鸡白痢沙门菌CRISPR-MVLST分型方法的建立与应用

Many prokaryotes contain CRISPR immunity system against foreign mobile genetic elements,including phage and plasmid. Like eukaryotic RNA interference,CRISPR system provides the bacteria with an efficient antiviral defence mechanism. CRISPR consists of identical repeated DNA sequences (repeats), interspaced by highly variable sequences named spacers. The spacers originate from invader DNA and comprise "immunological memory" of the host. As one of the fastest evolving genetic elements, the invader short DNA fragment was incorporated into the CRISPR array for defending the bacteria against the same invaders. CRISPRs in Salmonella are evolving due to plasmids and/or phages present in the environment. The insertion and deletion of spacers in CRISPRs show the changing of the environment during different time. Multilocus sequence typing(MLST) based on housekeeping genes is widely used for Salmonella typing, but it has much lower discriminatory power than PFGE. So virulence genes have been included in MLST schemes for subtying Salmonella to increase discriminatory power. MLST schemes using both housekeeping and virulence genes have been used for subtyping Salmonella to the serovar level or even to the strain level of some Salmonella serovar. But the virulence genes alone are not discriminatory enough for differentiating strains from different resources or outbreaks. Therefore, additional genome targets are needed in order to creat an effective MLST scheme for Salmonella subtyping. Because of the potential value of CRISPR for epidemiologic investigations, a subtyping method-CRISPR-MVLST by combination of CRISPR and virulence genes (fimH,sseL) will be constructed for typing of Salmonella enterica seovar Pullorum strains. More than 300 isolates in eastern China from 1962 until now will be used for subtyping with construction of phylogenetic tree based on CST profile.As the PFGE profile has been finished for 237 isolates among them, the left strains will be finished in this project. By comparison with PFGE, the discriminatory power and the epidemiologic concordance will be calculated. Our previous study showed there was little corelation between PFGE and antibiotic resistance, some strains with same PFGE profile showed very different antibiotic resistance. But CRISPR has been proved to interpret the plasmid -mediated antibiotic resistance, and it was well-known that mobile genetic elements play important role in antibiotc resistance, so we want to reveal the relationship between CRISPR-MVLST subtyping method and antibiotic resistance.

作为细菌抵御噬菌体或质粒等外源DNA侵染的免疫系统- - CRISPR，显示了细菌在其生长繁殖环境中的不断进化过程。细菌利用CRISPR系过将外源DNA的部分短片段整合入自身基因组中而获得了免疫能力。由于其在分子分型上具有巨大的潜力，因此本研究期望联合CRISPR序列和毒力基因fimH、sseL的测序分析，建立针对我国家禽中流行的传染性鸡白痢沙门菌的CRISPR-MVLST分型方法，并对本室保存的1962年至今的300多株来源于不同地区的鸡白痢沙门菌进行分子分型，构建细菌CST型进化树，同时通过与本室已建立的传统沙门菌PFGE分型方法进行比较来评价新分型方法的分辨力、CRISPR在细菌分子分型中的应用价值及解释该菌流行病学规律的能力。在此基础上，综合前期对菌株的耐药性分析结果，及PFGE型与细菌耐药表型尚不存在明显的相关性，应用CRISPR-MVLST分型来评价耐药表型与CST型之间的相关性。

鸡白痢沙门菌是禽的重要致病菌之一，主要引发雏鸡的急性死亡，鸡白痢病仍是目前我国及部分发展中国家养禽业的主要危害之一。作为细菌抵御噬菌体或质粒等外源DNA 侵染的免疫系统——CRISPR，显示了细菌在其生长繁殖环境中的不断进化过程。细菌利用CRISPR 系过将外源DNA 的部分短片段整合入自身基因组中而获得了免疫能力。目前，CRISPR在分子分型上的巨大潜力使其在沙门菌、结核分枝杆菌等病原菌的分子分型中的得到应用。.本研究目的是基于鸡白痢沙门菌的CRISPR序列构建应用于鸡白痢沙门菌的CRISPR分子分型方法，并与传统PFGE分型或其他分型方法比较以评价该方法的分型能力，同时初步探讨CRISPR与耐药性之间的联系。.完成了鸡白痢沙门菌中国菌株S06004的基因组图谱，并对现有公布的鸡白痢沙门菌基因组中所含的CRISPR序列进行分析，建立了可以应用于鸡白痢沙门菌的CRISPR分子分型方法。并通过与PFGE和全基因组SNP分型方法比较，显示了该分型方法具有很好的分型能力及研究同一血清型不同菌株间的进化能力。.应用建立的CRISPR分型方法对615株分离自1962-2015年的菌株进行了分子分型，共获得了19个CRISPR型，其中451株属于同一CRISPR型，称为CRISPR I型，占总菌株数的73.3%；85株属于另一型即CRISPR II型，占总菌株数的13.8%，两者一起构建称为鸡白痢沙门菌的优势CRISPR型；其余13.9%的菌株分属17个不同的型别。与全基因组SNP分型之间存在很好的对应关系，证明该分型方法兼具分子分型和阐述细菌之间的进化关系的特征。.通过分析菌株之间耐药谱与CRISPR型别之间的关系，揭示CRISPR与细菌耐药之间并不存在相关性。但是CRISPR相关蛋白Cse3在鸡白痢沙门菌感染宿主过程中表达，并能刺激机体产生相应的抗体，表明CRISPR在细菌感染宿主过程中发挥了一定的作用。.CRISPR在沙门菌中广泛存在，其分子分型价值在沙门菌中早已体现出来，但是对于CRISPR在免疫作用外的其他功能仍然具有较好的研究前景。