Naresh ast

ANTIMICROBIAL
SUSCEPTIBILITYTESTING
Mr. Naresh Pokhrel
M.Sc Clinical Microbiology
Department of Microbiology
Father Muller Medical College,
Mangalore
ANTIBIOTIC SUSCEPTIBILITY TESTING

Modern era in Antibiotics begins with
Fleming.

Guidelines :
CLSI 2019
EUCAST

Overview:-
Introduction
Goal of AST
Types
Disc Diffusion Methods (Qualitative)
Medium (Mueller-Hinton Agar)
Kirby-Bauer Disk Diffusion Method
Troubleshooting the Reading of Disk Diffusion Tests
Dilution Tests
Epsilometer or E-test
Automated Antimicrobial Susceptibility Tests
Molecular Methods
Quality Control In AST

Antimicrobial Susceptibility Testing: 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.

Goal of AST
1.The identification of relevant antibiotics to specific 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.

Types:-

Disc Diffusion Methods (Qualitative)
Disk diffusion susceptibility tests became standardized in the United
States based on the work of Bauer, Kirby, and coworkers.
This method is still widely used because of its easy of use and lower
cost compared to other methods.

Method Description. The basic principle of the standardized disk diffusion
susceptibility test is illustrated below, All disk diffusion methods rely on
diffusion of drug released from an impregnated disk through agar.

The principle of antibiotic diffusion in agar. The concentration of antibiotic decreases
as the distance from the disk increases. ANTIBIOTIC SUSCEPTIBILITY TESTING

Why mueller-hinton agar is used in routine antibiotic
susceptibility testing (AST) ?
Antimicrobial Susceptibility testing using disk diffusion Mueller-
Hinton agar is the best medium for routine antibiotic susceptibility
testing (AST) because of the following reasons:
 It shows acceptable batch-to-batch reproducibility for susceptibility
testing.

It has minimal inhibitory effect on sulfonamide and trimethoprim.
Hence, these antibiotics are better tested in MHA than any other media.
It supports satisfactory growth of most nonfastidious pathogens.

Modifications of Mueller Hinton agar
Mueller Hinton agar medium supplemented with 5% sheep blood is
recommended for determining the antimicrobial susceptibility of
Streptococcus pneumoniae
Neisseria meningitidis
Haemophilus test medium (HTM) is the preferred medium for the antimicrobial
susceptibility testing of H. influenzae using modified Kirby Bauer disk diffusion
method. HTM medium consists of following ingredients: thymidine free MHA
supplemented with 15 μg/ml NAD, 15 μg/ml bovine hemin, and 5 mg/ml yeast extract.

Preparation Of MHA
Measure 38 g of medium (or the components listed above) in 1 liter of purified
water.
Mix thoroughly.
Heat with frequent agitation and boil for 1 minute to completely dissolve the
components.
Autoclave at 121°C for 15 minutes.
Cool to 45°C
Pour cooled Mueller Hinton Agar into sterile petri dishes on a level, horizontal
surface to give uniform depth.

Allow to solidify at room temperature.
Check prepared Mueller Hinton Agar to ensure the final pH is 7.3 ±1 at 25°C.
Note: If the pH is <7.2 certain drugs will appear to lose potency (aminoglycosides,
quinolones, macrolides), while other agents may appear to have excessive activity
(tetracycline). If the pH is >7.4, the opposite results may occur.
Prepared media can be stored at 4 to 8°C. Mueller-Hinton agar is stable for
approximately 70 days from the date of preparation.

Note: The plates must be poured to a depth of 4 mm (approximately 25 ml of liquid
agar for 100-mm plates and 60 ml of liquid agar for 150-mm plates, but in any case to
a measured depth of 4 mm).

Principle of MHA
Mueller Hinton Media contains Beef Extract, Acid Hydrolysate of Casein, Starch
and Agar.
 Beef Extract and Acid Hydrolysate of Casein provide nitrogen, vitamins,
carbon, amino acids, sulphur and other essential nutrients.
Starch is added to absorb any toxic metabolites produced.
Starch hydrolysis yields dextrose, which serves as a source of energy.
Agar is the solidifying agent.

lnoculum
Isolated pure colonies of the test organism are inoculated in a
suitable liquid medium (peptone water broth) and incubated at
35-37'C for 4- 6 hours.
The density of the organisms in broth is adjusted to 0.5
McFarland opacity standard tube. (1.5 * 108 cfu/mL)

Lawn culture: The broth is then inoculated on the medium by sterile swabs.
The ideal inoculum after overnight incubation gives an even semi confluent grow.
Too heavy inoculum reduces the size of inhibition zones.

Antibiotic Disk
Antibiotic disks are available commercially or prepared in house.
Sterile filter paper (whatman number 1) disks of 6 mm diameter are
impregnated with standard quantity of antibiotic solution.

Calculation of stock solution
1000
--------- * V * C = W
P
P – Potency of preparation
V – Volume required (mL)
C – Final Concentration of solution
W – Weight of antibiotic to be dissolved in V

Choice of Antibiotic Disk
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 pathogen.
Cost, toxicity, pharmacokinetics, and spectrum of
activity of an antimicrobial agent for the management
of illness in a particular patient.

First-line drugs: Those antibiotics that are commonly used in a
locality with respect to the organism isolated should
be tested first.
Second-line drugs: include the panel of those antibiotics for which the
prescription is restricted only to special circumstances.

Kirby-Bauer Disk Diffusion Method
A cotton swab is dipped into inoculum and squeezed to drain out the excess fluid.
Then the swab is inoculated on to the Mueller-Hinton agar plate by streaking the
swab three times over the entire agar surface.

After drying the surface of agar place for 3-5 minutes the antibiotic disks are
applied using either sterile forceps or multidisk dispenser.
 Disks should not to be placed closer than 20,mm(center co center) on the MHA
plate.
Ordinarily, maximum up to 6 disks can be applied on a 100 mm plate (five in the
periphery and one in the center)

The plates are then incubated at 37'C for 16- 18 hours. When tested for MRSA.
result should be read only after 24 hours of incubation.
 The zones of complete growth of inhibition around each of the disks are measured
using a ruler or Vernier caliper. The diameter of the disk is also included in this
measurement.
 The interpretation of zone size into sensitive, intermediate or resistant is based on
the standard zone size interpretation chart .

 Control strains should be tested each time when a new batch of disks
or Mueller-Hinton agar is used.

Storage of discs :
Refrigerate at 2 to 8 deg C in dessicators.
Beta lactam class drugs should be frozen.
The drugs should be kept outside at RT 1 to 2 hours before work
The dispensing apparatus which is used to deliver the drugs should also be
Refrigerated. Check the expiry of drugs

Stokes method
 Built in controls against many variables and provide dependable results
 A standard sensitive strain of the bacterium is inoculated in the middle third of the
culture plate

 The test bacterium is inoculated in the upper and lower third of the plate.
An uninoculated gap of 2-3 mm wide should separate the test and the control
area on which the antibiotic disks are applied so that zones of inhibition
formed around each disc are composed of standard and test bacteria.
 The plates are then incubated at 37 deg C for 16-18 hours.

 The 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 (S):Zone radius is wider than or equal to, or not more than 3 mm smaller
than the control.
Intermediate (I): Zone radius is >2 mm but smaller than the control by >3 mm.
 Resistant (R): No zone of inhibition or zone radius measures 2 mm or less.

Interpretation of Results. The zone size observed in a disk diffusion
test is compared to the interpretive standards provided by the CLSI.
Zone diameter sizes derived from testing a wide range of strains are
correlated with the known MICs of the species tested to assign
interpretive breakpoint criteria for susceptible, intermediate and
resistance zone sizes.
A prototype of a regression curve providing such a correlation is
illustrated in graph.

Troubleshooting the Reading of Disk Diffusion Tests

Limitations :-
This medium is recommended for susceptibility testing of pure cultures only.
Inoculum density may effect the zone size. Heavy inoculum may result in smaller
zones or too less inoculum may result in bigger zones.

Fastidious organisms may not grow on this medium and may require
supplementation of blood. Fastidious anaerobes may not grow on this medium.
As antimicrobial susceptibility is carried with antibiotic disc, proper storage of the
disc is desired which may effect the potency of the disc.
Under certain circumstances, the in vitro results of antibiotic susceptibility may not
show the same in vivo

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 (minimum inhibitory concentration) 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
Serial dilutions of the antimicrobial agent in Mueller Hinton 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.
 The MIC is determined by noting the lowest concentration of the drug at which
there is no visible growth, i.e. broth appears clear.

The minimum bactericidal concentration (MBC) can be obtained by sub
culturing from each tube (showing no growth) onto a nutrient agar plate without
any antimicrobial agent The tube containing the lowest concentration of the drug
that fails to show growth, on subculture, is the MBC of the drug for that test
Strain.
Broth dilution test can also be done using microtiter plates, such method is
called as micro dilution method.

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 following advantage:
 Several strains can to do the tested at the same time by using the same
plate.
 It directly measures the MBC; there is no need of sub culturing as it is
done with broth dilution method.

Epsilometer or E-test
This is a quantitative method of detecting MIC by using the principles of
both dilution and diffusion of antibiotic into the medium.
It uses an absorbent strip containing pre defined gradient (serial dilution) of
antibiotic concentration immobilized along its length.
It is applied to a lawn inoculum of a bacterium. Following incubation of the
test organism, an elliptical zone of inhibition is produced surrounding the
strip.

Result and Interpretation:
Read MIC at the point where ellipse intersects the scale. If a MIC value between
two two fold dilutions is seen, always round up to the highest value.
Read the MIC value at complete inhibition of all growth.
If the intersect differs on either side of the strip, read the MIC as the greater value.
Ignore any growth at the edge of the strip.

Troubleshooting the Reading of E-test Assays
Zone of inhibition includes pinpoint colonies and hazy growth within the zone of
inhibition.
 Check the purity of the culture when there are pinpoint colonies in the zone of
inhibition; do not ignore these colonies in determining the MIC.

If the zone of inhibition intersects the strip between two markings, read the higher
value. Similarly, if the zone intersects the strip slightly differently on the two sides,
read the higher value.
When testing β-hemolytic bacteria, assess the zone of inhibition and ignore the
hemolysis.
use a magnifying glass to visualize pinpoint colonies and hazes, especially for
enterococci, pneumococci, and Stenotrophomonas spp. Include a haze as growth when
determining inhibition, but ignore the swarming of Proteus spp.

Automated Antimicrobial Susceptibility Tests
Several automated systems are available now, such as :
 VITEK 2 bacterial identification and antimicrobial sensitivity system
(bioMerieux)
 Phoenix System (Becton Dickinson)
 Micro Scan Walk Away system

Molecular Methods
PCR based assay are available targeting specific drug resistant genes: for
example mecA gene for MRSA detection.

Quality Control In AST
 QC: a process in the laboratory designed to monitor the analytical phase
of testing procedures to ensure that tests are working properly
 CLSI recommends use of ATCC strains for QC in AST

 QC strains should be included daily with the test ideally; due to expense, it can
be switched as once weekly testing, with less than 10% inaccuracy
 Not more than 1 in 20 results should be outside the accuracy limits
 No zone should be more than 4 SD away from midpoint between the stated limits
 Test repeated for each new drug included
 All documentation maintained indefinitely

QC for MHA
 To ensure that the zone diameters are sufficiently reliable for testing
susceptibility to sulfonamides and trimethoprim, MHA must have low
concentrations of the inhibitors thymidine and thymine.
 Each new lot of MHA should therefore be tested with a control strain
of Enterococcus faecalis and a disc of co-trimoxazole.
 A satisfactory lot of medium will give a distinct inhibition zone of 20 mm or
more that is essentially free of hazy growth or fine colonies.

Reference:-
 Koneman’s Color Atlas and Textbook of Diagnostic Microbiology,
Seventh Edition.
 Essentials Of Medical Microbiology , Apurba Sankar Sastry.
 Kit Inserts, Hi Media.
 Manual of Antimicrobial Susceptibility Testing ,ASM.
 Image courtesy – Google search

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