AST

• What is the title of the document related to antimicrobial susceptibility testing?

  ANTIMICROBIAL SUSCEPTIBILITY TESTING (AST)
• What are the direct measures of antimicrobial activity?

  Conventional susceptibility testing methods, commercial susceptibility testing systems, and special screens and indicator tests
• What are the conventional susceptibility testing methods?


  • Tube/Microplate dilution
  • Agar dilution
  • Disk diffusion
• What is the purpose of the Minimum Inhibitory Concentration (MIC)?


  To determine the lowest concentration of an antibiotic that inhibits bacterial growth
• How is the Tube/Microplate dilution method performed?


  A serial dilution of antibiotic is prepared in each tube with a uniform amount of inoculum added
• What does MBC stand for in antimicrobial testing?


  Minimum Bactericidal Concentration
• What is the agar dilution method?

  Varying concentrations of antibiotic are incorporated into appropriate plating media
• What are the steps in the Disk Diffusion method?


  Inoculate MHA, apply antibiotic disks, incubate, measure zone of inhibition, interpret susceptibility
• What is the incubation time for the Disk Diffusion method?


  16 to 18 hours
• What could cause errors in antimicrobial susceptibility testing?

  Use of mixed culture, incorrect inoculum density, moisture issues, improper storage of disks, reading errors
• What are the possible sources of error in antimicrobial susceptibility testing?


  • Use of mixed culture
  • Inoculum too light or too heavy
  • Too much moisture on agar
  • Very dry agar surface
  • Improper storage of disk
  • Reading and clerical error
  • Deterioration of turbidity standard or control strains
• How does increased calcium and magnesium affect P. aeruginosa?


  It causes increased resistance to aminoglycosides
• What is the proper long-term storage temperature for antibiotic disks?


  • 20°C in a non-frost free freezer
• What is the purpose of the E-Test?


  To determine the MIC using test strips with an antimicrobial concentration gradient
• What are the components of the Vitek systems?


  Vitek legacy, Vitek 2 systems, MicroScan WalkAway system, and Phoenix system
• What are the main classes of antimicrobial agents and their targets?


  1. Cell wall synthesis

  • Beta lactams
  • Glycopeptides
  • Cycloserine
  • Bacitracin

  1. Protein Synthesis

  • Aminoglycosides
  • Tetracyclines
  • Erythromycin
  • Chloramphenicol
  • Fusidic acid

  1. Nucleic Acid Synthesis

  • Sulfonamides
  • Trimethoprim
  • Quinolones
  • Rifampicin

  1. Cell membrane function

  • Polymyxins
• What is the composition of TSI media?


  Protein source, carbohydrates, pH indicator, H2S indicator
• What does the TSI media test for?


  It is used for the initial step in the identification of Enterobacteriaceae
• What is the effect of aminoglycosides on bacterial growth?


  They inhibit protein synthesis
• What is the role of sulfonamides in antimicrobial therapy?


  They inhibit nucleic acid synthesis
• What is the function of polymyxins in antimicrobial therapy?


  They inhibit cell membrane function
• Contents of tube dilution:

  • MIC
  • MBC
  • Inoculum
• Method for Minimum Inhibitory Concentration (MIC)


  Broth Dilution method
• Example of MIC


  TSB compared to McFarland Standard
• Incubation period for TSB in compare to McFarland Standard


  3-5hrs
• Done in microplate then streak in the colored tube


  MBC
• Measurement of bacterial growth


  1.5x10^8C Fu/m^2
• Signifies bacterial growth for broth dilution

  Turbidity
• Second concentration with no turbidity and no signs of bacteria growth


  MBC
• Another name for disk diffusion is Kirby Bauer
• Steps for MHA

  1. Pick 4 to 5 colonies into TSB. Incubate at 37 degrees Celsius for 2 to 5 hours
  2. Compare turbidity to 0.5 McFarland Standard (Barium SO4) then do the inoculum.
  3. Inoculate MHA
  4. Apply antibiotic disks
  5. Invert plates and incubate 37 degrees Celsius for 16 to 18 hours
  6. Measure zone of growth inhibition
  7. Interpret susceptibility from standard chart zone.
• How many H2SO4 are there in the MHA inoculum


  9.95 mL 1% H2SO4
• How many BaCl2 are there in the MHA inoculum


  0.05 mL & 1.1752 BaCl2
• How many yeast in the MHA inoculum


  1.5x10^8CFu/mL
• 1.5x10^8CFu/mL yeast diffusion tube has the same turbidity with


  5x10^5CFu/ml yeast diffusion tube
• How to inoculate MHA


  Overlap method with the use of cotton swabs
• How to apply antibiotics disks


  24mm from disk center to center, stand for 3-5mins not exceeding 15 mins.
• AST is only done when there are isolated organisms whether it is from pure or unknown culture.
• Measurement for susceptible organism


  >16mm
• Measurement for resistant organisms


  <12mm