LED杀菌调控乳粉中亚致死态阪崎克罗诺杆菌致病能力的分子机制

Cronobacter sakazakii is a foodborne pathogen that has been implicated in some severe forms of neonatal infection including infant necrotizing enterocolitis, bacteraemia, and meningitis. Powdered infant formula is considered to be the most common sources and vehicles of C. sakazakii transmission. In recent years, light-emitting diodes (LEDs) holds considerable promise in food processing as an emerging nonthermal method for microbial inactivation. We have demonstrated that 405 nm LED had a good antibacterial effect against C. sakazakii, and it also can cause sublethal C. sakazakii, but data of its effect and mechanism on pathogenic potential of sublethal C. sakazakii has not been reported. In this proposal, changes in the resistance to environment stresses, specific bioflm formations, endotoxin production, the ability to overcome host barriers and evade the immune response, and in vivo pathogenicity of C. sakazakii will be determined. In addition, possible regulation mechanisms will also be investigated through fluorescent dyes, RT-PCR and western blot. The findings from this project will provide theoretic bases for future development of LED to control C. sakazakii in powdered infant formula.

阪崎克罗诺杆菌能够引起婴幼儿坏死性小肠结肠炎、菌血症、脑膜炎等多种严重疾病，婴幼儿配方乳粉是其污染及传播的主要途径。LED杀菌作为一种新型的非热杀菌方式受到食品行业的极大关注，申请人前期确证了405 nm的LED光照对阪崎克罗诺杆菌具有较好的杀菌作用，但也会形成亚致死状态的菌体，然而LED杀菌对亚致死态的阪崎克罗诺杆菌致病能力的影响尚未证实，且调控机制尚不明确。本项目拟通过研究LED杀菌对乳粉中阪崎克罗诺杆菌环境耐受能力、内毒素产生量、生物膜形成、粘附及侵入肠上皮细胞、巨噬细胞内的存活与增殖、体内感染能力的影响，全面揭示LED杀菌对亚致死态的阪崎克罗诺杆菌致病能力的影响规律，并通过荧光染料、RT-PCR和western blot技术手段建立LED杀菌调控乳粉中亚致死态阪崎克罗诺杆菌致病能力的分子机制，为LED杀菌应用于婴幼儿乳粉生产加工中控制阪崎克罗诺杆菌奠定理论基础。

阪崎克罗诺杆菌能够引起婴幼儿多种严重疾病，婴幼儿配方乳粉是其污染及传播的主要途径。LED杀菌作为一种新型的非热杀菌方式受到食品行业的极大关注，405 nm的LED光照对阪崎克罗诺杆菌具有较好的杀菌作用，但也会形成亚致死状态的菌体，然而LED杀菌对亚致死态的阪崎克罗诺杆菌致病能力的影响尚未证实，且调控机制尚不明确。针对该问题，本研究首先设计并制作了LED杀菌装置，构建了阪崎克罗诺杆菌污染的婴幼儿乳粉模型，在测定了对菌体的抑杀作用后确定了LED的亚致死辐照剂量。然后，本研究测定了405 nm LED对乳粉中阪崎克罗诺杆菌耐受不同不良环境能力的影响。研究发现，405 nm LED显著降低了乳粉中阪崎克罗诺杆菌对干燥、热、胃酸、胆盐等不良环境的耐受能力。其次，本研究探究了405 nm LED对阪崎克罗诺杆菌黏附与侵入Caco-2细胞及在巨噬细胞RAW 264.7内存活与增殖能力的影响。研究发现，经405 nm LED处理后的阪崎克罗诺杆菌对Caco-2细胞的黏附能力下降但其侵入能力却增强。同时405 nm LED可以显著提高阪崎克罗诺杆菌在巨噬细胞RAW 264.7内的存活及增殖能力。最后，通过激光共聚焦显微镜观察了405 nm LED对阪崎克罗诺杆菌细胞膜的影响，并利用RT-PCR技术测定了405 nm LED对阪崎克罗诺杆菌环境耐受及毒力相关基因转录水平的影响。研究发现，405 nm LED处理后的阪崎克罗诺杆菌的细胞膜完整性受到了破坏。8个与阪崎克罗诺杆菌环境耐受能力相关的基因均显著下调。3个与黏附侵入相关的毒力基因中，ompA显著上调，而ompX、uvrY转录水平下降。与阪崎克罗诺杆菌在巨噬细胞中存活增殖能力相关的基因sod显著上调。本研究全方位分析了LED对亚致死态阪崎克罗诺杆菌致病能力的调控作用及分子机制，为LED在婴幼儿乳粉生产加工中的应用提供了理论基础。