湛江地区患病对虾池塘中副溶血性弧菌的MLST分型和新型耐药毒株的分离鉴定  

MLST typing of Vibrio parahaemolyticus from diseased shrimp ponds in Zhanjiang area and isolation and identification of novel drug-resistant strains

作　　者：吴俊敏 王艺 郝婧薇 王青瑶 张晋 傅松哲 刘鹰 WU Junmin;WANG Yi;HAO Jingwei;WANG Qingyao;ZHANG Jin;FU Songzhe;LIU Ying(Key Laboratory of Environment Controlled Aquaculture(Dalian Ocean University),Ministry of Education,Dalian 116023,China;College of Marine Science and Environment,Dalian Ocean University,Dalian 116023,China;College of Fisheries and Life Science,Dalian Ocean University,Dalian 116023,China;College of Biosystems Engineering and Food Science,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]设施渔业教育部重点实验室(大连海洋大学),辽宁大连116023 [2]大连海洋大学海洋科技与环境学院,辽宁大连116023 [3]大连海洋大学水产与生命学院,辽宁大连116023 [4]浙江大学生物系统工程与食品科学学院,浙江杭州310058

出　　处：《大连海洋大学学报》2023年第1期59-67,共9页Journal of Dalian Ocean University

基　　金：国家重点研发计划“蓝色粮仓科技创新”项目(2019YFD0900503);国家自然科学基金(81903372);辽宁省教育厅科研项目(QL202005)。

摘　　要：为研究湛江地区患病对虾养殖池塘中副溶血性弧菌(Vibrio parahaemolyticus)的遗传多样性,选择副溶血性弧菌的7个管家基因对分离菌株进行MLST分型,采用PCR法检测tdh、trh、tlh、mam7、vcrD1、vcrD2和pirAB毒力基因的携带情况,用纸片扩散法对所有菌株进行药敏试验,选择其中1株多重耐药菌株进行全基因组测序,并通过IslandPath-DIOMB分析其耐药基因岛来源。结果表明:从患病的对虾池塘水样中共分离出58株副溶血性弧菌,通过MLST分型共分为34个ST型;58株菌株均携带毒力基因tlh、mam7和vcrD1,未发现tdh、trh和vcrD2基因存在,其中,8株菌株携带引起急性肝胰腺坏死病(AHPND)的毒力基因pirAB;药敏试验显示,58株菌株对青霉素耐药率均为100%,对氨苄西林、克拉霉素和庆大霉素的耐药率较高,其中ST1740型的4株菌株均为多重耐药,从中选择ZJ20-3/2020菌株进行全基因组测序,发现该菌株含有8个耐药基因,分别为blaCARB-41、aadA16、aph(6)-Id、aph(3″)-Ib、tet(A)、sul1、dfrA27和ARR-3,并对青霉素、卡那霉素、四环素和利福平耐药;用IslandPath-DIOMB分析发现,ZJ20-3/2020菌株存在4个基因岛。研究表明,湛江地区患病对虾养殖池塘中的副溶血性弧菌具有丰富的遗传多样性,发现了一个携带pirAB的新ST型(ST1740)菌株,其携带4种来自希瓦氏菌的基因岛且为多重耐药菌株,本研究首次在副溶血性弧菌中发现来自希瓦氏菌属的基因水平转移,这些结果为防治急性肝胰腺坏死病提供了科学依据。In order to evaluate the genetic diversity of Vibrio parahaemolyticus in shrimp ponds in Zhanjiang area,seven housekeeping genes dnaE,gyrB,recA,dtdS,pntA,pyrC and tnaA of pathogenic V.parahaemolyticus were selected in the experiment,and the isolated strains were subjected to MLST typing for genetic evolution analysis by MEGA 6.0 software.Also,the virulence genes of tdh,trh,tlh,mam7,vcrD1,vcrD2 and pirAB were detected by a PCR method.The drug susceptibility test was carried out on all strains by disc diffusion method,and one multi-drug resistant strain was selected using the Illumina Hiseq 2500 platform.Subsequently,the spliced sequences were detected by IslandPath-DIOMB software to analyze the origin of drug resistance gene islands.The 58 strains of V.parahaemolyticus were divided into 34 ST types by MLST,all of which contained virulence genes tlh,mam7 and vcrD1,without tdh,trh and vcrD2,8 strains containing the virulence gene pirAB causing AHPND.It was found that the 58 strains were 100%resistant to penicillin and high resistance rates to ampicillin,clarithromycin and gentamicin.Four strains of ST1740 were found to be multi-drug resistant.One of the 4 ST1740 strains(ZJ20-3/2020)was selected for whole genome sequencing,and contained 8 drug resistance genes,namely blaCARB-41,aadA16,aph(6)-Id,aph(3″)-Ib,tet(A),sul1,dfrA27 and ARR-3,and were resistant to penicillin,kanamycin,tetracycline and rifampicin.Four gene islands were found in ST1740 strain by IslandPath-DIOMB software,with 99%DNA sequence similarity with the corresponding sequences in Shewanella NCTC12093,indicating that the V.parahaemolyticus in the shrimp culture area in Zhanjiang area had rich genetic diversity.MLST typing identified 2 new pirA-containing genotypes,and subsequent genomic analysis identified a novel drug-resistant strain.A new ST strain(ST1740)carrying pirAB was identified,which carried 4 gene islands from Shiwanella and were multidrug-resistant strains.The horizontal gene transfer from Shewanella in V.parahaemolyticus was the first repo

关 键 词：副溶血性弧菌 凡纳滨对虾 药敏试验 多位点序列分型 

分 类 号：S917.1[农业科学—水产科学] Q93.3[生物学—微生物学]