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

食品加工环境和食品中活的但不可培养的(VBNC)病原体的存在和复苏对公众健康构成了重大风险。VBNC病原体可以通过传统的培养方法逃避检测，但保持毒力，在复苏时有可能导致食源性疾病。空肠弯曲杆菌是引起人类胃肠道疾病的主要病原菌，但空肠弯曲杆菌的形成、复苏及其在食品工业中的控制措施尚未得到很好的研究。这项建议的目的是调查食品加工和储存相关环境以及食品中空肠弯曲菌的形成和复苏。在空肠弯曲菌VBNC形成的背景下，我们将监测空肠弯曲菌在模拟食物加工和保存的各种胁迫条件下从正常可培养状态向VBNC状态转变过程中的动态。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状态的空肠弯曲菌细胞的生化和转录图谱。此外，我们还将开发微流控“芯片实验室”设备，结合激光镊拉曼光谱，从不同细菌亚群(即健康细胞、受损细胞和死亡细胞)的混合物中同时鉴定和分离单个VBNC空肠弯曲菌细胞，然后使用单细胞转录组测序进行鉴定。在空肠弯曲菌VBNC复苏的背景下，我们将利用代谢组学的方法分析能够促进VBNC空肠弯曲菌细胞复苏的农产品，并确定食品中潜在的复苏促进分子。然后，我们将使用RNA测序和蛋白质组学来研究复苏的空肠弯曲菌细胞的转录和翻译图谱。最后，我们将制定干预策略，减少环境和食物系统中正常和VBNC空肠弯曲菌的数量。研究结果可为食品工业中空肠弯曲菌的快速、灵敏、易操作的检测和鉴定方法的开发，以及降低环境和农业食品中弯曲杆菌流行率的缓解策略提供参考。

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.