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Antibiotic Sensitivity Tests

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Antibiotic Sensitivity Tests

Hari Krishnan K

Published in: Health & Medicine
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Antibiotic Sensitivity Tests

  1. 1. K Hari Krishnan II MBBS (2009-’11) Tirunelveli Medical College Tirunelveli, Tamilnadu, India
  2. 2. A test done to check the effectiveness of a drug against a bacterium and to select the best drug that acts against the bacterium. K Hari Krishnan Tirunelveli Medical College
  3. 3. The in vitro testing of bacterial cultures with antibiotics to determine . K Hari Krishnan Tirunelveli Medical College
  4. 4. Antibiotic Sensitivity Testing K Hari Krishnan Tirunelveli Medical College
  5. 5. To guide the clinician in selecting the best antibiotic agent for an individual patient. To control the use of 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. K Hari Krishnan Tirunelveli Medical College
  6. 6. 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. K Hari Krishnan Tirunelveli Medical College
  7. 7. AST is essential for the selection of the K Hari Krishnan Tirunelveli Medical College
  8. 8. – For the testing of isolates from “healthy” patients with intact immune defenses. – For such as uncomplicated urinary tract infections. – 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. K Hari Krishnan Tirunelveli Medical College
  9. 9. Antibiotic Sensitivity Tests Diffusion Kirby-Bauer Method Stokes Method Dilution Tube Dilution Agar Dilution Diffusion & Dilution E-Test Qualitative Methods Quantitative Methods K Hari Krishnan Tirunelveli Medical College
  10. 10. Staphylococcus Enterobacteriaceae Pseudomonas Blood & Tissues Intestinal Urinary aeruginosa Drugs Benzylpenicillin Oxacillin Erythromycin Tetracycline Chloramphenicol Ampicillin Chloramphenicol Cotrimoxazole Tetracycline Sulfonamides Trimethoprim Cotrimoxazole Ampicillin Nitrofurantoin Tetracycline Ampicillin Chloramphenicol Cotrimoxazole Tetracycline Cefalotin Gentamycin Piperacillin Gentamycin Tobramycin K Hari Krishnan Tirunelveli Medical College
  11. 11. K Hari Krishnan Tirunelveli Medical College
  12. 12. – 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. K Hari Krishnan Tirunelveli Medical College
  13. 13. The contained in a reservoir is allowed to 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 diffuses out of the disk to form the gradient. – The starts to divide and grow and progresses toward a critical mass of cells. K Hari Krishnan Tirunelveli Medical College
  14. 14. 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. K Hari Krishnan Tirunelveli Medical College
  15. 15. K Hari Krishnan Tirunelveli Medical College
  16. 16. Medium containing beef infusion, peptone, and starch. Used primarily for the disk-diffusion method. Robust red algae (Solieria robusta) Source of Agar K Hari Krishnan Tirunelveli Medical College
  17. 17. Mueller-Hinton agar is considered the for routine susceptibility testing of nonfastidious bacteria. 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. K Hari Krishnan Tirunelveli Medical College
  18. 18. to 45–50 ⁰C and pour into the plates. Allow to , to a depth of approximately 4 mm. – A 9-cm plate requires approximately 25 ml of medium. When the agar has solidified, 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. K Hari Krishnan Tirunelveli Medical College
  19. 19. 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. K Hari Krishnan Tirunelveli Medical College
  20. 20. K Hari Krishnan Tirunelveli Medical College
  21. 21. Prepared by pouring 0.6 ml of a 1% (10 g/l) solution of into a 100-ml graduated cylinder, and filling to 100ml with 1% (10 ml/l) . K Hari Krishnan Tirunelveli Medical College
  22. 22. 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. K Hari Krishnan Tirunelveli Medical College
  23. 23. K Hari Krishnan Tirunelveli Medical College
  24. 24. Application of Antibiotic Discs Incubation At 35⁰C for 16-18 hours Measurement of inhibition zone diameter K Hari Krishnan Tirunelveli Medical College
  25. 25. To prepare the inoculum from the primary culture plate, , of similar appearance, of the organism to be tested. K Hari Krishnan Tirunelveli Medical College
  26. 26. Transfer this growth to a tube of saline. K Hari Krishnan Tirunelveli Medical College
  27. 27. Compare the tube with the turbidity standard and of the test suspension to that of the standard by adding more bacteria or more sterile saline. K Hari Krishnan Tirunelveli Medical College
  28. 28. Inoculate the plates by 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. K Hari Krishnan Tirunelveli Medical College
  29. 29. the swab all over the surface of the medium three times, rotating the plate through an angle of 60⁰ after each application. Finally, pass the swab round the edge of the agar surface. K Hari Krishnan Tirunelveli Medical College
  30. 30. Leave the inoculum to for a few minutes at room temperature with the lid closed. K Hari Krishnan Tirunelveli Medical College
  31. 31. The may be placed on the inoculated plates using – sterile forceps. – a template. – a sterile needle tip. – antibiotic disc dispenser. K Hari Krishnan Tirunelveli Medical College
  32. 32. Application of Antibiotic Discs Incubation At 35⁰C for 16-18 hours Measurement of inhibition zone diameter K Hari Krishnan Tirunelveli Medical College
  33. 33. A maximum of 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 of preparation. Temperatures results for oxacillin/methicillin.  an atmosphere of . Disks should after diffusion. K Hari Krishnan Tirunelveli Medical College
  34. 34. K Hari Krishnan Tirunelveli Medical College
  35. 35. Application of Antibiotic Discs Incubation At 35⁰C for 16-18 hours Measurement of inhibition zone diameter K Hari Krishnan Tirunelveli Medical College
  36. 36. Using a ruler – on the under-surface of the plate containing transparent medium. Using a pair of calipers – on the plate containing opaque medium. K Hari Krishnan Tirunelveli Medical College
  37. 37. Using automated zone readers – BIOMIC – Aura – Protozone K Hari Krishnan Tirunelveli Medical College
  38. 38. K Hari Krishnan Tirunelveli Medical College
  39. 39. Standard templates are available for each antibiotic. K Hari Krishnan Tirunelveli Medical College
  40. 40. Result interpretation • When the edge of the zone of inhibition is the black circle. • When there is no zone, or when it lies the white circle. • When the edge of the zone of inhibition lies the black circle. K Hari Krishnan Tirunelveli Medical College
  41. 41. 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. K Hari Krishnan Tirunelveli Medical College
  42. 42. 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 <14 15-16 >17 Erythromycin <13 14-22 >23 Gentamycin <12 13-14 >15 K Hari Krishnan Tirunelveli Medical College
  43. 43. – 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. – 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. – 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. K Hari Krishnan Tirunelveli Medical College
  44. 44. K Hari Krishnan Tirunelveli Medical College
  45. 45. – Too 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 inoculum • Inhibition zones will be smaller Relatively susceptible strains may then be falsely reported as resistant. K Hari Krishnan Tirunelveli Medical College
  46. 46. • of disk application K Hari Krishnan Tirunelveli Medical College
  47. 47. of incubation – If the temperature is , the time required for effective growth is extended and result. Potency of – If the owing to deterioration during storage, the . K Hari Krishnan Tirunelveli Medical College
  48. 48. 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. K Hari Krishnan Tirunelveli Medical College
  49. 49. K Hari Krishnan Tirunelveli Medical College
  50. 50. Used to determine the of antibiotic to inhibit or kill the microorganism. Achieved by dilution of antibiotic in either agar or broth media. K Hari Krishnan Tirunelveli Medical College
  51. 51. K Hari Krishnan Tirunelveli Medical College
  52. 52. The lowest concentration of drug that 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. K Hari Krishnan Tirunelveli Medical College
  53. 53. 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 K Hari Krishnan Tirunelveli Medical College
  54. 54. MIC K Hari Krishnan Tirunelveli Medical College
  55. 55. The lowest concentration of drug that the bacteria isolated from the patient. K Hari Krishnan Tirunelveli Medical College
  56. 56. Serial dilutions of the drug are prepared in agar and poured into plates. – Many strains can be inoculated on each plate containing an antibiotic dilution. K Hari Krishnan Tirunelveli Medical College
  57. 57. K Hari Krishnan Tirunelveli Medical College
  58. 58.  Broth microdilution plate contains – Each row: • standard dilutions of eight in each row (denoted by letters A-H). – Each column • contains a standard 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. K Hari Krishnan Tirunelveli Medical College
  59. 59. K Hari Krishnan Tirunelveli Medical College
  60. 60. K Hari Krishnan Tirunelveli Medical College
  61. 61. Epsilometer Test Quantitative method of antibiotic sensitivity testing. Applies both dilution of antibiotic and diffusion of antibiotic into the medium. K Hari Krishnan Tirunelveli Medical College
  62. 62. Combines the principles of disk diffusion and agar dilution methods Diffusion Dilution E-Test K Hari Krishnan Tirunelveli Medical College
  63. 63. 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). K Hari Krishnan Tirunelveli Medical College
  64. 64. The intersection of the inhibitory zone edge and the calibrated carrier strip indicates the MIC with inherent precision and accuracy. K Hari Krishnan Tirunelveli Medical College
  65. 65. MIC K Hari Krishnan Tirunelveli Medical College
  66. 66. K Hari Krishnan Tirunelveli Medical College
  67. 67. Over 100 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 K Hari Krishnan Tirunelveli Medical College
  68. 68. Determining the MIC of , 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 . Testing an antibiotic not performed in routine use or a new, antibiotic agent.  an equivocal AST result. K Hari Krishnan Tirunelveli Medical College
  69. 69. K Hari Krishnan Tirunelveli Medical College
  70. 70. 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 K Hari Krishnan Tirunelveli Medical College
  71. 71. 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 – Agar dilution – Broth microdilution – Brucella agar (or) – Broth supplemented with vitamin K and hemin K Hari Krishnan Tirunelveli Medical College
  72. 72. – Disk diffusion – Broth microdilution Automated Vitek Test Machine K Hari Krishnan Tirunelveli Medical College
  73. 73. K Hari Krishnan Tirunelveli Medical College
  74. 74. K Hari Krishnan Tirunelveli Medical College
  75. 75. Antibiotic Sensitivity Testing K Hari Krishnan Tirunelveli Medical College

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