ANTIBIOTICS SUSCEPTIBILITY TESTING Prepared by: Miss Norzawani Jaffar Bsc Hons Biomedical Science, UKM MICROBIOLOGY II: TOPIC 2 MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2 Lecture’s Outcome By the end of the lecture student should be able to discuss the importance and limitation of antibiotics susceptibility test. able to define Resistant sensitive/susceptible MIC MBC Able to describe methods use for susceptibility test Disk diffusion Dilution methods E-TEST MICROBIOLOGY II: TOPIC 2

Antimicrobial Susceptibility Testing In the treatment and control of infectious diseases, especially when caused by pathogens that are often drug resistant, susceptibility (sensitivity) testing is used to select effective antimicrobial drugs. MICROBIOLOGY II: TOPIC 2 MICROBIOLOGY II: TOPIC 2

Limitation of Antimicrobial Susceptibility Testing Susceptibility testing measure antimicrobial activity against bacteria under laboratory condition (in vitro), not in the patient (in vivo activity). It cannot be assumed therefore, that an antimicrobial which kills or prevents an organism from growing in vitro will be a successful treatment. MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2 Antimicrobial treatment- considering the patient’s clinical condition (eg. Liver or kidney disease), the type of infection, any history of drug hypersensitivity, age of patient and whether a patient is pregnant. Necessary to know the activity of the different drugs including their rates of absorption, diffusion in the tissues, metabolism, excretion, and also possible toxicity and effects on the patient’s normal microbial flora. MICROBIOLOGY II: TOPIC 2

Sensitivity/Susceptibility Test Laboratory antimicrobial susceptibility testing can be performed using: Disk diffusion technique Broth / Agar Dilution technique E-test MICROBIOLOGY II: TOPIC 2 MICROBIOLOGY II: TOPIC 2

Minimum Inhibitory Concentration (MIC) Definition: The lowest antibiotic concentration of tested antibiotic that inhibit the growth of the bacteria. MICROBIOLOGY II: TOPIC 2 MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2 DISK DIFFUSION METHOD Paper discs containing known concentrations of antibiotics are applied to the surface, and the plate is incubated at 35°C for 16-18 hrs. The appearance of a zone of inhibition surrounding the disc is indicative of sensitivity. Strains susceptible to antimicrobial are inhibited at a distance from the disc whereas resistant strains have smaller zones of inhibition or grow up to edge of the disc. By comparing the diameter of the zones to a standard table, one may determine if the test organism is susceptible, or resistant to the antibiotic. If the organism is susceptible, it is likely to be killed in the blood stream of the patient if that concentration of the drug is reached. Resistance indicates that the antibiotic will not be effective at that concentration in the blood stream. MICROBIOLOGY II: TOPIC 2 MICROBIOLOGY II: TOPIC 2

Modified Kirby-Bauer susceptibility testing technique REQUIRED MUELLER HINTON AGAR Prepare and sterilize the medium. The pH of the medium should be 7.2-7.4. Pour into 90 mm diameter sterile petri dishes to a depth of 4 mm (about 25 ml per plate). Care must be taken to pour the plates on a level surface so that the depth of medium is uniform. MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2 ANTIMICROBIAL DISC Paper antimicrobial discs are commercially available from most manufacturers of culture media. Most paper disc can be used for 1 yr or longer from the date of manufacture providing they store correctly (-20°C, or working stock at 2-8°C in an airtight container with indicating desiccant. Expired disc should not be used. Quality control of disc is essential. -About 1 hr before use, the working stock of disc should be allowed to warm to room temperature, protected from direct sunlight. MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2 STANDARD INOCULUM/BACTERIA SUSPENSION 5 colonies from an agar plate culture grown overnight are inoculated into 5 ml of suitable liquid broth and the culture is incubated at 35°C until a turbidity equivalent to 0.5 McFarland (approx 108 colony-forming-unit (CFU)/ml is reached, which can occur within a few hours. The turbidity of the culture is adjusted with sterile saline serum or broth to reach 0.5-McFarland standard. This can be checked using an optical device or by visual inspection. MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2

Interpretation of zone sizes Using interpretative Chart, interpret the zones sizes of each antimicrobial, reporting the organism as; Resistant Intermediate Moderately Susceptible Susceptible. MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2

Quality Control when performing Kirby-Bauer disc Diffusion Technique Purchase Mueller Hinton agar from a reliable source. Check its pH. Ensure plates contain the correct amount of agar. Prepare carefully the inoculum of the test and control organism to ensure growth is confluent. Renew the turbidity standard every few months. Use disc containing the correct amount of antimicrobial. Store disc correctly and do not use them beyond their expiry date. Place disc or tables correctly on the plate, not too close to each other or to the rim of the plate. Use appropriate control strains Regularly check the temperature of the incubator to ensure test are incubated at 35°C. Measure inhibition zones carefully.. Zone diameters with control strains should be with in the limits published by NCCLS MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2 DILUTION METHOD A weakness of the diffusion method is that it does not determine whether a drug is bactericidal and not just bacteriostatic. A dilution method is often useful in determining the MIC and Minimal Bactericidal Concentration (MBC) of antimicrobial drug. The MIC is determines by making sequence of decreasing concentration of the drug in a broth, which is the inoculated with the test bacteria. MICROBIOLOGY II: TOPIC 2 MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2 After overnight incubation, the MIC is reported as the lowest concentration of antimicrobial required to prevent visible growth. The wells that do not show growth (higher concentration than the MIC) can be cultured in broth/agar free of the drug for MBC determination. If growth occurs in this broth, the drug was not bactericidal, and the MBC can be determined. MIC and MBC determination is important because it avoids the excessive use of expensive antibiotics and minimizes the chance of toxic reactions that larger-than-necessary doses might cause. MICROBIOLOGY II: TOPIC 2 MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2 E-TEST METHOD Confirmation of unusual resistance profiles can be performed using the E-test method. E-test consists of a predefined gradient of antibiotic concentrations on a plastic strip and is used to determine the Minimum Inhibitory Concentration (MIC) of antibiotics, antifungal agents and antimycobacterial agents E-test  is a more advance diffusion method that enables a lab technician to estimate the MIC. MIC is read directly from the scale in micrograms per milliliter (µg/ml) at the point where the inhibition elipse edge intersects the strips. MICROBIOLOGY II: TOPIC 2 MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2 Etest is recognized as a cost-effective tool for generating MICs across 15 dilutions. Over 100 antibiotics are now available in the product range for testing of aerobic bacteria and fastidious organisms such as pneumococci, haemophilus, H. pylori, meningococci, gonococci, anaerobes, fungi and mycobacteria.  Etest promotes the rational use of antibiotics by providing results to guide the therapy of individual patients and to validate empiric drug regimens. MICROBIOLOGY II: TOPIC 2 MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2 The E-test, a diffusion method that determines antibiotic sensitivity and estimates minimal inhibitory concentration (MIC). The plastic strip, which is places on agar surface inoculated with test bacteria, contains increasing gradient of antibiotic. MICROBIOLOGY II: TOPIC 2 MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2 END Q&A MICROBIOLOGY II: TOPIC 2

MICROBIOLOGY II: TOPIC 2 Take note from http://norazlicucst.weebly.com MICROBIOLOGY II: TOPIC 2