多位点序列分型应用于致病性黄疸出血群钩端螺旋体分析
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摘要
目的初步探讨MLST(multilocus sequence typing)分型技术在致病性钩端螺旋体中的应用。方法对我国3个省份的39株致病性钩端螺旋体黄疸出血群菌株提取DNA,采用7个位点进行PCR扩增、序列测定,应用eBURST和BioNumerics(Version5.10)软件进行分析。结果 39株菌株呈现5个ST型别,ST1为中国三省份中主要的基因型,占53.85%(21/39)。eBURST分析显示:39株菌株分为2个Clonal complexes和1个singleton,其中Group1占66.67%(26/39),Group2占20.51%(8/39),一个singleton占12.82%(5/39);BioNumerics软件聚类分析显示:39株菌株分为3个Group,与eBURST分析中的分群结构一致。MLST基因型别具有明显的地域性特征。结论 MLST方法可初步应用于致病性钩端螺旋体分子流行病学、种群结构、亲缘进化关系等研究。
In order to explore the genetic diversity of pathogenic Leptospira,a collection of 39 strains belonging to L.interrogans serogroup Icterohaemorrhagiae isolated from three provinces in China were analyzed using multilocus sequence typing(MLST)in this study.Seven loci were chosen for genotyping in MLST analysis and the results were analyzed by eBURST and BioNumerics(Version 5.10)softwares.Among 39 Leptospirastrains,a total of 5different sequence types(STs)were obtained.ST1 accounted for 53.85%(21/39)and was the predominant genotype in the three provinces in China.The eBURST analysis revealed that 39 isolates were classified into two clonal complexes and one singleton.Group1 accounted for 66.67%(26/39),Group2 accounted for 20.51%(8/39)and the remaining singleton accounted for 12.82%(5/39).Phylogenetic analysis using BioNumerics softwares demonstrated that three major clusters(Group1,Group2 and Group3)were identified among39 isolates.Results of phylogenetic analysis by eBURST and BioNumerics softwares were consistent with each other.Obvious epidemiological relationship was found between the distributions of STs and isolated location.The strains isolated from the same geographic regions belonging to the same serogroup shared a common ST type.In this work,the results demonstrated that this MLST typing technique could be used to explore the genetic diversity and population structure of Leptospira interrogans serogroup Icterohaemorrhag strains.The current MLST method may greatly contribute to a better knowledge of the molecular epidemiology,population structure and evolutionary studies of pathogenic Leptospira.
In order to explore the genetic diversity of pathogenic Leptospira,a collection of 39 strains belonging to L.interrogans serogroup Icterohaemorrhagiae isolated from three provinces in China were analyzed using multilocus sequence typing(MLST)in this study.Seven loci were chosen for genotyping in MLST analysis and the results were analyzed by eBURST and BioNumerics(Version 5.10)softwares.Among 39 Leptospirastrains,a total of 5different sequence types(STs)were obtained.ST1 accounted for 53.85%(21/39)and was the predominant genotype in the three provinces in China.The eBURST analysis revealed that 39 isolates were classified into two clonal complexes and one singleton.Group1 accounted for 66.67%(26/39),Group2 accounted for 20.51%(8/39)and the remaining singleton accounted for 12.82%(5/39).Phylogenetic analysis using BioNumerics softwares demonstrated that three major clusters(Group1,Group2 and Group3)were identified among39 isolates.Results of phylogenetic analysis by eBURST and BioNumerics softwares were consistent with each other.Obvious epidemiological relationship was found between the distributions of STs and isolated location.The strains isolated from the same geographic regions belonging to the same serogroup shared a common ST type.In this work,the results demonstrated that this MLST typing technique could be used to explore the genetic diversity and population structure of Leptospira interrogans serogroup Icterohaemorrhag strains.The current MLST method may greatly contribute to a better knowledge of the molecular epidemiology,population structure and evolutionary studies of pathogenic Leptospira.
引文
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[8]Thaipadungpanit J,Wuthiekanun V,Chierakul W,et al.A dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand[J].PLoS Negl Trop Dis,2007,1(1):e56.
[2]Zhang C,Wang H,Yan J.Leptospirosis prevalence in Chinese populations in the last two decades[J].Microbes Infect,2012,14(4):317-323.DOI:10.1016/j.micinf.2011.11.007
[3]Zhang CC,Nie YX,Li XW,et al.Application of multiple-locus variable-number tandem repeat analysis(MLVA)for molecular typing of Leptospira interrogans serogroup Icterohaemorrhag[J].Chin J Microbiol Immunol,2009,29(12):1144-1147.DOI:10.3760/cma.j.issn.0254-5101.2009.12.025(in Chinese)张翠彩,聂一新,李秀文,等.黄疸出血群钩端螺旋体MLVA分型研究.中华微生物学和免疫学杂志,2009,29(12):1144-1147.
[4]Li SJ,Zhang CC,Li XW,et al.Molecular typing of Leptospira interrogans strains isolated from Rattus tanezumi in Guizhou Province,Southwest of China[J].Biomed Environ Sci,2012,25(5):542-548.DOI:10.3967/0895-3988.2012.05.007
[5]Voronina OL,Kunda MS,Aksenova EI,et al.The characteristics of ubiquitous and unique Leptospirastrains from the collection of Russian centre for leptospirosis[J].Biomed Res Int,2014,2014:649034.DOI:10.1155/2014/649034
[6]Romero EC,Blanco RM,Galloway RL.Analysis of multilocus sequence typing for identification of Leptospiraisolates in Brazil[J].J Clin Microbiol,2011,49(11):3940-3942.DOI:10.1128/JCM.01119-11
[7]Caimi K,Varni V,Melendez Y,et al.A combined approach of VNTR and MLST analysis:improving molecular typing of Argentinean isolates of Leptospira interrogans[J].Mem Inst Oswaldo Cruz,2012,107(5):644-651.
[8]Thaipadungpanit J,Wuthiekanun V,Chierakul W,et al.A dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand[J].PLoS Negl Trop Dis,2007,1(1):e56.