产CTX-M-55型超广谱β-内酰胺酶大肠埃希菌全基因组序列耐药和毒力特征研究

Genetic characterization of two CTX-M-55 type extended-spectrum β-lactamase-producing Escherichia coli isolates

目的基于全基因组序列,对2株食源性产CTX-M-55型超广谱β-内酰胺酶(ESBLs)大肠埃希菌的耐药和毒力分子机制进行研究。方法采用微量肉汤稀释法对大肠埃希菌食品分离株进行药敏试验,设计产ESBLs基因引物并通过聚合酶链式反应(PCR)扩增筛选出2株产CTX-M-55型ESBLs大肠埃希菌(编号为EC001和EC002)进行全基因组测序,并进行序列型(ST)、质粒复制子类型、血清型、耐药基因和毒力因子识别。结果 2株大肠埃希菌均为头孢类和喹诺酮类抗生素双重耐药ESBLs菌株;全基因组序列分析结果显示,2株菌的血清型均为肠道致病性大肠埃希菌(EPEC)O119∶H8,ST型分别为ST21(EC001)和ST342(EC002),EC001菌株含有Inc FII、IncX1、IncY、Col156和IncI2 5个不相容群质粒,EC002菌株含有IncFII和IncX1 2个不相容群质粒,2株菌株染色体中均携带bla_(CTX-M-55)和bla_(TEM-141)2种ESBLs基因。此外,EC001菌株还携带sul2/3、tet(A)/(B)、dfrA12、strA/B、aph(3')-IIa、cml/cmlA1、floR和oqxA/B耐药相关基因,其染色体喹诺酮耐药决定区gyr A基因存在2个突变位点(S83L,D87H)、parC基因存在1个突变位点(S80I),多粘菌素耐药相关基因pmr E基因存在3个突变位点(D73Y,M185T,S225T);而EC002菌株携带fosA和qnrS1耐药相关基因。2株菌均携带致黏附与脱落(A/E)损伤的肠细胞脱落位点(LEE)毒力岛基因esc、esp、eae A和tir,且EC001菌株还携带增强菌株在血清中存活能力的iss基因。结论对2株食源性产CTX-M-55型ESBLs大肠埃希菌进行全基因组测序以及耐药和毒力的分子机制的研究,其结果可为后续开展大肠埃希菌耐药和毒力表型预测、指导食用畜禽养殖过程中抗生素合理使用及耐药菌株防控提供依据。

Objective To study the antimicrobial resistance and virulence mechanism of two foodborne CTX-M-55 type extended-spectrum β-lactamase（ ESBL）-producing Escherichia coli strains based on whole genome sequences. Methods The antimicrobial susceptibility testing of two CTX-M-55 type ESBL-producing Escherichia coli isolates（ EC001 and EC002） that were collected from foodborne pathogen surveillance system in 2011 was conducted by antimicrobial susceptibility test and PCR. Then two strains were sequenced using the Illunima Hiseq 2000 platform. Based on the assembled genome sequences of two strains,the annotation of sequence type,plasmid replicon type,serotype,and antimicrobial resistance/virulence genes was performed using Bio Numerics software. Results The two strains were identified to be ESBL-producing and fluoroquinolone-resistant. The genomic analysis result showed that strains EC001 and EC002 were ST21 and ST342,respectively,and serotyped to be enteropathogenic Escherichia coli（ EPEC） O119∶ H8. The plasmid of EC001 belonged to 5 incompatible groups including Inc FII,IncX1,IncY,Col156,and IncI2,and that of EC002 belonged to 2 incompatible groups including Inc FII and IncX1. Notably,both strains harbored 2 types of ESBL-encoding genes bla_（CTX-M-55） and bla_（TEM-141）. Additionally,the multi-antimicrobial resistance genes sul2/3,tet（ A）/（ B）,dfrA12,strA/B,aph（ 3＇）-IIa,cml/cmlA1,floR,and oqxA/B were identified on the genome of strain EC001,while fosA and qnrS1 were identified on the genome of strain EC002. Compared with the genome of Escherichia coli K12_ 1655,two mutations were found at the 83 th（ S83 L） and 87 th（ D87 H） codons of gyr A,and one mutation was found at the 80 th（ S80 I） codon of parC in QRDRs of strain EC001. Noteworthy,3 mutations were found at the 73 th（ D73 Y）,185 th（ M185 T） and 225 th（ S225 T） codons of pmr E in the chromosome of strain EC001,which might be responsible for its resistance to colistin. The virulence gene prediction result showed that both strains harbored classical locus of enterocyte and effacement（ LEE） genes esc,esp,eae A and tir,while EC001 contained iss gene which could increase serum survival capacity of this bacterium. Conclusion To our best knowledge,we applied whole genome sequencing in annotation of the resistance and virulence mechanism of two CTX-M-55-type ESBL-producing Escherichia coli strains in China. The data in this study will shed light on antimicrobial resistance and virulence mechanisms of Escherichia coli,and finally help prevent and control the contamination of this foodborne pathogen.