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drug susceptibility testing by Pranzly.pptx

This document discusses drug susceptibility testing, which determines if microbes like bacteria or fungi can be inhibited by antimicrobial drugs. There are two main types of testing - qualitative for less serious infections, and quantitative for serious infections. The Kirby-Bauer disk diffusion method is commonly used, involving placing disks with antibiotics on agar plated with a test organism. The size of the inhibition zone is measured to determine resistance or susceptibility. Factors like incubation time and temperature affect results. Minimum inhibitory concentration methods like broth dilution are also used but are more complex. Computer programs help analyze and track susceptibility data.

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DRUG SUSCEPTIBILITY
TESTING
SUBMITTED BY PRANZLY
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.
drug susceptibility testing by Pranzly.pptx
drug susceptibility testing by Pranzly.pptx
drug susceptibility testing by Pranzly.pptx
OBJECTIVE OF ANTIBIOTIC SUSCEPTIBILITY TESTING

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drug susceptibility testing by Pranzly.pptx

  • 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.
  • 6. OBJECTIVE OF ANTIBIOTIC SUSCEPTIBILITY TESTING
  • 7. 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)
  • 10. Disk diffusion method  Principle • Disk impregnated with a defined amount of antibiotic are placed on agar medium uniformly seeded with the test organism.
  • 11. KIRBY-BAUER DISC DIFFUSION METHOD  . Material required Mueller Hinton agar Antibiotic disc Turbidity standard swabs
  • 12. 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
  • 13. ANTIBIOTIC DISC  Commercially available  Stocks of antibiotic disc stored at - 14° C for 1 month  Equilibriate with room temperature before application
  • 14. Turbidity Standard 0.5 ml solution A (0.048 M BaCl2) 99.5 ml solution B (0.36 N H2SO4)  Mc Farland  0.5  Turbidity standard
  • 15. KIRBY-BAUER test / sold agar test 1. Fresh organism suspended in broth 2. Swab organism all over the plate evenly 3. Place disc containing specified concentration of different antibiotics on the plate
  • 16. . 4. Incubate at 37°C 5. Measure diameter of Inhibition zone 6. Use tables to assess if zone size indicates resistance or sensitivity to that antibiotic.
  • 17. PROCEDURE OF KIRBY-BAUER TEST 1. TO PREPARE THE INOCULUM FROM THE PRIMARY CULTURE PLATE, TOUCH WITH A LOOP ON THE TOP OF COLONIES
  • 18. 2. TRANSFER THIS GROWTH TO A TUBE OF SALINE OR BROTH
  • 19. 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
  • 20. 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.
  • 21. 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.
  • 22. 8. THE ANTIBIOTIC DISC ARE PLACED ONTO AGAR SURFACE USING -  STERILE FORECEPS • AUTOMATED DISC DISPENSER
  • 23. • 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
  • 24. 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
  • 26. FACTORS AFFECTING THE ZONE OF INHIBITION  Size of the inoculum o Turbidity of medium -0.5 Mc Farland opacity standard  Test medium o Mueller Hinton agar on its modification (isosensitest agar, oxoid) o Its has good batch to batch reproducibility o Low in sulphonamide, trimethoprim and tetracycline inhibitors o Satisfactory growth of pathogen.  Antimicrobial agent and its concentration in disc  Incubation conditions o 35C for 16-18h under aerobic conditions  Test bacterium – resistance or susceptibility  Effects of variation in divalent cations.
  • 27. 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
  • 29. 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
  • 31. DILUTION METHOD (REFERENCE METHOD)  AGAR DILUTION (Solid media)  BROTH DILUTION (Liquid media) o Microbroth test (petri plates) o Macrobroth test (test tubes)
  • 32. 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
  • 33. • 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.
  • 34. AGAR DILUTION (Solid media)  Medium – cation adjusted Muller Hinton agar Prepare serial dilution Add standard inoculum Spread it Look for lowest concentration of prevent the appearance of colonies. Advantage – fastidious bacteria can be tested - can be used for anaerobes Disadvantage- cumbersome procedure.
  • 35. 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
  • 36. • 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
  • 37. 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.
  • 38. 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