Rapid in vivo assessment of drug efficacy against Mycobacterium tuberculosis using an improved firefly luciferase

Abstract

ObjectivesIn vivo experimentation is costly and time-consuming, and presents a major bottleneck in anti-tuberculosis drug development. Conventional methods rely on the enumeration of bacterial colonies, and it can take up to 4 weeks for Mycobacterium tuberculosis to grow on agar plates. Light produced by recombinant bacteria expressing luciferase enzymes can be used as a marker of bacterial load, and disease progression can be easily followed non-invasively in live animals by using the appropriate imaging equipment. The objective of this work was to develop a bioluminescence-based mouse model of tuberculosis to assess antibiotic efficacy against M. tuberculosis in vivo.MethodsWe used an M. tuberculosis strain carrying a red-shifted derivative of the firefly luciferase gene (FFlucRT) to infect mice, and monitored disease progression in living animals by bioluminescence imaging before and after treatment with the frontline anti-tuberculosis drug isoniazid. The resulting images were analysed and the bioluminescence was correlated with bacterial counts.ResultsUsing bioluminescence imaging we detected as few as 1.7 × 103 and 7.5 × 104 reporter bacteria ex vivo and in vivo, respectively, in the lungs of mice. A good correlation was found between bioluminescence and bacterial load in both cases. Furthermore, a marked reduction in luminescence was observed in living mice given isoniazid treatment.ConclusionsWe have shown that an improved bioluminescent strain of M. tuberculosis can be visualized by non-invasive imaging in live mice during an acute, progressive infection and that this technique can be used to rapidly visualize and quantify the effect of antibiotic treatment. We believe that the model presented here will be of great benefit in early drug discovery as an easy and rapid way to identify active compounds in vivo.

A Bioluminescent Francisella tularensis SCHU S4 Strain Enables Noninvasive Tracking of Bacterial Dissemination and the Evaluation of Antibiotics in an Inhalational Mouse Model of Tularemia

“…One milliliter of Leibovitz's L-15 medium (Gibco Life Technologies) containing 10 6 CFU of either SCHU S4 or SCHU S4-lux was added to cell monolayers to achieve a multiplicity of infection (MOI) of 1. Each strain was added to wells in triplicate for each time point.…”

“…Noninvasive imaging techniques, such as bioluminescence imaging (BLI), can report spatial and temporal aspects of infectious diseases in vivo both longitudinally and in real time (9). BLI typically relies on the detection of light produced by luciferase-catalyzed oxidation reactions, which are encoded in the pathogen of interest, by use of a sensitive charge-coupled device (CCD) camera (10). Pivotal experiments by Contag et al have demonstrated the usefulness of BLI in the noninvasive study of bacterial pathogenesis and the evaluation of antibiotic treatments for Salmonella enterica serovar Typhimurium (11).…”

“…Pivotal experiments by Contag et al have demonstrated the usefulness of BLI in the noninvasive study of bacterial pathogenesis and the evaluation of antibiotic treatments for Salmonella enterica serovar Typhimurium (11). Subsequently, bioluminescent reporters have been introduced into other bacteria, including Mycobacterium tuberculosis (10,12), Staphylococcus aureus (13,14), Burkholderia mallei (15), and Yersinia pestis (16,17). Further, BLI has been used to evaluate vaccines (18), antibiotics (19)(20)(21), and supportive therapies (22) for a range of pathogens.…”

New Targets of Molecular Imaging in Atherosclerosis: Prehension of Current Status

“…70 Also, in vivo imaging of tuberculosis progression was tried in mice after infection of an M. tuberculosis strain carrying a red-shifted derivative of the firefly luciferase gene. 71 Recently, MRI imaging of S. aureus labeled with iron oxide particle was developed for detection of S. aureus colonies in infection models. 72 However, these methods are not suitable for clinical application because they involve pre-labeled/modified bacteria, where bacteria tracking in animal models were visualized by luminescence and MRI.…”

“…In addition, new putative targets of atherosclerosis that are non-macrophage-related, such as biofilm and amyloid fibrils, have been suggested as potential targets. Although bacteria/ bacteria film (biofilm) [113][114][115] and amyloid fibrils [69][70][71] are known to be components of atherogenesis, thus far there have been no development of non-invasive imaging techniques that are targeted at either biofilm or amyloid fibrils in the area of atherosclerosis imaging. Thus, it is clinically necessary to develop a non-invasive imaging modality to evaluate biofilm and amyloid load quantitatively, to monitor disease progression and response to treatment.…”