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

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

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

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

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

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

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

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

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

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.

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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.