Ant ib iot ic

Sen si tiv it y
Tes tin g

Dr.T.V.Rao MD
Modern era in Antibiotics begins
with Fleming.
History of Antibiotic Discovery
 Uses of Antibiotic Sensitivity
Testing
 Antibiotic sensitivity test: A laboratory
test which determines how effective
antibiotic therapy is against a bacterial
infections.
 Antibiotic sensitivity testing will control the
use of Antibiotics in clinical practice
 Testing will assist the clinicians in the
choice of drugs for the treatment of
infections.

 Dr.T.V.Rao MD
 Components of Antibiotic
Sensitivity Testing
 1.The identification of relevant pathogens
in exudates and body fluids collected from
patients
 2. Sensitivity tests done to determine the
degree of sensitivity or resistance of
pathogens isolated from patient to an
appropriate range of antimicrobial drugs
 3. Assay of the concentration of an
administered drug in the blood or body
fluid of patient required to control the
schedule of dosage.
Dr.T.V.Rao MD
Antibiotic Sensitivity Testing Is
Essential of selection of
Antibiotics
 Isolation and Identification of
Bacteria precedes the selection of
Antibiotic Testing Methods
 Uses of Antibiotic Sensitivity
Testing
 Helps to guide the Physician in choosing
Antibiotics
 The accumulated results on different
pathogens their sensitivity will guide the
physician in choosing empirical treatment
in serious patients before the individual’s
laboratory results are analyzed in the
Microbiology laboratory.
 Reveals the changing trends in the local
isolates.
 Helps the local pattern of antibiotic
 Why Need continues for testing
for Antibiotic Sensitivity
 Bacteria have the
ability to develop
resistance following
repeated or subclinical
(insufficient) doses, so
more advanced
antibiotics and
synthetic
antimicrobials are
continually required to
overcome them.

Dr.T.V.Rao MD
Testing for Antibiotic sensitivity
 The method includes several steps
including obtaining a bacterial
sample; identifying the type of
bacteria in the bacterial sample;
selecting a set of antibiotics based on
the identity of the bacteria in the
bacterial sample; obtaining a control
sample from the bacterial sample;
Dr.T.V.Rao MD
 Testing Antibiotic
Susceptibility
 Antibiotic sensitivity test: A
laboratory test which determines
how effective antibiotic therapy is
against a bacterial infections.
 Antibiotic sensitivity test: the in
vitro testing of bacterial cultures with
antibiotics to determine susceptibility
of bacteria to antibiotic therapy.
Bauer-Kirby test.
Dr.T.V.Rao MD
 How results to be Reported
after Sensitivity Testing
 One should follow guidelines in reporting the
results and make matters simple with clear words
as

Organism A isolated and sensitive ( or

resistant ) to Antibiotic B

Such a report is relevant to present clinical

condition “ that the minimum inhibitory
concentration( MIC ) of the antibiotic for it
has been measured in some way and that, if
the organism is reported as sensitive the
MIC is less than a half or quarter of the
concentration of antibiotics likely to be
found in the infected tissues of a patient
given the usual schedule of doses i.e. that
 What is Resistance in Antibiotic
Sensitivity Testing

 Resistance implies that the infection is not

treatable with the tested Antibiotic
because its MIC exceeds achievable safe
tissue or urine levels.
 Intermediate sensitivity means they show
a unimodal distribution, but they can
often be reclassified as sensitive or
resistant if retested.
 Dr.T.V.Rao MD
 Kirby-Bauer methods
A commonly used method in basic
laboratories
 Kirby-Bauer antibiotic testing
(KB testing or disk diffusion
antibiotic sensitivity testing) is a
test which uses antibiotic-
impregnated wafers to test whether
particular bacteria are susceptible to
specific antibiotics

 Dr.T.V.Rao MD
 How to perform Kirby- Bauer
testing
 The basics are easy: The bacterium is
swabbed on the agar and the antibiotic
discs are placed on top. The antibiotic
diffuses from the disc into the agar in
decreasing amounts the further it is away
from the disc. If the organism is killed or
inhibited by the concentration of the
antibiotic, there will be NO growth in the
immediate area around the disc: This is
called the zone of inhibition .
 Dr.T.V.Rao MD
Steps in Antibiotic sensitivity
testing
 Streaking the Inoculum
 Kirby-Bauer (also
Bauer-Kirby) disk
diffusion antibiotic
susceptibility testing
applies a defined
inoculum (compared
to
McFarland 0.5 OD standard
streaked as a lawn
onto a large Mueller-
Hinton agar or Blood
agar plate (in 3
directions to ensure
confluence).

Dr.T.V.Rao MD
 Making proper inoculum
 Swab a Mueller-Hinton
plate with each of the
bacteria. Dip a sterile
swab into the broth
and express any
excess moisture by
pressing the swab
against the side of the
tube.

Dr.T.V.Rao MD
Bacteria are inoculated as
lawn culture
 Method of
inoculation- Good
results are
obtained by placing
a standard loopful
of inoculum
suspension on the
plate and then
spreading it with a
dry sterile swab.
 Disk Diffusion Method
 After completely swabbing the plate,
turn it 90 degrees and repeat the
swabbing process. (It is not
necessary to re-moisten the swab.)
Run the swab around the
circumference of the plate before
discarding it in the discard bag.

 Dr.T.V.Rao MD
 Placing the Antibiotic disks
 Then, using a
dispenser such as
the one pictured,
antibiotic-
impregnated disks
are placed onto the
agar surface. As the
bacteria on the lawn
grow, they are
inhibited to varying
degrees by the
antibiotic diffusing
from the disk
 Dr.T.V.Rao MD
 Zone sizes differ on sensitivity
pattern
 It has been
determined that
zones of inhibition
of a certain
diameter (varies
for antibiotic and to
a lesser extent,
bacterial species)
correlate with
sensitivity or
resistance to the
antibiotic tested
 Look at the Charts for
establishing the zones of
Sensitivity
 The zone sizes are looked up on a
standardized chart to give a result of
sensitivie, resistant, or intermediate.
Many charts have a corresponding
column that also gives the MIC
(minimal inhibitory concentration) for
that drug.
 Interpretation
 Place the metric ruler across the zone of
inhibition, at the widest diameter, and
measure from one edge of the zone to the
other edge. HOLDING THE PLATE UP TO
THE LIGHT MIGHT HELP.
 The disc diameter will actually be part of
that number. If there is NO zone at all,
report it as 0---even though the disc itself
is around 7 mm.
 Zone diameter is reported in millimetres,
looked up on the chart, and result
reported as S (sensitive), R (resistant), or
I (intermediate).
 The area of Inhibition is
measured with a Scale
 Record the
results for
everyone on
your table in
the table
below.
 The disk diffusion methods are
commonly used for routine testing
Read the plates in
traanmitted light
 The zone of inhibition
guides the right choice of
Antibiotic
 How results to be Reported
after Sensitivity Testing
 One should follow guidelines in reporting the
results and make matters simple with clear words
as

Organism A isolated and sensitive ( or

resistant ) to Antibiotic B

Such a report is relevant to present clinical

condition “ that the minimum inhibitory
concentration( MIC ) of the antibiotic for it
has been measured in some way and that, if
the organism is reported as sensitive the
MIC is less than a half or quarter of the
concentration of antibiotics likely to be
found in the infected tissues of a patient
given the usual schedule of doses i.e. that
 Stokes’ Method
 In original Stokes’
method the inoculum
of the control strain is
evenly spread over
the upper and lower
thirds of a plate and
that of the test strain
over the central
third;uninoculated
gaps 2 – 3 mm wide
are left to test from
the control areas.
 Dr.T.V.Rao MD
 Control strains
in Stokes’ Method
 The Microbiology laboratories should
always practice to test the quality
control of their work with the use of
standard strains in Stokes method
with following standard strains and
results are compared
1 Escherichia coli NCTC 10418
2 Pseudomonas aeruginosa NCTC
10662
3 Staphylococcus aureus NCTC
 Stokes Method for Antibiotic
Sensitivity testing
 In the Stokes controlled
sensitivity test, a control
organism is inoculated on
part of a plate and the test
organism is plated on the
remainder. Disks are
placed at the interface and
the zones of inhibition are
compared. The use of a
sensitive control shows
that the antibiotic is active,
so that if the test organism
grows up to the disk it
may safely be assumed
that the test organism is
resistant to that drug.
 Dr.T.V.Rao MD
 The strips with multiple
Antibiotics can be tested in one
go
 Other methods of Antibiotic
susceptibility testing
 Other methods to test antimicrobial
susceptibility include the Stokes
method, E-test (also based on
antibiotic diffusion). Agar and Broth
dilution methods for
Minimum Inhibitory Concentration
determination.
 Testing Minimum Inhibitory
Concentration
 In alternative measure of susceptibility is
to determine the Minimum Inhibitory
Concentration (MIC) and the Minimum
Bactericidal Concentration (MBC) of a
drug. A series of broths are mixed with
serially diluted antibiotic solutions and a
standard inoculum is applied. After
incubation, the MIC is the first broth in
which growth of the organism has been

 Dr.T.V.Rao MD
 What is Minimum Inhibitory
concentration
 Minimum inhibitory
concentration (MIC), in
microbiology, is the lowest
concentration of an antimicrobial that
will inhibit the visible growth of a
micro organism after overnight
incubation. Minimum inhibitory
concentrations are important in
diagnostic laboratories to confirm
resistance of micro organisms to an
antimicrobial agent and also to
monitor the activity of new
The Antibiotics are diluted to various
dilution to test the minimum
inhibitory concentration
MIC test results graphed
 What is E Test
 Etest is an antimicrobial gradient
technique in which 15 reference MIC
dilutions of an antibiotic have been
repackaged with innovative dry chemistry
technology onto a plastic strip. The
predefined gradient provides precise and
accurate assessment of antimicrobial
activity against both fastidious and non-
fastidious microorganisms.

 Dr.T.V.Rao MD
The strips are impregnated
with various concentration
of Antibiotics
E = testi ng on v ar ious
isolates
 Multiple drug resistant
organisms

Multiple drug resistant organisms are

resistant to treatment with several, often
unrelated, antimicrobial agents as
described above in Shigella. Some of the
most important types of multiple drug
resistant organisms that have been
encountered.

Dr.T.V.Rao MD
 Examples of Bacteria of Clinical
Importance
 MRSA - methicillin/oxacillin-resistant
Staphylococcus aureus
VRE - vancomycin-resistant
enterococci
ESBLs - extended-spectrum beta-
lactamases (which are resistant to
cephalosporins and monobactams)
PRSP - penicillin-resistant
Streptococcus pneumoniae
 Dr.T.V.Rao MD
Methicillin resistance in
Staphylococcu aureus
Detection of MRSA made simple
with Disk Diffusion Methods
 The MRSA among the
Staphylococcus aureus needs
detection in special condtions either
by carrying out the test on medium
containing 5 % Nacl with Methicillin
disk with 5 or 10 micrograms
 Recently the detection was made
simple with testing on Oxacillin or
Cefotixin disks
 Newer Methods in MRSA
detection
 Recently developed chromogenic
media combine primary growth and
selectivity with differentiation from
coagulase negative staphylococci.
These media show improved
specificity when compared with
traditional media. Sensitivity is also
improved but requires 48hrs
incubation to achieve >85%.
Chromagar for detection of MRSA
 BBL™ CHROMagar™
MRSA is a selective and
differential medium for the
qualitative direct detection
of nasal colonization by
Methicillin resistant
Staphylococcus aureus
(MRSA) to aid in the
prevention and control of
MRSA infections in
healthcare settings. The
test is performed on
anterior nares swab
specimens from patients
and healthcare workers to
screen for MRSA
colonization. BBL
CHROMagar MRSA is not
intended to diagnose MRSA
infection nor to guide or
monitor treatment for
Detection of ESBL strains in
Enterobacteriaceae
 Methods to detect ESBL
producers
 Various
combination of
Antibiotic disks
are used for
the detection
of ESBL
producers in
Gram negative
bacilli.
 Antibiotic disks helps in
detection of ESBL producers
 Various Antibiotic
disks kept at
specified place
helps in detection
of ESBL producers
 Limitation of Disk Diffusion
Methods
 Disk diffusion methods
are not suitable for
slow grwoing bacterial
pathogens
 The great limitation
being for testing on
Mycobacterium
tuberculosis which
needs varied,
technically demanding
methods
 Antibiotic Assays
 The amount of antibiotic in the Blood or other
body fluids of patients measured for following
reasons
1 To ensure adequate theraputic concentrations
are reached – in serious infections with parental
administration
2 To avoid toxic concentration
3 To study pharmacokinetics of Antibiotic
4 To identify the clinical response with identified
concentration
At present Automated methods are
popularly used as the testing protocols are
technically demanding
Dr.T.V.Rao MD
Antibiotic Sensitivity testing can
be done with automation
 There is a growing need for
Automation in Antibiotic sensitivity
testing
 Limitation of Antibiotic
Sensitivity usage

Both Microbiologists and Clinician

should however bear in mind that
the response therapy in vivo may not
always reflect the results of testing
the sensitivity of patient's pathogen
in vitro.
 Various Methods of Antibiotic
sensitivity testing are used for
quality control studies
 Created for awareness
to Medical and
Paramedical staff in the
Developing world
Dr.T.V.Rao MD ‘s Basic learning
series
Email
doctortvrao@gmail.com