ANTIBIOTIC SUSCEPTIBILITY TESTING (AST)

ANTIBIOTIC SUSCEPTIBILITY TESTING (AST)

Performed on bacteria isolated from clinical specimens 

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

antibiogram

refers to the susceptibility profile

TRUE

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

• 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

• Body Site

• Presence of other Bacteria and Quality of Specimens

• Host Status

FACTORS TO CONSIDER IF AST IS NEEDED/WARRANTED

DO NOT perform AST on bacteria isolated from its natural habitat

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

E. coli

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

TRUE

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

Viridans cocci

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

>2 species at >105 CFU/mL

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

pure culture

When doing AST make sure that __ culture is used

standardized

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

• 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

host status

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

• 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

• Bacterial inoculum size

• Growth medium

• Incubation atmosphere

• Incubation temperature

• Incubation duration

• Antimicrobial concentrations

standardized components of AST

• pH

• Cation concentration

• blood and serum supplements

• Thymidine content

examples of growth medium

Clinical Laboratory and Standards Institute (CLSI)

Standards are updated and published by __

• Traditional AST Methods

• Commercially Available Methods

• Special Screens and Indicator Tests

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

• disk diffusion

• quantitative dilution susceptibility testing - determination of MIC

What are the traditional AST Methods

• phenotypic methods

• genotypic methods

METHODS THAT DIRECTLY DETECT THE PRESENCE OF A SPECIFIC RESISTANCE MECHANISM

• 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

genotypic

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

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

• iodometric

• acidtometric methods

Other methods for Beta-Lactamase

red coloration; reaction occurs within 30 seconds

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

Chloramphenicol Acetyltransferase Detection

Determination of chloramphenicol resistance

Detection of Methicillin (Oxacillin) resistant S. aureus

Use of chromogenic agar (ex: CHROM agar)

MIC od >/= 4ug/mL

For Reliable detection of MRSA: MIC

Inducible Clindamycin Resistance Test

D-zone test - for Staphylococcus

Detection of mecA gene

gold standard for confirmation of phenotypic oxacillin resistance

• Detection of mecA gene

• Detection of vanA gene

Molecular Methods to determine the presence of resistant genes

mecA gene

both methods are for the detection of __ gene

• Latex Agglutination Test

• Molecular Methods

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

antimicrobial concentration test

measures amount of antimicrobial agent in serum of body fluid

minimum bactericidal concentration (MBC)

measure lowest concentration of antimicrobial agent that kills a bacterial isolate

minimum bactericidal concentration (MBC)

predictive of bactericidal activity level

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

serum bactericidal test (schlichter test)

predictive of response to current antibiotic regimen

synergy

measures susceptibility to a combination of antimicrobial agents

time kill assay

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

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.

Aliquots

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

molecular detection

detection of resistance genes

MONITOR EFFECTIVENESS

IS THIS A TEST FOR MONITORING/PREDICTING EFFECTIVENESS OF THERAPY

• Antibiotic Synergy Test

MONITOR EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

• Molecular detection

PREDICT EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

• Disk diffusion

PREDICT EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

• MIC

PREDICT EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

• Etest

PREDICT EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

• Growth Curve Calculation

MONITOR EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

• Serum Bactericidal Titers

MONITOR EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

• MBC

MONITOR EFFECTIVENESS

IS THIS A TEST THAT MONITOR/PREDICT EFFECTIVENESS OF THERAPY

• Disk Diffusion

agar dilution method

reference method of anaerobes (susceptibility testing)

• Supplemented Brucella

• laked sheep blood agar

recommended agar for anaerobes

• 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)

peak level

therapeutic indicator

trough level

toxicity indicator

quality control

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

QUALITY CONTROL

• __ strains should represent varying degrees of susceptibility or resistance

daily or weekly

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

4 to 5

TRADITIONAL AST METHODS

• Preparation of the Standardized Inoculum

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

inoculum standardization

• comparison is done visually

• nephelometric or spectrophotometric methods can be done for more precision

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

0.5 MCFARLAND STANDARD

• Corresponds to 1.5 x 10⁸ CFU/ml

monthly

McFarland Standard should be checked ___

0.08 - 0.1

At 625nm, the absorbance should be ranging from __

0.5 MCFARLAND STANDARD

• Preparation for __:

  • 99.5ml 1% Sulfuric Acid +

  • 0.5 ml 1.175% Barium chloride

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.

0.5 MCFARLAND STANDARD

wicker ham card is used in what standard

10-15

Selection of Test Batteries/Panel (usually __ antimicrobials)

Kirby Bauer Method

__ Method is used for aerobic and non-fastidious organisms

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

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

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

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

resistance

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

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

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.

DISK DIFFUSION METHOD - KIRBY BAUER 

commonly used in vitro tests in the laboratory

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

• Stoke’s Diffusion

• Kirby Bauer Disk Diffusion method

inexpensive, easy to perform disk diffusion methods

Mueller Hinton Agar (MHA)

Kirby Bauer method involves inoculating a standardized bacterial suspension on

• SUSCEPTIBLE

• RESISTANT

• INTERMEDIATE or MODERATELY SUSCEPTIBLE

disk diffusion method- Kirby Bauer method is reported as

susceptible

• antibiotic is effective choice for treatment

Resistant

• antibiotic is not appropriate for the identified organism

Intermediate

• less effective than susceptible

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

directly

Diameter of inhibition is __ proportional to the susceptibility

inverse linear relationship

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

• Bacterial suspension

• Swab plate

• Select Panel of antibiotics

• Measure the Zone of Inhibition

procedure of Kirby Bauer

• simple streak

• 60 degrees

• repeat streaking 3 times

Kirby Bauer Method: Swab Plate

• type of streak

• degree of rotation

• streak repetition for swabbing plate

12

a 150 mm petri dish can accommodate __ antibiotic disks

24 mm

 distance between each antibiotic

vernier caliper

To measure the zone of inhibition use a ruler or a

• 6mm

KIRBY BAUER METHOD

• Standard disk width: __

• 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

Mueller Hinton

KIRBY BAUER METHOD

• Culture Medium: __

15 minutes

• Inoculum and Inoculation

  after __ minutes, the disks are then placed

  • let it be absorbed by the MHA plate