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 FormularyDrug Enterococcus Staphylococcus spp.

Ampicillin X

Cefazolin X

Clindamycin

Erythromycin X

Linezolid X X

Oxacillin X

Penicillin G X X

Rifampin X

Streptomycin-2000 X

Tetracycline X X

Trimeth/ Sulfa X

Vancomycin X X

Example of Drug FormularyDrug Enterobacteriaceae Ps. aeruginosa

Ampicillin X

Piperacillin/ Tazo. X X

Cefepime X X

Imipenem X X

Gentamycin X X

Tobramycin X X

Ciprofoxacin X X

Levofloxacin X X

Nitrofurantoin X

Trimethoprim/Sulfa X

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/clinic

al-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.