鸡蛋贮藏过程中肠炎沙门氏菌生存与增殖的抗逆分子机制

Salmonella enterica serovar Enteritidis is the only bacterium routinely transmitted to humans through intact chicken eggs, and it means that this bacterium must be able to persist inside chicken eggs. Therefore, the survival and multiplication mechanism of S. Enteritidis against those stress conditions inside chicken eggs has become a key scientific issue. In this study, the monitoring, isolation, identification and genotyping of S. Enteritidis isolates from stored chicken eggs and clinic children patients will be conducted in order to find out the genetic relationship between the two kinds of isolates. The typical subtype isolates will be selected and involved in the cultivation and tests for their abilities of survival and multiplication against effective components of albumen so that the dynamic model of the bacterial survival and multiplication in chicken eggs and the inhibition model of chicken egg albumen to the bacterial isolates will be established. Those typical survival isolates and non-survival isolates (control), epidemic clone strains and multi-drug resistant isolates will be selected for the full-genome sequencing and analysis in gene expression microarray. Possible anti-stress genes (loci) related to the survival and multiplication of S. Enteritidis in chicken egg will be found out by screening with a mining tool for specific gene (loci) in microbial pathogens (SMM-system) developed in our lab. Those possible anti-stress genes (loci) and related genes previously hypothesized will be further characterized and confirmed for their functions by deletion or site-directed mutagenesis. The interaction network among those anti-stress genes (loci) will be developed based on the results of two-dimensional protein electrophoresis and gene expression microarray. Consequently, the anti-stress molecular mechanism for survival and multiplication of S. Enteritidis in chicken eggs will be systematically disclosed at the genomic level.

肠炎沙门氏菌是迄今发现的唯一能够在完整鸡蛋内部生存与增殖的沙门氏菌血清型，它们应对鸡蛋中抑（杀）菌物质的抗逆机制已成为一个重要的科学问题。本项目通过对鸡蛋贮藏过程中和临床儿童病例中肠炎沙门氏菌进行监测、分离鉴定与基因分型，探索二者之间的遗传相关性；将各亚型代表菌株分别采用全蛋液和蛋清组分进行培养与抑菌测试，结合蛋清蛋白质组学研究结果，建立肠炎沙门氏菌的生存与增殖动力学模型和蛋清组分抑菌模型；选取高生长率和不存活的代表菌株、流行克隆株和多重耐药菌株进行全基因组测序和表达芯片分析，并利用自主研发的特异靶点发掘平台筛选出抗逆候选基因和差异位点；结合前人预测的相关基因，采用自主优化的稳定沙门氏菌定点突变系统，实现抗逆基因的突变，以验证其功能；借助蛋白质双向电泳和表达芯片技术，分析抗逆基因间的协同作用，从而在基因组水平上较系统地揭示肠炎沙门氏菌在鸡蛋中生存与增殖的抗逆分子机制。

肠炎沙门氏菌是迄今发现的唯一能够在完整鸡蛋内部生存与增殖的沙门氏菌血清型，它们应对鸡蛋中抑（杀）菌物质的抗逆机制已成为一个重要的科学问题。经过5年的努力，本项目组采用自主优化建立的“肠炎沙门氏菌蛋清生存能力评价体系”，对从鸡蛋相关食品样品和儿童食物中毒临床样本中分离获得的600株肠炎沙门氏菌进行蛋清存活能力的评价，获得蛋清抗性菌株33株，蛋清敏感菌株30株；采用PFGE、MLVA等方法对它们进行分子分型和聚类分析，得到10个PFGE群和5个MLVA类；结合分型数据，以蛋清抗逆株、敏感菌株和标准菌株各1株为代表，分析它们在全蛋、蛋清和蛋清抑菌组分纯化物中的生存与增殖规律，分别建立了肠炎沙门氏菌生存与增殖动力学模型和蛋清组分抑菌模型；通过全基因组测序、RNAseq和iTRAQ等高通量技术，对抗逆与敏感两组肠炎沙门氏菌代表菌株共22株进行组学分析；综合基因组、转录组和蛋白质组的测序结果，采用自主优化建立的稳定沙门氏菌定点突变系统，实现了17个抗逆基因的突变，并从存活能力、脂多糖结构、细胞形态结构、运动能力等角度评价其功能，从而阐明了抗逆基因各自的作用及其相互间的协同关系，同时揭示了铁吸收、生物素合成、壁膜损伤修复、细菌趋化等途径在肠炎沙门氏菌蛋清抗逆中发挥着重要功能；结合前人结果和抗逆基因间上述的协同关系，绘制了这些关键生物途径间相互关系网络。这些结果较为详细地阐述了困惑科学家几十年的肠炎沙门氏菌在完整鸡蛋内部存活并增殖以及对抗鸡蛋蛋清逆境环境的分子机制，对解决鸡蛋中肠炎沙门氏菌带来的重大食品安全问题具有理论指导意义。项目实施期间，以第一标注在食品安全领域的主流期刊上发表论文17篇（其中SCI 论文11篇），其中包括Food Microbiology、Food Control等5篇。培养研究生15名。由于在食品安全领域做出的创新性科学贡献，项目负责人于2014年当选为国际食品保护学会中国分会副理事长，2016年被遴选为国际食品科学院（IAFoST）Fellow。