基于高通量测序的平均核苷酸一致性在1株弗朗西斯菌鉴定中的应用

摘要

目的 对一溺水性肺炎患者血液标本中分离的弗朗西斯菌(编号Wenzhou1)进行菌种鉴定.方法 采用Illumina二代测序平台2000/miSeq系统对实验菌株进行高通量测序,MicrobeTrakr Plus软件对测得的全基因组草图进行序列拼接.在GenBank数据库中,对实验菌株的16S rRNA基因、mdh基因、rpoB基因和sdhA基因序列进行NCBI BLAST分析,获得与实验菌株遗传关系接近的近缘菌种信息.Java 8运行环境,OrthoANI软件对实验菌株和近缘菌种进行全基因组序列NCBI BLAST搜索和全基因组平均核苷酸一致性(average nucleotide identity,ANI)分析.结果 测得的弗朗西斯菌Wenzhou1的基因组大小为1.96×106 bp,含74个重叠群(contigs).基因组G+C mol％为32.1％,与其他弗朗西斯菌和另弗朗西斯菌一致.GenBank数据库NCBI BLAST分析结果显示,Wenzhou1的16S rRNA基因、mdh基因、rpoB基因和sdhA基因序列与西班牙弗朗西斯菌FSC454和“土拉弗朗西斯菌新凶手亚种”3523最为近缘.ANI分析显示,实验菌株Wenzhou1与西班牙弗朗西斯菌FSC454的ANI为97.8％,与“土拉弗朗西斯菌新凶手亚种”3523的ANI在97.5％～97.6％之间,而与土拉弗朗西斯菌的4个亚种的ANI在91.3％～91.5％之间.结论 基于全基因组序列的ANI分析是一种准确和有效的菌种鉴定方法.Wenzhou1可明确鉴定为西班牙弗朗西斯菌.%Objectives To identify the Francisella strain isolated from blood of a patient with drowning-associated pneumonia.Methods The whole genome of the strain,designated Wenzhou1,was sequenced using the high throughput sequencing technology by 2000/miSeq system of Illumina platform,and the obtained genome draft was assembled by MicrobeTrakr Plus software.The phylogenetic neighbors of Wenzhou1 were obtained by NCBI BLAST analysis from GenBank database for the gene sequences of 16S rRNA,malate dehydrogenase(mdh),DNA-directed RNA polymerase subunit beta (rpoB) and succinate dehydrogenase subunit alpha (sdhA).The average nucleotide identity(ANI) between Wenzhou1 and its phylogenetic neighbors was analyzed by the software OrthoANI using NCBI BLAST search under the Java Runtime Environment Version 8.Results The genome size of Wenzhou1 was 1.96 × 106 bp,containing 74 contigs.The genomic G + C mol％ of Wenzhou1 was 32.1％,which was similar to the other species of genus Francisella and Allofranicella.Based on the analysis of NCBI BLAST of GenBank for the similarities of 16S rRNA gene,mdh gene,rpoB gene and sdbA gene sequences,Wenzhou1 was most closely related to F.hispaniensis FSC454 and Francisella cf.novicida 3523.The ANI of Wenzhou1 was 97.8％ to F.hispaniensis FSC454,97.5％ to 97.6％ to Francisella cf.novicida 3523,but only 91.3％ to 91.5％ to the four subspecies of F.tularensis.Conclusion ANI analysis based on whole genome sequence should be an accurate,effective method for bacterial identification.Wenzhou1 could be identified as F.hispaniensis by ANI with high-throughput whole genome sequencing technology.