ANTIBIOTIC SUSCEPTIBILITY TESTING (AST)

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ANTIBIOTIC SUSCEPTIBILITY TESTING (AST)

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ANTIBIOTIC SUSCEPTIBILITY TESTING (AST)

Performed on bacteria isolated from clinical specimens 

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To provide in-vitro testing of a bacterial pathogen to a set of a available antibiotics to determine its “antibiogram” in order to predict the in vivo effectiveness of a particular antibiotic orantibiotic regimen

primary goal of AST

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antibiogram

refers to the susceptibility profile

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TRUE

TRUE OR FALSE:  Implicated/probable bacteria causing the infection should only be tested

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  • Performed on a bacterial isolate that is the probable cause of infection

  • not performed on bacterial isolates that are predictably susceptible to a certain agent

  • Provides information on the decrease of the antimicrobial susceptibility

REASONS WHY AST IS PERFORMED

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  • Body Site

  • Presence of other Bacteria and Quality of Specimens

  • Host Status

FACTORS TO CONSIDER IF AST IS NEEDED/WARRANTED

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DO NOT perform AST on bacteria isolated from its natural habitat

TRUE OR FALSE: perform AST on bacteria isolated from its natural habitat

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E. coli

Normally a stool sample is a specimen wherein we can isolate ___ because it is a normal flora of the GI tract

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TRUE

TRUE OR FALSE: there is no need to check AST pattern when E coli is isolated from the stool sample

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Viridans cocci

___are normal flora of the upper respiratory tract. Therefore, there is no need perform AST

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>2 species at >105 CFU/mL

The presence of __species at __CFU/ml in urine is possibly due to contamination

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pure culture

When doing AST make sure that __ culture is used

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standardized

 if we want to associate the activity to the antimicrobial agent, the procedure for doing the AST has to be ___

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  • To optimize bacterial growth conditions to ensure that the inhibition of growth can be attributed to the antimicrobial agent.

  • To optimize conditions for maintaining antimicrobial integrity and activity, attributing the failure to inhibit bacterial growth to organism-associated resistance.

  • To maintain reproducibility and consistency in the resistance profile of an organism, regardless of what laboratory performs the test.

3 important purposes of standardization of AST

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host status

Normal Flora might be the cause of the infection in cases of immunocompromised patients

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  • activity

  • specific resistance mechanism

  • complex antimicrobial-organism

METHODS OF AST

  • Methods that directly measure the __ of one or more antimicrobial agents

  • Methods that directly detect the presence of a __

  • Special methods that measure ___ interactions

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  • Bacterial inoculum size

  • Growth medium

  • Incubation atmosphere

  • Incubation temperature

  • Incubation duration

  • Antimicrobial concentrations

standardized components of AST

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  • pH

  • Cation concentration

  • blood and serum supplements

  • Thymidine content

examples of growth medium

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Clinical Laboratory and Standards Institute (CLSI)

Standards are updated and published by __

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  • Traditional AST Methods

  • Commercially Available Methods

  • Special Screens and Indicator Tests

METHODS THAT DIRECTLY MEASURE THE ACTIVITY OF ONE OR MORE ANTIMICROBIAL AGENTS

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  • disk diffusion

  • quantitative dilution susceptibility testing - determination of MIC

What are the traditional AST Methods

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  • phenotypic methods

  • genotypic methods

METHODS THAT DIRECTLY DETECT THE PRESENCE OF A SPECIFIC RESISTANCE MECHANISM

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  • Test for the presence of Beta-Lactamase

  • Chloramphenicol Acetyltransferase Detection

  • Detection of Methicillin (Oxacillin) resistant S. aureus

  • Inducible Clindamycin Resistance Test

what are the phenotypic methods

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genotypic

genotypic or phenotypic methods: Molecular Methods to determine presence of resistant genes

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cephalosporinase test (cefinase test)

  • Use of filter paper disks impregnated with nitrocefin

  • Moisten with water or NSS and add a loopful of the colony onto the disk

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  • iodometric

  • acidtometric methods

Other methods for Beta-Lactamase

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red coloration; reaction occurs within 30 seconds

(+) result for Cefinase test; reaction occurs within __ seconds

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Chloramphenicol Acetyltransferase Detection

Determination of chloramphenicol resistance

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Detection of Methicillin (Oxacillin) resistant S. aureus

Use of chromogenic agar (ex: CHROM agar)

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MIC od >/= 4ug/mL

For Reliable detection of MRSA: MIC

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Inducible Clindamycin Resistance Test

D-zone test - for Staphylococcus

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Detection of mecA gene

gold standard for confirmation of phenotypic oxacillin resistance

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  • Detection of mecA gene

  • Detection of vanA gene

Molecular Methods to determine the presence of resistant genes

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mecA gene

both methods are for the detection of __ gene

  • Latex Agglutination Test

  • Molecular Methods

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complex antimicrobial-organism interactions

the ff. are methods that measure ___

  • antimicrobial concentration test

  • minimum bactericidal concentration (MBC)

  • serum bactericidal test (schlichter test)

  • synergy

  • time kill assay

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antimicrobial concentration test

measures amount of antimicrobial agent in serum of body fluid

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minimum bactericidal concentration (MBC)

measure lowest concentration of antimicrobial agent that kills a bacterial isolate

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minimum bactericidal concentration (MBC)

predictive of bactericidal activity level

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serum bactericidal test (schlichter test)

measure the highest dilution or titer of a patient’s serum that is inhibitory to the bacterium and the highest dilution or titer that is bactericidal

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serum bactericidal test (schlichter test)

predictive of response to current antibiotic regimen

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synergy

measures susceptibility to a combination of antimicrobial agents

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time kill assay

measure the rate of killing of bacteria by measuring the number of remaining viable bacteria

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time kill assay

In the ___, the test bacteria are inoculated to two tubes with varying concentration of antibiotic, incubated, then small aliquots are obtained at different time intervals.

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Aliquots

__ are diluted for easy counting to the plate and then you plot the count of the colonies present over the time of incubation.

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molecular detection

detection of resistance genes

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MONITOR EFFECTIVENESS

IS THIS A TEST FOR MONITORING/PREDICTING EFFECTIVENESS OF THERAPY

  • Antibiotic Synergy Test

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MONITOR EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

  • Molecular detection

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PREDICT EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

  • Disk diffusion

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PREDICT EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

  • MIC

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PREDICT EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

  • Etest

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PREDICT EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

  • Growth Curve Calculation

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MONITOR EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

  • Serum Bactericidal Titers

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MONITOR EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

  • MBC

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MONITOR EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

  • Disk Diffusion

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agar dilution method

reference method of anaerobes (susceptibility testing)

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  • Supplemented Brucella

  • laked sheep blood agar

recommended agar for anaerobes

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  • trough

  • peak

  • Antimicrobial Levels

    • Concentration of antibiotic in serum just before the antibiotic is given (__ level), and 15-20 mins after the dose is administered (__ level)

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peak level

therapeutic indicator

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trough level

toxicity indicator

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quality control

  • Use of standard reference strains that are susceptible to antimicrobial agents

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QUALITY CONTROL

  • __ strains should represent varying degrees of susceptibility or resistance

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daily or weekly

QC procedures must be done___ if there is acceptable performance with the OC strains 

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4 to 5

TRADITIONAL AST METHODS

  • Preparation of the Standardized Inoculum

    • Picking of __ similar-looking colonies from a non-inhibitory medium

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inoculum standardization

  • comparison is done visually

  • nephelometric or spectrophotometric methods can be done for more precision

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0.5 MCFARLAND STANDARD

  • Standard used to be able to determine if the broth is already same with that of the turbidity of the McFarland Standard

  • Commercially available and widely used standard

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0.5 MCFARLAND STANDARD

  • Corresponds to 1.5 x 108 CFU/ml

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monthly

McFarland Standard should be checked ___

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0.08 - 0.1

At 625nm, the absorbance should be ranging from __

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0.5 MCFARLAND STANDARD

  • Preparation for __:

    • 99.5ml 1% Sulfuric Acid +

    • 0.5 ml 1.175% Barium chloride

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wicker ham card

  • Made up of 2 by 3 plastic laminated card with thick black and white lines

  • To facilitate the preparation of bacterial and yeast suspension

  • Check the intensity of the lines in the background.

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0.5 MCFARLAND STANDARD

wicker ham card is used in what standard

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10-15

Selection of Test Batteries/Panel (usually __ antimicrobials)

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Kirby Bauer Method

__ Method is used for aerobic and non-fastidious organisms

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Criteria for Antimicrobial Battery Content and Use

  • Organism Identification or Group

  • Acquired Resistance Patterns Common to Local Microbial Flora

  • Antimicrobial Susceptibility Testing Method Used

  • Site of Infection

  • Availability of Antimicrobial Agents in the Formulary

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vancomycin vs gram-negative bacilli

Criteria for Antimicrobial Battery Content and Use: Organism Identification or Group

  • example of antimicrobials to which the organism is intrinsically resistant are routinely excluded from the test battery

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ceftazidime for use against Pseudomonas aeruginosa but not against Staphylococcus aureus

Criteria for Antimicrobial Battery Content and Use: Organism Identification or Group

  • antimicrobials developed specifically for use against particular organisms, but not against others

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more potent; more potent against

Criteria for Antimicrobial Battery Content and Use: Acquired Resistance Patterns Common to Local Microbial Flora

antimicrobials are then included in the test battery. Conversely, ___may not need to be in the test battery if susceptibility to less potent agents is highly prevalent

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resistance

Depending on the testing method, some agents do not reliably detect __ and should not be included in the battery.

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nitrofurantoin

Criteria for Antimicrobial Battery Content and Use: Site of Infection

Some antimicrobial agents, such as __, achieve effective levels only in the urinary tract and should not be included in batteries tested against bacterial isolated from other body sites

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Availability of Antimicrobial Agents in the Formulary

Under what criteria for Antimicrobial Battery Content and Use:

Antimicrobial test batteries are selected for their ability to detect bacterial resistance to agents used by the medical staff and accessible in the pharmacy.

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DISK DIFFUSION METHOD - KIRBY BAUER 

commonly used in vitro tests in the laboratory

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DISK DIFFUSION METHOD - KIRBY BAUER 

  • Advantages

    • Convenient and User Friendly

  • Disadvantages

    • Not all organisms have interpretative criteria

    • Unable to provide more precise data about the level of resistance

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  • Stoke’s Diffusion

  • Kirby Bauer Disk Diffusion method

inexpensive, easy to perform disk diffusion methods

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Mueller Hinton Agar (MHA)

Kirby Bauer method involves inoculating a standardized bacterial suspension on

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  • SUSCEPTIBLE

  • RESISTANT

  • INTERMEDIATE or MODERATELY SUSCEPTIBLE

disk diffusion method- Kirby Bauer method is reported as

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susceptible

  • antibiotic is effective choice for treatment

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Resistant

  • antibiotic is not appropriate for the identified organism

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Intermediate

  • less effective than susceptible

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DISK DIFFUSION METHOD - KIRBY BAUER 

these are the principles for which method

  • Gradient of antimicrobial agent is formed

  • as the agent diffuses farther, the concentration decreases until it reaches a point when the bacterial growth is not anymore inhibited

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directly

Diameter of inhibition is __ proportional to the susceptibility

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inverse linear relationship

  • zone of inhibition diameter and the logarithm of the minimum inhibitory concentration (MIC) has an __ relationship

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  • Bacterial suspension

  • Swab plate

  • Select Panel of antibiotics

  • Measure the Zone of Inhibition

procedure of Kirby Bauer

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  • simple streak

  • 60 degrees

  • repeat streaking 3 times

Kirby Bauer Method: Swab Plate

  • type of streak

  • degree of rotation

  • streak repetition for swabbing plate

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12

a 150 mm petri dish can accommodate __ antibiotic disks

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24 mm

 distance between each antibiotic

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vernier caliper

To measure the zone of inhibition use a ruler or a

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  • 6mm

KIRBY BAUER METHOD

  • Standard disk width: __

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  • 2-8C

  • -20c in a non frost freezer

KIRBY BAUER METHOD

  • Disk storage

    • working supply (1 week): __

    • stocks (long term): __

    • store in a tightly sealed container with desiccant

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Mueller Hinton

KIRBY BAUER METHOD

  • Culture Medium: __

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15 minutes

  • Inoculum and Inoculation

    after __ minutes, the disks are then placed

    • let it be absorbed by the MHA plate

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