Mod 7

• Antimicrobial susceptibility testing


  The process of determining the sensitivity of a microorganism to antimicrobial agents
• Overview of Antibiotic Susceptibility Testing

  1. Isolate and identify causative agent

  2. Determine susceptibility and resistance of organism to antimicrobial agents

  3. Effectively treat the infection

  
• Pharmacokinetic properties


  The active form of the agent, which takes into account the route by which the patient receives the agent
• Pharmacodynamic design


  The antibiotic must be able to achieve sufficient levels or concentrations at the site of infection so that it has a chance to exert an antibacterial effect

  
• Mode of action


  Direct interactions between the antibacterial agent and the bacterial cell
• Categories and types of antibiotics

  • Cell wall synthesis inhibitors
  • Protein synthesis inhibitors
  • Nucleic acid synthesis inhibitors
  • Cell membrane function inhibitors
  • Other metabolic process inhibitors
• Beta-lactams


  • Effective against Gram (+) and Gram (-) organisms, usually bactericidal, low toxicity
• Glycopeptides


  • Effective against Gram-positive resistant to other beta lactams, usually ineffective against Gram (-)
• Aminoglycosides


  • Effective against Gram (+) and Gram (-) organisms, Chlamydia and rickettsiae, toxic to kidneys and ears
• Tetracyclines


  • Effective against a wide variety Gram (+) and Gram (-) organisms, not widely used due to toxicity
• Macrolides-Lincosamides-Streptogramin group


  • Effective against most Gram (+) and Gram (-) organisms
• Fluoroquinolones

  • Inhibit DNA gyrase, effective against Gram (+) and Gram (-) organisms
• Rifampicin


  • Inhibits DNA-dependent RNA polymerase, effective against Gram (+) and certain Gram (-), resistance develops quickly
• Metronidazole


  • Reduction products cause altered DNA synthesis, bactericidal effect on some Gram (+), many Gram (-) & anaerobes
• Sulfonamides

  • Inhibit folic acid synthesis, target and bind to dihydropteroate synthase
• Trimethoprim


  • Inhibits dihydrofolate reductase, for Gram (+) & many Gram (-), frequently combined with sulfonamide
• Nitrofurantoin


  • Inhibits bacterial enzymes, used only to treat UTI
• Polymyxins


  • More active against Gram (-), risk of toxicity
• Amphotericin B


  • An antifungal used to treat fungal infections
• General considerations for susceptibility testing

  1. Inoculum preparation

  2. Use of pure culture

  3. Use of standard-sized inoculum

  4. Selection of antimicrobial agent
• Antimicrobial battery

  A panel of antimicrobial agents tested against a bacterial isolate
• Antimicrobial battery examples

  • Escherichia coli and Other Gram Negative Bacilli
  • Pseudomonas aeruginosa and Other Nonfermenting Gram Negative Bacilli
  • Staphylococcus species
• Antimicrobial susceptibility test methods

  • Quantitative dilution tests
  • Qualitative diffusion tests
• Agar dilution method

  1. Add various concentrations of antimicrobial agents into heated agar

  2. Spot-inoculate a standard inoculum of bacteria onto agars containing antimicrobial agents

  3. Incubate and examine for visible growth

  4. Determine the MIC
• Macrobroth dilution susceptibility testing

  1. Prepare two-fold dilutions of antibiotics in a liquid growth medium in test tubes

  2. Inoculate the antibiotic-containing tubes with a standardized bacterial suspension

  3. Incubate and examine for visible bacterial growth

  
• Procedure for antimicrobial susceptibility testing

  1. Add various concentrations of antimicrobial agents into heated agar

  2. Spot-inoculate a standard inoculum of bacteria onto agars containing antimicrobial agents

  3. Incubate the plates overnight and examine for visible growth

  4. Determine the MIC

  
• Antimicrobial susceptibility testing


  • Routinely used in anaerobic susceptibility testing and in research facilities
  • Advantageous for testing one or more bacterial isolates (max. 32)
• Macrobroth Dilution Susceptibility Testing

  1. Prepare two-fold dilutions of antibiotics in a liquid growth medium

  2. Inoculate the antibiotic-containing tubes with a standardized bacterial suspension

  3. Incubate overnight and examine for visible bacterial growth

  
• Broth-Macrodilution Method

  1. Add 1 ml of test bacteria (1 × 10^8 CFU/ml) to tubes containing 1 ml broth and concentration of antibiotic (mg/l)

  2. Determine MIC (tube with lowest concentration of antibiotic that shows no turbidity)

  3. Subculture non-turbid tubes to agar plates

  4. Determine MBC (lowest concentration that kills 99.9% bacteria)

  
• Microbroth Dilution Susceptibility Testing


  Utilizes the principle of broth dilution method except that it utilizes "microdilutions"

  
• Gradient Diffusion Test

  Also known as Epsilometer test, a variation to the disk diffusion test that provides quantitative value and can identify the presence of antimicrobial resistance
• Gradient Diffusion Test Procedure

  1. Inoculate agar plate with standardized inoculum

  2. Place the antibiotic strips on the agar surface

  3. Incubate overnight

  4. Examine for elliptical zone of inhibition relative to the antibiotic concentration
• Disk Diffusion Method (Kirby-Bauer Technique)

  Standardized bacterial isolate is spread on an agar plate and then paper disks containing specific concentration of antibiotics are placed
• Disk Diffusion Method

  • Provides greatest flexibility and cost-effectiveness
  • Limited to aerobic and facultatively anaerobic bacteria, not recommended for slow-growing organisms and anaerobes
• Kirby-Bauer Disc Diffusion Susceptibility Test Procedure

  1. Preparation of Mueller-Hinton plate

  2. Preparation of Inoculum

  3. Inoculation of the Mueller-Hinton Plate

  4. Placement of the Antibiotic Discs

  5. Incubation of the Mueller-Hinton Plates

  6. Measuring zone sizes
• Zone diameter interpretative standards for Staphylococcus species
• w inches above a black, nonreflecting surface

  Illuminated with reflected light
• View the plate


  Using a direct, vertical line of sight to avoid any parallax that may result in misreading
• Record the zone size

  On the recording sheet
• If the placement of the disc or the size of the zone does not allow you to read the diameter of the zone

  Measure from the center of the disc to a point on the circumference of the zone where a distinct edge is present (the radius) and multiply the measurement by 2 to determine the diameter