乳制品加工中克诺罗杆菌生物膜形成关键基因及蛋白组学研究

Cronobacter sakazakii is an important pathogen in the powder infant formula, and the biofilm formation make it more difficult to prevent and control. We choose the genus or species representative strain of Cronobacter sakazakii and strains isolated from dairy products or processing process. The research is as following: (1)Establishment of the molecular typing and traceability system based on the evaluation of existing classification system, (2) Establishment the Vi and KdpD genes knock out and knoch in model to verify its function in bacterial biofilm formation, (3) We will study the gene and protein expression difference in wild type strain and mutation strain by using iTraq and RNA-seq techniques to find the key genes and proteins. The expected results is to find the VI and KdpD gene function mechanism in Cronobacter sakazakii biofilm formation, to establish biofilm formation and regulation model, to provide the molecular basis for prevention and control of Cronobacter sakazakii pollution. It can rich the study of Cronobacter sakazakii biofilm formation, invasion and virulence reseach, providing theoretical support for higher sensitative detection method.

克诺罗杆菌是婴幼儿配方奶粉中的重要生物危害，其生物膜形成的特性增加了防控的难度。针对该特性，以克诺罗杆菌种属代表菌株以及分离株为研究对象，重点研究以下内容：（1）采用新的克诺罗杆菌种属分类体系，对196株克诺罗杆菌食源性分离菌株进行分子分型，建立婴幼儿配方粉生产加工过程中克诺罗杆菌分子分型溯源模型；（2）建立克诺罗杆菌VI和kpd基因缺失、回补基因功能学模型，验证关键基因功能；（3）采用比较蛋白组学iTraq和转录组测序技术（RNA-seq）比较克诺罗杆菌野生型和缺失突变株基因和蛋白表达的差异，寻找生物膜形成的关键基因和蛋白；预期研究结果将找出VI和KdpD基因在克诺罗杆菌菌膜形成中的作用机制，建立生物膜形成调控模型，为通过控制生物膜形成调控克诺罗杆菌污染提供分子基础。本研究将丰富克诺罗杆菌生物膜形成、侵袭性及毒力研究的理论，为实际应用指导克诺罗杆菌防控，建立高灵敏度的检测技术打下基础。

项目以实验室分离保存的52株进口食品克诺罗杆菌（Cronobacter）分离株和42株国内市售食品克诺罗杆菌（Cronobacter.spp）分离株为研究对象，采用生理生化鉴定和全基因组测序（whole genome sequencing，WGS），在获取菌株DNA序列的基础上进行物种鉴定，基因分型，泛基因组和功能注释方面的分析，在全基因组测序和抗生素敏感性实验基础上，还对克罗诺杆菌的耐药及毒力相关基因进行了预测；项目比较了多位点序列分型（multilocus sequence typing，MLST），CRISPR-cas分析，核心基因组多位点序列分型（core genome multilocus sequence typing，cgMLST）及全基因组共线性分析等方法的差异和优势，将PubMLST公共数据库中两千余株克罗诺杆菌下载建立本地数据库，以数据库中的样本和分离菌株样本互为参照对本项目的菌株进行溯源分析。