drug susceptibility testing by Pranzly.pptx

antibiotic susceptibility testing
disk diffusion method
Kirby Bauer disc diffusion method
Stokes method
diluted method
agar dilution
test tube dilution
epsilometer test (E test)

1. DRUG SUSCEPTIBILITY TESTING SUBMITTED BY PRANZLY

2. INTRODUCTION Drug Susceptibility- Susceptibility is a term used when microbe such as bacteria and fungi are unable to grow in the presence of one or more antimicrobial drugs. Susceptibility testing is performed on bacteria or fungi causing an individual’s infection after they have been recovered in a culture of the specimen . Bacteria and fungi have the potential to develop resistance to antibiotics and antifungal drugs at any time. This means that antibiotics once used to kill or inhibit their growth may no longer be effective.

3. OBJECTIVE OF ANTIBIOTIC SUSCEPTIBILITY TESTING

4. TYPES QUALITATIVE For testing of isolates from “healthy” patients with intact immune defences. 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 patients groups such as immunocompromised patients ( eg. Transplant patients)

5. Diffusion methods

6. Disk diffusion method Principle Disk impregnated with a defined amount of antibiotic are placed on agar medium uniformly seeded with the test organism.

7. KIRBY-BAUER DISC DIFFUSION METHOD .

8. MUELLER HINTON AGAR Non selective, Non- differential medium Used primarily for the disk diffusion method Medium containing beef extract casein hydrolysate, starch, and agar. Starch absorb toxin released from bacteria, so that they cannot interfere with the antibiotics. It is a loose agar: better diffusion of the antibiotics. Robust red agar ( Solieria robusta ) Source of agar

9. ANTIBIOTIC DISC Commercially available Stocks of antibiotic disc stored at -14° C for 1 month Equilibriate with room temperature before application

10. Turbidity Standard 0.5 ml solution A (0.048 M BaCl 2 ) 99.5 ml solution B (0.36 N H 2 SO 4 ) Mc Farland 0.5 Turbidity standard

11. KIRBY-BAUER test / sold agar test

12. .

13. PROCEDURE OF KIRBY-BAUER TEST 1. TO PREPARE THE INOCULUM FROM THE PRIMARY CULTURE PLATE, TOUCH WITH A LOOP ON THE TOP OF COLONIES

14. 2. TRANSFER THIS GROWTH TO A TUBE OF SALINE OR BROTH

15. 3. COMPARE THE TUBE WITH THE 0.5 MAC FARLAND TURBIDITY STANDARD AND ADJUST THA TURBIDITY OF THE TEST SUSPENSION BY ADDING MORE BACTERIA OR MORE SALINE

16. 4. INOCULATE THE PLATE 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 fluid level.

17. 5. STREAK THE SWAB ALL OVER THE SURFACE IN THREE DIRECTION. 6. FINALLY PASS THE SWAB AROUND THE EDGE OF AGAR SURFACE 7. LEAVE THE INOCULATED PLATES TO STAND FOR 3-5 MINUTES FOR ABSORPTION OF EXCESS MOISTURE.

18. 8. THE ANTIBIOTIC DISC ARE PLACED ONTO AGAR SURFACE USING - STERILE FORECEPS AUTOMATED DISC DISPENSER

19. Each disc is gently pressed to ensure complete contact with the agar surface Centre to centre distance between disc 24mm 6 disc per standard 90mm petri disc . 9. THE PLATES SHOULD BE PLACED IN AN INCUBATOR AT 35°C WITHIN 15 MINUTES OF PREPARATION. Incubated aerobically for 16-18 hours

20. MEASUREMENT OF INHIBITION ZONE DIAMETER USING RULER USING A PAIR OF CALLIPERS. TRANSPARENT MEDIUM FROM THE BACK OF THE PLATE OPAQUE MEDIUM OVER THE SURFACE OF AGAR

21. FACTORS AFFECTING THE ZONE OF INHIBITION Size of the inoculum Turbidity of medium -0.5 Mc Farland opacity standard Test medium Mueller Hinton agar on its modification (isosensitest agar, oxoid) Its has good batch to batch reproducibility Low in sulphonamide, trimethoprim and tetracycline inhibitors Satisfactory growth of pathogen. Antimicrobial agent and its concentration in disc Incubation conditions 35C for 16-18h under aerobic conditions Test bacterium – resistance or susceptibility Effects of variation in divalent cations.

22. Results Results of the testing are usually reported as: Susceptible — likely, but not guaranteed to inhibit the pathogenic microbe; may be an appropriate choice for treatment Intermediate — may be effective at a higher dosage, or more frequent dosage, or effective only in specific body sites where the antibiotic penetrates to provide adequate concentrations Resistant — not effective at inhibiting the growth of the organism in a laboratory test; may not be an appropriate choice for treatment

23. STOKES METHOD Interpretation based on comparison between zones seen with test organism & those of the known sensitive control. Only followed in certain European countries. On the same plate- antibiotic disc, control strain and test strain placed. Incubated at same conditions Therefore, no need of any tables to compare Advantage – easy to do Disadvantage- MIC cannot be determined T = zone of inhibition of test organism C= zone of inhibition of control organism

24. DILUTION METHOD (REFERENCE METHOD) AGAR DILUTION (Solid media) BROTH DILUTION (Liquid media) Microbroth test (petri plates) Macrobroth test (test tubes)

25. BROTH DILUTION METHOD MEDIUM- NUTRIENT BROTH E.g. UTI Escherichia coli overnight broth culture in peptone standard inoculum In macrobroth dilution method ,we will take same amount of nutrient broth in series of test tubes Serial dilution of antibiotics prepared in nutrient broth control with no antibiotics Add 1 ml of standard inoculum to all test tubes Incubate overnight at 37 °C

26. Control – maximum growth (maximum turbidity) As the concentration of antibiotics increases, turbidity decreases At a specific conc. – no turbidity – minimum inhibitor concentration (MIC) MIC- lowest concentration of drug at which there is no visible growth. If the whole process is done in petri dish- microbroth dilution Disadvantage -cumbersome procedure -Fastidious organisms cannot ne tested.

27. AGAR DILUTION (Solid media) Medium – cation adjusted Muller Hinton agar Advantage – fastidious bacteria can be tested - can be used for anaerobes Disadvantage- cumbersome procedure.

28. E-TEST/ EPSILOMETER TEST Combination of dilution and disc diffusion method MEDIUM - Muller Hinton Agar STANDARD solution of 0.5 mc farland turbidity Instead of discs, plating strips impregnated with graded concentration of antibiotics (serial dilution) along its length

29. The concentration at which the zone of inhibition intersect the plastic will determine the mic Advantage - Easy to do, quantitative method Disadvantage - Sometimes results may confuse- go for dilution or disc diffusion

30. APPLICATION OF COMPUTERS IN ANTIBACTERIAL SUSCEPTIBILITY TESTING WHONET – software developed for the management of routine laboratory results by WHO. Useful in supplying current guidelines, protocols to local laboratories, in identifying the clusters of resistant isolates and emerging outbreaks, research studies.

31. BIBLIOGRAPHY Street, T. (Updated 2014 March 13). Antimicrobial Susceptibility. Medscape. Available online at https://emedicine.medscape.com/article/2103786-overview ? L.Barth Reller, Melvin Weinstein, James H. Jorgensen, Mary Jane Ferraro ANTIMICROBIAL SUSCEPTIBILITY TESTING: A review of general principle and contemporary practices. (Updated 2009 December 01) Issue 11, Volume 49.()PG. 11749-1755 IMAGES SOURCE Reseachgate.net