffusion method
Kirby- Bauer
Gradient diffusion method (E-Test)

10. Disk Diffusion/ Kirby- Bauer
Procedure
Use a well-isolated, hour old organism
Transfer organism to a broth
Either tryptic soy/sterile saline
Ensure a turbidity of 0.5 McFarland
Inoculate MH agar by swabbing in three different directions “Lawn of growth”
Place filter paper disks impregnated with antimicrobial agents on the agar
Invert and incubate for hours at35 oC in non-CO2
McFarland standards. No. 0.5, 1 and 2.

11. Disk Diffusion/ Kirby-Bauer (cont’d)
During incubation, drug diffuses into agar
Depending on the organism and drug, areas of no growth form a zone of inhibition
Zones are measured to determine whether the organism is susceptible, intermediate, or resistant to the drug

12. E- test/ Gradient Diffusion Method
“MIC on a stick”
Plastic strips impregnated with antimicrobial on one side
MIC scale on the other side
Read MIC where zone of inhibition intersects E strip scale

13. Common interpretation problems
Results depends on the technique used
Many factors influence results
Lack of standardization of the inoculums
Thickness and quality of the culture media
Quality and conservation of the disks
Quality control with standardized strains
Condition and duration of incubation

14. Common interpretation problems
An agar gel that is too thick leads to smaller zones

15. Common interpretation problems
Problem with the size of the inoculums Solution:
Use McFarland 0.5 photometer
Scale -> same tubes

16. Common interpretation problems
Contamination with another organism

17. Common interpretation problems
Bad manipulation
Inoculation of the Muller Hinton
Swabbing
Not by flooding

18. Common interpretation problems
Problems with E-test reading

19. Kirby-Bauer disc testing
Antibiotic-impregnated discs placed on an agar plate at the
interface between test organism and susceptible control organism
Resulting zones of inhibition compared, use of controls
Susceptibility is inferred (standard tables)

20. Table of zone diameter breakpoint for Staphylococcus aureus
Q: Zone of inhibition of Staphylococcus aureus was 20 mm when bacteria treated with Methicillin. Comment
Comment: S.aureus is Sensitive to Penicillin

21. E-test Plastic strips with a predefined gradient of One antibiotic
One antifungal
Only one manufacturer
One strip per antibiotic
Wide range of antibiotics
Easy to use
Storage at -20°C
Short shelf life, expensive

22. Reading E-tests Ciprofloxacin for Yersinia pestis
Resistant > 4 ug/ml
Intermediate 1-4 ug/ml
Susceptible < 1
Upper reading

23. Dilution in liquid broth
Tubes containing increasing antibiotic concentrations
Incubation during 18 hr at 37°C
Tedious
MIC
Bacterial growth
Inhibition
0 (Control) 0, , mg/l

24. Turbid
.25 .5 1 2 4 8
Clean
Bacterial growth
Inhibition
The MIC is 2 mg/l ( The first visible clean tube with lowest concentration of the Antibiotic)
The MBC is 4 mg/l ( The lowest concentration of the Antibiotic without growth of bacteria on agar media)