Antibiotic Sensitivity Tests

K Hari Krishnan
II MBBS (2009-’11)
Tirunelveli Medical College
Tirunelveli, Tamilnadu, India

A test done to check the
effectiveness of a drug against a
bacterium and to select the best
drug that acts against the
bacterium.
K Hari Krishnan
Tirunelveli Medical
College

The in vitro testing of bacterial cultures
with antibiotics to determine
.
K Hari Krishnan
Tirunelveli Medical
College

Antibiotic
Sensitivity
Testing
K Hari Krishnan
Tirunelveli Medical
College

To guide the clinician in selecting the best
antibiotic agent for an individual patient.
To control the use of
in clinical practice.
To accumulate epidemiological information on
the resistance of microorganisms of public
health importance within the community.
To reveal the changing trends in the
local isolates. K Hari Krishnan
Tirunelveli Medical
College

Bacteria have the ability to develop
resistance following repeated or subclinical
(insufficient) doses, so more advanced
antibiotics and synthetic antibiotics are
continually required to overcome them.
K Hari Krishnan
Tirunelveli Medical
College

AST is essential for the selection of the
K Hari Krishnan
Tirunelveli Medical
College

– For the testing of isolates from “healthy” patients with
intact immune defenses.
– For such as uncomplicated
urinary tract infections.
– In the treatment of serious infections such as
endocarditis or osteomyelitis.
– For infections in high-risk patient groups such as
immunocompromised patients (e.g.. transplant
patients).
– Those who are critically ill. K Hari Krishnan
Tirunelveli Medical
College

Antibiotic Sensitivity Tests
Diffusion
Kirby-Bauer
Method
Stokes
Method
Dilution
Tube
Dilution
Agar
Dilution
Diffusion &
Dilution
E-Test
Qualitative Methods Quantitative Methods
K Hari Krishnan
Tirunelveli Medical
College

Staphylococcus
Enterobacteriaceae
Pseudomonas
Blood &
Tissues
Intestinal Urinary aeruginosa
Drugs
Benzylpenicillin
Oxacillin
Erythromycin
Tetracycline
Chloramphenicol
Ampicillin
Chloramphenicol
Cotrimoxazole
Tetracycline
Sulfonamides
Trimethoprim
Cotrimoxazole
Ampicillin
Nitrofurantoin
Tetracycline
Ampicillin
Chloramphenicol
Cotrimoxazole
Tetracycline
Cefalotin
Gentamycin
Piperacillin
Gentamycin
Tobramycin
K Hari Krishnan
Tirunelveli Medical
College

K Hari Krishnan
Tirunelveli Medical
College

– A paper disk with a defined amount of antibiotic is
used to generate a dynamically changing gradient
of antibiotic concentrations in the agar in the
vicinity of the disk.
K Hari Krishnan
Tirunelveli Medical
College

The contained in a reservoir is
allowed to and
interact in a plate freshly seeded with the test
organisms.
The disk is applied to the surface of an agar
plate inoculated with the test organism.
– The diffuses out of the disk to form the
gradient.
– The starts to divide and grow and
progresses toward a critical mass of cells.
K Hari Krishnan
Tirunelveli Medical
College

is formed at the critical
time where a particular concentration of the
antibiotic is just able to inhibit the organism
before it reaches an overwhelming cell mass
or critical mass.
K Hari Krishnan
Tirunelveli Medical
College

Medium containing beef
infusion, peptone, and
starch.
Used primarily for the disk-diffusion
method.
Robust red algae (Solieria robusta)
Source of Agar K Hari Krishnan
Tirunelveli Medical
College

Mueller-Hinton agar is considered the for
routine susceptibility testing of nonfastidious bacteria.
It shows acceptable batch-to-batch reproducibility
for susceptibility testing.
It is low in sulfonamides, trimethoprim, and
tetracycline inhibitors.
It gives satisfactory growth of most nonfastidious
pathogens.
A large body of data and experience has been
collected concerning susceptibility tests performed
with this medium.
K Hari Krishnan
Tirunelveli Medical
College

to 45–50 ⁰C and pour into
the plates. Allow to , to a
depth of approximately 4 mm.
– A 9-cm plate requires approximately 25 ml of
medium.
When the agar has solidified,
for 10–30 minutes at 35 ⁰C by placing them in
the upright position in the incubator with the
lids tilted.
– If it is not to be used immediately, the agar
medium can be stored in a refrigerator (2 to 8C)
for 2 weeks. K Hari Krishnan
Tirunelveli Medical
College

Any commercially available discs with the
proper diameter and potency can be used.
Stocks of antibiotic discs can be stored at
-20 ⁰C for 1 month.
– On removal from the refrigerator, the containers
should be left at room temperature for about 1
hour to allow the temperature to equilibrate.
K Hari Krishnan
Tirunelveli Medical
College

Prepared by pouring 0.6 ml of
a 1% (10 g/l) solution of
into a 100-ml graduated
cylinder, and filling to 100ml
with 1% (10 ml/l)
.
K Hari Krishnan
Tirunelveli Medical
College

A supply of cotton wool swabs on wooden
applicator sticks should be prepared.
They can be sterilized in tins, culture tubes, or
on paper, either in the autoclave or by dry
heat.
K Hari Krishnan
Tirunelveli Medical
College

Application of Antibiotic Discs
Incubation
At 35⁰C for 16-18 hours
Measurement of inhibition zone diameter
K Hari Krishnan
Tirunelveli Medical
College

To prepare the inoculum from the primary
culture plate,
, of similar appearance,
of the organism to be tested.
K Hari Krishnan
Tirunelveli Medical
College

Transfer this growth to a tube of saline.
K Hari Krishnan
Tirunelveli Medical
College

Compare the
tube with the
turbidity standard
and
of the test
suspension to that
of the standard by
adding more
bacteria or more
sterile saline.
K Hari Krishnan
Tirunelveli Medical
College

Inoculate the plates by
into the inoculum.
Remove excess inoculum by pressing and rotating
the swab firmly against the side of the tube above
the level of the liquid.
K Hari Krishnan
Tirunelveli Medical
College

the swab all over the surface of the
medium three times, rotating the plate
through an angle of 60⁰ after each application.
Finally, pass the swab round the edge of the
agar surface.
K Hari Krishnan
Tirunelveli Medical
College

Leave the inoculum to for a few
minutes at room temperature with the lid
closed.
K Hari Krishnan
Tirunelveli Medical
College

The may be placed on the
inoculated plates using
– sterile forceps.
– a template.
– a sterile needle tip.
– antibiotic disc dispenser.
K Hari Krishnan
Tirunelveli Medical
College

A maximum of can be placed
on a 9–10 cm plate.
– Six discs may be spaced evenly, approximately
15 mm from the edge of the plate, and 1 disc
placed in the centre of the plate.
The plates should be
of
preparation.
Temperatures
results for oxacillin/methicillin.
 an atmosphere of
.
Disks should after diffusion. K Hari Krishnan
Tirunelveli Medical
College

Using a ruler
– on the under-surface
of the plate containing
transparent medium.
Using a pair of
calipers
– on the plate
containing opaque
medium.
K Hari Krishnan
Tirunelveli Medical
College

Using automated zone readers
– BIOMIC
– Aura
– Protozone
K Hari Krishnan
Tirunelveli Medical
College

Standard templates are available for each
antibiotic.
K Hari Krishnan
Tirunelveli Medical
College

Result interpretation
• When the edge of the zone of inhibition is the
black circle.
• When there is no zone, or when it lies the
white circle.
• When the edge of the zone of inhibition lies the
black circle.
K Hari Krishnan
Tirunelveli Medical
College

The diameter of the
zone of inhibition is
measured using a
ruler or a pair of
calipers.
– This diameter is
interpreted according
to the critical
diameters.
K Hari Krishnan
Tirunelveli Medical
College

Interpretative chart of zone sizes
Antibiotic
Diameter of zone inhibition (mm)
Resistant Intermediate Susceptible
Tetracycline <14 15-18 >19
Chloramphenicol <12 13-17 >18
Cotrimoxazole <10 11-15 ≥16
Nitrofurantoin <14 15-16 >17
Erythromycin <13 14-22 >23
Gentamycin <12 13-14 >15
K Hari Krishnan
Tirunelveli Medical
College

– An organism is called susceptible to an antibiotic when the
infection caused by it is likely to respond to treatment
with this antibiotic, at the recommended dosage.
– An organism is called resistant if it is expected not to
respond to a given antibiotic, irrespective of the
dosage and of the location of the infection.
– Strains that are “moderately susceptible” to an antibiotic
that can be used for treatment at a higher dosage
(e.g. b-lactams) because of its low toxicity.
– Strains that show “intermediate susceptibility” to a more
toxic antibiotic (e.g. aminoglycoside) that cannot be used
at a higher dosage. K Hari Krishnan
Tirunelveli Medical
College

– Too inoculum
• Inhibition zones will be larger even though the
sensitivity of the organism is unchanged
Relatively resistant strains may be falsely reported as
susceptible.
– Too inoculum
• Inhibition zones will be smaller
Relatively susceptible strains may then be falsely
reported as resistant.
K Hari Krishnan
Tirunelveli Medical
College

• of disk application
K Hari Krishnan
Tirunelveli Medical
College

of incubation
– If the temperature is , the time
required for effective growth is extended and
result.
Potency of
– If the owing to
deterioration during storage, the
.
K Hari Krishnan
Tirunelveli Medical
College

Standardised inoculum is replaced by the
pathological specimen itself, e.g. urine, a
positive blood culture, or a swab of pus.
Advantage
– Results are obtained 24 hours earlier.
Disadvantage
– Density of the inoculum cannot be properly
controlled.
• The results of the primary test should be verified by
testing the isolates subsequently. K Hari Krishnan
Tirunelveli Medical
College

Used to determine the
of antibiotic to inhibit or kill the
microorganism.
Achieved by dilution of antibiotic in either
agar or broth media.
K Hari Krishnan
Tirunelveli Medical
College

The lowest concentration of drug that
of the bacteria
isolated from the patient.
The MIC is determined by inoculating the
organism isolated from the patient into a
series of tubes or cups containing progressive
dilutions of the drug.
K Hari Krishnan
Tirunelveli Medical
College

Patient's organism is added to tubes
containing decreasing amounts of the
antibiotic
Incubation
At 37°C overnight
Lowest concentration of drug that
inhibits growth is the MIC
K Hari Krishnan
Tirunelveli Medical
College

MIC
K Hari Krishnan
Tirunelveli Medical
College

The lowest concentration of drug that
the bacteria isolated from the patient.
K Hari Krishnan
Tirunelveli Medical
College

Serial dilutions of the drug are prepared in
agar and poured into plates.
– Many strains can be inoculated on each plate
containing an antibiotic dilution.
K Hari Krishnan
Tirunelveli Medical
College

 Broth microdilution plate contains
– Each row:
• standard dilutions of eight in each row (denoted
by letters A-H).
– Each column
• contains a standard concentration that doubles when moving
from right to left.
 The minimum inhibitory concentration (MIC) is determined by
the first well where there is no visible growth.
K Hari Krishnan
Tirunelveli Medical
College

Epsilometer Test
Quantitative method of antibiotic sensitivity
testing.
Applies both dilution of antibiotic and
diffusion of antibiotic into the medium.
K Hari Krishnan
Tirunelveli Medical
College

Combines the principles of disk diffusion and
agar dilution methods
Diffusion
Dilution
E-Test
K Hari Krishnan
Tirunelveli Medical
College

A predefined stable antibiotic gradient is
present on a thin inert carrier strip.
Using innovative dry chemistry technology,
E-Test is used to determine the on-scale
Minimum Inhibitory Concentration (MIC).
K Hari Krishnan
Tirunelveli Medical
College

The intersection of the inhibitory zone edge and the
calibrated carrier strip indicates the MIC with inherent
precision and accuracy. K Hari Krishnan
Tirunelveli Medical
College

Over 100 are now available in the
product range for testing of aerobic bacteria
and fastidious organisms such as
– Pneumococci
– Haemophilus
– Helicobacter pylori
– Meningococci
– Gonococci
– Fungi
– Mycobacteria
K Hari Krishnan
Tirunelveli Medical
College

Determining the MIC of
, or for
a specific type of patient or infection.
Detecting
– Glycopeptide-resistant Enterococci (GRE)
– Glycopeptide-intermediate S. aureus (GISA)
– Resistant Mycobacterium tuberculosis
– Extended spectrum beta lactamases (ESBL)
Detecting .
Testing an antibiotic not performed in routine use
or a new, antibiotic agent.
 an equivocal AST result.
K Hari Krishnan
Tirunelveli Medical
College

Most fastidious organisms do not grow well
enough in routine antibiotic testing systems
and require some type of supplementation.
Pathogen Medium
Streptococcus pneumoniae Mueller-Hinton sheep blood agar
Haemophilus sp. Haemophilus Test Medium (Mueller-
Hinton Agar, β NAD, bovine hematin,
yeast extract)
Neisseria gonorrheae Thayer-Martin agar K Hari Krishnan
Tirunelveli Medical
College

Antibiotic resistance among many clinically important
species of anaerobes has increased, which has made
empiric therapy choices unpredictable.
– E.g.. Metronidazole resistance in Propionibacterium and
Bacteroides
– Agar dilution
– Broth microdilution
– Brucella agar
(or)
– Broth supplemented with vitamin K and hemin K Hari Krishnan
Tirunelveli Medical
College

– Disk diffusion
– Broth microdilution
Automated Vitek Test Machine K Hari Krishnan
Tirunelveli Medical
College

Antibiotic
Sensitivity Testing
K Hari Krishnan
Tirunelveli Medical
College

Antibiotic Sensitivity Tests

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