Antimicrobial Susceptibility Testing

• MIC


  Minimum inhibitory concentration
• MBC

  Minimum bactericidal concentration
• ZOI


  Zone of inhibition
• CLSI


  Clinical and Laboratory Standards Institute
• EUCAST


  European Committee on Antimicrobial Susceptibility Testing
• AST is performed on bacteria and fungi isolated from clinical specimens to determine which antimicrobial agents might be effective in treating infections caused by these organisms
• Major goal of AST

  To ID organisms that are the TRUE pathogens causing the clinical infection and to arrive at the earliest possible time frame to initiate antimicrobial therapy to the patient
• Currently more than 50 antimicrobial agents are in use for treating bacterial and fungal infections
• The limiting factor for the number of drugs tested is usually the methodology
• Primary tenet of antimicrobial therapy

  To use the least toxic, most cost-effective, and most clinically effective antimicrobial
• CLSI provides a cascade reporting protocol for reporting susceptibility test results
• Inoculum preparation


  1. Add cells from 4-5 colonies to broth medium

  2. Suspend thoroughly using vortex mixer

  3. Allow to grow to log phase at 35°C for 2-6 hours until 0.5 McFarland standard is reached

  4. Use fresh (16-24 hr) colonies

  
• 0.5 McFarland standard


  Provides turbidity comparable with a bacterial suspension containing approximately 1.5 x 10^8 CFU/ml
• Manual visual comparison of inoculum to 0.5 McFarland standard


  1. Position tube side by side with 0.5 McFarland standard against a white card with black lines

  2. If too dense, dilute with saline

  3. If too light, add more colonies or reincubate until similar turbidity

  
• Nephelometer


  Measures the concentration of suspended particles in a solution by measuring the intensity of scattered light

  
• Nephelometer


  • Scattered light is measured at an angle from the light source to avoid interference of possible transmitted light
  • The light scattered is directly proportional to the density of the solution

  
• Broth Dilution Test


  1. Broth Microdilution

  2. Broth Macrodilution

  
• Disk Diffusion Test (Kirby-Bauer)


  Measure ZOI of 12 antimicrobials

  
• Rapid Diagnostic Test (RDT)


  Goal: to arrive at an earliest possible TTD (time to detection) of the etiologic agent to allow earliest possible TTA (time to administration) of antimicrobials
• Broth Macrodilution MIC Test

  1. Performed in 12-15 tubes using 2-fold serial dilution series

  2. Add standardized bacterial suspension to each dilution to obtain 5 x 10^5 CFU/ml

  3. Use growth control and sterility control tubes

  4. Determine MIC as lowest concentration that inhibits growth

  
• Broth Microdilution MIC Test

  1. Performed in multi-well microdilution trays

  2. Wells filled with 2-fold dilution concentrations of antimicrobial agent in broth

  3. Inoculate wells with 0.01 ml of standardized bacterial suspension to obtain 5 x 10^5 CFU/ml

  4. Growth seen as turbidity, haze, or pellet in well

  
• Agar Dilution Tests


  1. Dispense antimicrobial solutions into molten agar, pour into plates

  2. Spot-inoculate 10^4 CFU/ml of test bacteria onto plates

  3. MIC is lowest concentration that inhibits visible growth

  
• Disk Diffusion Test (Kirby-Bauer)

  1. Swab bacterial suspension in MH broth (0.5 McFarland) onto MH agar plate

  2. Place antimicrobial discs onto inoculated surface

  3. After incubation, measure diameters of ZOI around each disc

  
• The relationship between ZOI and MICs is used to interpret disk diffusion test results
• Disk Diffusion Test


  1. Prepare bacterial suspension in MH broth and compare to 0.5 McFarland Standard

  2. Swab suspension over MH agar plate

  3. Place antimicrobial discs onto inoculated surface

  4. Incubate 16-18 hours

  5. Measure diameters of zones of inhibition around each disc

  
• Disk Diffusion Test


  • Standardized for common, rapidly growing bacteria
  • Should not be used for slow-growing bacteria and obligate anaerobes
• Disk Diffusion Test is widely used in clinical labs since 1966
• Zone of Inhibition (ZOI)


  The clear circular area around antimicrobial discs in which bacteria are unable to grow
• ZOI are related to Minimum Inhibitory Concentrations (MICs), and this relationship is used to determine breakpoints for interpreting susceptibility
• Preparing inoculum for Disk Diffusion Test

  1. Compare turbidity of bacterial suspension to 0.5 McFarland Standard

  2. Swab suspension evenly over MH agar plate using overlapping streak method

  3. Inoculate purity plate from same swab

  
• A 150 mm diameter plate can accommodate testing of 12 antimicrobial discs, while a 90 mm plate can hold 8 discs
• Placement of more than 12 discs can result in overlapping ZOI, which are difficult to measure and may produce erroneous results
• Applying antimicrobial discs


  1. Apply discs individually with sterile forceps or multiple disk-dispenser within 15 minutes of inoculation

  2. Press discs firmly to ensure contact with agar

  
• Incubating and reading Disk Diffusion Test

  1. Invert plates and incubate at 35°C for 16-18 hours

  2. Inspect purity plate to confirm pure culture was tested

  3. Measure diameter of ZOI for each antimicrobial using ruler or caliper

  
• Presence of individual colonies usually indicates contamination or a resistant subpopulation
• Interpreting Disk Diffusion Test results

  Compare ZOI measurements to interpretive tables to determine susceptible, nonsusceptible, intermediate, or resistant
• Factors affecting Disk Diffusion Test

  • Agar depth
  • Agar pH
  • Supplements for fastidious organisms
  • Inoculum age and turbidity
  • Incubation conditions
  • Reading method
  • Inoculum cell density
  • Disk placement distance
  • Use of expired disks
• 1. Test (Epsilometer Test)


  • Uses an antimicrobial concentration gradient in agar to determine susceptibility
  • Plastic test strips are impregnated with antimicrobial gradient and placed on inoculated agar plate
  • MIC is determined where growth ellipse intersects the strip

  
• 1. Test is more expensive than Disk Diffusion but has flexibility of drug selection
• Rapid/Automated Antimicrobial Susceptibility Test Methods

  • BD Phoenix System
  • Beckman Coulter Microscan Walkaway System
  • Thermo Fisher Scientific Sensititre ARIS 2X
  • Biomerieux Vitek