Analysis of growth and death kinetics at single-cell resolution is a key step in understanding the complexity of the nonreplicating growth phenotype of the bacterial pathogen Mycobacterium tuberculosis. Here, we developed a single-cell-resolution microfluidic mycobacterial culture device that allows time-lapse microscopy-based long-term phenotypic visualization of the live replication dynamics of mycobacteria. This technology was successfully applied to monitor the real-time growth dynamics of the fast-growing model strain Mycobacterium smegmatis (M. smegmatis) while subjected to drug treatment regimens during continuous culture for 48 h inside the microfluidic device. A clear morphological change leading to significant swelling at the poles of the bacterial membrane was observed during drug treatment. In addition, a small subpopulation of cells surviving treatment by frontline antibiotics was observed to recover and achieve robust replicative growth once regular culture media was provided, suggesting the possibility of identifying and isolating nonreplicative mycobacteria. This device is a simple, easy-to-use, and low-cost solution for studying the single-cell phenotype and growth dynamics of mycobacteria, especially during drug treatment.

在单细胞分辨率下分析生长和死亡动力学是了解细菌病原体结核分枝杆菌的非复制生长表型的复杂性的关键一步。在这里，我们开发了一种单细胞分辨率的微流体分枝杆菌培养装置，它允许基于延时显微镜的分枝杆菌实时复制动态的长期表型可视化。该技术成功应用于监测快速生长模型菌株耻垢分枝杆菌（M. smegmatis）的实时生长动态) 同时在微流体装置内连续培养 48 小时期间接受药物治疗方案。在药物治疗期间观察到导致细菌膜两极显着肿胀的明显形态变化。此外，观察到一小部分在一线抗生素治疗中存活下来的细胞，一旦提供常规培养基，就会恢复并实现强劲的复制生长，这表明识别和分离非复制性分枝杆菌的可能性。该设备是一种简单、易于使用且成本低廉的解决方案，用于研究分枝杆菌的单细胞表型和生长动态，尤其是在药物治疗期间。