Antibiotic Sensitivity Tests Dr. Baha Eldin Khalid Elamin PhD Molecular Biology & Biotechnology

A test done to check the effectiveness of a drug against a bacterium and to select the best drug that acts against the bacterium.

Antibiotic Sensitivity Test The in vitro testing of bacterial cultures with antibiotics to determine susceptibility of bacteria to antibiotic therapy.

Antibiotic Sensitivity Testing Right Drug Right Microbe Right Cure

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.

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.

AST is essential for the selection of the appropriate antibiotic

Types Qualitative Quantitative 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.

Antibiotic Sensitivity Tests Diffusion Kirby-Bauer Method Stokes Method Dilution Tube Dilution Agar Dilution Diffusion & Dilution E-Test Qualitative Methods Quantitative Methods

Antibiotics for routine testing Staphylococcus Enterobacteriaceae Pseudomonas aeruginosa Intestinal Urinary Blood & Tissues Drugs Benzylpenicillin Oxacillin Erythromycin Tetracycline Chloramphenicol Ampicillin Cotrimoxazole Sulfonamides Trimethoprim Nitrofurantoin Cefalotin Gentamycin Piperacillin Tobramycin

Diffusion methods

Disk Diffusion Method 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.

The basics 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.

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

Kirby-Bauer Method

Mueller-Hinton Agar Antibiotic Disks Turbidity Standard Swabs MATERIALS

Mueller-Hinton Agar Medium containing beef infusion, peptone, and starch. Used primarily for the disk-diffusion method. Robust red algae (Solieria robusta) Source of Agar

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.

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.

Antibiotic Disks 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

Turbidity Standard 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.

Cotton Swabs 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

Procedure Application of Antibiotic Discs Incubation At 35⁰C for 16-18 hours Measurement of inhibition zone diameter

Kirby-Bauer Method Procedure 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.

2. Transfer this growth to a tube of saline.

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.

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.

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.

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

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

Application of Antibiotic Discs Incubation At 35⁰C for 16-18 hours Measurement of inhibition zone diameter

! 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.

Strips of multiple antibiotics can be tested in one go

Application of Antibiotic Discs Incubation At 35⁰C for 16-18 hours Measurement of inhibition zone diameter

Measurement of diameter 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 Using automated zone readers BIOMIC Aura Protozone

Interpretation of results

Using a template Standard templates are available for each antibiotic.

Using a template Result interpretation Susceptible Resistant 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 ruler 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.

Interpretative chart of zone sizes Antibiotic Diameter of zone inhibition (mm) Resistant Intermediate Susceptible Tetracycline <14 15-18 >19 Chloramphenicol <12 13-17 >18 Cotrimoxazole <10 11-15 ≥16 Nitrofurantoin 15-16 >17 Erythromycin <13 14-22 >23 Gentamycin 13-14 >15

Definitions Susceptible Resistant Intermediate 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.

Methicillin resistance in Staphylococcus aureus

size of zone Factors influencing 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.

Timing of disk application Plates 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

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.

Primary disk diffusion 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.

Dilution methods

Dilution methods 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.

MIC Tube dilution Agar dilution

Minimum inhibitory concentration 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.

Tube dilution Incubation At 37°C overnight Patient's organism is added to tubes containing decreasing amounts of the antibiotic Incubation At 37°C overnight Lowest concentration of drug that inhibits growth is the MIC

MIC

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

Agar dilution 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.

Tube and Agar Dilution Limitations Not easily automated Fresh reagents required Time-consuming

Broth microdilution 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.

E-Test

What is E-Test? Epsilometer Test Quantitative method of antibiotic sensitivity testing. Applies both dilution of antibiotic and diffusion of antibiotic into the medium.

Combines the principles of disk diffusion and agar dilution methods E-Test

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).

Procedure E-Test 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.

MIC

K Hari Krishnan Tirunelveli 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

E-Test Uses 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 Simple Accurate Reliable

AST of Fastidious organisms 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 Anaerobes 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

Automated Vitek Test Machine AST of Fungi Methods Disk diffusion Broth microdilution Automated Vitek Test Machine

AST can be done with automation

There is a growing need for Automation in Antibiotic sensitivity testing