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

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•Right Drug

•Right Microbe

•Right Cure

Antibiotic Sensitivity

Testing

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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.

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AST is essential for the selection of the appropriate antibiotic

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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.

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Antibiotic Sensitivity Tests

Diffusion

Kirby-Bauer

Method

Stokes Method

Dilution

Tube Dilution

Agar Dilution

Diffusion & Dilution

E-Test

Qualitative Methods Quantitative Methods

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Antibiotics for routine testing

Staphylococcus

EnterobacteriaceaePseudomonas

aeruginosaIntestinal Urinary Blood & Tissues

BenzylpenicillinOxacillinErythromycinTetracyclineChloramphenicol

AmpicillinChloramphenicolCotrimoxazoleTetracycline

SulfonamidesTrimethoprimCotrimoxazoleAmpicillinNitrofurantoinTetracycline

AmpicillinChloramphenicolCotrimoxazoleTetracyclineCefalotinGentamycin

PiperacillinGentamycinTobramycin

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Diffusion methods

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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

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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

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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

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Kirby-Bauer Method

Mueller-Hinton Agar

Antibiotic Disks

Turbidity Standard

MATERIALS

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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

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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

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Antibiotic Disks

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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

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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

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Cotton Swabs

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Application of Antibiotic Discs

Incubation At 35⁰C for 16-18 hours

Measurement of inhibition zone diameter

Procedure

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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

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2. Transfer this growth to a tube of saline.

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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.

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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.

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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.

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7. Leave the inoculum to dry for a few minutes at room temperature with the lid closed.

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8. The antibiotic discs may be placed on the inoculated plates using– sterile forceps.– a template.– a sterile needle tip.– antibiotic disc dispenser.

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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

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Strips of multiple antibiotics can be tested in one go

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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

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Using automated zone readers– BIOMIC– Aura– Protozone

Measurement of diameter

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Interpretation of results

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Standard templates are available for each antibiotic.

Using a template

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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

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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

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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

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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

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Methicillin resistance in Staphylococcus aureus

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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

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• 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

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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.

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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

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Dilution methods

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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

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Tube dilution Agar dilution

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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

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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

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MICK Hari KrishnanTirunelveli Medical

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The lowest concentration of drug that kills the bacteria isolated from the patient.

Minimum bactericidal concentration

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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

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Tube and Agar DilutionLimitations

Not easily automated

Fresh reagents required

Time-consumingK Hari KrishnanTirunelveli Medical

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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

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E-TestK Hari KrishnanTirunelveli Medical

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Epsilometer Test Quantitative method of antibiotic sensitivity

testing. Applies both dilution of antibiotic and

diffusion of antibiotic into the medium.

What is E-Test?

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Combines the principles of disk diffusion and agar dilution methods

Diffusio

Dilution

E-Test

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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).

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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

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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

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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

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Simple Accurate Reliable

E-Test

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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

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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

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METHODS– Disk diffusion– Broth microdilution

Automated Vitek Test Machine

AST of Fungi

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AST can be done with automation

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There is a growing need for

Automation in

Antibiotic sensitivity

testing

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Choose the right drug!Get faster cure!

Prevent drug resistance!

Antibiotic Sensitivity Testing

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Antibiotic Sensitivity Tests

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