Antimicrobial

susceptibility testing

Dr. Ni Made Adi Tarini, SpMK

Clinical Microbiology Department

Aims of Presentation
Answers to the following questions:-
(1) Why do we test antimicrobial
susceptibility?
(2) How do we perform
antimicrobial susceptibility tests?
(3) How can we detect resistance
mechanisms?
(4) Why & how do we assay
antimicrobial serum levels?
Antibiotics and the
Laboratory
If there was no antibiotic resistance, there
would be no need for a microbiology lab!

Infections could be treated syndromically

Why do we test antimicrobial
susceptibility?

To direct & predict antimicrobial

chemotherapy.
To review & monitor epidemiological trends.
To set national & local antibiotic policies.
To test the activity of a new antimicrobial

agent.
To presumptively identify isolates.
But remember
Other factors are very important when we
choose an antibiotic
Will it get to where the infection is?
Bioavailability
Cost
Toxicity
Likelihood of development of resistance
Etc
What Does the Laboratory Need to
Know
about Antimicrobial Susceptibility
Testing (AST) ?

Which organisms to test?

What methods to use?
What antibiotics to test?
How to report results?
What Does a Laboratory Need to Know
about AST? (cont)

How to determine the clinical

significance of results?
How to ensure accuracy of results?
Quality control / quality assurance
When to call the MD, infection
control, public health?
Brief Review of Routine
AST Methods

AST
Routine Susceptibility
Tests
Disk diffusion (Kirby Bauer)
Broth micro-dilution MIC
NCCLS reference method
Etest
Disc Susceptibility Tests
Agar surface evenly inoculated with the test
organism.
Antibiotic filter paper discs applied to the
plate.
Plates are incubated & antibiotics diffuse
into the agar.
Antibiotic concentration decreases at
increasing distance from disc.
Circular zone of growth appears.
Size of zone of inhibition indicates
susceptibility of organism.
Kirby-Bauer Method
Developed in the USA in 1966.
Based on NCCLS ( National Committee for

Clinical Laboratory Standards ) data.

Use Mueller-Hinton agar.
Use standard 0.5 McFarland inoculum.
Streak inoculum in 3 directions or rotary

plate.
Kirby-Bauer Method
Use standard discs & incubation conditions.
Use standard NCCLS tables to interpret zone

sizes as S, I or R.
Interpretation based on regression line

analysis of zone diameter size to MIC.

Interpretation based on confluent growth of

the organism.
Disk Diffusion
Test
 Measure Zones
Transmitted Reflected
Light Light
Disk Diffusion Test
Qualitative results
Susceptible
Intermediate may respond if
infection is at body site where
drug concentrates (e.g. urine) or if
higher than normal dose can be
safely given
Resistant
Minimum Inhibitory
Concentration (MIC)
The MIC is the lowest concentration of the
antimicrobial required to inhibit growth of
the organism.
It is used to determine the quantitative
activity of an antimicrobial.
It is used to confirm resistance or
equivocal results.
It is used in cases of prolonged treatment
or endocarditis to adjust the dose of
therapy.
It is used to determine the susceptibility of
slow-growing organisms e.g anaerobes
Tube MIC
Set up a series of antibiotic doubling
dilutions in tubes containing liquid media
For example 128mg/l, 64, 32, 16, 8, 4, 2, 1,

0.5, 0.25, 0.125, 0.

Set up tubes for test organism & control

organism.
Tube MIC
Add standard organism inoculum to each
tube.
Include an antibiotic free tube i.e organism
only.
Include an organism free tube i.e antibiotic
only.
Incubate tubes overnight.
Examine for presence of growth by shaking
each tube & observing turbidity.
Tube MIC
Check antibiotic free tube has growth.
Check organism free tube has no growth.
Check the organisme control gives the

recommended MIC.
The MIC is the first tube dilution without

visible growth.
The tube MIC is very labour intensive,

difficult to get right & prone to error.

E-tests (AB
Biodisk(Sweden))
A commercial alternative to tube MIC.
Consists of a plastic strip 6cm by 0.5cm in

size.
Exponential gradient of antimicrobial dried

on one side.
MIC scale printed on the other side.
The range corresponds to fifteen 2-fold

dilutions.
E-tests
Follow manufacturers instructions for
inoculum preparation, media
recommendations & incubation conditions.
MIC interpretation made where growth of

inhibition ellipses the strip.

Most E-test require examination with a

hand-lens to look for minute colonies

intersecting the strip.
Examples of E-tests
Automated Methods
Three main methods are in use in the U.K.
These include the Vitek (Biomerieux), the

Phoenix (Becton-Dickinson) & the

Mastascan Elite (Mast).
Host factors affecting
treatment
Diffusion in tissues
Serum protein binding
Drug interactions
Immune system
Multiple simultaneous infections
Virulence of organism
Site and severity of infection
Common interpretation
problems
Results depends on the technique used
Many factors influence results
Lack of standardization of the inoculums
Thickness and quality of the culture media
Quality and conservation of the disks
Quality control with standardized strains
Condition and duration of incubation
Common interpretation
problems
An agar gel that is too thick leads to smaller zones

Source: http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/
Common interpretation problems

Problem with the size of the

inoculums

Solution:
Use McFarland 0.5 photometer

Scale -> same tubes

Common interpretation problems

Contamination with
another organism
Common interpretation problems

Bad manipulation
Inoculation of the Muller
Hinton
Common interpretation
problems
Problems with E-test reading
Cost of anti-microbial
resistance
Cheap antimicrobials become ineffective
Individual treatment failure
Prolonged illness, hospitalization
Need to switch to more expensive, complex

drugs
that are often not even available in resource-
poor
settings
Need to develop new antimicrobials
Good antimicrobial susceptibility testing saves

lives and money

THANK YOU