Download Antibiotic sensitivity test

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Definition and uses of antibiotic
sensitivity testing

 Antibiotic sensitivity test : a laboratory test use to
measure the ability of an antimicrobial agent to
inhibite the growth of m.o in vitro this test will assist
the clinicians in the choice of drug for treatment of
infections in vivo.
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According to this the m.o classified
into 3 categories

 Sensitive
 Intermediate
 Resistant
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
 Sensitive: An organism is called sensitive to an antimicrobial
agent when the infection caused by it is likely to respond to
treatment with that particular antimicrobial agent at the
recommended dose.
 Intermediate: This term is applicable to strains that are
moderately suscepitable to an antimicrobial agent that can be
used for treatment at higher dose (if it has a low toxicity or the
antimicrobial agent is concentrated in the focus of infection as
in urine). But if the antimicrobial agent was toxic in high dose
then other measure should be use as it can use in combination
with other agent.
 Resistant :An organism is not to respond to a given
antimicrobial agent irrespective of the dose and the location of
the infection.
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The main purposes of
susceptibility (sensitivity) test

 As a guide for treatment
 It helps the physician in selecting the best
antimicrobial agent for patients .
 As an epidemiological tool
 The emergence of resistant strains of major
pathogens (e. g. Shigella, Salmonella typhi)
 Continued surveillance of the susceptibility
pattern of the prevalent strains (e. g.
Staphylococci, Gram-negative bacilli)
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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?
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Susceptibility Tests

There are 4 methods
to perform the
antibiotic sensitivity
test :
1- disc diffusion
2- broth dillution
3- E- test
4- vitek 2 test
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1- Disc agar –diffusion (Kirby-Bauer)
test

 The principle of disc agar –diffusion method:
Dependent upon the inhibition of reproduction of a
m.o on the surface of a solid medium by an
antimicrobial agents which diffuses into the medium
from a filter paper disc into the medium . 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 .
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Materials and reagents

1-The media: Muller –Hinton , nutrient agar or blood agar
2-Antimicrobial discs: commercially available disc are used
but chossing the appropriate antibiotic for routine
susceptibility tests is as follow:
 Drugs for routine susceptibility tests:
 Set 1: the drugs that are available in most hospitals and for
which routine testing should be carried out for every strain
 Set 2: the drugs that are tested only:
 at the special request of the physician
 or when the causative organism is resistant to the firstchoice drugs
 or when other reasons (allergy to a drug, or its
unavailability) make further testing justified
3- the sample: a bacterial sample ,after isolation and
identification of bacteria precede the selection of antibiotic
testing method
4- sterile cotton swab
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Table 1: Basic sets of drugs for routine susceptibility
Set 1
Set 2
Staphylococcus
Benzyl penicillin
Oxacillin
Erythromycin
Tetracycline
Chloramphenicol
Gentamicin
Amikacin
Co-trimoxazole
Clindamycin
Intestinal
Ampicillin
Chloramphenicol
Co-trimoxazole
Nalidixic acid
Tetracycline
Norfloxacin
Enterobacteriaceae
Urinary
Sulfonamide
Trimethoprim
Co-trimoxazole
Ampicillin
Nitrofurantoin
Nalidixic acid
Tetracycline
Norfloxacin
Chloramphenicol
Gentamicin
Blood and tissues
Ampicillin
Chloramphenicol
Cotrimoxazole
Tetracycline
Gentamicin
Cefuroxime
Ceftriaxone
Ciprofloxacin
Piperacillin
Amikacin
Pseudomonas aeruginosa
Piperacillin
Gentamicin
Tobramycin
Amikacin
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Disc Diffusion Method
Procedure

1- Prepare the inoculum : 108 CFU/ml bacterial inoculum in
nutrient broth which is prepared by Picking 3-5 isolated
colonies from the plate
2-Adjust the turbidity to the same as the McFarland No.
0.5 standard.*
3-Dipping a sterile swab into the broth
4-Streak the swab on the surface of the Mueller-Hinton agar or
nutrient agar (3 times in 3 quadrants)
5-Leave 5-10 min to dry the surface of agar which allowing the
bacteria to establish themselves on the media.
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
6-Place the appropriate drug immpregnant disc on the
surface of the inoculated agar plate.
7-Invert the plate and incubate them at 37ºC for (18-24
h).
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Disk Diffusion
Test

Prepare inoculum
suspension
Prepare inoculum
Select
colonies
suspension
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Prepare the Material for
Inoculation

Standardize inoculum
Suspension as per Mac farland
standard
Mix well
Nadia A. Hussin
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Swab the plate with optimal
sample

Remove sample
Swab plate
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Select the Disks and Apply

Select disks
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Incubate Overnight

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8-Reporting the Results
By comparing the diameters with “standard tables” as the
bacteria on the media grow, they are inhibited to vary
degree by the diffusing from the disc.
1-zone size differ on sensitivity pattern (varies for antibiotic
correlated with sensitivity or resistant to the antibiotic
tested.
2-look at the chart for establishing the zone of sensitivity: the
zone size are looked up on a standerdized chart to give a
result of sensitivity ,resistane, or intermediate .
3-Read the result : Place the metric ruler across the zone of
inhibition, at the widest diameter and measure from one one
edge of the zone to the other edge. The disc diameter will
actually be part of the number . Zone diameter result
reported in milimeter , looked up on the chart and result
reported as sensitive (S), resistant (R) ,or intermediate (I).
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
Transmitted
Light
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
Limitation of disc diffusion methods
Disc diffusion methods are not suitable for slow
growing bacterial pathogens.
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Dilution antibiotic sensitivity test

 a laboratory test used to determine the minimum
concentration of antibiotic that required to
control the infection (complete inhibition of the
m.o)
 Minimum Inhibition Concentration (MIC)
 The lowest concentration of antimicrobial agent that inhibits
bacterial growth/ multiplication
 Minimum Bactericidal Concentration (MBC) or
Minimum Lethal Concentration (MLC)
 The lowest concentration of antimicrobial agent
that allows less than 0.1% of the original inoculum
to survive
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Broth Dilution Method
Procedure

Making dilutions (2-fold) of antibiotic in broth Mueller-Hinton,
or
nutrient broth (e.g , 1,2,4,8,16 µg/ml) in a liquid medium dispensed in
test tube.
1-Transfer 2 ml of each antibiotic dispensed in test tubes.
2-The antibiotic containing tubes were inoculate with standardized
bacterial suspension of the McFarland No. 0.5 standard.*
3-Following overnight incubation at 37ºC ,the tubes were for visible
bacterial growth as evidence by turbidity.
4-The lowest concentration of antibiotic that prevented growth
represented the minimal inhibitary concentration
5-Controls: no inoculum, no antibiotic
6- read the result :by culturing the tube of no turbidity on a suitable agar
plate overnight.
 Turbidity visualization  MIC
 Sub culturing of non-turbid tubes, overnight
 Growth (bacterial count)  MBC
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Broth Dilution Method
64
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16
8
4
2
1 C1 C2
0.01 ml (spread plate), Incubate 35 oC, o/n
64
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16
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Broth Dilution
Advantage

 More accurate than the disc diffusion method
 Can be used for academic purpose
Disadvantages :
Only one antibiotic & one organism can be
tested each time
Time-consuming
Broth dilution method are technically
difficult
It is very expensive
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3-E-test

 E-test is a well established
AST
method
in
microbiology
laboratories
around the world. The Etest
technique
comprises
a
predefined gradient of
antibiotic concentrations on
a plastic strip, and can be
used to determine the
Minimum
Inhibitory
Concentration (MIC) of
antibiotics.
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MIC of the Bacteria can be read
Directly

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Antibiotic Sensitivity testing
can be done with automation

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4-VITEK 2 system Reporting of
Resistance

 VITEK 2 system is the
Advanced Expert System
(AES™),
a
software
which
interprets
susceptibility test results,
and detects antibiotic
resistance
mechanisms.
The AES Expert System is
the
most
developed
software system in this
field, and is capable of
identifying even low-level
resistance.
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