对1株疑似鼠疫耶尔森氏菌的系统鉴定

Systematic identification of a suspicious Yersinia pestis isolate

目的对1株可疑菌株进行系列验证实验,以确定其是否为鼠疫耶尔森氏菌(以下简称鼠疫菌)。方法用鼠疫细菌学常规方法和分子生物学手段确定其生物学表型特征、特异性基因及基因组特征。结果该菌株具备鼠疫菌的典型形态特征;能被鼠疫噬菌体完全裂解;主要生化特性为阿胶糖(+)、鼠李糖(-)、麦芽糖(+)、蜜二糖(-)、甘油(+)、脱氮(+),与典型鼠疫菌一致。毒力因子检查结果为均为阴性;对实验动物小白鼠完全无致死能力。全基因组芯片杂交实验和PCR扩增表明55023菌株没有鼠疫菌的三个质粒;也不具鼠疫标识基因;pgm位点代表性基因YPO1954扩增阳性,YPO1908扩增阴性,表明其pgm位点不完整;差异片段(DFR)分型结果表明该菌株缺失了14个DFR,不符合鼠疫菌的特征;多位点序列分型(MLST)分析结果表明该菌株与鼠疫存在16个碱基的差异,而与血清Ⅲ型假结核耶尔森氏菌仅相差两个碱基。结论尽管55023菌株具备鼠疫菌的一些表型特征,但基因组特征表明其不是鼠疫菌,而可能是血清III型的假结核耶尔森氏菌;噬菌体裂解结果等表型不能作为鼠疫菌鉴定的最终标准。

A handful of traditional and molecular assays were applied to decipher the phenotypic and genomic characteristics of a suspicious Yersinia pestis strain 55023.Strain 55023 showed typical colony morphism and microscopic features of Y.pestis.It can be lysed by the diagnostic phage of Y.pestis at 22℃,28℃ and 37℃,which resembles typical Y.pestis isolates.Strain 55023 was positive for utilizing arabinose,glucosylglucose,glycerine and denitrification,while negative for rhamnose and melibiose,which was also identical to reference Y.pestis strains.Known virulence factors for Y.pestis（F1 antigen,VW antigen,pgm and Pst 1） were all absent from strain 55023,and all mice survived even challenged by 17.5 billion CFU of 55023.Both microarray and PCR verified the absence of three known plasmids of Yersinia pestis（pPCP,pMT1 and pCD）,and microarray analysis also revealed the missing of 12% Y.pestis genes in strain 55023.Two signature genes failed to get amplified in this stain.One of representative genes of pgm locus,YPO1954,is present,while the other one YPO1908 absent in 55023,which suggests a partial pgm locus in it.Different regions（DFR） profiling disclosed the absence of 14 out of 23 DFRs from this strain,which tended to reject it as Y.pestis strain.Six-gene multi-locus sequencing typing（MLST） analysis identified 16 different nucleotides out of 2,509 bp between 55023 and Yersinia pestis,while only 2 variations between 55023 and Yersinia pseudotuberculosis serovars III.Although stain 55023 shared quite a few phenotypic characteristics with Y.pestis,the detailed molecular data identified it as Yersinia pseudotuberculosis（probably serovars III）.It＇s suggested that phenotypic analysis,like phage lysis,is not capable of providing solid data for the identification of Y.pestis.