PFGE和质粒图谱分析人和猪来源德尔卑沙门菌多重耐药株的同源性  

Study on molecular homology of multi-drug resistant Salmonella derby isolated from human and pig by PFGE and plasmid profile analysis

作　　者：郭仲辉 陈冬雅 黎毓光 林晓晖 严智敏 

机构地区：[1]广州市番禺区中心医院,广州511400

出　　处：《中国抗生素杂志》2014年第10期758-763,共6页Chinese Journal of Antibiotics

基　　金：广东省科技厅2011B061300001;广州市番禺区科技和信息化局2013-专-01-030

摘　　要：目的分析人和猪来源的德尔卑沙门菌的分子同源性，为研究多重耐药性在人畜之间传播提供分子生物学技术依据。方法药敏试验检测人和猪来源的德尔卑沙门菌多重耐药株对10种抗菌药物的敏感性；用脉冲凝胶电泳方法（PFGE）检测菌株间的分子同源性；结合实验或电转移试验获得目标质粒，质粒酶切图谱检测质粒的同源性。PCR分析染色体介导喹诺酮耐药基NgyrA~@arC，质粒可介导的喹诺酮耐药基因qnrA／qnrB／qnrC／qnrS、aac（6）-I b—cr、qepA和oqxAB。结果共收集48株德尔卑沙门菌，经药敏试验筛选获得11株多重耐药株（MDR），人源性8株，猪源性3株。11株菌除对氨苄青霉素、氯霉素、链霉素、磺胺和四环素（R型：ACSSuT）耐药外，所有菌株也对萘啶酸和环丙沙星耐药，6株对左氧氟沙星耐药，5株对头孢曲松耐药。PFGE分析，11株菌共分为6个克隆，3株动物源性菌株与8株人源性菌株无克隆相关性。喹诺酮耐药基因分析显示，有5株和3株动物源株检NNgyrA的Ser83Leu或／和Asp87Asn突变，6株人源株和3株动物源株检测到parC的Ser80Ile或／和Thr57Ser突变。质粒电泳分析显示，所有菌株都存在1．4条质粒条带，大小介于2～180kb，其中3株人源性菌株和l株动物源性株都存在4kb左右大小的质粒；2株动物源性菌株仅存在20kb大小质粒。用结合实验和电转移方法，分析4kb质粒结构，证实4个质粒的酶切图谱完全一致，质粒中均存在介导外排基NoqxAB~l喹诺酮耐药基因aac（6’）-Ib-cr。结论人和猪来源的德尔卑沙门菌多重耐药株中质粒介导的外排因oqxAB和诺酮耐药基因Naac（6）-Ib-c是喹诺酮重要的耐药机制，可能在沙门菌间传递，导致对喹诺酮类高耐药性。通过人和猪的接触或间接途径，沙门菌有传播喹诺酮耐药性的风险，同一种质粒可在人和动物中转移而传播该耐药机制。Objective To investigate the molecular homology of Salmonella derby strains isolated from human and pigs, as a molecular base for further study of multidrug resistance transmission between two different sources, Methods Antimicrobial susceptibility was determined by broth microdilution minimal inhibitory concentration for multi-drug-resistant S. derby isolates against 10 antimicrobial agents. The clonally relationship of isolates and plasmids was performed by PFGE and restriction enzyme mating respectively, following the conduction of conjugative test or electro transformation. Details of quinolone resistant genes was detected by PCR for both chromosome- mediated genes （gyrA and parC） and plasmid mediated ones including qnrA/qnrB/qnrC/qnrS, aac（6＇）- I b-cr, and oqxAB. ResuLts Including 8 isolates of human sources and 3 of pig sources were conferred as MDR S. derby among a total of 48 isolates resulting from the antimicrobial susceptibility test. All of the 11 MDR S. derby strains showed a resistant-profile against ampicillin, cefuroxime, kanamycin, gentamicin, trimesulf, chloramphenicol, nalidixic acid and ciprofloxacin, still resistant to levofloxacin in 6 strains and ceftriaxone in 5. The 11 MDR S. derby isolates were classified into six genotypes, and no close relationship was found between human isolates and pig isolates in PFEG analysis. Mutations in gyrA of Ser83Leu or/and Asp87Asn and parC of Ser80Ile or/and Thr57Ser occurred in 8 isolates （5 human and 3 animal） and 9 isolates （6 human and 3 animal） respectively. Plasmids varied in amount of 1 to 4 were found in each MDR S. derby isolate, with size ranging from 2kb to 180kb. Two isolates of animal source had a lonely plasmid of 20kb, while the same sized plasmid at 4kb was found in both isolates of two sources （3 human and 1 animal） and revealed exactly the same restriction enzyme maps in the plasmid structure study, harboring oqxAB and aac（6＇）- I b-er responsible for efflux pump and resistance to quinolone. Conclusion Resistanc

关 键 词：德尔卑沙门菌 脉冲凝胶电泳 质粒 同源性 喹诺酮药物 

分 类 号：R378[医药卫生—病原生物学]