一株杀白细胞素基因阳性耐甲氧西林金黄色葡萄球菌的分子分型及肠毒素检测  

Molecular characteristics and Staphylococcal enterotoxins of a Panton-Valentine leucocidin(PVL)-positive ethicillin-resistant Staphylococcus aureus isolates

作　　者：苏雅静 乔霞 康宇婷 汪澎涛 杨宁爱 贾伟 赵志军 SU Ya-jing;QIAO Xia;KANG Yu-ting;WANG Peng-tao;YANG Ning-ai;JIA Wei;ZHAO Zhi-jun(Ningxia Key Laboratory of Clinical and Pathogenic Microbiology,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Laboratory Center of Medicine,General Hospital of Ningxia Medical University)

机构地区：[1]宁夏医科大学总医院宁夏临床病原微生物重点实验室,宁夏银川750004 [2]宁夏医科大学总医院医学实验中心

出　　处：《中国病原生物学杂志》2022年第6期649-653,659,共6页Journal of Pathogen Biology

基　　金：宁夏自然科学基金项目(No.2022AAC03548);第四批宁夏青年科技人才托举工程项目(No.TJGC2019089)。

摘　　要：目的 对宁夏某医院临床分离的1株杀白细胞素基因阳性耐甲氧西林金黄色葡萄球菌(PVL^(+) MRSA)菌株进行分子分型和肠毒素检测分析,以了解该菌株的遗传学特征。方法 按AxyPrep质粒小量制备试剂盒说明书提取细菌DNA,利用葡萄球菌染色体基因盒mec(staphylococcal cassette chromosome mec,SCCmec)分型、多位点序列分型(multilocus sequence typing,MLST)以及金黄色葡萄球菌表面蛋白A基因多态性分型(staphylococcus protein A,Spa)技术对PVL^(+) MRSA分离株进行分子分型。采用PCR法检测金黄色葡萄球菌7个管家基因(arcC,aroE,tpi,pta,glpF,gmk和yqiL),将测序结果与MLST数据库进行比对。采用微孔板酶联免疫法(ELISA)检测PVL^(+) MRSA分离株中5种传统肠毒素(staphylococcal enterotoxins A-E,SEA-SEE)的表达情况。结果 对PVL^(+) MRSA分离株进行SCCmec基因的多重PCR扩增,扩增产物经测序比对分析,确定该菌株为SCCmecⅣa型。PCR扩增金黄色葡萄球菌7个管家基因后测序比对,获得PVL^(+) MRSA分离株的序列型(sequence types,STs)为ST 3191。PCR扩增spa基因并测序,测序结果与Spa Serve数据库比对,未检出PVL^(+) MRSA分离株的Spa型别(non-typeable,NT)。ELISA检测PVL^(+) MRSA分离株产B和C混合型葡萄球菌肠毒素。结论 PVL^(+) MRSA分离株的分子型为ST 3191-MRSA-SCCmecⅣa型,且产B和C混合型葡萄球菌肠毒素,属于社区获得性耐甲氧西林金黄色葡萄球菌(CA-MRSA)。Objective The present study aimed to investigate the genetic characteristics of a Panton-Valentine leucocidin-positive(PVL^(+)) methicillin-resistant Staphylococcus aureus(MRSA) isolated from a hospital of Ningxia province through detection of molecular typing and staphylococcal enterotoxins.Methods Genomic DNA was extracted from S.aureus isolates PVL^(+) MRSA by AxyPrep Plasmid Miniprep Kit according to the manufacturer’s instruction.The isolates PVL^(+) MRSA was then characterized for the determination of the molecular typing by staphylococcal cassette chromosome mec(SCCmec) typing,multilocus sequence typing(MLST),and staphylococcus protein A(Spa) typing.We also used PCR assays to detect seven housekeeping genes(arcC,aroE,glpF,gmk,pta,tpi,and yqiL) and,the result of the sequence was compared with the MLST database.Meanwhile,5 traditional staphylococcal enterotoxins A-E(SEA-SEE) in isolates PVL^(+) MRSA were determined by enzyme-linked immunosorbent assay(ELISA).Results Multiplex PCR method was done for the determination of SCCmec types and a fragment of 750 base pairs(bp) was amplified.The isolates PVL^(+) MRSA was identified as SCCmec Ⅳa after testing and ontrasted sequence.MLST revealed the isolates PVL^(+) MRSA belonging to type ST 3191.The spa typing of the isolates PVL^(+) MRSA was not identified(non-typeable,NT) according to the sequenced and deduced using the Ridom Spa Server database.Further,ELISA howed the isolates PVL^(+) MRSA was found to produce enterotoxins type B and C.Conclusion The isolates PVL^(+) MRSA was ST 3191-MRSA-SCCmec Ⅳa type,which belonging to community-associated methicillin-resistant staphylococcus aureus(CA-MRSA).The enterotoxins SEB and SEC were produced in the isolates PVL^(+) MRSA.

关 键 词：耐甲氧西林金黄色葡萄球菌 分子分型 PCR 肠毒素 

分 类 号：R378.11[医药卫生—病原生物学]