Multiparametric Flow Cytometry and Cell Sorting for the Assessment of Viable, Injured, and Dead Bifidobacterium Cells during Bile Salt Stress

Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC(4)(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.

本研究采用基于流式细胞术(flow cytometry)的检测方法，评估了乳酸双歧杆菌（Bifidobacterium lactis）DSM 10140与青春双歧杆菌（Bifidobacterium adolescentis）DSM 20083在胆盐胁迫条件下的存活能力。本研究选用羧基荧光素二乙酸酯(Carboxyfluorescein diacetate, cFDA)、碘化丙啶(propidium iodide, PI)以及氧杂蒽染料[DiBAC(4)(3)]分别监测酯酶活性、细胞膜完整性与膜电位，以此作为细菌存活能力的评价指标。单独使用上述探针进行单染色时，可快速且清晰地反映两种菌株在胁迫过程中的生理状态变化。然而，与平板菌落计数法(plate counts)相比，流式细胞术的检测结果往往会高估两种菌株的存活率，这一现象与胆盐胁迫造成的亚致死损伤导致部分菌体丧失培养能力密切相关。当菌体同时采用cFDA与PI进行双染色时，流式细胞术与细胞分选技术(cell sorting)可揭示受胁迫双歧杆菌群体中存在显著的生理异质性。依据对探针的摄取差异，可将该群体划分为三个亚群：仅cF染色阳性亚群、cF与PI双染色阳性亚群以及仅PI染色阳性亚群，分别对应存活菌体、受损伤菌体与死亡菌体。经分选与复苏后，双染色阳性亚群中有40%的菌体可在琼脂平板上重新恢复生长。本研究结果表明，采用多参数流式细胞术(multiparameter flow cytometry)与细胞分选技术对受胁迫双歧杆菌的生理活性进行原位检测，可为益生菌的存活能力与稳定性评估提供一种高效且灵敏的检测手段。