一株肠致病性大肠杆菌的全基因组序列分析

Complete genomic fine sequence analysis of 1 strain of the enteropathogenic Escherichia coli

目的分析并找出肠致病性大肠杆菌Deng(Enteropathogenic Escherichia coli Deng,EPEC Deng)的全基因组序列特点。方法 EPEC Deng来源于我国婴幼儿腹泻患者的粪便标本,菌株采用诊断血清鉴定血清型,通过自动细菌鉴定仪,使用微量肉汤法进行药敏试验。进一步提取细菌总DNA,构建测序文库,通过Illumina 2000仪器测序,然后利用多引物PCR等方法进行补缺口及拼接,最终获得完整的全基因组。采用相关软件获得基因序列,通过对比已知的蛋白数据库进行基因功能注释及生物信息学分析。结果 EPEC Deng菌株归属O119:H6,药敏结果显示该菌株对环丙沙星、左氧氟沙星、氨苄西林及氨苄西林/舒巴坦耐药,对其余的抗菌药物均敏感。EPEC Deng基因组包含5 233 046 bp,GC含量50.48%。基因组通过注释后总共有5 347个编码蛋白序列,80个t RNA的编码基因,以及22个完整的r RNA基因编码的操纵子。EPEC染色体基因组中含有致病岛,大小约为40Kb左右,与数据库中的一个37Kb的致病岛(AF200363.1)序列极为相似,相似度达到99%。结论完成了肠致病性大肠杆菌基因组精细测序分析,PAI是EPEC致病的关键,为进一步研究菌株的进化关系提供了基因数据。

Objective To analyze the complete genomic characteristic of an enteropathogenic Escherichia coli Dengisolated from the native infants. Methods Enteropathogenic Escherichia coli Deng was isolated from native infant stool.Serotype was detected using diagnostic serum protocol. Bacterial susceptibility testing was carried out according to a unifiedprotocol using broth microdilution method by BD- Automatic identification of bacterial analyzer. Total DNA were extractedusing Wizard genomic DNA purification kit, then the sequence of EPEC Deng DNA was determined by high- throughputIllumina 2000 sequencing approach. Contigs were generated and assembled into scaffolds by using the SPAdes software.Subsequently, gap closures were performed by Gapcloser and Gap Filler software. The annotation was performed by RSATsoftware, and predicted genes were analyzed compared to known databases. Results The serotype of EPEC DENG strain wasO119：H6, it was resistant to ciprofloxacin, levofloxacin and ampicillin. The completed enteropathogenic E. coli Deng circularchromosome contains 5233046 bp with a GC content of 50.48 %. Annotation created a total of 5347 coding sequences（CDS）,80 t RNA- encoding genes, and 22 entire r RNA-encoding operons. There was an pathogenicity island（PAI）in the genome,which was about 40 Kb,and the familiarity to the PAI in Pubmed database（Genebank No. AF200363.1） was 99%.Conclusion The complete genomic sequence of an EPEC strain was completed. PAI is the key point of pathogenicity ofEPEC, which provided a genetic data for evolution analysis of EPEC strain.