中国汉赛巴尔通体的分子分型研究

发布时间：2018-07-27 14:48

【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。
[Abstract]:Han Sabar general body is an important zoonosis pathogen of the genera of Barr. It has extensive pathogenicity and can involve all tissues and organs of the whole body. Cat scratch disease is the main pathogenic species and generally shows a benign self restriction. But once the immune function is reduced, it may cause serious systemic disease, and it may cause death in a few cases. It is considered that the distribution of some subtypes of Han Sabar's common body is geographically specific and is related to the species and intensity of the disease. Researchers in other countries have applied a variety of molecular methods to the subtype analysis of hhsbarall body. In China, there is no report on the variety of the species of the Han Sabar body. The differences in genotypes, population structure and distribution of pathogens in China are still lack of understanding, and the differences in the domestic and foreign isolates are not clear. To answer the above questions, this study has been analyzed by multipoint sequence typing (MLST), multipoint variable number tandem repeat sequence analysis (MLVA) and genomic single nucleotide polymorphism (SNP) analysis. Based on the data of the pulsed field gel electrophoresis (PFGE) in the laboratory, the molecular typing of Han Sabar's whole body in China was analyzed.
80 Han Sabar bodies preserved by the MLST method in recent years were divided into 3 sequence types (ST), of which ST1 accounted for 90% (72/80) of the total number of isolated strains, ST9 and ST30 accounted for 8.25% (7/80) and 1.25% (1/80). Phylogenetic analysis showed that 3 sequences came from a clon group. On the one hand, the Chinese Han Sabar body was highly cloned. The evolution of the strain evolved and accumulated some of the variation with time. On the other hand, the current MLST scheme was not suitable for the investigation of the epidemic situation of the Chinese Han Sabar. The widely existing ST1 strains need a more discernable method to find out.
At the same time, we have established a higher resolution MLVA method in the laboratory to compare the genotypic differences between Chinese and British Han Sabar isolates. In the 6 VNTR loci selected, the respective resolution Nei's index is 0.54 to 0.90, and the 84 strains are divided into 53 different MLVA types (MT) and the total resolution index after the combination of the loci. The 0.98. clustering analysis showed that the MLVA method could divide the analyzed strains into two groups of Group A and Group B, of which all the 7 strains of Group A were from the UK. The remaining strains belonged to Group B.Group B and could be divided into two industry groups and 5 small classes. The minimum spanning tree map was divided into 6 groups of the 62 ST1 strains of ST1, suggesting that 1 strains of the complex diversity of ST9 bacteria within the ST1 strain were divided into the group of ST1, reflecting the possible evolutionary evolutionary pathways of the strain.
In order to further understand the variation among ST1 strains, we re sequenced 4 ST1 strains isolated from different locations and times in China by using the Bio System Inc SOLiD sequencer, do genomic level SNP analysis, and the MT and PFGE results of 4 strains of bacteria are not the same. In the same time, 8 strains were isolated from the UK. The results showed that 9566 SNP loci were distributed in 12 sequenced strains, and the number of SNP in ST1 was 374-1932. The phylogenetic tree based on SNP could separate all ST1 strains and showed the strong resolution of genomic SNP analysis. At the same time, an interesting image was found, and 4 ST1 bacteria in China were divided into 2 branches. One strain was clustered with the ST1 as a reference strain, while the rest of the 3 strains became one. Suggesting the existence of a variant in ST1, but the variation is limited, and another subgroup may be formed, but the current MLST scheme has not been found.
Using genomic SNP data to improve the MLST scheme, we applied it to isolate the strains. We found the subtype hidden in the ST1, and made the MLST results consistent with the SNP analysis. Compared with the other two types, the improved MLST scheme was partly consistent with the MLVA clustering results, and the complete corresponding to the PFGE was divided into 2 groups and integrated the methods. It is recommended to use the method of MLST and PFGE to investigate the epidemiological characteristics of Han Sabar's common body. At the same time, genome sequencing data provide the variation characteristics of gene expression and protein coding in the genostrains of Han Sabar, and need to be further analyzed.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R378

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