Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence

Abstract

Objectives: Tuberculosis drug development is hampered by the slow growth of Mycobacterium tuberculosis. Bioluminescence, light produced by an enzymatic reaction, constitutes a rapid and highly sensitive measurement of cell metabolic function that can be used as an indirect marker of cell viability in drug screening assays. The aim of this work was to validate and standardize the use of luminescent M. tuberculosis strains to test the activity of antibacterial drugs in vitro and inside macrophages in a 96-well format.

Methods: We have used strains that express the bacterial lux operon and therefore do not require exogenous substrate to produce light, as well as strains expressing the firefly luciferase that need luciferin substrate. Results were compared with those obtained using the resazurin reduction assay and cfu plating.

Results: Using bioluminescence we were able to reduce the time required to measure the MIC and bactericidal concentrations of antimicrobials to just 3 and 6 days, respectively. Furthermore, antibacterial activity against intracellular mycobacteria was detected within 2 days post-infection. Results were comparable to those obtained by conventional methods.

Conclusions: We have developed a simple and rapid method for screening antimycobacterial drugs in culture and in macrophages. The use of autoluminescent bacteria also facilitates the determination of growth and inhibition kinetics. The method is cost-effective, can easily be adapted to a larger scale and is amenable to automation. Current efforts are directed towards applying this technology to drug screening in vivo.

Figure 1.

Effect of isoniazid (INH), chloramphenicol (CHL) and streptomycin (STR) on bioluminescence of H37Rv LuxG13 expressed as a function of time for all the concentrations tested (top graphs), and as a dose–response curve after 2–8 days of incubation (bottom graphs). The vertical dotted line in the dose–response curves indicates the MIC measured using the bioluminescence results. Data shown are the mean and standard deviation from one representative experiment.

Figure 2.

Bioluminescence of H37Rv LuxG13 incubated for 3 days in the presence of the indicated antimicrobial agents (MIC plates), followed by incubation in the absence of antibiotics (MBC plates). The vertical dotted line indicates the time when the antibiotic was removed. Data shown are the mean and standard deviation from one representative experiment. Bioluminescence was corrected for the dilution applied to set up the MBC plates. Readings for the non-luminescent H37Rv hsp strain represent the background bioluminescence, which is indicated as the shaded area. INH, isoniazid; CHL, chloramphenicol; STR, streptomycin.

Figure 3.

Effect of isoniazid (INH) and chloramphenicol (CHL) on intracellular H37Rv LuxG13, as determined by bioluminescence reading (top graphs) and by cfu counts (bottom graphs). Data are plotted as a function of time and as dose–response curve, and are the mean and standard deviation of 2–3 independent experiments.

Figure 4.

Effect of isoniazid (INH) and chloramphenicol (CHL) on intracellular H37Rv hspFFluc, as determined by bioluminescence reading and by cfu counts. Data are plotted as a function of time and as dose–response curve, and are the mean and standard deviation of 2–3 independent experiments.