Tuberculosis is the most important bacterial infection worldwide, killing approximately 2 million people annually. Antibiotic treatment is available, but regimens are prolonged and effectiveness relies on high patient compliance. Increasing numbers of multi-drug resistant strains are emerging and therefore new and more effective antibiotics are urgently needed. Conventional assays to test the efficacy of new drugs in vivo rely on culturing bacteria from organ homogenates and enumeration of colony forming units (CFU). This method is well established and reliable, but labour intensive, with high animal usage, and time consuming, as it can take up to 6 weeks for mycobacterial colonies to form. Furthermore, enumeration of bacteria by CFU assay is a retrospective end point measurement. To expedite the development and testing of new drugs, we developed a method for imaging tuberculosis, using fluorescent reporter strains of Mycobacterium tuberculosis (Mtb) in an immune-compromised mouse model of infection. This method can provide detailed information on the bacterial location in a living animal or exteriorised organ of interest, together with an instant assessment of the bacterial load. Repeated imaging of the same animals also allows the collection of longitudinal data for individual animals. We have constructed several Mtb strains expressing red fluorescent proteins, and have evaluated their suitability for use in fluorescence imaging assays. Mtb Charge3, a strain expressing Turbo-635 as a marker, retained virulence in vivo when compared to the Mtb wild type H37rv, and stably expressed the marker protein for the duration of the infection. Using a commercial IVIS® and a custom built FMT system to characterise Mtb Charge3, the detection limits in the lungs in vivo and ex vivo were determined, and compared between the two imaging systems. We found that using the IVIS® system, the detection limit of Mtb Charge3 in infected mice is approx. 10⁸ CFU/lung and the measured fluorescence is significantly higher than that measured from Mtb H37rv WT infected mice. Ex vivo imaging of whole organs showed an improved detection limit of approx. 10⁷ CFU/lung. Imaging organs ex vivo using the FMT system yielded significantly higher spatial resolution of the fluorescent signal as compared to the IVIS® system. Furthermore, bacterial load, as determined by CFU plating, correlated to the fluorescence measured ex vivo. Proof-of-principle experiments to investigate the effect of the antibiotic moxifloxacin on the fluorescent signal are currently ongoing. By avoiding the need for the slow time course required for CFU counts, fluorescent imaging could be adapted as a quick and efficient method for the testing of new antibiotics against tuberculosis.

Mice infected with M. tuberculosis (Mtb) expressing Turbo-635 or Mtb wild type (WT) and imaged in an IVIS® system. Smaller images in the bottom row show exteriorised lungs from each mouse.