致病性大肠杆菌全基因组测序及毒力基因分析

Whole genome sequencing and virulence gene analysis of pathogenic Escherichia coli

为了研究导致致病性大肠杆菌致病力差异的主要因素,本试验以分离自仔猪腹泻样的五株致病性大肠杆菌(P211、P555、P32、P444和P111)和两株参考大肠杆菌(S10670、E24190)为实验材料,通过小鼠感染试验鉴别菌株的致病力,并对菌株进行全基因组测序,分析菌株基因组。结果显示,强毒株S10670和E24190病死率为100%,中等毒株P211和P555为40%,弱毒株P32、P444和P111为20%。七株菌基因组拼接后长度为4.6 Mbp~5.3 Mbp之间,COG注释到3183~3483个功能基因,VFDB注释到38~126个毒力基因。菌株间毒力基因分析发现,强毒株S10670含有志贺毒素(Stx),溶血素(hlyA,hlyB,hlyC,hlyD,hlyE/A),紧密黏附素(eae,tir)强毒素基因以及大量铁转运吸收系统和分泌系统相关基因。强毒株E24190含有肠毒素基因(LT)、溶血素基因(hlyA)、CFA/I菌毛基因(cfaA,cfaB,cfaC,cfaD,cfaE)。中等毒株P211也有CFA/I菌毛基因,此外还含有P菌毛基因(papA,papC,papD,papH)和铁摄取系统基因(sitA,sitB,sitC,sitD);P555含有12个VI分泌系统基因(aec15~aec19,aec22~aec32)。弱毒株P32、P444和P111含有少部分与毒力有关的基因(如黏附素Fim、侵袭素Ibe等)在其他菌株中均存在。综上,可以认为菌株间毒力基因的差异是导致大肠杆菌致病力差异的重要原因,该结果为大肠杆菌病导致的腹泻等疾病的防治提供理论依据。

In order to investigate the main factors leading to differences in pathogenicity of pathogenic E.coli,five pathogenic E.coli strains(P211,P555,P32,P444,P111)isolated from piglet diarrhea samples and two reference E.coli strains(S10670,E24190)were used as experimental materials to identify the pathogenicity of the strains by mouse infection test,and whole genome of the strain was sequenced.Meanwhile,the genomes of the strains were analyzed.The results showed that the disease mortality rate was 100%for the strong virulent strains S10670 and E24190,40%for the moderately virulent strains P211 and P555,and 20%for the weak virulent strains P32,P444 and P111.The genomes of the seven strains were between 4.6 Mbp~5.3 Mbp in length after splicing,3183~3483 functional genes was annotated by COG and 42~131 virulence genes was annotated by VFDB.Inter-strain virulence gene analysis revealed that the strong virulence strain S10670 contained strong toxin genes for shiga toxin(Stx),hemolysin(hlyA,hlyB,hlyC,hlyD,hlyE/A),tight adhesin(eae,tir)and a large number of genes related to iron transport absorption system and secretion system.The strong virulent strain E24190 contained enterotoxin genes(LT),hemolysin genes(hlyA),and CFA/I hair genes(cfaA,cfaB,cfaC,cfaD,cfaE).The moderate virulent strain P211 also had CFA/I bacterial hair genes,and in addition,contained P bacterial hair genes(papA,papC,papD,papH)and iron uptake system genes(sitA,sitB,sitC,sitD);P555 contained 12 VI secretion system genes(aec15~aec19,aec22~aec32).Weak virulent strains P32,P444 and P111 contained a small number of genes related to virulence(e.g.,adhesin Fim,invasin Ibe,etc.)existed in other strains.In conclusion,it could be considered that the differences in virulence genes among strains were important reasons of the differences in pathogenicity of E.coli,which provided a theoretical basis for the prevention and control of diseases such as diarrhea caused by E.coli disease.