Antibiotic Sensitivity Testing

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A presentation on the methods for testing the sensitivity of organisms to antibiotics

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Antibiotic Sensitivity Tests : 

Antibiotic Sensitivity Tests K Hari Krishnan II MBBS (2009-’11) Tirunelveli Medical College Tirunelveli, Tamilnadu, India

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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

Antibiotic Sensitivity Test : 

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

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Antibiotic Sensitivity Testing K Hari KrishnanTirunelveli Medical College

Purposes : 

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 KrishnanTirunelveli Medical College

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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 : 

AST is essential for the selection of the appropriate antibiotic K Hari KrishnanTirunelveli Medical College

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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. Types K Hari KrishnanTirunelveli Medical College

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Qualitative Methods Quantitative Methods K Hari KrishnanTirunelveli Medical College

Antibiotics for routine testing : 

Antibiotics for routine testing K Hari KrishnanTirunelveli Medical College

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Diffusion methods K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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Kirby-Bauer Method

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K Hari KrishnanTirunelveli Medical College

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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 KrishnanTirunelveli Medical College

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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

Mueller-Hinton Agar : 

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 8C) for 2 weeks. Mueller-Hinton Agar K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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Antibiotic Disks K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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Cotton Swabs K Hari KrishnanTirunelveli Medical College

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Procedure K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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2. Transfer this growth to a tube of saline. K Hari KrishnanTirunelveli Medical College

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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. K Hari KrishnanTirunelveli Medical College

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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. K Hari KrishnanTirunelveli Medical College

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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. K Hari KrishnanTirunelveli Medical College

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7. Leave the inoculum to dry for a few minutes at room temperature with the lid closed. K Hari KrishnanTirunelveli Medical College

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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. K Hari KrishnanTirunelveli Medical College

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K Hari KrishnanTirunelveli Medical College

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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 College

Strips of multiple antibiotics can be tested in one go : 

Strips of multiple antibiotics can be tested in one go K Hari KrishnanTirunelveli Medical College

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K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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Using automated zone readers BIOMIC Aura Protozone Measurement of diameter K Hari KrishnanTirunelveli Medical College

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Interpretation of results K Hari KrishnanTirunelveli Medical College

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Standard templates are available for each antibiotic. Using a template K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

Methicillin resistance in Staphylococcus aureus : 

Methicillin resistance in Staphylococcus aureus K Hari KrishnanTirunelveli Medical College

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Inoculum density Too light inoculum Inhibition zones will be larger even though the sensitivity of the organism is unchanged Relatively resistant strains may be falsely reported as susceptible. Too heavy inoculum Inhibition zones will be smaller Relatively susceptible strains may then be falsely reported as resistant. Factors influencing size of zone K Hari KrishnanTirunelveli Medical College

Timing of disk application : 

Timing of disk application K Hari KrishnanTirunelveli Medical College

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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. K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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Dilution methods K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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Tube dilution K Hari KrishnanTirunelveli Medical College

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MIC K Hari KrishnanTirunelveli Medical College

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The lowest concentration of drug that kills the bacteria isolated from the patient. Minimum bactericidal concentration K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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K Hari KrishnanTirunelveli Medical College

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E-Test K Hari KrishnanTirunelveli Medical College

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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? K Hari KrishnanTirunelveli Medical College

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Combines the principles of disk diffusion and agar dilution methods K Hari KrishnanTirunelveli Medical College

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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). K Hari KrishnanTirunelveli Medical College

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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

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MIC K Hari KrishnanTirunelveli Medical College

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K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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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 Uses K Hari KrishnanTirunelveli Medical College

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Simple Accurate Reliable E-Test K Hari KrishnanTirunelveli Medical College

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Most fastidious organisms do not grow well enough in routine antibiotic testing systems and require some type of supplementation. AST of Fastidious organisms K Hari KrishnanTirunelveli Medical College

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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 K Hari KrishnanTirunelveli Medical College

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Methods Disk diffusion Broth microdilution Automated Vitek Test Machine AST of Fungi K Hari KrishnanTirunelveli Medical College

AST can be done with automation : 

AST can be done with automation K Hari KrishnanTirunelveli Medical College

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There is a growing need for Automation in Antibiotic sensitivity testing K Hari KrishnanTirunelveli Medical College

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Choose the right drug! Get faster cure! Prevent drug resistance! Antibiotic Sensitivity Testing K Hari KrishnanTirunelveli Medical College

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Thank You