纹带棒状杆菌多位点序列分型及应用

Establishment and application of a multilocus sequence typing assay for Corynebacterium striatum

目的建立纹带棒状杆菌的多位点序列分型(MLST)方法,探讨临床分离纹带棒状杆菌的种群结构和遗传进化关系。方法筛选出7个管家基因(gyrA、gyrB、hsp65、sodA、secA1、rpoB、16S rRNA),设计引物并进行PCR扩增和测序,测序所得序列通过SeqMan软件进行拼接。采用DnaSP 5.10.01软件、Splits tree 4.14.2软件对管家基因的多样性及基因重组特征进行评价;采用MEGA 7.0.14软件基于序列型别(ST)采用M-L法构建系统发育树,采用BioNumerics软件基于ST特征值构建最小生成树,并用eBURST软件分析ST间遗传进化关系。结果所选的7个位点在所有试验菌株中均获得了预期的扩增产物;Splits tree表明所有纹带棒状杆菌的聚类一致,提示基因重组是推动纹带棒状杆菌进化的潜在动力;MLST将344株纹带棒状杆菌分成72个STs,85.7%的菌株形成克隆复合体(CC)结构,CC19形成了优势克隆复合体,但包含菌株数最多的ST为该克隆复合体中的ST16。ST具有一定的地域聚集性且与分离年份具有一定的相关性。结论我国纹带棒状杆菌呈现高度的遗传多样性,CC19为优势克隆复合体。本研究建立的MLST分型方案可用于纹带棒状杆菌的分型,但尚需优化改进。

Objective To establish a multilocus sequence typing(MLST)assay for Corynebacterium(C.)striatum,explore the population structure and evolution relationship of clinical isolates of C.striatum.Methods Seven housekeeping genes(gyrA,gyrB,hsp65,sodA,secA1,rpoB,16S rRNA)were amplified with PCR by using self-designed specific primers and sequenced.Then,the sequences were assembled with software SeqMan.The gene diversity and gene recombination characteristics were evaluated by using software DnaSP 5.10.01 and Splits tree 4.14.2.The phylogenetic tree and the minimum spanning tree were constructed based on the sequence types(ST)characteristics by using software MEGA 7.0.14 and BioNumerics,respectively.In addition,the genetic evolutionary relationship among STs were analyzed by using software eBURST 3.0.Results The expected amplification products of seven sites selected in all the test strains were obtained.Splits tree showed that the clustering of all C.striatum strains was consistent,suggesting that gene recombination is the potential driving force for the evolution of C.striatum.All of the 344 C.striatum strains were divided into 72 STs by MLST and 85.7%of the strains formed clonal complexes.CC19 was the predominant clonal complex,whereas ST16 in the clonal complex was detected in the most strains.ST had a certain geographic clustering and a certain correlation with the isolation time.Conclusions C.striatum showed high genetic diversity in China and CC19 was the predominant clonal complex.The MLST assay established in this study can be used for the typing of C.striatum,but further improvement is needed.