Antibiotic Susceptibility

• Antibiotic susceptibility test

  Performed on bacteria isolated from clinical specimens to determine which antimicrobial agents might be effective in treating infections caused by the bacteria
• Only bacteria that are likely to be contributing to an infection should be tested
• Standard conditions for antimicrobial susceptibility testing methods have been established based on numerous laboratory investigations. Guidelines and recommendations for their use are published by the Clinical and Laboratory Standards Institute (CLSI)

  
• Components of antimicrobial susceptibility testing that are standardized and controlled

  • Bacterial inoculum size
  • Growth medium
  • pH
  • Cation concentration
  • Blood and serum supplements
  • Thymidine content
  • Incubation atmosphere
  • Incubation temperature
  • Incubation duration
  • Antimicrobial concentrations tested

  
• Manual methods

  • Dilution methods
  • Diffusion methods
  • Alamar system
• Automated methods

  • Turbidimetry
  • Nephelometry
• Macrobroth dilution

  Dilution method
• Microbroth dilution

  Dilution method
• Agar dilution


  Dilution method
• 1. test or Espolometer test

  Diffusion method
• Alamar system

  Automated method
• Vitek systems

  Automated method
• Conventional testing methods

  • General considerations
  • Inoculum preparation
  • Selection of antimicrobial agents for testing
• Inoculum preparation

  Properly prepared inocula are key to any of the antimicrobial susceptibility testing methods. Inconsistencies in inoculum preparation often lead to inconsistencies and inaccuracies in susceptibility test results.
• Requirements for appropriate inoculum preparation

  • Use of a pure culture
  • Standardized inoculum
• Obtaining pure inocula

  Selecting 4-5 colonies of the same morphology, inoculating them to a broth medium, and allowing the culture to achieve good active growth, as indicated by observable turbidity in the broth. This requires 3-5 hours of incubation
• Alternatively obtaining pure inocula

  Selecting 4-5 colonies 16 to 24 hours of age from an agar plate and suspending them in broth or 0.85% saline solution to achieve a turbid suspension

  
• Standardized inoculum size

  As important as culture purity, accomplished by comparison of the turbidity of the organism suspension with a turbidity standard

  
• McFarland standard

  99.5 mL of 1% Sulfuric Acid & 0.5 mL of 1.175% Barium Chloride, provides an optical density comparable to the density of a bacterial suspension of 1.5 × 108 colony forming units (CFU)/Ml

  
• Matching turbidity using the unaided eye

  Holding the bacterial suspension and McFarland tubes side by side and viewing them against a black-lined background

  
• Once standardized, the inoculum suspensions should be used within 15 minutes of preparation
• If the bacterial suspension initially does not match the standard's turbidity, the suspension may be diluted or supplemented with more organisms as needed

  
• Antimicrobial battery or panel


  The antimicrobial agents that are chosen for testing against a particular bacterial isolate
• Criteria for antimicrobial battery content and use

  • Organism identification or group
  • Acquired resistance patterns common to local microbial flora
  • Antimicrobial susceptibility testing method used
  • Site of infection
  • Availability of antimicrobial agents in formulary
• Organism groups for which individual testing batteries are commonly considered

  • Enterobacteriaceae
  • Pseudomonas aeruginosa and Acinetobacter spp.
  • Staphylococcus spp
  • Enterococcus spp.
  • Streptococcus spp. (not including S. pneumoniae)
  • Streptococcus pneumoniae
  • Haemophilus influenzae
  • Neisseria gonorrhoeae
• Disk diffusion testing (Kirby Bauer test)

  A McFarland 0.5 standardized suspension of bacteria is swabbed over the surface of a Mueller-Hinton agar plate

  2. Paper disks containing specific concentrations of antimicrobial agent are placed onto the inoculated surface

  3. After overnight incubation, the diameters of the zones produced by antimicrobial inhibition of bacterial growth are measured

  4. Result is interpreted as nonsusceptible, susceptible, intermediate, or resistant to a particular drug according to preset criteria

  
• Most clinical laboratories follow the protocol specified by the CLSI for disk diffusion testing
• Disk storage

  • Long term storage: -20oC or below in a non–frost-free freezer

  Working supply: 2°C to 8°C for at least 1 week

  Stored in a tightly sealed container with desiccant, allowed to warm to room temperature before opening
• Inoculation and incubation

  A sterile cotton swab is dipped into the suspension, pressed and rotated firmly against the side of the tube to express excess liquid, and then swabbed evenly across the surface of a Mueller-Hinton agar plate

  The disks are pressed firmly to ensure contact with the agar. Within 15 minutes of disk placement, plates are inverted and placed in a 35°C ambient air incubator for 16 to 18 hours

  Mueller-Hinton agar containing 5% sheep blood is used for testing streptococci
• Reading plates and test interpretation

  The lawn of growth must be confluent or almost confluent

  The diameter of each inhibition zone is measured using a ruler or calipers

  Plates are placed a few inches above a black, nonreflecting surface, and zones are examined from the back side (agar side) of the plate illuminated with reflected light

  Transmitted light (plate held up to light source) is used for tests with the penicillinase-resistant penicillins, linezolid, and vancomycin when testing staphylococci and for vancomycin when testing enterococci

  For plates containing blood, the zone of inhibition of growth, not hemolysis, is read
• Susceptible


  The antimicrobial agent in question may be an appropriate choice for treating the infection caused by the bacterial isolate tested
• Intermediate


  Indicates a number of possibilities, including: utility of the antimicrobial agent in body sites where it may be concentrated or if high concentrations of the drug are used, the antimicrobial agent may still be effective against the tested isolate but possibly less so than against a susceptible isolate, or as an interpretive safety margin to prevent relatively small changes in test results from leading to major swings in interpretive category
• Resistant


  The antimicrobial agent in question may not be an appropriate choice for treating the infection caused by the bacterial isolate tested