焦磷酸测序检测产ESBLs革兰阴性菌的SHV基因型

检验医学 ›› 2012, Vol. 27 ›› Issue (10): 829-834.

焦磷酸测序检测产ESBLs革兰阴性菌的SHV基因型

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

出版日期:2012-10-30 发布日期:2012-10-12
作者简介:黄源芳，女，1986年生，硕士，主要从事抗菌药物及耐药研究。

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

1. West China Preclinical and Forensic Medical College，Sichuan University，Sichuan Chengdu 610041，China; 2. Department of Clinical Laboratory，Chengdu Second People′s Hospital，Sichuan Chengdu 610017，China

Online:2012-10-30 Published:2012-10-12

摘要/Abstract

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噁唑的耐药性较高，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。

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

Abstract: 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.

Key words: SHV genotype, Pyrosequencing, Extended-spectrum beta-lactamase , Single nucleotide polymorphism

黄源芳1，宋晓红1，尹亚非2，扎拉嘎白乙拉1，姬小丽1，刘小康1. 焦磷酸测序检测产ESBLs革兰阴性菌的SHV基因型[J]. 检验医学, 2012, 27(10): 829-834.

HUANG Yuanfang 1，SONG Xiaohong 1，YIN Yafei 2，ZHA Lagabaiyila 1，JI Xiaoli 1，LIU Xiaokang 1. Detection of SHV genotype of ESBLs-producing Gram-negative bacterium by pyrosequencing[J]. , 2012, 27(10): 829-834.

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