【作者】 赵帆；

【导师】 刘起勇；

【作者基本信息】 中国疾病预防控制中心 ， 流行病与卫生统计学， 2011， 博士

【摘要】 汉赛巴尔通体是巴尔通体属中重要的人兽共患病病原菌,具有广泛的致病性,可累及全身各组织和器官。猫抓病是其主要致病种类,一般表现为良性自限性。但是一旦机体免疫功能降低,则可能发生严重的全身病症,极个别还会导致死亡。研究认为汉赛巴尔通体某些亚型的分布具有地理特异性,并与其所致疾病的种类和强度有关,其他国家的研究者已将多种分子方法应用于汉赛巴尔通体的亚型分析,而在中国,目前还没有关于汉赛巴尔通体种类多样性的报道,我们对该病原体在中国的基因型别差异、群体结构特征和分布情况还缺乏了解,对国内外同类分离株的差异还不清楚。为回答上述问题,本研究通过多位点序列分型(MLST)、多位点可变数目串联重复序列分析(MLVA)和基因组单核苷酸多态性(SNP)分析等方法,并结合实验室前期的脉冲场凝胶电泳(PFGE)分型数据对中国汉赛巴尔通体作了全面的分子分型分析。应用MLST方法将近几年实验室保藏的80株汉赛巴尔通体分为3个序列型(ST),其中ST1占分离株总数的90%(72/80),ST9和ST30分别占8.25%(7/80)和1.25%(1/80)。系统发育分析表明,3个序列型来自一个克隆群,一方面提示中国汉赛巴尔通体由高度克隆化的菌株进化演变而来,并随着时间的变迁,积累一些散在变异；另一方面,提示目前的MLST方案不适合于中国汉赛巴尔通体菌的流行情况调查,广泛存在的ST1菌株间的变异需要分辨能力更强的方法来发现。同时,我们在实验室建立了分辨力更高的MLVA方法,用以比较中国和英国汉赛巴尔通体分离株的基因型别差异。在选用的6个VNTR位点中,各自的分辨力Nei’s指数在0.54～0.90间,位点联合之后将84株菌分为53种不同的MLVA型(MT),总的分辨力指数达到0.98。聚类分析显示该MLVA方法能将参与分析的菌株分为Group A和Group B两个类群,其中Group A包含的7株菌全部来自英国,余下所有菌株属于Group B。Group B又可被分为Group B1和Group B2两个业群及B2中的5个小类。不同的业群和小类表现出菌株不同的地理分布特征。最小生成树图将参与分析的62株ST1汉赛巴尔通体分为6个类群,提示ST1菌株内部的复杂多样性。7株ST9细菌也有1株分到ST1所在类群中,反映出菌株可能的进化演变途径。为进一步了解ST1菌株间的变异情况,我们用应用生物系统公司的SOLiD测序仪对分离自中国不同地点和时间的4株ST1细菌进行重测序,做基因组水平的SNP分析,4株细菌的MT和PFGE结果也互不相同,同时参与分析比较的还有8株分离自英国的不同ST菌株。结果显示,一共有9566个SNP位点分布于12个测序菌株,ST1的SNP数目范围在374-1932间。基于SNP的系统发育树能将所有ST1菌株分开,显示出基因组SNP分析的强大分辨力。同时发现一个有趣的现象,中国的4株ST1细菌被分为2个分支,其中一株与作为参考菌株的ST1聚到一起,而其余3株自成一支。提示ST1内部存在变异体,但变异有限,可能形成另一个亚克隆群,而当前的MLST方案未能发现。通过基因组SNP数据对MLST方案进行改进,应用于分离菌株,我们找出了隐藏于ST1中的亚型,使MLST结果与SNP分析一致。同其它两种分型方法比较,发现改进后的MLST方案与MLVA聚类结果部分吻合,而完全对应于PFGE分出的2大类群,综合各方法的分型能力,推荐使用MLST与PFGE联合使用的办法调查汉赛巴尔通体的流行病学特征。同时,基因组测序数据提供了关于汉赛巴尔通体菌株内部的基因表达和蛋白编码方面的变异特征,还需要进一步分析。更多还原

【Abstract】 Bartonella henselae is an important zoonosis pathogen in Bartonella, which can affect the whole body tissues and organs with a wide range of pathogenicity. Cat scratch disease is the main pathogenic species, usually expressed as a benign self-limiting. But once the body’s immune function was limited, it would became serious systemic disease, even lead to death. Some studies suggest that geographic distribution of B.henselae is subtype-specific, and some subtypes have a relation to the disease and the intensity. Some attemps of molecular methods have been used to analysis B.henselae subtype in many countries. But in China, there were no reports on intra-species diversity of B.henselae. Also, it is not clear about the pathogen in intra-genotype situation, population structure and geographic distribution. To answer these questions, in this research we used molecular typing analysis such as multilocus sequence typing (MLST), multilocus variable-number tandem repeat analysis (MLVA) and genomeic single nucleotide polymorphism (SNP) analysis for B.henselae intra-species typing.Using MLST method, we divided 80 isolates into three sequence types (ST), in which the total number of ST1 accounted for 90%(72/80), ST9, and ST30, respectively,8.25%(7/80) and 1.25%(1/80). Phylogenetic analysis showed that the three sequence types come from a same clonal complex, suggesting Chinese isolates evovled from a single clonal complex and had little mutation, indicating the established MLST scheme is not suitable for the prevalent investigation of B.henselae in China. And a widespread variation of ST1 strains need to be distinguished using more discrimination approach.At the same time, we established MLVA, a more powerful method in our lab to compare the difference between Chinese and British B.henselae isolates. For the 6 selected VNTR loci, the Nei’s index is from 0.54 to 0.90 respectively, and the discrimination index is up to 0.98 while combine them together which could divided 84 isolates into 53 different MLVA types (MTs). Cluster analysis showed that the MLVA method can devided the strains into Group A and Group B.7 isolates contented in Group A which are from the United Kingdom, and all remaining strains belonging to Group B. Group B can be divided into Group B1 and Group B2, and B2 contains 5 small subsets. Different groups and subsets show different geographic distribution. Minimum spanning tree diagram divided 62 ST1 into six groups, suggesting the intra-ST diversity among ST1 isolates.6 of 7 ST9 were from one group while the remaining one have grouped to ST1, suggesting the evolution posibility routine of B.henselae isolates.To further understand the diversity among the ST1 strains, we resequenced four ST1 strains isolated from different loci and time to analysis the genomic SNPs using the next-genaration sequence ehchnology-SOLiDTM sequencer of Apply Biosystem. An additional eight strains from UK were included for sequencing and comparative analysis. Result showed a total 9566 SNPs loci were distributed during 12 isoaltes and for ST1 strain, the SNP range from 374 to 1932. Phylogenetic tree besed on SNP concatenation could separate all ST1 strains but formed two branches, one clustered with reference strain and the other just including Chinese ST1, indicating diversity existed in ST1 strains, and the variant could form another cluster but the diversity is limited, and the established MLST scheme failed to describe it.The MLST scheme has been improved according to genomic SNP data and applied in isolates. In the improved MLST scheme, we identified the subtype hidden in ST1, and make the cluster result consistent with SNP-based phylogenetic tree. Comparing with the other two methods, we found that the improved MLST scheme was partly congruous with MLVA clustering, and completely congruous with the PFGE main groups. Considring all the result in this study, we recommended a combination of MLST and pulsed-field gel electrophoresis (PFGE) method to investigate the epidemiological characteristics of B.henselae. Genome analysis including the protein coding and variation information in the B.henselae would proceed in the futhur research.更多还原