养殖环境及贝源溶藻弧菌MLST分型及其毒力基因、耐药性分析

Study on Multi-Locus Sequence Typing,Virulence Genes,and Drug Resistance of Vibrio alginolyticus from Shellfish and Culture Environment

溶藻弧菌(Vibrio alginolyticus)是近年来贝类病害中最常见的细菌性病原之一,对贝类养殖产业的健康发展构成严重威胁。本研究旨在分析不同养殖环境水体和贝类组织中,溶藻弧菌的基因变异、毒力基因、耐药性及其分布规律。对12株溶藻弧菌分离株开展多位点序列分型(multilocus sequence typing,MLST)、毒力因子以及菌株耐药性分析,结果显示,12株溶藻弧菌的序列型(sequence typing,ST)分型互不相同,7株为Pub MLST数据库已经收录的ST型,5株因管家基因的等位基因位点变化而形成新的ST型,贝类养殖环境中的溶藻弧菌具有较高的遗传多样性。12株溶藻弧菌都携带tlh、fur和collagenase三种毒力基因,但均未检测到tdh、trh、toxR和tcpA毒力基因。溶藻弧菌携带毒力因子的种类和数量受地区分布等因素的影响。不同来源的溶藻弧菌均具有多重耐药特征,对青霉素和氨苄西林产生抗性。本研究表明,贝类养殖环境中的溶藻弧菌具有种群复杂、遗传多样性高的特点;不同来源菌株在毒力基因携带和耐药性方面存在较大差异。本研究通过探究不同区域内不同来源的溶藻弧菌遗传变异及耐药性差异,对贝源溶藻弧菌的有效防控提供一定理论参考。

China is the most productive country in the world in terms of shellfish farming,with seawater shellfish occupying a dominant position in China´s shellfish farming industry.In the past two decades,China´s marine shellfish culture has been enriched in terms of species and culture methods.Moreover,its scale has expanded,with annual production remaining above 10 million tons and the total economic output exceeding 220 billion yuan.However,the economic losses caused by epidemic diseases in the shellfish aquaculture industry are also increasing annually,exceeding 10 billion yuan in 2021.These diseases have become some of the primary limiting factors for the healthy development of the shellfish aquaculture industry.Epidemiological surveys in recent years have shown that vibriosis is the most prevalent bacterial disease and the leading cause of mass mortality in shellfish farming.Vibrio alginolyticus is one of the most common Vibrio pathogens in shellfish diseases,posing a grave threat to the healthy development of the shellfish farming industry.However,effective methods for preventing and controlling V.alginolyticus are still lacking.The pathogenicity of V.alginolyticus is frequently closely related to its virulence factors and biological characteristics,and it is unclear how the virulence factors and biological characteristics of V.alginolyticus vary according to different sources and regions.Therefore,in this study,we aimed to gain a deeper understanding of the pathogenicity and scientific control of V.alginolyticus by analyzing the genetic variation and distribution patterns of V.alginolyticus in different aquaculture environments and shellfish tissues.The study was conducted to observe the external morphological characteristics and conduct 16S identification of twelve V.alginolyticus isolates collected from four regions:Qingdao,Weifang,Weihai,and Yantai.These were isolated and purified in TCBS selective medium;MLST typing of the strains by four housekeeping genes;the distribution of thirteen Vibrio virulence genes in V.alginolyticus;and the resistance of V.alginolyticus to 10 common antibiotics.The findings showed that all V.alginolyticus colonies were yellow or yellowish in color,round and transparent in shape with a raised center and smooth edges,moist,and difficult to harvest.16S rRNA sequencing showed a homology of greater than 99%with V.alginolyticus,which was initially verified as V.alginolyticus and was consistent with the initial identification results.The ST typing of the twelve V.algolyticus strains differed from each other.Seven strains(A_(2),A_(3),B_(2),B_(3),C_(3),D_(2),and D_(3))contained ST types already included in the PubMLST database,with ST types 45,87,156,56,125,96,and 57;five strains(A_(1),B_(1),C_(1),C_(2),and D_(1))formed new ST types owing to allelic locus changes in housekeeping genes,and four of the new ST types were isolated from shellfish tissue.The Qingdao isolate(A1)has similar ST types 38,131,134,46,and 56 in the database;the Weihai isolate(C_(1),C_(2))has similar ST types 111,322,and 61 in the database;and the Yantai isolate(D1)has similar ST types 268,275,341,344,351,and 358 in the database.These results suggested that V.alginolyticus in the shellfish culture environment had a high genetic diversity and that V.alginolyticus of shellfish origin might be more easily typed than V.alginolyticus from water sources.The MLST typing phylogenetic tree showed that there were four distinct branches:Group 1,Group 2,Group 3,and Group 4.All V.alginolyticus from shellfish tissues were predominantly observed in Group 1;isolates from Group 2 and Group 3 were mainly from marine environments and had closer evolutionary relationships with V.alginolyticus from the same region of the aquatic environment.The evolutionary relationships between V.alginolyticus from different areas of the aquatic environment and V.alginolyticus from shellfish tissues showed different characteristics.All V.alginolyticus strains carried three virulence genes:tlh,fur,and collagenase;VscB,Ompw,FlaA,and toxS virulence genes were present in most strains;UreB and AspA virulence genes were only present in a few strains;and tdh,trh,toxR,and tcpA virulence genes were not detected in any of the strains.The variety and number of virulence factors carried by V.alginolyticus were influenced by factors such as regional distribution.V.alginolyticus of different origins were characterized by multiple drug resistance interactions,but there were differences in the types of antibiotics to which resistance was developed.All V.alginolyticus species showed high susceptibility to cotrimoxazole and chloramphenicol,while they were resistant to penicillin and ampicillin.The majority of V.alginolyticus developed intermediate or high susceptibility to antibiotics such as butyraminecarbana,gentamicin,erythromycin,norfloxacin,and ciprofloxacin.Among all V.alginolyticus,one strain was resistant to ceftolozoline,five strains were intermediated,and six strains were highly sensitive.In addition,five strains were intermediated,and six were highly sensitive to butyraminecarbana,gentamicin,and erythromycin.Two strains were intermediated,and ten were highly sensitive to norfloxacin.Four strains were intermediated,and eight were highly sensitive to ciprofloxacin.Combining the MLST typing results,drug resistance results,and virulence genes in this study,no significant correlation between the three was found at this time.This study showed that V.alginolyticus in a shellfish culture environment was characterized by complex populations and high genetic diversity.There were large differences in virulence gene carriage and drug resistance among strains from different sources.The study provides a theoretical reference framework for understanding the pathogenicity of V.alginolyticus and assisting in the effective control of V.alginolyticus of shellfish origin by investigating the genetic variation and drug resistance of V.alginolyticus from different sources in various regions.