【作者】 李伟；

【导师】 徐建国；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2009， 博士

【摘要】 猪链球菌(Streptococcus suis)是一个重要的人兽共患病病原。猪链球菌MLST序列7型(ST7)菌株是引起1998年和2005年猪链球菌暴发疫情的菌株。既往研究中使用脉冲场凝胶电泳分析技术(PFGE),所有ST7型菌株的PFGE型别相同,因而需要建立一个更高区分能力的分型方法开展分子流行病学研究。本文建立了猪链球菌的多位点可变数目串联重复序列分析方法(Multiple-Locus Variable-number tandem-repeatAnalysis,MLVA)。利用Tandem Repeats Finder software(TRF)软件,通过比较和筛选4个猪链球菌全基因组中的串联重复位点,确定了9个指标用于MLVA分型。其中,8个位点(TR1～TR8)为中等多态性指标,第九个位点(TR9)为高变异度的指标。利用我们建立的MLVA分型方法,分析了199株猪链球菌,包括33个血清型参考菌株和166株血清2型菌株(154株中国菌株,12株其他国家菌株)。上述菌株可分为两大类(Cluster),85个MLVA型别。Cluster-Ⅰ包括34株菌,33个MLVA型别,Cluster-Ⅱ包含血清2型和血清14型、15型菌株,海外血清型2型菌株同中国菌株分属不同的MLVA型别。利用MLVA方法可以将167个血清2型菌株分为53个MLVA型,其中144个ST7菌株分为35个MLVA型,10个ST1菌株分为9个MLVA型别。利用我们建立的MLVA方法,对中国人感染猪链球菌暴发疫情进行分子流行病学分析。8株1998年江苏分离的ST7菌株分为5个MLVA型别,其中的四个型别在2005年四川菌株中也有分布,提示1998年和2005年的暴发疫情可能是同一个来源。另外2005年江西病人是由于搬运源于江苏的病死猪肉而感染,病人和猪肉中分离的菌株MLVA型别一致,说明2005年江西菌株来源于江苏,流行病学调查也证实了该来源关系。这些都说明我们建立的MLVA方法具有良好的流行病学相关性,可以对MLST和PFGE不能区分的菌株精细分型,可以用以调查感染来源和追踪感染途径。MLVA分型方法具有简便,信息数字化、易于标准化等特点。我们建立的MLVA方法,可以和MLST、PFGE方法一起用于猪链球菌监测和传染源、传播途径的调查。更多还原

【Abstract】 Streptococcus suis is an important zoonotic agent in humans, often linking to occupational exposure to pig or porcine products, resulting with meningitis, septicemia, permanent hearing loss. A largest recorded outbreak of S.suis serotype 2 infection occurred in 202 villages in Sichuan Province, China, during the period from July to August of 2005. The sequence type 7 (ST7) S. suis was recognized as the causative pathogen for the Sichuan outbreak in 2005 and Jiangsu in 1998. So far, S. suis ST7 has not been isolated in any other country except China. For the reason that S.suis serotype 2 strains involved in two outbreaks of China can not be differentiated by MLST or PFGE method. This indicated a more descreminative method is required to discriminate strains from different origins.In this study we identified and assessed polymorphic tandem repeat loci within the genomes of four S.suis serotype 2 strains and developed a multiple-locus variable number of tandem repeat analysis (MLVA) method. The complete genome sequence of four S. suis serotype 2 strains were analyzed for potential VNTR loci using the Tandem Repeat Finder software (TRF). By assessing the polymorphism of loci, ultimately, 9 VNTR loci were selected to subtype S. suis in MLVA analyses. The markers we selected in MLVA analysis, eight (TR1-8) belongs to moderately diversity markers. The TR9 locus belongs to higher sensitive marker.A total of 199 S. suis isolates were analyzed in this study including 33 serotype reference strains and 166 clinical isolates in serotype 2 (154 from China and 12 from other countries). A total of 85 MLVA types were defined, in which 33 MLVA types were identified for the 33 serotypes reference strains. 85 MLVA types were clustered into two clusters (Cluster-I, II). S.suis Serotype 2 strains were divided into 53 MLVA types. S.suis serotype2 strains of China and some European country clustered together and shared Cluster-II with serotype 14 and 15 strain.The investigation of outbreak-associated ST7 S. suis strain analyzed by MLVA Scheme showed that 144 ST7 strains were discriminated into thirty-five MLVAs, of which 100 ST7 strains isolated in 2005 were divided into thirty MLVAs. Noteworthy, eight isolates from Jiangsu Province in 1998 were divided into five MLVA types, of which four subtypes were also detected in isolates in Sichuan in 2005, which provide molecular clues that the pathogens responsible for these two outbreaks in China were closely associated or from the same origin. In addition, the MLVA subtypes of ST7 strains isolated from Chongqing, Guangdong, Jiangxi, and Jiangsu Province in 2005 were also detected in strains from Sichuan in 2005, which provided evidence that the causative agents of non-Sichuan provinces in 2005 were originated from strains of 2005 Sichuan outbreak. These isolates isolated in 2006-2007 shared some identical MLVA profiles with Chinese major epidemic S. suis serotype 2 strains in 1998 and 2005, which supported the inference that the isolates collected in Sichuan Province in 2006-2007 were derived from some clones with the former outbreaks strains.The MLVA scheme described here has a higher discriminative typing power than pulsedfield gel electrophoresis (PFGE), which suitable in outbreak surveillance and investigation of S. suis to detect the source of infection and transmission route. Such as one strain, isolated from a Jiangxi patient who processed pork transported from Jiangsu Province, had same MLVA subtypes with anther three strains isolated from pork he processed. These data conclusively demonstrates the Jiangxi S. suis was transmitted from Jaingsu Province in 2005 and S.suis can transmitted from one province to another by porcine products communication; The route of some S.suis infections confirmed by MLVA were also proofed by the epidemiology observation.The MLVA developed here shows many advantages have many necessary characteristics of an efficient typing method and represent a promising tool in molecular subtyping of Streptococcus suis /serotype 2 strains. To our knowledge, this is the first report of MLVA method applied to S. suis. It is believed that this technique can be efficiently used in future phylogenetic investigation, forensic tracking, or epidemiological investigations of S. suis infections.更多还原