我国主要沿海地区O1群霍乱弧菌分子分型特征分析

Analysis of molecular typing characteristics of Vibrio cholerae O1 in major coastal areas of China

目的采用MLST方法及基质辅助激光解析电离飞行时间质谱(Matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF MS)技术研究我国主要沿海地区192株O1群霍乱弧菌的种群结构、进化趋势及流行特性。方法PCR扩增192株O1群霍乱弧菌的7个管家基因adk、gyrB、mdh、metE、pntA、purM和pyrC,根据核酸序列信息获得其等位基因型及序列型(Sequence Type,ST)。利用DnaSP 5.0及eBURST Version 3.0分析序列多样性,SplitsTree 4.0、STRUCTURE 2.2等软件分析霍乱弧菌种群结构特征,同时数据库中非O1/O139群对应的STs也纳入分析,以比较其与O1/O139群对应STs在克隆群和种群结构方面的关系。此外还应用BioTyper软件,对霍乱弧菌蛋白指纹图谱数据进行聚类分析。结果192株O1群霍乱弧菌共22个序列型,其中11个为本研究新发现的序列型;ST189、ST191和ST184存在2个不同组别间的重组现象。SplitsTree 4.0种群结构分析结果显示,22个序列型共分成2个分支,STRUCTURE种群结构分析将22个序列型分为3个亚群,不同的16S rDNA序列存在共有的ST,如ST69、ST75和ST173;非O1/O139群对应的STs大部分以单体形式存在,仅形成4个克隆群,在种群结构分析中大部分STs与O1/O139群对应的STs划分到同一个亚群中;MALDI-TOF MS聚类分析结果显示,当距离为200时,22个STs被分为两组,当距离为100时,被分成3组;霍乱弧菌整体的重组率(包括O1/O139群和非O1/O139群)要远远高于O1/O139群的重组率。结论192株O1群霍乱弧菌MLST分析表明ST69和ST75是O1群霍乱弧菌最主要的2个序列型。7个管家基因重组/突变的比值差异较大,但整体而言,重组比突变更容易发生。O1/O139群和非O1/O139群霍乱弧菌间仍存在较大差异。霍乱弧菌MLST分型与基于蛋白指纹图谱的分型结果间尚未发现明显的联系。

To research the population structure,evolutionary trends,and epidemic characteristics of 192 strains of the Vibrio cholerae O1 group,we performed MLST and matrix-assisted laser desorption/ionization-time of flight mass spectrometry(MALDI-TOF MS)in this study.The seven housekeeping genes adk,gyrB,mdh,metE,pntA,purM,and pyrC from 192 strains of V.cholerae O1 were amplified by PCR,and their allelic genotypes and sequence types(ST)were obtained on the basis of the nucleic acid sequence information.DnaSP 5.0 and eBURST Version 3.0 were used to analyze sequence diversity,and SplitsTree 4.0 and STRUCTURE 2.2 were used to analyze the structural characteristics of V.cholerae populations.The STs corresponding to the non-O1/O139 group in the database were also included in the analysis to compare their relationship with the STs corresponding to the O1/O139 group in terms of clonal group and population structure.In addition,Biotyper software was used for cluster analysis of the V.cholerae protein fingerprint data.Finally,we identified 22 STs in 192 strains of V.cholerae O1,11 of which were newly discovered.ST189,ST191,and ST184 showed recombination between two groups.Population structure analysis with SplitsTree 4.0 revealed that the 22 sequence types were divided into two branches,and population structure analysis with STRUCTURE 2.2 divided the 22 sequence types into three subgroups.Different 16 S rDNA sequences had shared STs,such as ST69,ST75,and ST173.Most of the STs corresponding to the non-O1/O139 group existed in the form of monomers,and only four clonal groups formed.In population structure analysis,most of the STs and the STs corresponding to the O1/O139 group were divided into the same subgroup.The results of cluster analysis with MALDI-TOF MS indicated that when the distance was 200,the 22 STs were divided into two groups,and when the distance was 100,they were divided into three groups.The overall recombination rate of V.cholerae(including the O1/O139 group and non-O1/O139 group)was much higher than that of the O1/O139 group.MLST analysis of 192 strains of the V.cholerae O1 group indicated that ST69 and ST75 were the two main STs.The recombination/mutation ratio of the seven housekeeping genes differed,but recombination was overall more likely to occur than mutation.Large differences were observed between the O1/O139 group and the non-O1/O139 group of V.cholerae.No clear relationship was observed between the MLST typing of V.cholerae and the typing results based on protein fingerprinting.