【作者】 韩辉；

【导师】 祁国明； 阚飙；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2010， 博士

【摘要】 目的：伤寒(Salmonella Typhi, S. Typhi)和甲型副伤寒沙门菌(Salmonella Paratyphi A, S. Paratyphi A)引起伤寒、副伤寒。近年来我国和一些东南亚国家出现了新的流行趋势,表现为伤寒菌株的多变和甲型副伤寒的出现以及迅速流行,在局部地区甲型副伤寒甚至超过伤寒成为优势菌感染,并表现为流行地区逐渐扩大。脉冲场凝胶电泳(PFGE)的分子分型资料显示,尽管两者传播机制相同,但同时期、同地区伤寒菌株菌型多变而甲副菌株相对稳定得多。是否由于二者存在基因组稳定性差异导致这种流行特征的出现成为我们关注的焦点。因此,我们从疾病监测的角度,应用基因分子分型技术检测经过实验室进化的伤寒、甲型副伤寒沙门菌遗传物质稳定性,并分析了伤寒基因组发生变异的机制。方法：选择伤寒沙门菌基因组测序菌株CT18和甲型副伤寒沙门菌的基因组测序菌株ATCC9150,以及本室保存的90年代中期以来引起伤寒和甲型副伤寒流行的PFGE带型优势株各两株。将上述菌株平行依次在实验室条件下连续培养传代。然后,对子代菌株分别进行了PFGE、rRNA操纵子重排以及噬菌体功能和原噬菌体相关基因的检测分析。结果：三株伤寒沙门菌的子代PFGE带型均发生改变,带型变化的百分比分别是：66.7%、63.5%、5.21%；三株甲型副伤寒沙门菌子代克隆的PFGE带型均未发生变化。分析伤寒基因组变异的机制,发现子代菌CT18-43的基因组发生了rRNA操纵子重排；GZ-902115的子代GZ-902115-85等的基因组发生原噬菌体prophage3区域部分染色体片段缺失。结论：1、在实验室连续传代过程中：三株伤寒沙门菌PFGE带型均发生改变,表明伤寒沙门菌经过不断传代基因组发生变异,不同菌株带型变化的百分比差别较大,揭示不同菌株基因组稳定性差别较大；甲型副伤寒沙门菌PFGE结果未发生变化,表明它的基因组经XbaI酶切后形成的大片段,在实验室连续传代中非常稳定。2、在一定实验室时间内,伤寒沙门菌和甲型副伤寒沙门菌基因组经过不断连续传代,在基因组水平发生了变异,伤寒沙门菌的部分子代基因组发生了rRNA操纵子重排和原噬菌体基因组的缺失,这些基因组水平发生的变异可直接或间接阐明PFGE带型的改变。目的：分析我国甲型副伤寒流行以来分离株的分子分型以及病原进化上的特征。方法：应用以Spel为限制性内切酶的脉冲场凝胶电泳方法和基于9个管家基因位点(aroC、thrA、hisD、purE、sucA、dnaN、hemD、adk和purA)的多位点序列分型方法,对我国近期甲型副伤寒流行以来的2000-2008年分离自10个省份的118株甲型副伤寒沙门菌进行分析。结果：PFGE将118株甲型副伤寒沙门菌分为32个型,而MLST只分出了2个ST型,显示各菌株的管家基因序列高度保守,应用MLST这种分型方法很难区分,MLST更适用于不同血清型菌株之间的分型研究。结论：目前中国的甲型副伤寒是由高度克隆化的菌株引起全国范围的扩散流行。随着年份的变迁,也积累了散在的变异。更多还原

【Abstract】 Objective Typhoid fever was caused by Salmonella Typhi or Paratyphi A, B, and C. It is a public health problem worldwide. Recently, paratyphoid fever became more severe than typhoid fever in China and some Southeast Asian countries, and the performance of the endemic area gradually expanded. Although S. Typhi and S. Paratyphi A has the same host specificity, similar mechanism of transmission and clinically similar enteric fever, Paratyphi A became predominant pathogen of typhoid fever in some regions. We focused on that if the different genetic stability leading to the emergence of paratyphoid epidemic. According to the perspective of disease surveillance, we monitored the S. Typhi and S. Paratyphi A after laboratory evolution by molecular typing. We observed the genetic stability of S. Typhi and S. Paratyphi in experiment microevolution and explored the possible mechanisms of genomic mutations.Methods CT18, ATCC9150, two S. Typhi strains and two S. Paratyphi A strains provided by Institute of Communicable Disease Prevention and Control Center for Disease Control and Prevention, China, were selected because CT18 and ATCC9150 have been sequenced, and the other 4 strains are predominant strains which caused typhoid fever in China in the mid-1990s. The 6 strains were divided into S. Typhi and S. Paratyphi A subgroups. The isolated colony of the 6 strains was incubated at 37℃in a rotating water bath. After 12 h,100μl of a 10-4 dilution was aliquoted into 9.9 ml of LB. This 12h incubation-aliquoting into LB cycle was done for 20 times. The isolated colonies of the end point (20 times) were analyzed by pulsed field gel electrophoresis (PFGE), rRNA rearrangement and prophage detecting.Results The 96x6 isolated strains of the end point (20 times) have been analyzed by PFGE. The colonies of three S. Typhi strains showed different changes, the rates of CT18, GX-039 and GZ-902115 were 66.7%,63.5% and 5.21%, respectively, while no change was observed for the colonies of three S. Paratyphi A. Furthermore, we determined the rrn arrangement of CT18-43 which was a isolated colony of CT18 and its rrn was rearranged during laboratory evolution. In addition, we also found some genes in prophage3 were deleted during laboratory evolution. The positive isolated colony is from GZ-902115 (GZ-902115-85).Conclusion During laboratory evolution, the PFGE patterns of isolated colonies from three S. Typhi changed. The change of PFGE pattern indicated there were some mutations on the genome. Each S. Typhi strain’s percentage of changed isolates varied greatly because their genome had many different variations. But the colonies of three S. Paratyphi A strains were very conserved and their PFGE patterns is unchanged. Some variations such as rRNA rearrangement and gene deletion among prophage area were detected on the colonies’genome which can be responsible for the changes of PFGE patterns partly. Objective To analyze molecular typing and characteristic of pathogen evolution of Salmonella paratyphi A strains.Methods Pulsed-field gel electrophoresis (PFGE) with SpeⅠas the restriction enzyme was used to analyze Salmonella paratyphi A strains from 10 provinces during 2000-2008. The genomic DNA in different Salmonella paratyphi A strains were also identified by multilocus sequence typing (MLST), under PCR products of housekeeping genes aroC、thrA、hisD、purE、sucA、dnaN、hemD、adk and purA.Results One hundred and eighteen Salmonella paratyphi A strains isolated in China in different years were quite different in PFGE fingerprints, and there were 32 kinds of PFGE fingerprints in total. However, data from MLST revealed lack of diversity among the strains of the same serotype and the number of variable nucleotide sites ranged from 1 to 20 between Salmonella paratyphi A and other serotypes of Salmonella. Conclusion Currently in China, high degree clonal strains caused paratyphoid fever epidemic spreading throughout the country. With the year changing, a number of sporadic mutations occurred.更多还原