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