Antibiotic Sensitivity Tests
K Hari KrishnanII MBBS (2009-’11)
Tirunelveli Medical CollegeTirunelveli, 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 KrishnanTirunelveli Medical
College
The in vitro testing of bacterial cultures with antibiotics to determine susceptibility of bacteria to antibiotic therapy.
Antibiotic Sensitivity Test
K Hari KrishnanTirunelveli Medical
College
•Right Drug
•Right Microbe
•Right Cure
Antibiotic Sensitivity
Testing
K Hari KrishnanTirunelveli Medical
College
Purposes To guide the clinician in selecting the best
antibiotic agent for an individual patient. To control the use of inappropriate
antibiotics 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 KrishnanTirunelveli Medical
College
AST is essential for the selection of the appropriate antibiotic
K Hari KrishnanTirunelveli Medical
College
Qualitative– For the testing of isolates from “healthy” patients with
intact immune defenses.
– For less serious infections such as uncomplicated urinary tract infections.
Quantitative– 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.
Types
K Hari KrishnanTirunelveli 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 KrishnanTirunelveli Medical
College
Antibiotics for routine testing
Staphylococcus
EnterobacteriaceaePseudomonas
aeruginosaIntestinal Urinary Blood & Tissues
Drugs
BenzylpenicillinOxacillinErythromycinTetracyclineChloramphenicol
AmpicillinChloramphenicolCotrimoxazoleTetracycline
SulfonamidesTrimethoprimCotrimoxazoleAmpicillinNitrofurantoinTetracycline
AmpicillinChloramphenicolCotrimoxazoleTetracyclineCefalotinGentamycin
PiperacillinGentamycinTobramycin
K Hari KrishnanTirunelveli Medical
College
Diffusion methods
K Hari KrishnanTirunelveli Medical
College
Principle– 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.
Disk Diffusion Method
K Hari KrishnanTirunelveli Medical
College
The antibiotic contained in a reservoir is allowed to diffuse out into the medium 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 antibiotic diffuses out of the disk to form the
gradient.– The test organism starts to divide and grow and
progresses toward a critical mass of cells.
The basics
K Hari KrishnanTirunelveli Medical
College
Inhibition zone edge 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.
INHIBITION ZONE EDGE
K Hari KrishnanTirunelveli Medical
College
Kirby-Bauer Method
Mueller-Hinton Agar
Antibiotic Disks
Turbidity Standard
Swabs
MATERIALS
K Hari KrishnanTirunelveli Medical
College
Medium containing beef infusion, peptone, and starch.
Used primarily for the disk-diffusion method.
Mueller-Hinton Agar
Robust red algae (Solieria robusta)Source of Agar K Hari Krishnan
Tirunelveli Medical College
Mueller-Hinton agar is considered the BEST for routine susceptibility testing of nonfastidious bacteria.
Why? 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 KrishnanTirunelveli Medical
College
Cool the medium to 45–50 ⁰C and pour into the plates. Allow to set on a level surface, to a depth of approximately 4 mm.– A 9-cm plate requires approximately 25 ml of
medium. When the agar has solidified, dry the plates 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 8C) for 2 weeks.
Mueller-Hinton Agar
K Hari KrishnanTirunelveli 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.
Antibiotic Disks
K Hari KrishnanTirunelveli Medical
College
Antibiotic Disks
K Hari KrishnanTirunelveli Medical
College
Prepared by pouring 0.6 ml of a 1% (10 g/l) solution of barium chloride dihydrate into a 100-ml graduated cylinder, and filling to 100ml with 1% (10 ml/l) sulfuric acid.
Turbidity Standard
K Hari KrishnanTirunelveli 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.
Cotton Swabs
K Hari KrishnanTirunelveli Medical
College
Cotton Swabs
K Hari KrishnanTirunelveli Medical
College
Application of Antibiotic Discs
Incubation At 35⁰C for 16-18 hours
Measurement of inhibition zone diameter
Procedure
K Hari KrishnanTirunelveli Medical
College
1. To prepare the inoculum from the primary culture plate, touch with a loop the tops of each of 3–5 colonies, of similar appearance, of the organism to be tested.
Kirby-Bauer Method
Procedure
K Hari KrishnanTirunelveli Medical
College
2. Transfer this growth to a tube of saline.
K Hari KrishnanTirunelveli Medical
College
3. Compare the tube with the turbidity standard and adjust the density of the test suspension to that of the standard by adding more bacteria or more sterile saline.
K Hari KrishnanTirunelveli Medical
College
4. Inoculate the plates by dipping a sterile swab 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 KrishnanTirunelveli Medical
College
5. Streak the swab all over the surface of the medium three times, rotating the plate through an angle of 60⁰ after each application.
6. Finally, pass the swab round the edge of the agar surface.
K Hari KrishnanTirunelveli Medical
College
7. Leave the inoculum to dry for a few minutes at room temperature with the lid closed.
K Hari KrishnanTirunelveli Medical
College
8. The antibiotic discs may be placed on the inoculated plates using– sterile forceps.– a template.– a sterile needle tip.– antibiotic disc dispenser.
K Hari KrishnanTirunelveli Medical
College
Application of Antibiotic Discs
Incubation At 35⁰C for 16-18 hours
Measurement of inhibition zone diameter
K Hari KrishnanTirunelveli Medical
College
A maximum of seven discs 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 placed in an incubator within 30 minutes of preparation.
Temperatures above 35⁰C invalidate results for oxacillin/methicillin.
Do not incubate in an atmosphere of carbon dioxide.
Disks should not be moved after diffusion.
!K Hari KrishnanTirunelveli Medical
College
Strips of multiple antibiotics can be tested in one go
K Hari KrishnanTirunelveli Medical
College
Application of Antibiotic Discs
Incubation At 35⁰C for 16-18 hours
Measurement of inhibition zone diameter
K Hari KrishnanTirunelveli 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.
Measurement of diameter
K Hari KrishnanTirunelveli Medical
College
Using automated zone readers– BIOMIC– Aura– Protozone
Measurement of diameter
K Hari KrishnanTirunelveli Medical
College
Interpretation of results
K Hari KrishnanTirunelveli Medical
College
Standard templates are available for each antibiotic.
Using a template
K Hari KrishnanTirunelveli Medical
College
Result interpretation– Susceptible• When the edge of the zone of inhibition is OUTSIDE the
black circle.
– Resistant• When there is no zone, or when it lies WITHIN the
white circle.
– Intermediate• When the edge of the zone of inhibition lies ON the
black circle.
Using a template
K Hari KrishnanTirunelveli 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.
Using a ruler
K Hari KrishnanTirunelveli Medical
College
Interpretative chart of zone sizes
AntibioticDiameter 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 KrishnanTirunelveli Medical
College
Susceptible– 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.
Resistant– 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.
Intermediate– 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.
Definitions
K Hari KrishnanTirunelveli Medical
College
Methicillin resistance in Staphylococcus aureus
K Hari KrishnanTirunelveli Medical
College
Inoculum density– Too light inoculum• Inhibition zones will be larger even though the
sensitivity of the organism is unchangedRelatively resistant strains may be falsely reported as
susceptible.
– Too heavy inoculum• Inhibition zones will be smallerRelatively susceptible strains may then be falsely
reported as resistant.
Factors influencingsize of zone
K Hari KrishnanTirunelveli Medical
College
• Timing of disk applicationPlates seeded with test strain
Left at room temperature for long periods
Multiplication of inoculum before disks are applied
Reduction in zone diameter
Susceptible strain reported as resistant
K Hari KrishnanTirunelveli Medical
College
Temperature of incubation– If the temperature is lowered, the time required
for effective growth is extended and larger zones result.
Potency of antibiotic disks– If the potency of the drug is reduced owing to
deterioration during storage, the inhibition zone will show a corresponding reduction in size.
K Hari KrishnanTirunelveli 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.
Primary disk diffusion
K Hari KrishnanTirunelveli Medical
College
Dilution methods
K Hari KrishnanTirunelveli Medical
College
Used to determine the minimal concentration of antibiotic to inhibit or kill the microorganism.
Achieved by dilution of antibiotic in either agar or broth media.
Dilution methods
K Hari KrishnanTirunelveli Medical
College
MIC
Tube dilution Agar dilution
K Hari KrishnanTirunelveli Medical
College
The lowest concentration of drug that inhibits the growth 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.
Minimum inhibitory concentration
K Hari KrishnanTirunelveli Medical
College
Patient's organism is added to tubes containing decreasing amounts of the antibiotic
IncubationAt 37°C overnight
Lowest concentration of drug that inhibits growth is the MIC
Tube dilution
K Hari KrishnanTirunelveli Medical
College
MICK Hari KrishnanTirunelveli Medical
College
The lowest concentration of drug that kills the bacteria isolated from the patient.
Minimum bactericidal concentration
K Hari KrishnanTirunelveli Medical
College
Serial dilutions of the drug are prepared in agar and poured into plates.
Advantage– Many strains can be inoculated on each plate
containing an antibiotic dilution.
Agar dilution
K Hari KrishnanTirunelveli Medical
College
Tube and Agar DilutionLimitations
Not easily automated
Fresh reagents required
Time-consumingK Hari KrishnanTirunelveli Medical
College
Broth microdilution plate contains– Each row:
• standard dilutions of eight bacterial organisms in each row (denoted by letters A-H).
– Each column• contains a standard antibiotic 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.
Broth microdilution
K Hari KrishnanTirunelveli Medical
College
K Hari KrishnanTirunelveli Medical
College
E-TestK Hari KrishnanTirunelveli Medical
College
Epsilometer Test Quantitative method of antibiotic sensitivity
testing. Applies both dilution of antibiotic and
diffusion of antibiotic into the medium.
What is E-Test?
K Hari KrishnanTirunelveli Medical
College
Combines the principles of disk diffusion and agar dilution methods
Diffusio
n
Dilution
E-Test
K Hari KrishnanTirunelveli 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 KrishnanTirunelveli Medical
College
Apply E-Test strip on an inoculated agar plate
Immediate release of drug
Incubation of plate
Symmetrical inhibition ellipse is produced
The intersection of the inhibitory zone edge and the calibrated carrier strip indicates the MIC with inherent precision and accuracy.
E-Test
Procedure
K Hari KrishnanTirunelveli Medical
College
MIC
K Hari KrishnanTirunelveli Medical
College
K Hari KrishnanTirunelveli Medical
College
Over 100 antibiotics 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 KrishnanTirunelveli Medical
College
Determining the MIC of fastidious, slow-growing or nutritionally deficient micro-organisms, 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 low levels of resistance. Testing an antibiotic not performed in routine use or a
new, recently introduced antibiotic agent. Confirming an equivocal AST result.
E-Test
Uses
K Hari KrishnanTirunelveli Medical
College
Simple Accurate Reliable
E-Test
K Hari KrishnanTirunelveli 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
AST of Fastidious organisms
K Hari KrishnanTirunelveli 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
METHODS– Agar dilution– Broth microdilution
MEDIA– Brucella agar (or)– Broth supplemented with vitamin K and hemin
AST of Anaerobes
K Hari KrishnanTirunelveli Medical
College
METHODS– Disk diffusion– Broth microdilution
Automated Vitek Test Machine
AST of Fungi
K Hari KrishnanTirunelveli Medical
College
AST can be done with automation
K Hari KrishnanTirunelveli Medical
College
There is a growing need for
Automation in
Antibiotic sensitivity
testing
K Hari KrishnanTirunelveli Medical
College
Choose the right drug!Get faster cure!
Prevent drug resistance!
Antibiotic Sensitivity Testing
K Hari KrishnanTirunelveli Medical
College