Luiz Pedro Carvalho, PhD (@luizcarvalholab.bsky.social)

Mtb-Timer: a fluorescent reporter to visualize Mycobacterium tuberculosis replication and antibiotic responses | mSystems Tuberculosis treatment is challenging in part because Mycobacterium tuberculosis (Mtb) populations are phenotypically heterogeneous. Within the same infection, bacterial cells can exist in different physiological states, from actively replicating to slow-growing or non-replicating forms. To better study these processes, we developed and validated Mtb-Timer, a fluorescent reporter that enables visualization of bacterial growth dynamics. The system is based on a fluorescent protein that changes color over time: newly synthesized protein emits green fluorescence (GFP) and gradually shifts to red (DsRed) as it matures. The green-to-red fluorescence ratio, therefore, reflects bacterial replication activity. Using microscopy and flow cytometry in both in vitro cultures and infected macrophages (in cellulo), Mtb-Timer enables the quantification of distinct bacterial subpopulations and the real-time monitoring of the green-to-red fluorescence ratio and bacterial burden during treatment with first-line antibiotics. https://journals.asm.org/doi/10.1128/msystems.01796-25