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.