1. Antimicrobial Susceptibility Testing (AST)
MLAB 2434 – Microbiology
Keri Brophy-Martinez

2. Reasons and Indications for Antimicrobial Susceptibility Testing (AST)
Goal
Offer guidance to physician in selecting effective antibacterial therapy for a pathogen in a specific body site
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. Factors to Consider When Determining Whether Testing is Warranted
Body site of infection
Susceptibility not performed on bacteria isolated from body site where they are normal flora
Ex. Susceptibility for E. coli is NOT performed when isolated from stool, but IS performed when isolated from blood

4. Factors to Consider When Determining Whether Testing is Warranted (cont’d)
Presence of other bacteria and quality of specimen
Ex. Two or more organisms grown in a urine specimen
Host status
Immunocompromised patients
Allergies to usual antimicrobials

5. Selecting Antimicrobial Agents for Testing and Reporting
Clinical & Laboratory Standards Institute (CLSI)
Develop standards, methods, QC parameters, and interpretive criteria for sensitivity testing
If necessary, can alter the breakpoints of the SIR ( susceptible, intermediate, resistant) based on emerging resistance

6. Selecting Antimicrobial Agents for Testing and Reporting (cont’d)
There are approximately 50 antibacterial agents
Follow CLSI recommendations
Each laboratory should have a battery of antibiotics ordinarily used for testing
Drug formulary decided by medical staff, pharmacists, and medical technologists

7. Selection of Test Batteries
Generally, labs choose antibiotics to test susceptibility for GP organisms and another for GN 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

8. Example of Drug Formulary
Enterococcus
Staphylococcus spp.
Ampicillin X
Cefazolin
Clindamycin
Erythromycin
Linezolid
Oxacillin
Penicillin G
Rifampin
Streptomycin-2000
Tetracycline
Trimeth/ Sulfa
Vancomycin

9. Example of Drug Formulary
Enterobacteriaceae
Ps. aeruginosa
Ampicillin X
Piperacillin/ Tazo.
Cefepime
Imipenem
Gentamycin
Tobramycin
Ciprofoxacin
Levofloxacin
Nitrofurantoin
Trimethoprim/Sulfa

10. Definitions 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 results in the death of the organism.

11. Definitions (cont’d) 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.

12. Methods of Performing AST
Agar dilution method
Broth macrodilution / Tube dilution
Broth microdilution
Disk diffusion method
Gradient diffusion method (E-Test)

13. Standardization of Antimicrobial Susceptibility Testing
Inoculum Preparation
Use 4-5 colonies NOT just 1 colony
Inoculum Standardization
using 0.5 McFarland standard

14. Methods of Performing AST
Agar Dilution
Dilutions of antimicrobial agent added to agar
Growth on agar indicates MIC
Broth macrodilution/Tube Dilution Tests
Two-fold serial dilution series, each with 1-2 mL of antimicrobial
Too expensive and time consuming
Microdilution Tests
plastic trays with dilutions of antimicrobials

15. 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 anitmicrobial agents on the agar
Invert and incubate for hours at35 oC in non-CO2

16. 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

17. 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

18. Automated Antimicrobial Susceptibility Test Methods
Detect growth in microvolumes of broth with various dilutions of antimicrobials
Detection via photometric, turbidimetric, or fluorometric methods
Types
BD Phoenix
Microscan Walkaway
TREK Sensititre
Vitek 1 and 2

19. Automated Antimicrobial Susceptibility Test Methods
Advantages
Increased reproducibility
Decreased labor costs
Rapid results
Software
Detects multi-drug resistances
ESBLs
Correlates bacterial ID with sensitivity
Disadvantages
Cost

20. Quality Control in Susceptibility Testing
Reflects types of patient isolates & range of susceptibility
Frequency of quality control depends on method, CLSI, or manufacturer
Reference strains of QC material
American Type Culture Collection(ATCC)
E. coli ATCC* 25922
S. aureus ATCC* 25923

21. The Superbugs Organisms resistant to previously effective drugs MRSA
methicillin-resistant Staphylococcus aureus
mecA gene codes for a PBP that does not bind beta-lactam antibiotics
Resistant to oxacillin
Vancomycin
VRE –Enterococcus species
VISA/VRSA- Staphylococcus aureus

22. The Superbugs: The Beta-Lactamases
Gram negative rods that have genes on chromosomes that code for enzymes against certain antimicrobials
ESBLs-extended spectrum beta lactamase
Resistant to extended spectrum cephalosporins, penicillins, aztreonam
Examples: E. coli, Klebsiella
Carbapenemases (CRE)
Klebsiella pneumoniae- KPC- Class A
Class B (NDM, VIM, IMP)- metallo beta lactamases
Resistant to penicillins, cephalosporins, carbapenems, and aztreonam
Cephalosporinases
AmpC enzyme
inducible
“SPACE” organisms

23. Controlling the Superbugs
Lab’s Role
Recognize and report isolates recovered from clinical specimens
Methods for identification include automated systems and screening agars

24. Controlling the Superbugs
Role of Health Care Workers/Facilities
Hand hygiene with the use of alcohol-based hand rubs or soap and water after patient care
Contact precautions for patients identified as colonized or infected with a superbug
Healthcare personnel education about the methods of transmission, contact precautions, and proper use of hand hygiene
Minimization of invasive devices (catheters, etc.)
Proper administration of antimicrobial agents where therapy is selected for susceptible organisms for the proper duration

25. References
Kiser, K. M., Payne, W. C., & Taff, T. A. (2011). Clinical Laboratory Microbiology: A Practical Approach . Upper Saddle River, NJ: Pearson Education.
Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.
Murray, P. R. (2013, May). Carbapenem-resistant Enterobacteriaceae: what has happened, and what is being done. MLO, 45(5),