非 O157产志贺毒素大肠埃希菌分离株eae 基因分型分析

摘要

目的：了解我国不同来源非O157产志贺毒素大肠埃希菌（ Shiga toxin-producing Esche-richia coli， STEC）分离株携带的eae基因型别。方法 PCR扩增eae全长基因，扩增产物经测序并拼接后在美国国立生物技术信息中心（ NCBI）中进行BLASTn比对，判定其eae基因型别，并与GenBank中已公布的30种 eae 不同型别的序列及产志贺毒素大肠埃希菌主要流行血清型（ O157∶H7， O26∶H11， O103∶H2， O111∶H8， O145∶H28， O45∶H2， O121∶H19）代表性基因组测序菌株的eae序列用Neighbor-Joining法构建系统发生树，分析其进化关系。根据mlst．ucc．ie数据库提供的大肠埃希菌多位点序列分型（ multilocus sequence typing， MLST）方案对菌株进行MLST分型分析，确定菌株等位基因型及序列类型，并用BioNumerics软件构建最小生成树（ minimum spanning tree， MST），分析携带eae基因的非O157 STEC分离株与溶血性尿毒综合征相关大肠埃希菌（ HUS-associated en-terohemorrhagic E．coli，HUSEC）、E．coli MLST数据库中所有人源流行血清群O26、O103、O111、O121、O145、O157 STEC菌株的进化关系。结果10株不同来源的非O157产志贺毒素大肠埃希菌分离株经扩增拼接后均获得了约2．8 kb的eae基因全长序列，分为3种已知的eae基因型别，其中以β1型为主（6／10），2株θ型，2株γ1型。某些eae序列与主要流行血清型STEC的eae序列完全一致。10株菌分为7个ST型，与HUSEC、人源流行血清群STEC菌株具有相近的进化关系。结论我国非O157产志贺毒素大肠埃希菌分离株eae基因存在一定程度的多样性，eae阳性菌株具有潜在的高致病性。%Objective To analyze the subtypes of eae genes in various non-O157 Shiga toxin-pro-ducing Escherichia coli ( STEC) strains isolated in China.Methods The complete nucleotide sequences of 10 eae genes were amplified by PCR and sequenced.The BLASTn software was used to analyze the se-quences for eae gene subtyping.A phylogenetic tree was constructed based on the10 ea e gene sequences to-gether with the gene sequences of 30 different subtypes in GenBank and those of STEC strains of 7 prevalent serotypes (O157 ∶H7, O26 ∶H11, O103 ∶H2, O111 ∶H8, O145 ∶H28, O45 ∶H2 and O121 ∶H19) using MEGA 5.0.Multilocus sequence typing (MLST) was performed on the 10 STEC strains with reference to the Escherichia coli ( E.coli) MLST website ( http://mlst.warwick.ac.uk/mlst/dbs/Ecoli) for the typing of multiple loci.A minimum spanning tree ( MST) was constructed using the BioNumerics software to inves-tigate the phylogenetic relationships between the 10 eae gene-positive STEC strains in this study and hemolyt-ic uremic syndrome-associated enterohemorrhagic E.coli ( HUSEC) strains as well as all human STEC strains of O157, O26, O45, O103, O111, O121 and O145 serotypes submitted to the E.coli MLST website data-base.Results The complete nucleotide sequences of eae genes in 10 non-O157 STEC strains were 2.8 kb in length and belonged to 3 known subtypes.The predominant subtype wasβ1, accounting for 60%of the 10 STEC strains (6/10), followed byθandγ1 subtypes with two strains in each type.The eae gene sequences in certain strains were identical to those of the prevalent serotypes.Seven sequence types ( STs) were identi-fied from the 10 STEC strains carrying eae gene.Conclusion The eae genes harbored by the non-O157 STEC strains isolated from different specimens in China were diverse and had close phylogenetic relationships with the highly pathogenic and prevalent STEC strains.This study implied that the STEC strains harboring eae gene had high pathogenic potential.