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Naresh ast

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Antimicrobial Susceptibility Testing

Publicado en: Salud y medicina
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Naresh ast

  1. 1. ANTIMICROBIAL SUSCEPTIBILITYTESTING Mr. Naresh Pokhrel M.Sc Clinical Microbiology Department of Microbiology Father Muller Medical College, Mangalore ANTIBIOTIC SUSCEPTIBILITY TESTING
  2. 2. Modern era in Antibiotics begins with Fleming. ANTIBIOTIC SUSCEPTIBILITY TESTING
  4. 4. Overview:- Introduction Goal of AST Types Disc Diffusion Methods (Qualitative) Medium (Mueller-Hinton Agar) Kirby-Bauer Disk Diffusion Method Troubleshooting the Reading of Disk Diffusion Tests Dilution Tests Epsilometer or E-test Automated Antimicrobial Susceptibility Tests Molecular Methods Quality Control In AST ANTIBIOTIC SUSCEPTIBILITY TESTING
  5. 5. Antimicrobial Susceptibility Testing: A laboratory test which determines how effective antibiotic therapy is against a bacterial infections. Antibiotic sensitivity testing will control the use of Antibiotics in clinical practice. Testing will assist the clinicians in the choice of drugs for the treatment of infections. ANTIBIOTIC SUSCEPTIBILITY TESTING
  6. 6. Goal of AST 1.The identification of relevant antibiotics to specific pathogens in exudates and body fluids collected from patients. 2. Sensitivity tests done to determine the degree of sensitivity or resistance of pathogens isolated from patient to an appropriate range of antimicrobial drugs. 3. Assay of the concentration of an administered drug in the blood or body fluid of patient required to control the schedule of dosage. ANTIBIOTIC SUSCEPTIBILITY TESTING
  8. 8. Disc Diffusion Methods (Qualitative) Disk diffusion susceptibility tests became standardized in the United States based on the work of Bauer, Kirby, and coworkers. This method is still widely used because of its easy of use and lower cost compared to other methods. ANTIBIOTIC SUSCEPTIBILITY TESTING
  9. 9. ANTIBIOTIC SUSCEPTIBILITY TESTING Method Description. The basic principle of the standardized disk diffusion susceptibility test is illustrated below, All disk diffusion methods rely on diffusion of drug released from an impregnated disk through agar.
  10. 10. The principle of antibiotic diffusion in agar. The concentration of antibiotic decreases as the distance from the disk increases. ANTIBIOTIC SUSCEPTIBILITY TESTING
  11. 11. Why mueller-hinton agar is used in routine antibiotic susceptibility testing (AST) ? Antimicrobial Susceptibility testing using disk diffusion Mueller- Hinton agar is the best medium for routine antibiotic susceptibility testing (AST) because of the following reasons:  It shows acceptable batch-to-batch reproducibility for susceptibility testing. ANTIBIOTIC SUSCEPTIBILITY TESTING
  12. 12. It has minimal inhibitory effect on sulfonamide and trimethoprim. Hence, these antibiotics are better tested in MHA than any other media. It supports satisfactory growth of most nonfastidious pathogens. ANTIBIOTIC SUSCEPTIBILITY TESTING
  13. 13. Modifications of Mueller Hinton agar Mueller Hinton agar medium supplemented with 5% sheep blood is recommended for determining the antimicrobial susceptibility of Streptococcus pneumoniae Neisseria meningitidis Haemophilus test medium (HTM) is the preferred medium for the antimicrobial susceptibility testing of H. influenzae using modified Kirby Bauer disk diffusion method. HTM medium consists of following ingredients: thymidine free MHA supplemented with 15 μg/ml NAD, 15 μg/ml bovine hemin, and 5 mg/ml yeast extract. ANTIBIOTIC SUSCEPTIBILITY TESTING
  14. 14. Preparation Of MHA Measure 38 g of medium (or the components listed above) in 1 liter of purified water. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the components. Autoclave at 121°C for 15 minutes. Cool to 45°C Pour cooled Mueller Hinton Agar into sterile petri dishes on a level, horizontal surface to give uniform depth. ANTIBIOTIC SUSCEPTIBILITY TESTING
  15. 15. Allow to solidify at room temperature. Check prepared Mueller Hinton Agar to ensure the final pH is 7.3 ±1 at 25°C. Note: If the pH is <7.2 certain drugs will appear to lose potency (aminoglycosides, quinolones, macrolides), while other agents may appear to have excessive activity (tetracycline). If the pH is >7.4, the opposite results may occur. Prepared media can be stored at 4 to 8°C. Mueller-Hinton agar is stable for approximately 70 days from the date of preparation. ANTIBIOTIC SUSCEPTIBILITY TESTING
  16. 16. ANTIBIOTIC SUSCEPTIBILITY TESTING Note: The plates must be poured to a depth of 4 mm (approximately 25 ml of liquid agar for 100-mm plates and 60 ml of liquid agar for 150-mm plates, but in any case to a measured depth of 4 mm).
  17. 17. Principle of MHA Mueller Hinton Media contains Beef Extract, Acid Hydrolysate of Casein, Starch and Agar.  Beef Extract and Acid Hydrolysate of Casein provide nitrogen, vitamins, carbon, amino acids, sulphur and other essential nutrients. Starch is added to absorb any toxic metabolites produced. Starch hydrolysis yields dextrose, which serves as a source of energy. Agar is the solidifying agent. ANTIBIOTIC SUSCEPTIBILITY TESTING
  18. 18. lnoculum Isolated pure colonies of the test organism are inoculated in a suitable liquid medium (peptone water broth) and incubated at 35-37'C for 4- 6 hours. The density of the organisms in broth is adjusted to 0.5 McFarland opacity standard tube. (1.5 * 108 cfu/mL) ANTIBIOTIC SUSCEPTIBILITY TESTING
  19. 19. Lawn culture: The broth is then inoculated on the medium by sterile swabs. The ideal inoculum after overnight incubation gives an even semi confluent grow. Too heavy inoculum reduces the size of inhibition zones. ANTIBIOTIC SUSCEPTIBILITY TESTING
  20. 20. Antibiotic Disk Antibiotic disks are available commercially or prepared in house. Sterile filter paper (whatman number 1) disks of 6 mm diameter are impregnated with standard quantity of antibiotic solution. ANTIBIOTIC SUSCEPTIBILITY TESTING
  21. 21. Calculation of stock solution 1000 --------- * V * C = W P P – Potency of preparation V – Volume required (mL) C – Final Concentration of solution W – Weight of antibiotic to be dissolved in V ANTIBIOTIC SUSCEPTIBILITY TESTING
  22. 22. Choice of Antibiotic Disk Antibiotics should likely to be used for therapy. Organism against which the drug has to be tested. ANTIBIOTIC SUSCEPTIBILITY TESTING
  23. 23. ANTIBIOTIC SUSCEPTIBILITY TESTING Local prescribing habits of the antimicrobial agents. Resistant pattern of the locally prevalent pathogen. Cost, toxicity, pharmacokinetics, and spectrum of activity of an antimicrobial agent for the management of illness in a particular patient.
  24. 24. First-line drugs: Those antibiotics that are commonly used in a locality with respect to the organism isolated should be tested first. Second-line drugs: include the panel of those antibiotics for which the prescription is restricted only to special circumstances. ANTIBIOTIC SUSCEPTIBILITY TESTING
  25. 25. Kirby-Bauer Disk Diffusion Method A cotton swab is dipped into inoculum and squeezed to drain out the excess fluid. Then the swab is inoculated on to the Mueller-Hinton agar plate by streaking the swab three times over the entire agar surface. ANTIBIOTIC SUSCEPTIBILITY TESTING
  26. 26. ANTIBIOTIC SUSCEPTIBILITY TESTING After drying the surface of agar place for 3-5 minutes the antibiotic disks are applied using either sterile forceps or multidisk dispenser.  Disks should not to be placed closer than 20,mm(center co center) on the MHA plate. Ordinarily, maximum up to 6 disks can be applied on a 100 mm plate (five in the periphery and one in the center)
  27. 27. The plates are then incubated at 37'C for 16- 18 hours. When tested for MRSA. result should be read only after 24 hours of incubation.  The zones of complete growth of inhibition around each of the disks are measured using a ruler or Vernier caliper. The diameter of the disk is also included in this measurement.  The interpretation of zone size into sensitive, intermediate or resistant is based on the standard zone size interpretation chart . ANTIBIOTIC SUSCEPTIBILITY TESTING
  28. 28. ANTIBIOTIC SUSCEPTIBILITY TESTING  Control strains should be tested each time when a new batch of disks or Mueller-Hinton agar is used.
  30. 30. Storage of discs : Refrigerate at 2 to 8 deg C in dessicators. Beta lactam class drugs should be frozen. The drugs should be kept outside at RT 1 to 2 hours before work The dispensing apparatus which is used to deliver the drugs should also be Refrigerated. Check the expiry of drugs ANTIBIOTIC SUSCEPTIBILITY TESTING
  31. 31. Stokes method  Built in controls against many variables and provide dependable results  A standard sensitive strain of the bacterium is inoculated in the middle third of the culture plate ANTIBIOTIC SUSCEPTIBILITY TESTING
  32. 32.  The test bacterium is inoculated in the upper and lower third of the plate. An uninoculated gap of 2-3 mm wide should separate the test and the control area on which the antibiotic disks are applied so that zones of inhibition formed around each disc are composed of standard and test bacteria.  The plates are then incubated at 37 deg C for 16-18 hours. ANTIBIOTIC SUSCEPTIBILITY TESTING
  33. 33.  The report is prepared by comparing the zones of inhibition of control and test bacterium.  The radius of the inhibition zone from the edge of the disk to the edge of the zone is measured. Interpretation :  Sensitive (S):Zone radius is wider than or equal to, or not more than 3 mm smaller than the control. Intermediate (I): Zone radius is >2 mm but smaller than the control by >3 mm.  Resistant (R): No zone of inhibition or zone radius measures 2 mm or less. ANTIBIOTIC SUSCEPTIBILITY TESTING
  34. 34. Interpretation of Results. The zone size observed in a disk diffusion test is compared to the interpretive standards provided by the CLSI. Zone diameter sizes derived from testing a wide range of strains are correlated with the known MICs of the species tested to assign interpretive breakpoint criteria for susceptible, intermediate and resistance zone sizes. A prototype of a regression curve providing such a correlation is illustrated in graph. ANTIBIOTIC SUSCEPTIBILITY TESTING
  36. 36. Troubleshooting the Reading of Disk Diffusion Tests
  40. 40. ANTIBIOTIC SUSCEPTIBILITY TESTING Limitations :- This medium is recommended for susceptibility testing of pure cultures only. Inoculum density may effect the zone size. Heavy inoculum may result in smaller zones or too less inoculum may result in bigger zones.
  41. 41. ANTIBIOTIC SUSCEPTIBILITY TESTING Fastidious organisms may not grow on this medium and may require supplementation of blood. Fastidious anaerobes may not grow on this medium. As antimicrobial susceptibility is carried with antibiotic disc, proper storage of the disc is desired which may effect the potency of the disc. Under certain circumstances, the in vitro results of antibiotic susceptibility may not show the same in vivo
  42. 42. Dilution Tests Here, the antimicrobial agent is serially diluted, each dilution is tested with the test organism for antimicrobial susceptibility test and the MIC is calculated. MIC (minimum inhibitory concentration) is the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation. Depending upon whether the dilutions of the antimicrobial agent are made in agar or broth ,there are two types of dilution tests. ANTIBIOTIC SUSCEPTIBILITY TESTING
  43. 43. Broth Dilution Method Serial dilutions of the antimicrobial agent in Mueller Hinton broth are taken in tubes and each tube is inoculated with a fixed amount of suspension of the test organism. A control organism of known sensitivity should also be tested. Tubes are incubated at 37'C for 18 hours.  The MIC is determined by noting the lowest concentration of the drug at which there is no visible growth, i.e. broth appears clear. ANTIBIOTIC SUSCEPTIBILITY TESTING
  44. 44. The minimum bactericidal concentration (MBC) can be obtained by sub culturing from each tube (showing no growth) onto a nutrient agar plate without any antimicrobial agent The tube containing the lowest concentration of the drug that fails to show growth, on subculture, is the MBC of the drug for that test Strain. Broth dilution test can also be done using microtiter plates, such method is called as micro dilution method. ANTIBIOTIC SUSCEPTIBILITY TESTING
  46. 46. Agar Dilution Method Here, the serial dilutions of the drug are prepared in molten agar and poured into Petri dishes. The test strain is spot inoculated. This method is more convenient than broth dilution and has the following advantage:  Several strains can to do the tested at the same time by using the same plate.  It directly measures the MBC; there is no need of sub culturing as it is done with broth dilution method. ANTIBIOTIC SUSCEPTIBILITY TESTING
  48. 48. Epsilometer or E-test This is a quantitative method of detecting MIC by using the principles of both dilution and diffusion of antibiotic into the medium. It uses an absorbent strip containing pre defined gradient (serial dilution) of antibiotic concentration immobilized along its length. It is applied to a lawn inoculum of a bacterium. Following incubation of the test organism, an elliptical zone of inhibition is produced surrounding the strip. ANTIBIOTIC SUSCEPTIBILITY TESTING
  50. 50. ANTIBIOTIC SUSCEPTIBILITY TESTING Result and Interpretation: Read MIC at the point where ellipse intersects the scale. If a MIC value between two two fold dilutions is seen, always round up to the highest value. Read the MIC value at complete inhibition of all growth. If the intersect differs on either side of the strip, read the MIC as the greater value. Ignore any growth at the edge of the strip.
  51. 51. ANTIBIOTIC SUSCEPTIBILITY TESTING Troubleshooting the Reading of E-test Assays Zone of inhibition includes pinpoint colonies and hazy growth within the zone of inhibition.  Check the purity of the culture when there are pinpoint colonies in the zone of inhibition; do not ignore these colonies in determining the MIC.
  52. 52. ANTIBIOTIC SUSCEPTIBILITY TESTING If the zone of inhibition intersects the strip between two markings, read the higher value. Similarly, if the zone intersects the strip slightly differently on the two sides, read the higher value. When testing β-hemolytic bacteria, assess the zone of inhibition and ignore the hemolysis. use a magnifying glass to visualize pinpoint colonies and hazes, especially for enterococci, pneumococci, and Stenotrophomonas spp. Include a haze as growth when determining inhibition, but ignore the swarming of Proteus spp.
  53. 53. Automated Antimicrobial Susceptibility Tests Several automated systems are available now, such as :  VITEK 2 bacterial identification and antimicrobial sensitivity system (bioMerieux)  Phoenix System (Becton Dickinson)  Micro Scan Walk Away system ANTIBIOTIC SUSCEPTIBILITY TESTING
  57. 57. Molecular Methods PCR based assay are available targeting specific drug resistant genes: for example mecA gene for MRSA detection. ANTIBIOTIC SUSCEPTIBILITY TESTING
  58. 58. Quality Control In AST  QC: a process in the laboratory designed to monitor the analytical phase of testing procedures to ensure that tests are working properly  CLSI recommends use of ATCC strains for QC in AST ANTIBIOTIC SUSCEPTIBILITY TESTING
  59. 59.  QC strains should be included daily with the test ideally; due to expense, it can be switched as once weekly testing, with less than 10% inaccuracy  Not more than 1 in 20 results should be outside the accuracy limits  No zone should be more than 4 SD away from midpoint between the stated limits  Test repeated for each new drug included  All documentation maintained indefinitely ANTIBIOTIC SUSCEPTIBILITY TESTING
  60. 60. QC for MHA  To ensure that the zone diameters are sufficiently reliable for testing susceptibility to sulfonamides and trimethoprim, MHA must have low concentrations of the inhibitors thymidine and thymine.  Each new lot of MHA should therefore be tested with a control strain of Enterococcus faecalis and a disc of co-trimoxazole.  A satisfactory lot of medium will give a distinct inhibition zone of 20 mm or more that is essentially free of hazy growth or fine colonies. ANTIBIOTIC SUSCEPTIBILITY TESTING
  61. 61. Reference:-  Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Seventh Edition.  Essentials Of Medical Microbiology , Apurba Sankar Sastry.  Kit Inserts, Hi Media.  Manual of Antimicrobial Susceptibility Testing ,ASM.  Image courtesy – Google search ANTIBIOTIC SUSCEPTIBILITY TESTING