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Antimicrobial sensitivity testing (AST)

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Atul Adhikari
Atul Adhikari

Antimicrobial sensitivity testing (AST) or Antibiotic Sensitivity Testing. Contents: 1. Need of AST 2. Bacterial Resistance 3. Preperation of test: selection of antibiotic and bacteria 4. Types of tests 5. Process of tests

Health & Medicine
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Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Antimicrobial Sensitivity Testing(AST) Atul Adhikari Fourth Semester, B. Pharm. School of Health and Allied Sciences Pokhara University, Dhungepatan, Lekhnath-12, Kaski , Nepal
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Introduction • A laboratory test which determines how effective antibiotic therapy is against a bacterial infections. • The goal of antimicrobial susceptibility testing is to predict the in vivo success or failure of antimicrobial therapy. • Tests are performed in vitro, and measure the growth response of an isolated organism to a particular drug or drugs. • The test results should be used to guide antibiotic choice. 2Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Why Need for Testing Antimicrobial Sensitivity?? • Bacteria have the ability to develop resistance following repeated or subclinical (insufficient) doses, so more advanced antibiotics and synthetic antimicrobials are continually required to overcome them. • Antibiotic sensitivity testing is essential part of Medical Care 3Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Resistance •intrinsic resistance and acquired resistance. •Intrinsic resistance means that the species was resistant to an antibiotic even before its introduction. •Acquired resistance means that the species was originally susceptible to an antibiotic, but later became resistant. •Bacteria can acquire antibiotic resistance either by mutation or through exchange of genetic material among same or closely related species (Transformation, transduction and conjugation) •The sudden acquisition of resistance to antibiotics poses difficulties in treating infections. •Resistance to several different antibiotics at the same time is even more significant problem.
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Bacterial Resistance Strategies •Several different mechanisms may work together to confer resistance to a single antimicrobial agent: (1) By prevention of the antimicrobial from reaching its target by reducing its ability to penetrate into the cell (2) via general or specific efflux pumps (3) By inactivation of antimicrobial agents via modification or degradation (4) By modification of the antimicrobial target within the bacteria
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Preparing for Testing • Inoculum preparation:- Suspension of usually log phase growth cells of bacteria - Number of test organisms can be determined using different methods: – Direct count (Microscopic examination) – The optical density (OD) at 600 nm (Spectrophotometry) – Plate count: making dilution first 6Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Sources of bacteria – Indirect source • cultured plate from pure culture – Direct source • Pathological specimen • e.g. urine, a positive blood culture, or a swab of pus Antimicrobial Sensitivity Testing (AST) 7
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences – Set 1: the drugs that are available in most hospitals and for which routine testing should be carried out for every strain – Set 2: the drugs that are tested only: • at the special request of the physician • or when the causative organism is resistant to the first- choice drugs • or when other reasons (allergy to a drug, or its unavailability) make further testing justified Antimicrobial Sensitivity Testing (AST) 8 Choosing the Appropriate Antibiotic
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Antimicrobial Sensitivity Testing (AST) 9 Set 1 Set 2 Staphylococcus Benzyl penicillin Oxacillin Erythromycin Tetracycline Chloramphenicol Gentamicin Amikacin Co-trimoxazole Clindamycin Intestinal Ampicillin Chloramphenicol Co-trimoxazole Nalidixic acid Tetracycline Norfloxacin Enterobacteriaceae Urinary Sulfonamide Trimethoprim Co-trimoxazole Ampicillin Nitrofurantoin Nalidixic acid Tetracycline Norfloxacin Chloramphenicol Gentamicin Blood and tissues Ampicillin Chloramphenicol Cotrimoxazole Tetracycline Gentamicin Cefuroxime Ceftriaxone Ciprofloxacin Piperacillin Amikacin Pseudomonas aeruginosa Piperacillin Gentamicin Tobramycin Amikacin
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Methods of antibiotic susceptibility testing : Various methods of antibiotic susceptibility testing are: 1. Diffusion methods/Qualitative Methods 2. Dilution methods/Quantitative Methods 3. Automated Susceptibility Tests 4.Newer Non-Automated Susceptibility Tests (E-Test) 5. Molecular/Genetic Techniques 10Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Disc diffusion method : The Kirby-Bauer test Antibiotic-impregnated filter disc Susceptibility test against more than one antibiotics by measuring size of “inhibition zone ” 11Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Disk Diffusion Test Prepare inoculum suspension Select colonies 12Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Standardize inoculum Suspension as per Mac farland standard Mix well Prepare the Material for Inoculation 13Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Remove sample Swab plate Swab the plate with optimal sample 14Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Select disks Select the Disks and Apply 15Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Incubate Overnight 16Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Disc Diffusion Method  Place the appropriate drug- impregnated disc on the surface of the inoculated agar plate  Invert the plates and incubate them at 35 oC, (18-24 h)  Measure the diameters of inhibition zone in mm  Measurement of the diameters of inhibition zone  Measure from the edge where the growth stats, 17Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Disc Diffusion Method Reporting the Results  Interpretation of results  By comparing with the diameters with “standard tables”  Susceptible  Intermediate susceptible  Resistant 18Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences • Minimum Inhibition Concentration (MIC) – The lowest concentration of antimicrobial agent that inhibits bacterial growth/ multiplication • Minimum Bactericidal Concentration (MBC) or Minimum Lethal Concentration (MLC) – The lowest concentration of antimicrobial agent that allows less than 0.1% of the original inoculum to survive Dilution Method 19Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Dilution methods/Quantitative Methods: • In these tests MIC is determined. • Bacterial isolate is subjected to various dilutions of antibiotics. • The highest dilution of antibiotic that has inhibited the growth of bacteria is considered as MIC. • These tests can be performed on broth or agar. 1. Broth dilution methods 2. Agar dilution methods 20Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences 1. Broth dilution methods a. Macrobroth dilution MIC tests b. Microbroth dilution MIC tests • The Broth dilution method involves subjecting the isolate to a series of concentrations of antimicrobial agents in a broth environment. • Microdilution testing uses about 0.05 to 0.1 ml total broth volume and can be conveniently performed in a microtiter format. • Macrodilution testing uses broth volumes at about 1.0 ml in standard test tubes. • For both of these broth dilution methods, the lowest concentration at which the isolate is completely inhibited (as evidenced by the absence of visible bacterial growth) is recorded as the minimal inhibitory concentration or MIC. 21Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Creating Dilutions Fig: Macrobroth dilution
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Select Micro titration plate and prepare inoculum Prepare inoculum suspension Micro dilution MIC tray 23Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Dilute & mix inoculum suspension 24Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Pour inoculum into reservoir and inoculate MIC tray 25Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Incubate overnight 26Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences - + 64 32 16 8 4 2 1 >64 0.5 >64 Read MICs 27Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences 2.Agar dilution • A procedure similar to broth dilution is agar dilution. • Agar dilution method follows the principle of establishing the lowest concentration of the serially diluted antibiotic concentration at which bacterial growth is still inhibited. • One concentration of antibiotic per plate • Possible for several different strains per plate 28Antimicrobial Sensitivity Testing (AST) 64 uGu/ml 32 ug/ml 16 ug/ml
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Antimicrobial Sensitivity Testing (AST) 29 Procedure • Inoculation of bacterial inoculum • Using a replicating inoculator device called “A Steers-Foltz replicator” • Delivers 0.001 ml of bacterial inoculum • Incubation Spot of growth MIC
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Broth Dilution Method are Technically Difficult • Disadvantages : – Only one antibiotic & one organism can be tested each time – Time-consuming • Solutions – Agar dilution method – Disc diffusion method – Micro broth dilution method 30Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Etest • Etest is a well established AST method around the world. The Etest technique comprises a predefined gradient of antibiotic concentrations on a plastic strip, and can be used to determine the Minimum Inhibitory Concentration (MIC) of antibiotics, antifungal agents and antimycobacterial agents. 31Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Antimicrobial Gradient Testing E-test Read plates after recommended Incubation Read MIC where elipse intersects scale 32Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences • The Etest system comprises a predefined and continuous concentration gradient of different antimicrobial agents, which when applied to inoculated agar plates and incubated, create ellipses of microbial inhibition. • The MIC is determined where the ellipse of inhibition intersects the strip, and is easily read off the MIC reading scale on the strip. 33Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Antimicrobial Sensitivity Testing (AST) 34 MIC on a strip
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Antibiotic Sensitivity testing can be done with automation 35Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences VITEK 2 Automates Reporting of Resistance • VITEK 2 system is the Advanced Expert System (AES), a software which validates and interprets susceptibility test results, and detects antibiotic resistance mechanisms. • The AES Expert System is the most developed software system in this field, and is capable of identifying even emerging and low-level resistance. 36Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences GENOTYPIC METHODS • Less practiced method • test for the specific genes that confer antibiotic resistance. • Polymerase chain reaction (PCR) • DNA hybridization. 37Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Interpretation of Susceptibility Test Results • Optimal interpretation of MICs requires knowledge of the pharmacokinetics of the drug in humans, and information on the likely success of a particular drug in eradicating bacteria at various body sites . • Both MIC values and disk diffusion zone diameters must be interpreted using a table of values that relate to proven clinical efficacy of each antibiotic and for various bacterial species. • Standard charts and values are provided by CLSI, EUCAST, etc. 38Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences REFERENCES • Stephen P and Norman A (2005) Pharmaceutical Microbiology, Replica Press Pvt. Ltd., India, pp 196- 201. • Michael E. Aulton (2011) the Design And Manufacture of Medicines, Churchill Livingstone Elsevier., London, pp 207-12. • URL: http://www.eucast.org/ 39Antimicrobial Sensitivity Testing (AST)
Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences THANKYOU 40Antimicrobial Sensitivity Testing (AST)

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Antimicrobial sensitivity testing (AST)

  • 1. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Antimicrobial Sensitivity Testing(AST) Atul Adhikari Fourth Semester, B. Pharm. School of Health and Allied Sciences Pokhara University, Dhungepatan, Lekhnath-12, Kaski , Nepal
  • 2. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Introduction • A laboratory test which determines how effective antibiotic therapy is against a bacterial infections. • The goal of antimicrobial susceptibility testing is to predict the in vivo success or failure of antimicrobial therapy. • Tests are performed in vitro, and measure the growth response of an isolated organism to a particular drug or drugs. • The test results should be used to guide antibiotic choice. 2Antimicrobial Sensitivity Testing (AST)
  • 3. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Why Need for Testing Antimicrobial Sensitivity?? • Bacteria have the ability to develop resistance following repeated or subclinical (insufficient) doses, so more advanced antibiotics and synthetic antimicrobials are continually required to overcome them. • Antibiotic sensitivity testing is essential part of Medical Care 3Antimicrobial Sensitivity Testing (AST)
  • 4. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Resistance •intrinsic resistance and acquired resistance. •Intrinsic resistance means that the species was resistant to an antibiotic even before its introduction. •Acquired resistance means that the species was originally susceptible to an antibiotic, but later became resistant. •Bacteria can acquire antibiotic resistance either by mutation or through exchange of genetic material among same or closely related species (Transformation, transduction and conjugation) •The sudden acquisition of resistance to antibiotics poses difficulties in treating infections. •Resistance to several different antibiotics at the same time is even more significant problem.
  • 5. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Bacterial Resistance Strategies •Several different mechanisms may work together to confer resistance to a single antimicrobial agent: (1) By prevention of the antimicrobial from reaching its target by reducing its ability to penetrate into the cell (2) via general or specific efflux pumps (3) By inactivation of antimicrobial agents via modification or degradation (4) By modification of the antimicrobial target within the bacteria
  • 6. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Preparing for Testing • Inoculum preparation:- Suspension of usually log phase growth cells of bacteria - Number of test organisms can be determined using different methods: – Direct count (Microscopic examination) – The optical density (OD) at 600 nm (Spectrophotometry) – Plate count: making dilution first 6Antimicrobial Sensitivity Testing (AST)
  • 7. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Sources of bacteria – Indirect source • cultured plate from pure culture – Direct source • Pathological specimen • e.g. urine, a positive blood culture, or a swab of pus Antimicrobial Sensitivity Testing (AST) 7
  • 8. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences – Set 1: the drugs that are available in most hospitals and for which routine testing should be carried out for every strain – Set 2: the drugs that are tested only: • at the special request of the physician • or when the causative organism is resistant to the first- choice drugs • or when other reasons (allergy to a drug, or its unavailability) make further testing justified Antimicrobial Sensitivity Testing (AST) 8 Choosing the Appropriate Antibiotic
  • 9. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Antimicrobial Sensitivity Testing (AST) 9 Set 1 Set 2 Staphylococcus Benzyl penicillin Oxacillin Erythromycin Tetracycline Chloramphenicol Gentamicin Amikacin Co-trimoxazole Clindamycin Intestinal Ampicillin Chloramphenicol Co-trimoxazole Nalidixic acid Tetracycline Norfloxacin Enterobacteriaceae Urinary Sulfonamide Trimethoprim Co-trimoxazole Ampicillin Nitrofurantoin Nalidixic acid Tetracycline Norfloxacin Chloramphenicol Gentamicin Blood and tissues Ampicillin Chloramphenicol Cotrimoxazole Tetracycline Gentamicin Cefuroxime Ceftriaxone Ciprofloxacin Piperacillin Amikacin Pseudomonas aeruginosa Piperacillin Gentamicin Tobramycin Amikacin
  • 10. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Methods of antibiotic susceptibility testing : Various methods of antibiotic susceptibility testing are: 1. Diffusion methods/Qualitative Methods 2. Dilution methods/Quantitative Methods 3. Automated Susceptibility Tests 4.Newer Non-Automated Susceptibility Tests (E-Test) 5. Molecular/Genetic Techniques 10Antimicrobial Sensitivity Testing (AST)
  • 11. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Disc diffusion method : The Kirby-Bauer test Antibiotic-impregnated filter disc Susceptibility test against more than one antibiotics by measuring size of “inhibition zone ” 11Antimicrobial Sensitivity Testing (AST)
  • 12. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Disk Diffusion Test Prepare inoculum suspension Select colonies 12Antimicrobial Sensitivity Testing (AST)
  • 13. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Standardize inoculum Suspension as per Mac farland standard Mix well Prepare the Material for Inoculation 13Antimicrobial Sensitivity Testing (AST)
  • 14. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Remove sample Swab plate Swab the plate with optimal sample 14Antimicrobial Sensitivity Testing (AST)
  • 15. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Select disks Select the Disks and Apply 15Antimicrobial Sensitivity Testing (AST)
  • 16. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Incubate Overnight 16Antimicrobial Sensitivity Testing (AST)
  • 17. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Disc Diffusion Method  Place the appropriate drug- impregnated disc on the surface of the inoculated agar plate  Invert the plates and incubate them at 35 oC, (18-24 h)  Measure the diameters of inhibition zone in mm  Measurement of the diameters of inhibition zone  Measure from the edge where the growth stats, 17Antimicrobial Sensitivity Testing (AST)
  • 18. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Disc Diffusion Method Reporting the Results  Interpretation of results  By comparing with the diameters with “standard tables”  Susceptible  Intermediate susceptible  Resistant 18Antimicrobial Sensitivity Testing (AST)
  • 19. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences • Minimum Inhibition Concentration (MIC) – The lowest concentration of antimicrobial agent that inhibits bacterial growth/ multiplication • Minimum Bactericidal Concentration (MBC) or Minimum Lethal Concentration (MLC) – The lowest concentration of antimicrobial agent that allows less than 0.1% of the original inoculum to survive Dilution Method 19Antimicrobial Sensitivity Testing (AST)
  • 20. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Dilution methods/Quantitative Methods: • In these tests MIC is determined. • Bacterial isolate is subjected to various dilutions of antibiotics. • The highest dilution of antibiotic that has inhibited the growth of bacteria is considered as MIC. • These tests can be performed on broth or agar. 1. Broth dilution methods 2. Agar dilution methods 20Antimicrobial Sensitivity Testing (AST)
  • 21. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences 1. Broth dilution methods a. Macrobroth dilution MIC tests b. Microbroth dilution MIC tests • The Broth dilution method involves subjecting the isolate to a series of concentrations of antimicrobial agents in a broth environment. • Microdilution testing uses about 0.05 to 0.1 ml total broth volume and can be conveniently performed in a microtiter format. • Macrodilution testing uses broth volumes at about 1.0 ml in standard test tubes. • For both of these broth dilution methods, the lowest concentration at which the isolate is completely inhibited (as evidenced by the absence of visible bacterial growth) is recorded as the minimal inhibitory concentration or MIC. 21Antimicrobial Sensitivity Testing (AST)
  • 22. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Creating Dilutions Fig: Macrobroth dilution
  • 23. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Select Micro titration plate and prepare inoculum Prepare inoculum suspension Micro dilution MIC tray 23Antimicrobial Sensitivity Testing (AST)
  • 24. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Dilute & mix inoculum suspension 24Antimicrobial Sensitivity Testing (AST)
  • 25. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Pour inoculum into reservoir and inoculate MIC tray 25Antimicrobial Sensitivity Testing (AST)
  • 26. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Incubate overnight 26Antimicrobial Sensitivity Testing (AST)
  • 27. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences - + 64 32 16 8 4 2 1 >64 0.5 >64 Read MICs 27Antimicrobial Sensitivity Testing (AST)
  • 28. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences 2.Agar dilution • A procedure similar to broth dilution is agar dilution. • Agar dilution method follows the principle of establishing the lowest concentration of the serially diluted antibiotic concentration at which bacterial growth is still inhibited. • One concentration of antibiotic per plate • Possible for several different strains per plate 28Antimicrobial Sensitivity Testing (AST) 64 uGu/ml 32 ug/ml 16 ug/ml
  • 29. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Antimicrobial Sensitivity Testing (AST) 29 Procedure • Inoculation of bacterial inoculum • Using a replicating inoculator device called “A Steers-Foltz replicator” • Delivers 0.001 ml of bacterial inoculum • Incubation Spot of growth MIC
  • 30. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Broth Dilution Method are Technically Difficult • Disadvantages : – Only one antibiotic & one organism can be tested each time – Time-consuming • Solutions – Agar dilution method – Disc diffusion method – Micro broth dilution method 30Antimicrobial Sensitivity Testing (AST)
  • 31. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Etest • Etest is a well established AST method around the world. The Etest technique comprises a predefined gradient of antibiotic concentrations on a plastic strip, and can be used to determine the Minimum Inhibitory Concentration (MIC) of antibiotics, antifungal agents and antimycobacterial agents. 31Antimicrobial Sensitivity Testing (AST)
  • 32. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Antimicrobial Gradient Testing E-test Read plates after recommended Incubation Read MIC where elipse intersects scale 32Antimicrobial Sensitivity Testing (AST)
  • 33. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences • The Etest system comprises a predefined and continuous concentration gradient of different antimicrobial agents, which when applied to inoculated agar plates and incubated, create ellipses of microbial inhibition. • The MIC is determined where the ellipse of inhibition intersects the strip, and is easily read off the MIC reading scale on the strip. 33Antimicrobial Sensitivity Testing (AST)
  • 34. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Antimicrobial Sensitivity Testing (AST) 34 MIC on a strip
  • 35. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Antibiotic Sensitivity testing can be done with automation 35Antimicrobial Sensitivity Testing (AST)
  • 36. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences VITEK 2 Automates Reporting of Resistance • VITEK 2 system is the Advanced Expert System (AES), a software which validates and interprets susceptibility test results, and detects antibiotic resistance mechanisms. • The AES Expert System is the most developed software system in this field, and is capable of identifying even emerging and low-level resistance. 36Antimicrobial Sensitivity Testing (AST)
  • 37. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences GENOTYPIC METHODS • Less practiced method • test for the specific genes that confer antibiotic resistance. • Polymerase chain reaction (PCR) • DNA hybridization. 37Antimicrobial Sensitivity Testing (AST)
  • 38. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences Interpretation of Susceptibility Test Results • Optimal interpretation of MICs requires knowledge of the pharmacokinetics of the drug in humans, and information on the likely success of a particular drug in eradicating bacteria at various body sites . • Both MIC values and disk diffusion zone diameters must be interpreted using a table of values that relate to proven clinical efficacy of each antibiotic and for various bacterial species. • Standard charts and values are provided by CLSI, EUCAST, etc. 38Antimicrobial Sensitivity Testing (AST)
  • 39. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences REFERENCES • Stephen P and Norman A (2005) Pharmaceutical Microbiology, Replica Press Pvt. Ltd., India, pp 196- 201. • Michael E. Aulton (2011) the Design And Manufacture of Medicines, Churchill Livingstone Elsevier., London, pp 207-12. • URL: http://www.eucast.org/ 39Antimicrobial Sensitivity Testing (AST)
  • 40. Department of Pharmacy Pokhara University School of Health and Allied Sciences Department of Pharmacy Pokhara University School of Health and Allied Sciences THANKYOU 40Antimicrobial Sensitivity Testing (AST)