Transcript Antimicrobial Susceptibility Testing (AST)

Chemical Agents that affect
microbial growth
Done by
Dania Qasrawi
Bacteriostatic and bactericidal antibiotics
• Antibiotics are categorized as bactericidal if they kill the
susceptible bacteria or bacteriostatic if they reversibly
inhibit the growth of bacteria.
• In general the use of bactericidal antibiotics is preferred
but many factors may dictate the use of a bacteriostatic
antibiotic.
• When a bacteriostatic antibiotic is used the duration of
therapy must be sufficient to allow cellular and humoral
defense mechanisms to eradicate the bacteria. If possible,
bactericidal antibiotics should be used to treat aggressive
infections.
ANTIBIOTIC SUSCEPTIBILITY TESTING(AST)
DEFINITION:
AST is the “study of the susceptibility of
bacteria to a specific antibiotic”
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.
Susceptibility testing is done using following
methods:
• Diffusion methods
Disc diffusion- Kirby-Bauer method
• Dilution methods
a) Agar dilution
b) Broth dilution
Kirby-Bauer Disk Diffusion method
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•
Preparation of inoculum
•
Standardize inoculum suspension
•
Inoculate Mueller Hinton Agar (MHA)
•
Dispense antibiotic disks
•
Incubate overnight at 37°C
Kirby-Bauer Disk Diffusion method
•Measure zone sizes
•Interpret the zone sizes with established chart:
- Sensitive (S)
- intermediate Sensitive (I) and
- Resistant (R)
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Kirby-Bauer Disk Diffusion: Factors affecting the
zone of inhibition
1. Thickness of medium: should be 4 mm
- Thicker medium – narrow zone
- Thin medium – larger zone
2. pH of medium: should be neutral pH
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Kirby-Bauer Disk Diffusion: Factors affecting the
zone of inhibition
3. Potency of antibiotic disks: Right concentration
- Zone size narrower if antibiotic
concentration not maintained
4. Inoculum density: based on McFarland standard
- Light inoculum: Large zone size
- Heavy inoculum: Small zone size
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Kirby-Bauer Disk Diffusion: Factors affecting the
zone of inhibition
5. Placing the disks: immediately
6. Spacing the disks: 2.5 cm
7. Incubation time: 16-18 hours
8. Incubation temperature: 35°C-37°C
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Standardization of Antimicrobial
Susceptibility Testing
• Inoculum Preparation
– Use 4-5 colonies
NOT just 1 colony
• Inoculum
Standardization
– using 0.5 McFarland
standard
0.5 McFarland standard
• McFarland standards are used as a reference to adjust
the turbidity of bacterial suspensions so that the number
of bacteria will be within a given range.
• Were mixing specified amounts of barium chloride and
sulfuric acid together.
• Mixing the two compounds forms a barium sulfate
precipitate, which causes turbidity in the solution.
• A 0.5 McFarland standard is prepared by mixing 0.1 mL
of 1% barium chloride (BaCl2), with 9.9 mL of 1% sulfuric
acid (H2SO4).Then mix 5ml from tube to 5ml normal
saline.
• Turbidity of 0.5 McFarland standard is approximately
equal 1.5X10^8 CFU/mL of bacterial suspension.
McFarland standards
Procedure:
1. Adjust the concentration of the inoculum by comparing the turbidity of Normal Saline
tube to that of a 0.5 McFarland standard.
Note: If the turbidity of the inoculum is greater than that of 0.5 McFarland
standard, dilute with sterile normal saline. If the turbidity of the standard is
greater than the inoculum, add more of the test organism.
2. Dip a sterile cotton swab into the adjusted inoculum tube and drain excess
fluid by pressing the swab against the walls of the test tube.
3. Hold Muller-Hinton plate half or partially open and streak the plate using the
wet cotton swab covering all the area even at the sides.
4. Place the plates aside for about 10 – 15 minutes at room temerature. Allow the
inoculum to dry.
Procedure: continue ……
5. Using a sterile forceps or needle, apply a set of suitable antibiotic disks. Five to six
disks for each plate, or 8 disks if you use the automatic dispenser.
6. incubate in inverted position at 37 oC for 18-24 hours.
Procedure: continue ……
7. Using a ruler or caliper, measure the zone of inhibition around each
antimicrobial disk and record it.
8. Consult the special chart provided by the manufacturer of the antimicrobial
disks and interpret results as Sensitive, Resistant, or Intermediate.
Mueller Hinton Agar Plate
Swabbing
Disc Placement
AST plate with zone of inhibition
Definitions
• 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.
 Several tests may be used to tell a physician which antimicrobial agent is most
likely to combat a specific pathogen:
1. Tube dilution tests:
A series of culture tubes are prepared, each containing a liquid medium and a •
different concentration of a chemotherapeutic agent. The tubes are then inoculated
with the test organism and incubated for 18-24 hours at 37 oC . After incubation, the
tubes are examined for turbidity (growth).
 Minimum Inhibitory Concentration (MIC):
Is the lowest concentration of chemotherapeutic agent capable of preventing
growth of the test organism.
 Minimum Bactericidal Concentration (MBC):
Is the lowest concentration of the chemotherapeutic agent that allows less than
0.1% of the inoculum to survive , also called “ Minimum Lethal Concentration
(MLC)”
Susceptibility Tests
1.
Broth dilution -
MIC test
MIC test
- Agar dilution
2.
Minimal Inhibitory Concentration (MIC)
vs.
Minimal Bactericidal Concentration (MBC)
32 ug/ml 16 ug/ml 8 ug/ml
Sub-culture to agar medium
4 ug/ml
2 ug/ml
1 ug/ml
MIC = 8 ug/ml
MBC = 16 ug/ml