Antibiotic sensitivity testing

Antibioic Sensitivity Testing
Aneesha K N

1. Antibiotic Sensitivity Testing Dr. ANEESHA.K.N MVSc SCHOLAR COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY

2. Synonyms Antibacterial Susceptibility Testing(ABST) Antibiotic Sensitivity Testing(AST) Antimicrobial Susceptibility Testing Antibiogram Test

3. OBJECTIVES To guide the clinician in selecting the best antibiotic To control the use of inappropriate antibiotics To distinguish the range of activity of an antibiotic To collect epidemiologic information on antibacterial resistance

4. Types

5. Diffusion methods

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

7. A concentration gradient of the antibiotic is formed by diffusion from the disk into the agar. Interact with the freshly seeded test organisms

8. If the organism is killed or inhibited by the concentration of the antibiotic ,there will be no growth in the immediate area around the disc:this is called the zone of inhibition Inhibition zone edge

9. Kirby –Bauer Disc D iffusion Method

10. 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 toxins released from bacteria, so that they cannot interfere with the antibiotics It is a loose agar: better diffusion of the antibiotics

11. Mueller-Hinton agar is considered best for routine susceptibility testing Reasons Batch to batch reproducibility Low in sulphonamides,trimethoprim and tetracycline inhibitors Satisfactory growth of most non fastidious pathogen

12. Antibiotic Disks Commercially available Stocks of antibiotic discs stored at - 14 C for 1 month Equilibriate with room temperature before application

13. Turbidity Standard

14. Kirby-Bauer Test /Sold agar test Fresh organism suspended in broth Swab organism all over the plate evenly Place discs containing specified concentration of different antibiotics on the plate

15. Incubate 37 C Measure diameter of Inhibition zone Use tables to assess if zone size indicates resistance or sensitivity to that antibiotic

16. Standard method 4-5 well isolated colonies are selected Top of the colonies are touched Growth transferred to broth Incubated at 35-37 C

17. Alternative method Colonies are selected Suspension is made in saline without pre –incubation Turbidity adjusted by comparison with 0.5 McFarland turbidity standard

18. Kirby-Bauer Method Procedure 1 . To prepare the inoculum from the primary culture plate,touch with a loop on the top of colonies

19. 2 . Transfer this growth to a tube of saline or broth

20. 3 .Compare the tube with the 0.5 McFarland turbidity standard and adjust the turbidity of the test suspension by adding more bacteria or more saline

21. 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 fluid level

22. 5 .Streak the swab all over the the surface in three directions 6 .Finally,pass the swab around the edge of agar surface

23. 7 .Leave the inoculated plates to stand for 3-5 minutes for absorption of excess moisture

24. 8 .The antibiotic discs are placed onto agar surface using -Sterile forceps -Antibiotic disc dispenser

25. Each disc is gently pressed to ensure complete contact with the agar surface Centre to centre distance between discs-24mm 6 discs per standard 90mm Petri dish

26. 9 .The plates should be placed in an incubator at 35 C within 15 minutes of preparation Incubated aerobically for 16-18 hours

27. Measurement of inhibition zone diameter Using a ruler Using a pair of calipers Transparent medium from the back of the plate Opaque medium over the surface of agar

28. Measurement of diameter Using automated zone readers -BIOMIC - Vitek

29. Factors affecting the zone of inhibition Size of the inoculum - Turbidity of medium-0.5 McFarland opacity standard Test medium - Mueller-Hinton agar or its modification( Isosensitest agar,Oxoid ) -It has good-batch-to-batch reproducibility, -Low in sulphonamide,trimethoprim and tetracycline inhibitors -Satisfactory growth of pathogen

30. Antimicrobial agent and its concentration in disc Incubation conditions -35 c for 16-18 hours under aerobic conditions Test bacterium -resistance or susceptibility Effects of Variation in Divalent Cations

31. Interpretation of Zone of Inhibition Double zone of inhibition Trimethoprim and sulphonamide Proteus –ignore the swarm

32. Results Susceptible Moderately susceptible Intremediate Resistant

33. Stokes Method Interpretation based on comparison between zones seen with the test organism & those of the known sensitive control.

34. T > C or T = C  Sensitive T< C  Resistance

35. Methicillin resistant Staphylococci Oxacillin disc is used Alternate method for standardisation Incubated at 30 C Multiple resistance can be used as an indication Confirmation -Inoculating the strain onto a quadrant of Mueller-Hinton agar supplemented with 4% NaCl and 6 microgram oxacillin per ml

36. A slight haze around the oxacillin disk indicates heteroresistance Heavy growth up to the oxacillin disk reflects homogenous resistance

37. Selection of antimicrobial discs Disc type Prediction Tetracycline Other tetracyclines Sulphisoxazole Sulphonamides Erythromycin Macrolides Clindamycin Lincomycin

38. Quality control To monitor precision and accuracy Potency of antimicrobial discs Satisfactory performance of the test medium

39. Control strains of bacteria Escherichia coli ATCC 25922 Staphylococcus aureus ATCC 25923 Pseudomonas aeruginosa ATCC 27853 Escherichia coli ATCC 35218 Enterococcus faecalis ATC 29212 or 33186-to test thymine or thymidine free medium

40. Antimicrobial discs Storage Routine use -Stored at 4 o C Beta lactam antibiotics-stored for upto one week only Long term storage- -14 c Don’t use expired discs Potency of new discs should be checked

41. Test medium Depth of agar -Mueller –Hinton agar plate depth-4mm pH -7.2 and 7.4 Sterility checking -30-35 c for 24hrs Storage -4 C for 7 days Ensure that medium is thymine or thymidine free Pseudomonas aeruginosa (ATCC 27853)-tested against tetracycline and aminoglycosides-to monitor calcium and magnesium ions

42. Zone size limits Control bacteria test should yield -No more than one zone size outside the control limits in 20 consecutive tests

43. 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

44. MINIMUM INHIBITORY CONCENTRATION Lowest concentration of drug that inhibits the growth of the bacteria Highest dilution of an antibiotic required to inhibit the growth of a bacterium

45. Tube dilution Organism is added to tubes containing decreasing amounts of the antibiotic Tubes are prepared by adding two fold dilutions of antibiotic in a series Each tube is inoculated with a suspension of test bacterium Incubation at 35-37 C for 24 hrs Lowest concentration of drug that inhibits growth is the MIC

46. Minimum bactericidal concentration The lowest concentration of drug that kills 99.9% of original inoculum Or Where a thousand fold reduction in bacterial number has occurred

47. Agar dilution/Agar incorporation tests 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

48. E-Test Epsilometer test Quantitative method of antibiotic sensitivity testing Applies both dilution of antibiotic and diffusion of antibiotic into the medium

49. Combines the principles of disk diffusion and agar dilution methods

50. PROCEDURE Apply E-test strip on an inoculated agar plate Immediate release of drug Incubation of plate Symmetrical inhibition of ellipse is produced The intersection of the inhibitory zone edge and the calibrated carrier strip indicates the MIC with inherent precision and accuracy

51. U ses Determining the MIC of fastidious ,slow –growing or nutritionally deficient micro- organisms,or for a specific type of patient or infection Detecting low levels of resistance Testing an antibiotic not performed in routine use or a new recently introduced antibiotic agent

52. AST of Fastidious organisms Most fastidious organisms do not grow well enough in routine antibiotic testing systems and require some type of supplementation 5-10% sheep blood agar - Streptococci or A ctinomyces pyogenes

53. AST of anaerobes Methods -Agar dilution - Broth dilution Medium - Brucella broth+vitamin K+Hemin+sheep blood Or - Brucella agar+vitamin K+ Hemin+sheep blood Incubation 48 hrs incubation at 35 C

54. Application of Computers in Antibacterial Susceptibility Testing ‘ WHONET ’- Software developed for the management of routine laboratory results by WHO U seful in supplying current guidelines, protocols to local laboratories, in identifying the clusters of resistant isolates and emerging outbreaks , research studies.

55. References Quinn PJ, Carter ME, Markey B & Carter GR. 1994. Clinical Veterinary Microbiology. Wolfe Publ. pp-95-102 NCCLS Manual of Antimicrobial Susceptibility Testing

56. THANK YOU..