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Susceptibility
• The opposite of resistance
Antimicrobial Susceptibility • Susceptibility is a term used
when microbe such as bacteria and fungi are
Testing unable to grow in the presence of one or
more antimicrobial drugs
Dr Mazhar Ul Haq
• It is a misleading name; since most useful is
Dr Asif Riaz
detection of resistant microorganisms

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Antimicrobial Susceptibility Testing Continued ………

• A generic term for the laboratory • Susceptibility testing is performed on bacteria or
measurement fungi causing an individual's infection after they
have been recovered in a culture of the specimen
• Any of a large number of tests used to • Testing is used to determine the potential
determine if bacteria are susceptible or effectiveness of specific antibiotics on the
resistant to an antimicrobial agent bacteria and/or to determine if the bacteria have
• Achievable through different methods like developed resistance to certain antibiotics
Disk Diffusion Method • The results of this test can be used to help select
the drug(s) that will likely be most effective in
treating an infection
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Zone of Inhibition Breakpoints

• Area of no bacterial growth around a disk • Agreed threshold criteria used to interpret MICs
containing antibiotic or disk diffusion zone sizes to define whenever a
• Used to measure the antibiotic susceptibility or bacterial isolate is a susceptible or a resistant to a
resistance particular antibiotic and aid in clinical decisions
• The size of the inhibition zone is the function of likely the outcomes of the treatment
the • An antibiotic breakpoint is the dilution where
• Rate of drug diffusion, bacteria begin to start showing resistance
• Thickness of media, • Breakpoints in susceptibility tests are designed in
• Concentration of drug in disk and part to distinguish between the populations
• Susceptibility of the microorganism (susceptible/resistant)
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Minimum Inhibitory Concentration

Continued …….
(MIC)
• MIC or zone size value used to categorize an • Lowest concentration of antibiotic that inhibits
organism visible bacterial growth
• These values are generated by a susceptibility • A numerical measure (expressed as mg/L or
test and can be interpreted based on accepted ug/ml) of the activity of a specific antibiotic
breakpoints against a particular bacterial isolate
• They are based on pharmacologically & clinically • It is determined in the lab and used with
rich datasets using in vitro and in vivo data breakpoints to decide whether the bacterial
• They are considered robust predictors of likely isolate is susceptible or resistant to that antibiotic
clinical outcome • Considered as a gold standard
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Minimum Bactericidal Concentration

MIC
(MBC)
• For effective antimicrobial therapy, the • The qualitative assay determines the minimal
clinically obtainable antibiotic concentration concentration of antibiotic that kills the
in body fluids should be greater than MIC bacteria under investigation
• MIC does not always represent an absolute • MBC is the lowest concentration of the
value antibacterial agent that results in a 99.9%
• The ‘True’ MIC is a point between the lowest decline in colony count after overnight broth
test concentration that inhibits the growth dilution incubations
and the next lowest test concentration • The tubes that show no growth in MIC assay
• MIC concentration does not kill the bacteria are subcultured into antiobiotic free media

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Antimicrobial Susceptibility Testing

• Two general methods
1. Diffusion Method
2. Dilution Method
• Both are phenotypic methods
• Both are performed in vitro on pure culture of
isolated pathogens

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Agar Disk Diffusion Method Continued …….

• Also known as Kirby Bauer Method • Application of this test in veterinary medicine
• A diffusion based categorical method represents an approximation, as drug
• Most commonly performed in veterinary labs
disposition in some species may differ
• Often misused ?
• It gives qualitative or semi-quantitative information on • The test is useful because breakpoints are
susceptability correlated with MICs
• This test applies only to fast growing aerobic bacteria • It is particularly designed to distinguish
• Under standard conditions it gives a linear inverse between sensitive & resistant members of
relationship between the diameter of ZOIs and MICs
bacterial population

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Figure A disk diffusion test with an isolate of Escherichia coli from

a urine culture. The diameters of all zones of ...
Disadvantages
• It underestimates bacterial susceptibility where
local administration of the drug like udder,
uterus, skin, GIT
• Also problem where rapid excretion like urine
• As both lead to drug concentration that greatly
exceeds usual blood/tissue concentration
• It overestimates susceptibility for some
antibiotics in sites like brain, prostate, eye where
drug may enter poorly
Clin Infect Dis, Volume 49, Issue 11, 1 December 2009, Pages 1749–1755, https://doi.org/10.1086/647952
The content of this slide may be subject to copyright: please see the slide notes for details. 16

Test Method McFarland Standard

• Principle: • Also known as turbidity standard
• A 0.5 ml of 0.048M BaCl2 (1.17% w/v BaCl2.2H2O) is
• antibiotic-impregnated disk, placed on agar added to 99.5 ml of 0.18M H2SO4 (1% w/v) in a tube
inoculated with the test bacterium, pick-up with constant stirring
moisture and the antibiotic diffuse radially • It is distributed to a screw cap tube
outward through the agar medium producing • Tube is sealed tightly to prevent loss by evaporation
an antibiotic concentration gradient • This should be kept at room temperature and
protected from light
• A clear zone or ring is formed around an • A newly formed bacterial culture is compared against a
antibiotic disk after incubation if the agent white background with contrasting black lines
inhibits bacterial growth • Vigorously agitate on vortex mixture before use

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Procedure Measurement
• Test is conducted on a 4-mm thick Muller-Hinton • Following incubation,
Agar (MHA) in Petri plates zone sizes are measured
• Drug is impregnated on filter paper disks to the nearest mm
• Plates are inoculated with a lawn of bacteria from using a ruler or caliper
a 0.5 McFarland suspension (approx. 1-2 x 108
CFU/ml) using a sterile swab • Diameter of the disk is
included in the
• Plates are allowed to dry for up to 15 min before
the disk is applied measurement
• These are incubated at 35 C overnight • Zone sizes are recorded
• After 24 h ZOIs are measured on a separate sheet

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Interpretation & Reporting of Results

• Using the published reference guidelines
(CLSI), susceptibility or resistance of the
organism for each tested drug is determined
• There are different charts for different
organisms
• For each drug, based on zone size the
outcome is determined whether it is
susceptible, intermediate or resistance

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Terms Resistant
• Susceptible: • A category defined by a breakpoint that
• A category that implies that the isolates are inhibited by
usually acheiveable concentrations of antimicrobial agents implies that isolates with an MIC at or above
when dosages recommended to treat the site of infection is or a zone diameter at or below the resistant
used
• Intermediate: breakpoint are not inhibited by the usually
• A category defined by a breakpoint that includes isolates achieveable concentration of the agent with
with MICs or zone diameters within the intermediate range
that approach usually an attainable blood & tissue levels normal dosage schedules and/or that
• The intermediate category implies clinical efficacy in demonstrate MIC or zone diameter that fall in
anatomical sites where the drugs are physiologically
concentrated the range in which specific microbial
resistance mechanisms are likely
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Dilution Methods Agar Dilution Method

• Agar dilution method • The agar dilution method is a manual method not
• Broth dilution method frequently used in the researched literature
• This technique is standardized by the National
• Dilution methods are reference methods
• Committee for Clinical Laboratory Standards (NCCLS)
• Both methods may be used to quantitatively
• The agar dilution method is performed by
measure the antimicrobial activity against incorporation of different concentrations of the
fastidious or nonfastidious bacteria, yeasts and antimicrobial agent into a molten agar medium
filamentous fungi • Serial twofold dilutions are employed
• Dilution methods have been used for the • A standardized microbial inoculum to the surface of
comparative testing of new antimicrobial agents the agar plate is innoculated

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Methods Broth Dilution Method

• Bacteria are seeded on nutrient agar medium • One of the oldest method aka tube dilution
method / broth macrodilution method
• The medium is supplemented with different
• This makes use of tubes containing two-fold serial
concentrations of antimicrobial agents dilutions of antibiotic agents in liquid growth
• CFU are then counted after 48 h of incubation medium
• Agar dilution is often recommended as a • Subsequently, a known quantity of suspended
standardised AST method for fastidious bacteria is added to each solution
organisms • After 24 h of incubation, the bacterial growth is
measured by the turbidity within the tubes,
which gives an MIC value
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Advantages /Disadvantages Microdilution Method

• The primary advantage of this method is the ability to • The miniaturization and mechanization of the test by
obtain quantitative MIC values use of small, disposable, plastic “microdilution” trays
• Also gives the minimum bactericidal concentration has made broth dilution testing practical and popular
(MBC) • Standard trays contain 96 wells, each containing a
• It is a reliable and standardised method for diagnostic volume of 0.1 mL that allows approximately 12
purposes antibiotics to be tested in a range of 8 two-fold
• A major disadvantage is that each antibiotic solution dilutions in a single tray
has to be prepared by hand
• Inoculation of panels with the standard 5×105CFU/mL
• It is a time consuming process
is accomplished using a disposable device that
• A considerable amount of reagents is needed for each transfers 0.01 to 0.05 mL of standardized bacterial
and every dilution
suspension into each well of the microdilution tray

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Advantages /Disadvantages Antimicrobial Gradient Method

• The advantages of the microdilution procedure include • The antimicrobial gradient diffusion method
• Generation of MICs uses the principle of establishment of an
• Reproducibility and convenience of having prepared
panels
antimicrobial concentration gradient in an
• Economy of reagents and space agar medium as a means of determining
• An assistance in generating computerized reports if an susceptibility
automated panel reader is used • The MIC is determined by the intersection of
• The main disadvantage of the microdilution method is the lower part of the ellipse shaped growth
some inflexibility of drug selections available in
standard commercial panels inhibition area with the test strip

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Figure 2 A Staphylococcus aureus isolate tested by the Etest

Etest gradient diffusion method with vancomycin (VA), ...

• The Etest (bioMérieux AB BIODISK) is a commercial version

available in the United States
• It employs thin plastic test strips that are impregnated on
the underside with a dried antibiotic concentration
gradient and are marked on the upper surface with a
concentration scale
• As many as 5 or 6 strips may be placed in a radial fashion
on the surface of an appropriate 150-mm agar plate that
has been inoculated with a standardized organism
suspension like that used for a disk diffusion test
• After overnight incubation, the tests are read by viewing
the strips from the top of the plate

Clin Infect Dis, Volume 49, Issue 11, 1 December 2009, Pages 1749–1755, https://doi.org/10.1086/647952
33 The content of this slide may be subject to copyright: please see the slide notes for details.

Advantages /Disadvantages Antifungal Susceptibility Testing

• The gradient diffusion method has intrinsic flexibility • In vitro antifungal susceptibility tests differ
by being able to test the drugs the laboratory chooses
from the susceptibility tests performed against
• This method is best suited to situations in which an
MIC for only 1 or 2 drugs is needed or when a bacteria
fastidious organism requiring enriched medium or • A given fungal species may be in the form of
special incubation atmosphere is to be tested
yeast or of filamentous fungi
• Etest strips expensive if more than a few drugs are
tested • Susceptibility of fungi, as with bacteria, is not
• There are some systematic biases toward higher or always predictable
lower MICs determined by the Etest when testing
certain organism-antimicrobial agent combinations

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