AST WPS Office.pdf ANTIMICROBIAL SUSCEPTIBILITY TESTING

ANTIMICROBIAL
SUSCEPTIBILITY
TESTING

1. ANTIMICROBIAL
SUSCEPTIBILITY
TESTING

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Bacteria exhibit great strain variation in
susceptibility to antimicrobial agents.It is
therefore , essential to determine the
susceptibility of pathogenic bacteria isolated
from the clinical specimens to antibiotics that
are likely to be used in the treatment.

AST is performed only for pathogenic bacteria
isolated from the specimen, and not for
commensal bacteria

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Disc diffusion methods
Kirby- Bauer disk diffusion method
Stokes disk diffusion method

Dilution tests
Broth dilution method
Agar dilution method

E- test

Automated methods

Molecular methods

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DISC DIFFUSION METHOD
Disc diffusion tests are the most widely used
method. They are suitable for rapidly growing
pathogenic bacteria; however, they are
unsuitable for slow growing bacteria.

The disk diffusion method is so named because :
It uses filter paper disks impregnated with
appropriate concentration of the antibiotic
solution
The test bacterium is inoculated ( lawn culture )
on the solid medium and then the antibiotic
disks are applied

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The antibiotic in the disk diffuses through the
solid medium , so that the concentration is
highest near the site application of the
antibiotic disk and decreases gradually away
from it
Sensitivity to the drug is determined by the
zone of inhibition of bacterial growth around
the disk

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Medium (Mueller - Hinton Agar )

MHA is considered as the best medium to
use for routine susceptibility testing of non
fastidious bacteria for the following reasons :

It supports satisfactory growth of most
nonfastidious pathogens

It has minimal inhibitory effect on sulfonamide
and trimethoprim. Hence , these antibiotics are
better tested in MHA than any other media

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Modifications of MHA

Lysed horse blood is added to MHA to support
the growth of fastidious organism , such as H.
influenzae


Sodium chloride (2-4%) should be added to the
medium for testing MRSA isolates

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Inoculum
Isolated pure colonies of the test organism are
inoculated in suitable liquid medium ( peptone
water ) and incubated at 35-37°C for 4-6hours

The density of the organisms in broth is adjusted
to approximately 1.5 ×10^8 CFU/ ml by comparing
its turbidity with that of 0.5 McFarland opacity
standard tube.

Lawn culture: The broth is then inoculated on the
medium by spreading with sterile swabs

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The ideal inoculum after overnight incubation gives an
even semiconfluent growth.Too heavy inoculum
reduces the size of inhibition zones

Control strains : similar to the test isolate , the
inoculum of control strain should also be made and
tested for AST.

The following ATCC ( American Type Control
Collection) strains are used as standard control
strains.
Escherichia coli ATCC 25922
Pseudomonas aeruginosa ATCC 27853
Staphylococcus aureus ATCC 25923
Enterococcus feacalis ATCC 29212

10. Antibiotic disk
Antibiotic disks are available commercially. Sterile
filter paper ( Whatman number 1) disks of 6 mm
diameter are impregnated with standard quantity of
antibiotic solution.

Choice of antibiotic disks
It is neither possible nor desirable to test the
susceptibility against all the drugs.

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The panel of the drugs to be tested against an
isolate depends upon various factors ;
Antibiotics should likely to be used for therapy
Organism against which the drug has to be
tested
Local prescribing habits of the antimicrobial
agents
Resistant pattern of the locally prevalent
pathogens
Cost,toxicity and spectrum of activity of an
antimicrobial agent for the management of
illness in a particular patient

12. Kirby - Bauer Disk Diffusion Method

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A cotton swab is dipped into inoculum and squeezed .
Then the swab is inoculated to MHA plate
After drying , for 3-5 minuted the antibiotic disk are
applied using sterile forceps
Disks should not be closer than 20 mm on the MHA
plate
Ordinarly, max up to 6 disks can be applied on a
100 mm plate
The plates are incubated at 37°C for 16-18 hours (for
MRSA ,result should read only after 24 hours of
incubation
Zone of complete growth of inhibition around each of
the disk are measured using vernier caliper
The interpretation of zone size into sensitive ,
intermediate , or resistant is based on standard zone
size interpretation chart

14. Stokes Disk Diffusion Method

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Here the MHA plate is divided into 3 parts. The
test organism is inoculated on the central 1/3 rd
and the control strain on upper and lower thirds
of the plate
In modified stokes method , the test bacterium
is inoculated over the upper and lower thirds of
the plate and control on central 1/3 rd
An uninoculated gap of 2-3 mm wide should
separate the test and control area on which the
antibiotic disk are applied
The plates are the incubated at 37°C for 16-18
hours

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Reporting in stokes method

The sensitivity report is prepared by
comparing the zones of inhibition of control and test
bacterium. The radius of the inhibition zone from
the edge of the disk to the edge of the zone is
measured
Interpretation
Sensitive : zone radius is wider than or equal to ,
or not more than 3mm smaller than the control
Intermediate: zone radius is > 2 mm but smaller
than the control by >3 mm
Resistant : No zone of inhibition or zone radius
measures 2mm or less

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DILUTION TESTS
Here, the antimicrobial agent is serially diluted ,
each dilution is tested with the test organism for
antimicrobial susceptibility test and the MIC is
calculated
MIC is the lowest concentration of an
antimicrobial agent that will inhibit the visible
growth of a microorganism after overnight
incubation
Depending upon whether the dilutions of the
antimicrobial agent are made in agar or broth ,
there are two types of dilution tests.
Broth dilution method
Agar dilution method

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Broth Dilution Method
Serial dilutions of the antimicrobial agent in MH broth
are taken in tubes and each tube is inoculated with a
fixed amount of suspension of the test organism. A
control organism of known sensitivity should also be
tested. Tubes are incubated at 37°C for 18 hours
MIC is determined by noting the low concentration of
the drug at which there is no visible growth , ie broth
appears clear
The minimum bactericidal concentration MBC can be
obtained by subculturing from each tube ( showing no
growth ) onto a nutrient agar plate without any
antimicrobial agent. The tube the lowest concentration
of the drug that fails to show growth , on subculture , is
the MBC of the drug for that test strain

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Agar Dilution Method

Here the serial dilutions of the drug are prepared
in molten agar and poured into petri dishes.The
test strain is spot inoculated. This method is more
convenient than broth dilution and has the
advantage of :
Several strains can be tested at the same time
by using the same plate
It directly measures the MBC ; there is no need
of subculturing as it is done with broth dilution
method.

22. EPSILOMETER OR E-TEST
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This is a quantitative method of detecting MIC by
using the principle of both dilution and diffusion of
antibiotic into the medium
It uses an absorbent strip containing predefined
gradient ( serial dilution ) of antibiotic
concentration immobilized along its length
It is applied to a lawn inoculum of a bacterium.
Following incubation , an elliptical zone of
inhibition is produced surrounding the strip
The antibiotic concentration at which the ellipse
edge intersects the strip, is taken as MIC value

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AUTOMATED AST
VITEK 2 bacterial identification and antimicrobial
sensitivity system ( bioMerieux )
Phoenix system ( Becton Dickinson )
Micro Scan Walk Away System

Most systems are computer assisted and have
sophisticated softwares to analyse the growth
rates and determine the antibiotic susceptibility
report.
They use commercially available panels that
contain added growth factors to speed organism
growth, thereby providing more rapid results
compared with traditional methods

25. MOLECULAR METHODS

•PCR, DNA microarray and DNA chips, and loop-
mediated isothermal amplification (LAMP) are
some of the genotypic techniques for the detection
of antibiotic resistance.
•Molecular or genotypic AST are the effective
direct methods
•Mutational assessment of methicillin resistance in
Staphylococcus spp., vancomycin resistance in
Enterococcus spp., and multi-antibiotic (isoniazid,
rifampin, streptomycin, pyrazinamide, and the
fluoroquinolones) resistance in Mycobacterium spp.
have been successfully estimated through various
genotypic techniques.

26. Emerging Methods for AST

Microfluidics-based diagnostics are one of
the most promising emerging tools for
AST.

27. THANK YOU...