Wang Shiwei, Doona Christopher J, Setlow Peter, Li Yong-Qing
Department of Physics, East Carolina University, Greenville, North Carolina, USA State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
U.S. Army-Natick Soldier RD&E Center, Natick, Massachusetts, USA.
Appl Environ Microbiol. 2016 Sep 16;82(19):5775-84. doi: 10.1128/AEM.01669-16. Print 2016 Oct 1.
Raman spectroscopy and phase-contrast microscopy were used to examine calcium dipicolinate (CaDPA) levels and rates of nutrient and nonnutrient germination of multiple individual Bacillus subtilis spores treated with cold atmospheric plasma (CAP). Major results for this work include the following: (i) >5 logs of spores deposited on glass surfaces were inactivated by CAP treatment for 3 min, while deposited spores placed inside an impermeable plastic bag were inactivated only ∼2 logs in 30 min; (ii) >80% of the spores treated for 1 to 3 min with CAP were nonculturable and retained CaDPA in their core, while >95% of spores treated with CAP for 5 to 10 min lost all CaDPA; (iii) Raman measurements of individual CAP-treated spores without CaDPA showed differences from spores that germinated with l-valine in terms of nucleic acids, lipids, and proteins; and (iv) 1 to 2 min of CAP treatment killed 99% of spores, but these spores still germinated with nutrients or exogenous CaDPA, albeit more slowly and to a lesser extent than untreated spores, while spores CAP treated for >3 min that retained CaDPA did not germinate via nutrients or CaDPA. However, even after 1 to 3 min of CAP treatment, spores germinated normally with dodecylamine. These results suggest that exposure to the present CAP configuration severely damages a spore's inner membrane and key germination proteins, such that the treated spores either lose CaDPA or can neither initiate nor complete germination with nutrients or CaDPA. Analysis of the various CAP components indicated that UV photons contributed minimally to spore inactivation, while charged particles and reactive oxygen species contributed significantly.
Much research has shown that cold atmospheric plasma (CAP) is a promising tool for the inactivation of spores in the medical and food industries. However, knowledge about the effects of plasma treatment on spore properties is limited, especially at the single-cell level. In this study, Raman spectroscopy and phase-contrast microscopy were used to analyze CaDPA levels and kinetics of nutrient- and non-nutrient-germinant-induced germination of multiple individual spores of Bacillus subtilis that were treated by a planar CAP device. The roles of different plasma species involved in spore inactivation were also investigated. The knowledge obtained in this study will aid in understanding the mechanism(s) of spore inactivation by CAP and potentially facilitate the development of more effective and efficient plasma sterilization techniques in various applications.
采用拉曼光谱和相差显微镜检测经冷大气等离子体（CAP）处理的多个枯草芽孢杆菌单个孢子中吡啶二羧酸钙（CaDPA）水平以及营养型和非营养型萌发率。这项工作的主要结果如下：（i）沉积在玻璃表面的孢子经CAP处理3分钟后，>5个对数级的孢子失活，而置于不透水塑料袋内的沉积孢子在30分钟内仅约2个对数级失活；（ii）经CAP处理1至3分钟的孢子中，>80%不可培养且核心中保留CaDPA，而经CAP处理5至10分钟的孢子中，>95%失去了所有CaDPA；（iii）对不含CaDPA的经CAP处理的单个孢子进行拉曼测量，结果显示在核酸、脂质和蛋白质方面与用L-缬氨酸萌发的孢子存在差异；（iv）CAP处理1至2分钟可杀死99%的孢子，但这些孢子仍能在营养物质或外源CaDPA作用下萌发，尽管比未处理的孢子萌发得更慢且程度更低，而经CAP处理>3分钟且保留CaDPA的孢子不能通过营养物质或CaDPA萌发。然而，即使经CAP处理1至3分钟后，孢子仍能正常在十二烷基胺作用下萌发。这些结果表明，暴露于当前的CAP配置会严重损害孢子的内膜和关键萌发蛋白，使得经处理的孢子要么失去CaDPA，要么无法启动或完成在营养物质或CaDPA作用下的萌发。对各种CAP成分的分析表明，紫外线光子对孢子失活的贡献最小，而带电粒子和活性氧物种的贡献显著。
许多研究表明，冷大气等离子体（CAP）是医疗和食品工业中用于孢子灭活的一种有前景的工具。然而，关于等离子体处理对孢子特性影响的知识有限，尤其是在单细胞水平。在本研究中，采用拉曼光谱和相差显微镜分析经平面CAP装置处理的多个枯草芽孢杆菌单个孢子的CaDPA水平以及营养型和非营养型萌发诱导剂诱导的萌发动力学。还研究了参与孢子失活的不同等离子体物种的作用。本研究获得的知识将有助于理解CAP使孢子失活的机制，并可能促进在各种应用中开发更有效和高效的等离子体灭菌技术。
