多位点分型技术应用于浙江省脑膜炎奈瑟菌基因分型研究

Application of Multilocus Sequence Typing on the Genotyping of the Neisseria meningitidis Isolates in Zhejiang Province

目的对浙江省2004~2013年分离的54株脑膜炎奈瑟菌（Neisseria meningitidis,Nm）进行基因分型研究,了解菌株间的遗传进化关系。方法参照细菌分子分型和基因组差异公共数据库（Pubmlst）提供的引物与方法,获得多位点序列分型（Multilocus Sequence Typing,MLST）序列型（Sequence Type,ST）。采用进化分析软件（Splitstree）4、聚类分析软件（e BURST）V3等处理所得序列数据,并构建遗传进化图谱。结果 54株Nm得到21个STs,其中11个STs为新发现,主要序列型ST-7和ST-4821分别占29.63%和27.78%。不同血清群其STs/ST-克隆群（Complex）有明显不同,A群菌株78.95%（15/19）为ST-7/ST-5Complex（15/19）;B群菌株STs分散,57.14%（8/14）呈单体存在;C群菌株82.35%（14/17）为ST-4821/ST-4821Complex。不同来源分离株其ST-Complex不同,来自病人及密切接触者分离株ST-5Complex分别占48.15%（13/27）及33.33%（4/12）、ST-4821Complex分别占40.74%（11/27）及25.00%（3/12）,而来自健康带菌者分离株ST-4821Complex克隆群占40.00%（6/15）。7个管家基因的核苷酸多态性（Pi）范围为0.01298（adk）~0.11100（aro E）。Splitstree 4构建径向系统进化树显示,浙江省Nm菌株分属5个分枝,主要序列型ST-7及ST-4821分别分布于3分枝及2分枝中,4分枝中均为浙江省STs。e BURST V3聚类分析发现,浙江省STs/ST-5Complex基因型相对稳定,但浙江省STs/ST-4821Complex基因型已具遗传多样性变化。结论浙江省Nm以A群78.95%（15/19）ST-7/ST-5Complex和C群82.35%（14/17）ST-4821/ST-4821Complex两大克隆群占绝对优势,B群57.14%（8/14）呈单体分散。浙江省A、B、C群均存在ST-4821Complex相同基因型菌株,可能不同血清群之间已产生荚膜转换,其种群间已显示基因重组进化特征,这是中国首次从A群Nm中检出ST-4821/ST-4821Complex及ST-5798/ST-4821Complex菌株。

Objective To determine the evolutionary relationship among 54 strains of Neisseria meningiti- des（Nm） isolated from 2004 to 2013 in Zhejiang province. Methods Using methods described in the Public database＇s for molecular typing and microbial genome diversity（ Pubmlst）, the nucleotide polymor- phism of the sequences was analyzed by DNAsp 5.0, and the genetic evolutionary profile of these isolates was constructed by Splits Tree 4and eBURST V3. Results Twenty-one sequence types （STs） were iden- tified by Muhilocus Sequence Typing （ MLST）, and eleven of them were first found in this study. The main sequence types were ST-7 （29. 63% ） and ST-4821 （27.78%）. Strains with different serotypes resulted in the difference of their STs/ST-Complex. Most group A strains belonged to the ST-7/ST-5 complex （78. 95%, 15/19） ; the group B strains belonged to different STs most of which were singletons （57. 14%,8/14） ; most group C strains belonged to ST-4821/ ST-4821 Complex （ 82. 35%, 14/17 ）. The STs/ ST-complex from different types of strains were dif- ferent. ST-5 Complexes isolated from patients and close contacts accounted for 48. 15% （13/27） and 33.33% （4/12） of complexes, respectively, while ST-4821 Complex was 40. 74% （ 11/27 ） and 25.00% （3/12）, respectively. ST-4821 Complex was isolated from healthy carriers and was 40. 00% （6/15） of these isolates. The Pi values for nucleotide polymorphism of the seven house-keeping genes ranged from 0. 01298 （adk） to 0. 11100 （aroE）. The phylogenetic tree constructed by the Splits Tree 4 showed that the Nm strains in this study were distributed in five branches; ST-7 and ST-4821 were distribu- ted in branch 3 and branch 2 respectively, and the strains in branch 4 were all isolated from Zhejiang prov- ince. Clustering analysis results of the STs/ST-5 complex showed stability. However, the STs/ST-4821 complex showed that the ST-9480 and the new ST-9802 were not present in the ST-4821 complex, which in- dicated the genetic diversity of the ST-4821 complex. Conclusion Group A of 78. 95% （15/19） ST-7/ ST-5Complex and group C of 82. 35% （14/17） ST-4821/ST-4821Complex were dominant Complexes, but 57. 14% （8/14） of group B were seen as singles. However, the strains with the same STs were found in groups A, B and C, showed that the capsular transformation may be transfered in different serotype strains, and that recombination and evolution among these strains was seen in this study. This was the first identifi- cation of the ST-4821/ST-4821Complex and ST-5798/ST-4821Complex strains in group A Nm in China.