Antimicrobial Susceptibility Testing (AST)

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 10-15 antibiotics to test susceptibility for GP organisms and another 10-15 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

9. Example of Drug Formulary

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, 18-24 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 16-18 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 • http://www.biomerieux-diagnostics.com/servlet/srt/bio/clinical-diagnostics/dynPage?doc=CNL_CLN_PRD_G_PRD_CLN_22 • http://www.cdc.gov/std/gonorrhea/lab/diskdiff.htm • http://www.who.int/drugresistance/Antimicrobial_Detection/en/index.html • 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), 26-30.