副猪嗜血杆菌链霉素耐药基因rpsL、rrs分析和关键位点的鉴定  

Analysis of Haemophilus parasuis streptomycin-resistant genes of rpsL and rrs and identification of the key sites

作　　者：王婉蓉 代科 马小雨 杨振 曹三杰 黄小波 赵勤 伍锐 文翼平 WANG Wan-rong;DAI Ke;MA Xiao-yu;YANG Zhen;CAO San-jie;HUANG Xiao-bo;ZHAO Qin;WU Rui;WEN Yi-ping(Sichuan Agricultural University Swine Disease Research Center,College of Veterinary Medicine Sichuan,Chengdu 611130,China)

机构地区：[1]四川农业大学动物医学院猪病研究中心,四川成都611130

出　　处：《中国预防兽医学报》2018年第11期1061-1065,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划"畜禽重大疫病防控与高效安全养殖综合技术研发"(2016YFD 500800)

摘　　要：为研究副猪嗜血杆菌(HPS)链霉素(SM)抗性的分子基础,本实验采用甲基磺酸乙酯(EMS)间断传代诱导HPS SC1401菌株,构建SM抗性突变株,测定其对SM的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),并对其耐药基因rpsL和rrs进行测序;通过对比分析筛选出rpsL基因中两个引起SM完全耐药的突变位点;为了进一步验证突变位点与抗性的关联性,设计位点突变引物以构建突变型质粒,并分别转化到亲本株中,获得单一位点突变菌株,测定其对SM的MIC和MBC。结果显示:EMS诱导的SM抗性突变菌株rpsL基因第43号(AAA→AGA)或第88号密码子(AAA→AGA)发生了突变,rrs基因未检测到突变;该抗性突变菌株与单一位点突变菌株1401D43和1401D88对SM的MIC和MBC均大于8 192μg/mL,远高于亲本株SC1401的SM抗性水平。实验结果表明,HPS的SM抗性主要由rpsL基因特定位点突变引起,其中,rpsL基因第43号和第88号密码子为主要突变位点。本实验为完善HPS耐药分子机制研究,特别是对氨基糖苷类药物产生耐药的分子基础提供一定的参考。To elucidate the candidate molecular mechanism of streptomycin (SM)resistance of Haemophilus parasuis,we successfully prepared SM-resistant derivatives from H.parasuis wild type strain SC1401by induction with ethyl methane sulfonate (EMS).Then,we determined the minimum inhibitory concentration (MIC)and minimum bactericidal concentration (MBC)of SM and screened the mutation sites by comparing the sequence of the rpsL gene and rrs gene.Afterwards,we utilized site-directed mutation primers to construct two mutation plasmids and transformed both plasmids into wild type strain HPS SC1401.We obtained specific site-point mutants strains and further determined their MIC and MBC.Upon sequencing and site-directed mutations,we found that the EMS-induced point mutation in rpsL at codon 43^rd (AAA→AGA)or at 88^th(AAA→AGA)confered the mutants a high SM resistance (MIC>8,192g/mL and MBC>8,192g/mL),which could not be found in rrs.Single mutants of 1401D43and 1401D88were completely resistant to SM,which were consistent with the corresponding EMS-induced mutant strains.Our studies demonstrated that the mutations in rpsL,mainly at codon 43^rd (AAA→AGA),were responsible for SM-resistance of H.parasuis.These data would help us to further understand the molecular mechanism of H.parasuis resistance, especially of the molecular basis of aminoglycoside drug resistance.

关 键 词：RPSL 链霉素抗性 点突变 副猪嗜血杆菌 甲基磺酸乙酯 

分 类 号：S852.61[农业科学—基础兽医学]