AST

• Antimicrobial Susceptibility


  The ability of an antibiotic or other microbial agent to inhibit bacterial growth
• Antimicrobial Susceptibility Testing

  1. Obtain pure inocula

  2. Suspend colonies in 0.85% NSS

  3. Compare bacterial suspension and McFarland solution

  4. Streak plates with overlapping streaks

  5. Apply antimicrobial agents within 15 minutes

  6. Incubate plates at 35*C for 16-18 hours

  7. Measure diameter of inhibition zones
• Primary Goal


  To determine whether the bacterial isolate is capable of expressing resistance to the antimicrobial agents selected for treatment
• Purpose


  • To guide the clinician in selecting appropriate antimicrobial agent
  • To gather epidemiological data on microbial resistance
• Standardization


  • To optimize bacterial growth conditions
  • To optimize conditions for maintaining antimicrobial integrity and activity
  • To maintain reproducibility and consistency in the resistance profile
• Disk Diffusion Method (Kirby-Bauer Test)


  • Medium: Mueller-Hinton Agar
  • Standard: 0.5 McFarland / Barium Sulfate Suspension
  • Standard inoculum: 1.5 x 10^8 organisms/mL
  • pH: 7.2 - 7.4
  • Depth: 4mm
  • Condition: Aerobic, No CO2
  • Temperature: 35 - 37*C
  • Incubation Time: 16 - 18 hours
  • Antibiotic Disc: 6 mm
  • Petroff-Hausser: Bacterial Count
• Factors Affecting Disk Diffusion

  • pH of the media
  • Thickness of the agar
  • Growth rate of the microbe
  • Amount of inoculum
  • Suitability of the medium used
  • Diffusability of the antibiotic
  • Discrepancy in inoculum and incubation
• Antimicrobial Susceptibility Testing

  • Disk Distance: 15 mm
  • 150 mm plate can house 12 discs
  • 100 mm petri dish can house 5-6 discs
• False Resistant

  • Increase moisture
  • Increase drying
  • Thick medium
  • Thin medium
  • Increase organisms = decrease ZOI
  • Decrease organisms = increase ZOI
• False Sensitive

  • 15 minutes (Delay of Disc Application)
  • 15 minutes (Delay of incubation)
• Confluent


  Equal distribution
• Susceptible


  Microorganism should respond to therapy with that antimicrobial agent
• Intermediate


  Microorganism falls into a range of susceptibility in which the minimum inhibitory concentration (MIC) approaches or exceeds the level of antimicrobial agent that can be achieved and for which clinical response is likely to be less than with a susceptible strain
• Resistant


  No zone or small zone of inhibition - antibiotic is not the appropriate choice of treatment
• Automated Systems

  • Vitek 2
  • Microscan Walkaway System by Beckman Coulter
  • Phoenix System by BD microbiology systems
• MRSA

  Methicillin-Resistant Staphylococcus aureus
• Types of MRSA

  • Health care-associated MRSA (HA-MRSA)
  • Community-associated MRSA (CA-MRSA)
• HA-MRSA


  Associated with invasive procedures or devices such as surgeries, intravenous tubing, or artificial joints. Usually contracted from the hospital
• CA-MRSA


  Often begins as a painful skin boil that can be spread by skin-to-skin contact. The community at risk for this infection are high school wrestlers, childcare workers, and people who live in crowded conditions
• Test for MRSA

  • Broth microdilution testing
  • Cefoxitin screen test
  • Latex agglutination test for PBP2a
  • Plate containing 6 ug/mL of oxacillin in Mueller-Hinton agar supplemented with 4% NaCl
  • Chromogenic agars
  • Nucleic acid amplification tests, e.g. polymerase chain reaction (PCR) to detect the mecA gene