Zhang Yifan, Delbrück Alessia I, Off Cosima L, Benke Stephan, Mathys Alexander
Sustainable Food Processing Laboratory, Institute of Food, Nutrition and Health, Department of Health Science and Technology, ETH Zürich, Zurich, Switzerland.
Cytometry Facility, University of Zurich, Zurich, Switzerland.
Front Microbiol. 2020 Jan 21;10:3118. doi: 10.3389/fmicb.2019.03118. eCollection 2019.
Isostatic high pressure (HP) of 150 MPa can trigger the germination of bacterial spores, making them lose their extreme resistance to stress factors, and increasing their susceptibility to milder inactivation strategies. However, germination response of spores within a population is very heterogeneous, and tools are needed to study this heterogeneity. Here, classical methods were combined with more recent and powerful techniques such as flow cytometry (FCM) and fluorescence activated cell sorting (FACS) to investigate spore germination behavior under HP. spores were treated with HP at 150 MPa and 37°C, stained with SYTO16 and PI, and analyzed via FCM. Four sub-populations were detected. These sub-populations were for the first time isolated on single cell level using FACS and characterized in terms of their heat resistance (80°C, 10 min) and cultivability in a nutrient-rich environment. The four isolated sub-populations were found to include (1) heat-resistant and mostly cultivable superdormant spores, i.e., spores that remained dormant after this specific HP treatment, (2) heat-sensitive and cultivable germinated spores, (3) heat-sensitive and partially-cultivable germinated spores, and (4) membrane-compromised cells with barely detectable cultivability. Of particular interest was the physiological state of the third sub-population, which was previously referred to as "unknown". Moreover, the kinetic transitions between different physiological states were characterized. After less than 10 min of HP treatment, the majority of spores germinated and ended up in a sublethally damaged stage. HP treatment at 150 MPa and 37°C did not cause inactivation of all geminated spores, suggesting that subsequent inactivation strategies such as mild heat inactivation or other inactivation techniques are necessary to control spores in food. This study validated FCM as a powerful technique to investigate the heterogeneous behavior of spores under HP, and provided a pipeline using FACS for isolation of different sub-populations and subsequent characterization to understand their physiological states.
150兆帕的等静压高压（HP）可引发细菌孢子萌发，使其失去对压力因素的极端抗性，并增加其对较温和灭活策略的敏感性。然而，群体内孢子的萌发反应非常不均一，因此需要工具来研究这种不均一性。在此，将经典方法与更新且强大的技术（如流式细胞术（FCM）和荧光激活细胞分选（FACS））相结合，以研究高压下的孢子萌发行为。将孢子在150兆帕和37°C下进行高压处理，用SYTO16和PI染色，并通过流式细胞术进行分析。检测到四个亚群。首次使用FACS在单细胞水平上分离出这些亚群，并根据其耐热性（80°C，10分钟）和在营养丰富环境中的可培养性进行表征。发现这四个分离出的亚群包括：（1）耐热且大多可培养的超级休眠孢子，即经过这种特定高压处理后仍保持休眠的孢子；（2）热敏且可培养的已萌发孢子；（3）热敏且部分可培养的已萌发孢子；（4）可培养性几乎无法检测到的膜受损细胞。特别令人感兴趣的是第三个亚群的生理状态，该亚群以前被称为“未知”。此外，还对不同生理状态之间的动力学转变进行了表征。在高压处理不到10分钟后，大多数孢子萌发并进入亚致死损伤阶段。在150兆帕和37°C下的高压处理并未导致所有已萌发孢子失活，这表明后续的灭活策略（如温和热灭活或其他灭活技术）对于控制食品中的孢子是必要的。本研究验证了流式细胞术是研究高压下孢子异质行为的强大技术，并提供了一种使用荧光激活细胞分选技术分离不同亚群并随后进行表征以了解其生理状态的流程。
