Application of luminescence-producing Mycobacterium tuberculosis strains for evaluating anti-tuberculosis drug and vaccine efficacy in vitro and in vivo

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains a major public health problem worldwide. The development of effective drugs and vaccines remains crucial but the prolonged culture period of M.tb hampers this process. This limitation has been addressed by developing a bioluminescent M.tb strain that can be used to measure bacterial burden in live animals non-invasively and in real time. We investigated whether bioluminescence real-time imaging technologies could be used to monitor the infection process in live mice, with the aim of facilitating TB research. In this study, we constructed bioluminescence-expressing M.tb strains. We compared the growth of these recombinant reporter and wild-type (WT) strains to confirm their stability in vitro. BALB/c mice were infected via intranasal or intravenous routes and monitored using an In Vivo Imaging System. The efficacy of isoniazid treatment was assessed using bioluminescence imaging and confirmed using colony-forming unit (CFU) assays. In a separate experiment, BCG-vaccinated and naïve control mice were infected with M.tb L5-Lux, a recombinant strain that expresses luxCDABE under the L5 promoter derived from the L5 mycobacteriophage. Vaccine efficacy was assessed by comparing bioluminescence and CFU counts. The M.tb L5-Lux strain showed stable growth and a strong bioluminescent signal comparable to the WT strain. In both infection models, bioluminescence intensity strongly correlated with bacterial load. Isoniazid treatment led to a significant reduction in luminescence and CFU. In the vaccine study, BCG-vaccinated mice exhibited lower in vivo and ex vivo bioluminescence and approximately 0.5 log₁₀ lower CFU counts than naïve controls, demonstrating the utility of the M.tb L5-Lux strain for vaccine efficacy assessment. The M.tb L5-Lux bioluminescent reporter strain provides a sensitive and efficient tool for monitoring M.tb infection and evaluating anti-TB drug and vaccine efficacy in real time. This imaging-based platform facilitates faster and more ethical preclinical testing by reducing the number of animals and experimental duration.