10.5 Testing the Effectiveness of Antimicrobials

Learning Objectives

  • Describe how the Kirby-Bauer disk diffusion test determines the susceptibility of a microbe to an antibacterial drug.
  • Explain the significance of the minimal inhibitory concentration and the minimal bactericidal concentration relative to the effectiveness of an antimicrobial drug.

Testing the effectiveness of antimicrobial drugs against specific organisms is important in identifying their spectrum of activity and the therapeutic dosage. This type of test, generally described as antimicrobial susceptibility testing (AST), is commonly performed in a clinical laboratory. In this section, we will discuss common methods of testing the effectiveness of antimicrobials.

The Kirby-Bauer Disk Diffusion Test

The Kirby-Bauer disk diffusion test has long been used as a starting point for determining the susceptibility of specific microbes to various antimicrobial drugs. The Kirby-Bauer assay starts with a Mueller-Hinton agar plate on which a confluent lawn is inoculated with a patient’s isolated bacterial pathogen. Filter paper disks impregnated with known amounts of antibacterial drugs to be tested are then placed on the agar plate. As the bacterial inoculum grows, antibiotic diffuses from the circular disk into the agar and interacts with the growing bacteria. Antibacterial activity is observed as a clear circular zone of inhibition around the drug-impregnated disk, similar to the disk-diffusion assay depicted in Figure 10.10. The diameter of the zone of inhibition, measured in millimeters and compared to a standardized chart, determines the susceptibility or resistance of the bacterial pathogen to the drug.

There are multiple factors that determine the size of a zone of inhibition in this assay, including drug solubility, rate of drug diffusion through agar, the thickness of the agar medium, and the drug concentration impregnated into the disk. Due to a lack of standardization of these factors, interpretation of the Kirby-Bauer disk diffusion assay provides only limited information on susceptibility and resistance to the drugs tested. The assay cannot distinguish between bacteriostatic and bactericidal activities, and differences in zone sizes cannot be used to compare drug potencies or efficacies. Comparison of zone sizes to a standardized chart will only provide information on the antibacterials to which a bacterial pathogen is susceptible or resistant.

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  • How does one use the information from a Kirby-Bauer assay to predict the therapeutic effectiveness of an antimicrobial drug in a patient?

Dilution Tests

As discussed, the limitations of the Kirby-Bauer disk diffusion test do not allow for a direct comparison of antibacterial potencies to guide selection of the best therapeutic choice. However, antibacterial dilution tests can be used to determine a particular drug’s minimal inhibitory concentration (MIC), the lowest concentration of drug that inhibits visible bacterial growth, and minimal bactericidal concentration (MBC), the lowest drug concentration that kills ≥99.9% of the starting inoculum. Determining these concentrations helps identify the correct drug for a particular pathogen. For the macrobroth dilution assay, a dilution series of the drug in broth is made in test tubes and the same number of cells of a test bacterial strain is added to each tube (Figure 10.10). The MIC is determined by examining the tubes to find the lowest drug concentration that inhibits visible growth; this is observed as turbidity (cloudiness) in the broth. Tubes with no visible growth are then inoculated onto agar media without antibiotic to determine the MBC. Generally, serum levels of an antibacterial should be at least three to five times above the MIC for treatment of an infection.

The MIC assay can also be performed using 96-well microdilution trays, which allow for the use of small volumes and automated dispensing devices, as well as the testing of multiple antimicrobials and/or microorganisms in one tray (Figure 10.11). MICs are interpreted as the lowest concentration that inhibits visible growth, the same as for the macrobroth dilution in test tubes. Growth may also be interpreted visually or by using a spectrophotometer or similar device to detect turbidity or a color change if an appropriate biochemical substrate that changes color in the presence of bacterial growth is also included in each well.

In a dilution test, the lowest dilution that inhibits turbidity (cloudiness) is the MIC. In this example, the MIC is 8 μg/mL. Broth from samples without turbidity can be inoculated onto plates lacking the antimicrobial drug. The lowest dilution that kills ≥99.9% of the starting inoculum is observed on the plates is the MBC.
Figure 10.10 In a dilution test, the lowest dilution that inhibits turbidity (cloudiness) is the MIC. In this example, the MIC is 8 μg/mL. Broth from samples without turbidity can be inoculated onto plates lacking the antimicrobial drug. The lowest dilution that kills ≥99.9% of the starting inoculum is observed on the plates is the MBC. (credit: modification of work by Suzanne Wakim)

 

A microdilution tray can also be used to determine MICs of multiple antimicrobial drugs in a single assay. In this example, the drug concentrations increase from left to right and the rows with clindamycin, penicillin, and erythromycin have been indicated to the left of the plate. For penicillin and erythromycin, the lowest concentrations that inhibited visible growth are indicated by red circles and were 0.06 μg/mL for penicillin and 8 μg/ mL for erythromycin. For clindamycin, visible bacterial growth was observed at every concentration up to 32 μg/mL and the MIC is interpreted as >32 μg/mL.
Figure 10.11 A microdilution tray can also be used to determine MICs of multiple antimicrobial drugs in a single assay. In this example, the drug concentrations increase from left to right and the rows with clindamycin, penicillin, and erythromycin have been indicated to the left of the plate. For penicillin and erythromycin, the lowest concentrations that inhibited visible growth are indicated by red circles and were 0.06 μg/mL for penicillin and 8 μg/ mL for erythromycin. For clindamycin, visible bacterial growth was observed at every concentration up to 32 μg/mL and the MIC is interpreted as >32 μg/mL. (credit: modification of work by Centers for Disease Control and Prevention)
  • Compare and contrast MIC and MBC.

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