An Assay to Evaluate Drug Efficacy against a Mycobacterium tuberculosis Infection Model

Take a multiwell plate containing an established Mycobacterium tuberculosis or Mtb infection model.

The wells contain cell aggregates comprising a core of densely packed Mtb and Mtb-infected necrotic macrophages, surrounded by uninfected macrophages — mimicking in vivo infection.

Take a serial dilution of the antibiotics rifampicin and moxifloxacin.

Discard media from the infection plate, transfer antibiotic dilutions to the wells, and incubate.

The antibiotics diffuse through the cell aggregates and enter the bacterial cells.

Rifampicin binds bacterial RNA polymerase, inhibiting RNA synthesis and causing bacterial death.

Moxifloxacin binds bacterial DNA topoisomerase, inhibiting replication and arresting bacterial growth. The binding also induces stress responses, including the accumulation of reactive oxygen species, killing the bacteria.

Post-incubation, add resazurin — a redox indicator. Inside living and metabolically active bacteria, the oxidoreductase enzyme reduces blue resazurin to pink fluorescent resorufin.

Measure the fluorescence to detect viable bacteria.

Bacterial survival decreases with increasing antibiotic concentration, exhibiting their efficacy against Mtb.

For drug testing, prepare two anti-tuberculosis drugs in triplicate on a new 96-well plate as follows. First, add 125 microliters of 7H9-C medium to wells B2 through G10. Next, prepare the two drugs at double the highest desired final concentration in 1 milliliter of 7H9-C.

Using a pipette, add 250 microliters of each drug to wells B, C, and D-11, and then E, F, and G-11 respectively for triplicate treatments. Next, using a multichannel pipette, serially dilute the test drugs two-fold by moving 125 microliters from B11 through G11 into B10 through G10. Mix by pipetting five times at each step.

Continue to move 125 microliters from column to column, right to left across the plate, and stop after column 4. After mixing column 4, discard 125 microliters into a waste container. Columns 2 and 3 should not contain any drugs. This will allow them to be used as a background and for positive growth controls.

Next, retrieve the 96-well plate containing the Mtb-infected macrophages from the incubator. Without tilting the plate, use a multichannel pipette to remove 150 microliters of medium from wells B2 through G11. Then, tilt the plate as shown here, and insert the pipette tips into the bottom edge of the wells, and remove the remaining medium, about 50 microliters.

As Mtb macrophage aggregates adhere to the bottom of the well, no material should be lost. However, removal of the medium should be as gentle as possible and care must be taken to avoid resuspension during this process.

Gently, add 100 microliters of 7H9-C medium to wells B2 through G11 of the plate, which contain the Mtb macrophage aggregates. Using a multichannel pipette, transfer 100 microliters of the drug-containing 96-well plate to the corresponding wells of the infection plate. Then, place the plate in a sealed bag and incubate it for three days at 37 degrees Celsius.

The resazurin assay relies on the oxidative species produced by metabolically active Mtb to convert the blue resazurin to fluorescent pink resorufin. The change in color and fluorescence can be used as a surrogate marker to determine the amount of bacterial growth.

Prepare a stock solution of resazurin at a final concentration of 0.8 milligrams per milliliter in water. Filter through a 0.22-micrometer pore-size PVDF membrane for sterilization. Prepare a working solution in resazurin by mixing the stock solution, water, and Tween-80 in a 2 to 1 to 1 ratio. Final concentrations are 0.4 milligrams per milliliter resazurin in 5% Tween-80.

Using a plate reader, set up a program to read the fluorescence at 530-nanometer excitation and 590-nanometer emission for 24 hours every 30 minutes at 37 degrees Celsius. Pre-warm the plate reader to 37 degrees Celsius.

Using a multichannel pipette, add 20 microliters of resazurin working solution to wells B2 through G11 of the drug-treated plate. Place the plate on the reader and start the program.