Real-Time Bioluminescence Imaging of Mixed Mycobacterial Infections

Abstract

Molecular analysis of infectious processes in bacteria normally involves construction of isogenic mutants that can then be compared to wild type in an animal model. Pathogenesis and antimicrobial studies are complicated by variability between animals and the need to sacrifice individual animals at specific time points. Live animal imaging allows real-time analysis of infections without the need to sacrifice animals, allowing quantitative data to be collected at multiple time points in all organs simultaneously. However, imaging has not previously allowed simultaneous imaging of both mutant and wild type strains of mycobacteria in the same animal. We address this problem by using both firefly (Photinus pyralis) and click beetle (Pyrophorus plagiophthalamus) red luciferases, which emit distinct bioluminescent spectra, allowing simultaneous imaging of two different mycobacterial strains during infection. We also demonstrate that these same bioluminescence reporters can be used to evaluate therapeutic efficacy in real-time, greatly facilitating our ability to screen novel antibiotics as they are developed. Due to the slow growth rate of mycobacteria, novel imaging technologies are a pressing need, since they can they can impact the rate of development of new therapeutics as well as improving our understanding of virulence mechanisms and the evaluation of novel vaccine candidates.

Dual-Color Bioluminescence Imaging for Simultaneous Monitoring of the Intestinal Persistence of Lactobacillus plantarum and Lactococcus lactis in Living Mice

“…In vivo, the bioluminescence of CBRluc was greater than the one from CBGluc in both strains, demonstrating that CBRluc is a more sensitive reporter than CBGluc for tracking bacteria in the GIT. Similar observations were made by Chang et al with CBRluc and Fluc (with an emission spectrum similar to CBGluc's) in the lungs of mice infected with bioluminescent mycobacteria (10). Similar conclusions also have been made by Mezzanotte et al on imaging the liver and prostate using a red-optimized Fluc with CBGluc (9).…”

“…Similar conclusions also have been made by Mezzanotte et al on imaging the liver and prostate using a red-optimized Fluc with CBGluc (9). The lungs, the liver, the prostate, and the GIT are difficult sites to image optically due to the great tissue depth, and the use of redemitting luciferases is more adapted to imaging in these tissues, as the longer wavelength enables better penetration through living tissues (4,(8)(9)(10).…”

“…Dual-color bioluminescence has been described with red and green luciferases expressed by mammalian cells that can be separated in vivo (9). Very recently, Chang et al showed that Fluc and CBRluc, which emit distinct light wavelengths, allow simultaneous imaging of two mycobacterial strains during infection (10).…”

New directions using reporter enzyme fluorescence (REF) as a tuberculosis diagnostic platform

“…In our own laboratory, we have targeted diagnostic strategies, since they have the potential to change the TB management landscape due to the fact that current diagnostic methods often cannot make a diagnosis prior to transmission [1,15]. Recent optical imaging technological developments have created more sensitive ways to visualize and quantify mycobacteria within cells and in vivo [2,3,6–9,12,21]. Despite these advances, reduction in signal due to tissue depth make application to diagnosis in humans extremely difficult.…”

Polyelectrolyte Multilayer Nanocoating Dramatically Reduces Bacterial Adhesion to Polyester Fabric

“…35,37 In the present study, bioluminescence is used to provide a fast and easy method to quantify bacterial concentration on polyester fabric substrates. This technique has been used to image living laboratory animals in vivo, 42–44 which speaks to its ability to evaluate bacterial growth in a complex environment. Ten bilayers of PDDA/PAA on polyester fabric removed 99% of S. aureus after simple rinsing with water.…”