Wellcome Centre for Infectious Diseases Research in Africa (CIDRI-Africa), Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, Cape Town, 7925, South Africa.
Institute of Infection and Global Health, University of Liverpool, Liverpool, L7 3EA, UK.
Sci Rep. 2021 Sep 20;11(1):18661. doi: 10.1038/s41598-021-98176-5.
Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology-including the tendency to clump, and "differential" culturability-and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA + cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis bacillus Calmette-Guérin (BCG) time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique.
检测和准确定量有活力的结核分枝杆菌对于理解分枝杆菌的致病性、结核病（TB）的疾病进展和结果、TB 传播、药物作用、疗效和耐药性至关重要。尽管如此，由于分枝杆菌细胞生物学的固有特征——包括聚集的趋势和“差异”可培养性，以及在生物安全条件下处理感染性病原体的技术挑战，用于确定活细菌数量的方法在准确性和精密度方面受到限制。我们使用台式流式细胞仪开发了一种用于液体培养物中的分枝杆菌绝对计数方法，使用廉价的荧光染料 Calcein-AM（CA）和 SYBR-gold（SG）。在指数生长期，CA+细胞计数与 CFU 计数高度相关，可以作为一种实时替代方法，简化实验中接种物的准确标准化。与 CFU 计数相比，该方法可以检测和计数样品中的细胞聚集体，我们表明，当使用现有方法时，这些聚集体是变异性和偏差的潜在来源。我们表明，CFUs 是液体培养物中完整、代谢活跃的分枝杆菌细胞的一个亚群，CFU 比例因生长条件而异。流式细胞术方法的药效学应用，探索与 CFU 相比荧光探针定义的亚群的动力学，得到了证明。利福平和卡那霉素与异烟肼和乙胺丁醇相比，牛分枝杆菌卡介苗（BCG）的流式细胞术时间杀伤曲线不同，这种高通量单细胞技术揭示的杆菌离散形态独特亚群的相对动力学也不同。
