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

Abstract

ObjectivesTuberculosis 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.MethodsWe 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.ResultsUsing 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.ConclusionsWe 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.

The Efflux Pump Inhibitor Timcodar Improves the Potency of Antimycobacterial Agents

“…An M. tuberculosis strain (H37Ra) was transfected with pVV261 expressing firefly luciferase by methods similar to those previously described (18) and used to infect a human macrophage-like cell line (THP-1; ATCC TIB-202). Briefly, THP-1 cell stocks were maintained at a culture density of between 2 ϫ 10 5 and 8 ϫ 10 5 cells/ml in RPMI 1640 medium (with phenol red, 25 mM HEPES, and 2 mM L-glutamine; Gibco) supplemented with 10% fetal bovine serum (Gibco) and 0.05 mM ␤-mercaptoethanol (Invitrogen) in 96-well tissue culture plates (Costar 3903; Corning).…”

Doxycycline and HIV Infection Suppress Tuberculosis-induced Matrix Metalloproteinases

“…Finally, to determine the minimum inhibitory concentration (MIC), we cultured bioluminescent Mtb with increasing concentrations of doxycycline and monitored bioluminescence over time, as previously described (27). Using this method, the MIC is defined as the lowest antibiotic concentration causing a 1 log drop in relative light units after 2 to 3 days of incubation, compared with the luminescence in the antibiotic-free controls (27). Doxycycline caused a dose dependent reduction in luminescence ( Figure 7C) with an MIC of 2.5 mg/ml.…”

Rapid Whole-Cell Assay of Antitubercular Drugs Using Second-Generation Fluoromycobacteriophages

“…In comparison with other whole-cell assays for activity testing of compounds (13)(14)(15)(16)(17)(18), results using fluoromycobacteriophages can be obtained in hours in contrast to days; no addition of substrate is needed (13,14,19), and, since mycobacterial cultures can be maintained constantly growing, they are readily available when needed. All these features make mCherry bomb ⌽ in combination with automated fluorimetric detection a convenient tool for activity testing of new antitubercular drugs and a potential rapid drug susceptibility testing (DST) assay of M. tuberculosis clinical isolates.…”