Antimicrobial Susceptibility Testing (AST) MLAB 2434 – Microbiology Keri Brophy-Martinez.
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Transcript of Antimicrobial Susceptibility Testing (AST) MLAB 2434 – Microbiology Keri Brophy-Martinez.
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/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.