U2.1.2 AST Flashcards by EJ E

Performedonbacteriaisolatedfromclinical specimens that are clinically significant

AST

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T/F The goal of AST is to provide STANDARDIZED in-vitro testing of a bacterial pathogen to a set of available antibiotics to determine its “antibiogram” in order to predict the in vivo effectiveness of a particular
antibiotic or antibiotic regimen

True

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T/F In AST, the normal flora and the implicated/probable bacteria causing the infection should be tested.

False, Implicated/probable bacteria causing the infection should only be tested !

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T/F All organisms are subject to AST

False

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Odd One Out

Organisms that are not subject to AST
a. S. pyogenes
b. H. influenzae
c. N. meningitidis
d. L. monocytogenes

b. H. influenzae
- Testing occasionally required

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Odd One Out

Organisms that are subject to AST
a. S. pyogenes
b. S. pneumoniae
c. Enterococci
d. P. aeruginosa

a. S. pyogenes
- not subject to AST

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Odd One Out

Organisms that are subject to AST
a. N. gonorrhoeae
b. H. influenzae
c. N. meningitidis
d. M. cattarhalis

c. N. meningitidis
- not subject to AST

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T/F

Standardization of AST is important to optimize bacterial growth conditions to ensure that the inhibition of growth can be attributed to the antimicrobial agent.

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T/F

Standardization of AST is important to optimize conditions for maintaining antimicrobial integrity and activity, attributing the failure to inhibit bacterial growth to organism-associated resistance.

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T/F

Standardization of AST is important to maintain reproducibility and consistency in the resistance profile of an organism, regardless of what laboratory performs the test.

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T/F

Methods used in AST directly measure the activitv of only one antimicrobial agents

F; one or more

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T/F

Methods of AST directly detect the absence of a specific resistance mechanism

F; directly detect the presence

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T/F

Methods of AST use special methods that measure complex antimicrobial-organism interactions

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Standardization of AST

Bacterial inoculum size

1.5x10^8 CFU/mL

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Standardization of AST

Colonies

should not be > 1 day old

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Standardization of AST

If colony is too thin, _____
If colony is too thick, ____

too thin = incubate
too thick = add broth, sterile NSS

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Standardization of AST

Growth medium : pH

7.2-7.4

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Standardization of AST

Growth medium : Cation concentration

Ca : 25 mg/L
Mg : 12.5 mg/L

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Standardization of AST

Incubation atmosphere

humidified, ambient air; no CO2

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Standardization of AST

Incubation temperature

35-37 C

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Standardization of AST

Incubation duration

16-18 hrs

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Standardization of AST

Antimicrobial concentrations

Short term : 2-8C
Long term : -70C

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Standardization of AST

Plate Diameter
100 mm = # of disks

10 disks

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Standardization of AST

Plate Diameter
150 mm = # of disks

12 disks

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3 Methods that directly measure the activity of one or more antimicrobial agents

1. Traditional AST Methods 2. Commercially Available Methods 3. Special Screens & Indicator Tests

Traditional AST Methods Preparation of the Standardized Inoculum : Picking of #-# similar looking colonies from a non-inhibitory medium

4-5 similar looking colonies

Traditional AST Methods Preparation of the Standardized Inoculum : Picked colonies are then transferred to a broth medium and allowed to grow to ____ phase (___ hrs)

Log phase; 3-5 hrs

Traditional AST Methods Preparation of the Standardized Inoculum : Alternative Method for Fastidious Organisms

1. Pick 4-5 colonies from a fresh (16-24hr) culture 2. Suspend in broth or NSS diluted to proper density

Traditional AST Methods Preparation of Standardized Inoculum : Tubirdity of broth is standardized by comparing it with ______

0.5 MacFarland Standard

Traditional AST Methods Preparation of MacFarland Standard

0.5mL 1.175% Barium Chloride 99.5 mL 1.5% Sulfuric Acid

Traditional AST Methods Inoculum Standardization : Standardized Inoculum must be used within ____ minutes

15 minutes

Traditional AST Methods Comparison is done ____. Methods done for precision are ___

comparison : visually methods : nephelometric or spectrophotometric

Traditional AST Methods Commercially available and widely used standard

0.5 McFarland Standard

Traditional AST Methods QC : McFarland Standard should be checked _____. At _____nm

checked montly; 625nm

Traditional AST Methods: Choice of Antimicrobials Selection of Test Batteries/ Panel depends on :

- Protocol of hospital - ID of organism - any known resistance patterns - method of AST - availability of antimicrobial agents

Traditional AST Methods Other name for disk diffusion Method

Kirby Bauer

Traditional AST Methods Commonly used in vitro tests in the laboratory

Disk Diffusion Method

Traditional AST Methods Agar used in Kirby Bauer

Mueller Hinton Agar

Traditional AST Methods Measured in Kirby Bauer

Diameters of zones of inhibitions

Traditional AST Methods Findings in Kirby Bauer is reported as

SIR Susceptible, Intermediate, Resistant

T/F Kirby Bauer Method : As the agent diffuses farther, the concentration increases until it reaches a point when the bacterial growth is not anymore inhibited.

F, concentration decreases

Traditional AST Methods Based on the inverse linear relationship between the zone of inhibition diameter and the logarithm of the minimum inhibitory concentration (MIC)

Kirby Bauer Method

Kirby Bauer Method Incubation

35C for 16-18 hours at ambient atmosphere

Kirby Bauer Method For Fastidious Organisms

5-10% CO2

Kirby Bauer Method Characteristic of reading plates

should be confluent, no individual colonies should be present

Kirby Bauer Method Factors ignored during reading (3)

1. Proteus : hazy/swarming growth within the zone 2. Sulfonamides & TMP : hazing 3. Beta-hemolytic bacteria : hemolysis produced

Traditional AST Methods Determine Minimum Inhibitory Concentration (MIC)

Dilution Susceptibility Testing

Traditional AST Methods - Use of serial 2-fold dilutions of the antimicrobial agent (expressed as ug or mcg/ml) - Once MIC is determined, ti is interpreted as S, I, R

Dilution Susceptibility Testing

Dilution Susceptibility Testing 2 Types of Broth Dilution

Macrodilution & Microdilution

Dilution Susceptibility Testing : Broth Dilution Use of test tubes, Volume: 1-2 ml

Macrodilution

Dilution Susceptibility Testing : Broth Dilution Use of wells, Volume: 0.05-0.1mL

Microdilution

Broth Dilution Report the MIC and is translated into the 3 general interpretative categories :

SIR Susceptible, Intermediate, Resistant

Broth Dilution NB: Recently added categories include

1. Susceptible Dose Dependent 2. Non susceptible

Broth Dilution specific concentrations that separate or define the different categories

Breakpoints

Dilution Susceptibility Testing Antimicrobial Concentrations and organisms to be tested are placed together on an agar medium

Spot Inoculation

Dilution Susceptibility Testing Series of dilutions, with one dilution per plate

Agar Dilution

Dilution Susceptibility Testing Standard Inoculum for Agar Dilution

1x10^4 CFU/spot

Dilution Susceptibility Testing lowest concentration of an antimicrobial agent ni agar that completely inhibits visible growth

MIC

Dilution Susceptibility Testing Advantage: Anaerobic OrganismsUseful for determining the MIC of N. gonorrhoeae

Agar Plate Dilution

Commercially Available Methods (2)

1. Diffusion in Agar Derivation 2. E-test

Commercially Available Methods also known as Gradient Diffusion Susceptibility Test

E-test

Commercially Available Methods Principle: Establishment of an antimicrobial density gradient

E-test

E-test Uses _____________ placed in a radial fashion on an inoculated plate.

thin plastic strips (with gradations)

E-test Interpretation : MIC is read wherein the ____________-

growth ellipse intersects the Etest strip

E-test used for ____ organisms

fastidious organisms