Formation and resuscitation of viable but non-culturable Campylobacter in agri-food industry

食品加工环境和食品中不可培养病原菌（VBNC）的存在和复苏对公众健康构成重大威胁。VBNC病原菌可以逃避常规培养方法的检测，但在复苏时保持毒力并有可能引起食源性疾病。空肠弯曲杆菌（Campylobacter jejuni）是世界范围内引起人类胃肠道疾病的主要细菌，但对其形成、复苏及其在食品工业中的控制措施的研究尚不充分。本研究旨在探讨空肠梭菌在食品加工、贮藏相关环境及食品中的形成与复苏。以空肠C. jejuni VBNC的形成为背景，在模拟食品加工和保存的不同胁迫条件下，监测空肠C. jejuni种群从正常可培养状态向VBNC状态过渡的动态。然后，我们将应用先进的基础设施（例如，全细胞指纹图谱和rna测序分析）来表征诱导进入VBNC状态的空肠梭菌细胞的生化和转录谱。此外，我们将开发与激光镊子拉曼光谱相结合的微流控“芯片实验室”设备，用于同时鉴定和分离来自不同细菌亚群（即健康细胞，损伤细胞和死亡细胞）的纯单个VBNC C.空肠细胞，然后使用单细胞转录组测序进行表征。在C. jejuni VBNC复苏的背景下，我们将利用代谢组学分析能够促进VBNC C. jejuni细胞复苏的农产品，并鉴定食品中潜在的促进复苏的分子。然后，我们将使用rna测序和蛋白质组学研究复苏的空肠梭菌细胞的转录和翻译谱。最后，我们将制定干预策略，以减少正常和VBNC空肠在环境和食物系统。研究结果可为食品工业中快速、灵敏、易于操作的空肠弯曲杆菌检测和鉴定方法的开发，以及减少弯曲杆菌在环境和农业食品中的流行策略提供参考。

The presence and resuscitation of viable but non-culturable (VBNC) pathogens in the food processing environment and food products pose a significant risk to the public health. VBNC pathogens can evade detection by the conventional culturing methods, but maintain virulence and have the potential to cause foodborne illnesses when resuscitated. Campylobacter jejuni is a leading bacterial cause of human gastrointestinal disease worldwide, but the formation and resuscitation of VBNC C. jejuni and its control measures in the food industry have not been well studied. The aim of this proposal is to investigate the formation and resuscitation of C. jejuni in food processing and storage-related environment as well as in food products. In the context of C. jejuni VBNC formation, we will monitor the dynamics of C. jejuni population during the transition from the normal culturable state to the VBNC state under various stress conditions that mimic food processing and preservation. We will then apply advanced infrastructures (e.g., whole cell fingerprinting spectroscopy and RNA-sequencing analysis) to characterize the biochemical and transcriptional profiles of C. jejuni cells induced into the VBNC state. Moreover, we will develop microfluidic “lab-on-a-chip” device coupled with laser tweezers Raman spectroscopy for the simultaneous identification and separation of pure single VBNC C. jejuni cells from a mixture of different bacterial subpopulations (i.e., healthy cells, injured cells, and dead cells), followed by characterization using single-cell transcriptome sequencing. In the context of C. jejuni VBNC resuscitation, we will use metabolomics to analyze agri-food products that can promote the resuscitation of VBNC C. jejuni cells and identify the potential resuscitation-promoting molecules in foods. We will then study the transcriptional and translational profiles of resuscitated C. jejuni cells using RNA-sequencing and proteomics. Finally, we will develop intervention strategies for the reduction of normal and VBNC C. jejuni in the environment and food system. The research outcome can provide an insight to the development of rapid, sensitive and easy-to-operate methods for the detection and characterization of VBNC C. jejuni in the food industry, and mitigation strategies to reduce the prevalence of Campylobacter in the environment and agri-foods.