焦磷酸测序检测产ESBLs革兰阴性菌的SHV基因型  

Detection of SHV genotype of ESBLs-producing Gram-negative bacterium by pyrosequencing

作　　者：黄源芳[1] 宋晓红[1] 尹亚非[2] 扎拉嘎白乙拉 姬小丽[1] 刘小康[1] 

机构地区：[1]四川大学华西基础医学与法医学院,四川成都610041 [2]成都市第二人民医院检验科,四川成都610017

出　　处：《检验医学》2012年第10期829-834,共6页Laboratory Medicine

摘　　要：目的探讨一种快速、准确检测产超广谱β-内酰胺酶(ESBLs)革兰阴性菌的ESBLs基因分型方法。方法双纸片法确定产ESBLs的临床分离菌,聚合酶链反应(PCR)扩增ESBLs的SHV基因片段,用焦磷酸测序技术对29株成都市区临床分离的产ESBLs肺炎克雷伯菌和大肠埃希菌进行SHV基因分型研究,检测SHV基因片段中编码35位氨基酸和编码43位氨基酸位点的基因多态性。同时,采用纸片扩散法进行药物敏感性试验。结果焦磷酸测序发现,本地区分离出的29株产ESBLs临床分离菌有21株扩增出SHV基因片段,且在43位氨基酸密码子均没有多态性,35位密码子有基因多态性,核苷酸由T突变为A,亮氨酸变为谷氨酰胺,突变发生率达到42.9%(9/21)。29株产ESBLs的菌株对亚胺培南全部敏感;对头孢西丁、头孢吡肟、头孢他啶耐药率分别为:大肠埃希菌29.4%、11.8%、41.2%;肺炎克雷伯菌50.0%、8.3%、33.3%;对氨苄西林、哌拉西林、头孢唑啉、头孢呋辛和复方磺胺甲口恶唑的耐药性较高,均达到75%以上,对其他药物均有不同程度的耐药性。结论焦磷酸测序技术可快速对临床分离菌产生的ESBLs耐药基因分型,具有准确、快速、实时和高通量等优点。Objective To investigate a method which can rapidly and accurately detect the genotyping extended-spectrum beta-lactamases（ESBLs）-producing Gram-negative bacterium.Methods Clinical isolated ESBLs-producing strains were detected by double-disk method.SHV gene fragments of ESBLs were amplified by polymerase chain reaction（PCR）.Pyrosequencing was used to study SHV genotyping of the 29 strains of ESBLs-producing Escherichia coli and Klebsiella pneumoniae isolated from hospitals in Chengdu.The gene polymorphism of amino acids site encoding 35 and 43 of SHV gene fragments were investigated.Meanwhile,antimicrobial susceptibility was determined by disk diffusion method.Results The results of pyrosequencing were that 21 of the 29 locally isolated ESBLs-producing strains had SHV gene fragments.Codon 43 amino acids had no polymorphism,while codon 35 had gene polymorphisms,nucleotide mutated from T to A,amino acids mutated from Leu to Glu,and the mutation rate reached 42.9%（9/21）.All of the 29 ESBLs-producing stains were sensitive to imipenem.The resistance rates to cefoxitin,cefepime and ceftazidime were 29.4%,11.8% and 41.2% for Escherichia coli and 50.0%,8.3% and 33.3% for Klebsiella pneumoniae,respectively.More than 75% of the stains were highly resistant to ampicillin,piperacillin,cefazolin,cefuroxime and cotrimoxazole,while all of them were resistant to other drugs with variable degrees.Conclusions Pyrosequencing can be used to rapidly detect resistant genotyping of clinical isolated ESBLs-producing strains with advantages of accuracy,speediness,real-time implementation and high-throughput.

关 键 词：SHV基因型 焦磷酸测序 超广谱β-内酰胺酶 单核苷酸多态性 

分 类 号：R446.5[医药卫生—诊断学]