鳜源维氏气单胞菌的分离鉴定及毒力基因分析

从发病鳜鱼中分离到1株优势菌(ZQ202010),进行剖检、细菌分离培养、革兰氏染色、生化鉴定、16S rRNA及gyr B基因测序、动物致病性试验、毒力检测和药敏试验.结果显示,分离株ZQ202010为革兰氏阴性小杆菌,具有运动性,该分离菌赖氨酸脱羧酶、氧化酶、VP、吲哚等反应呈阳性;精氨酸水解酶、硫化氢等反应呈阴性...     

黄金鲫致病性维氏气单胞菌的分离鉴定及耐药性分析

为确定吉林省黄金鲫鱼发病死亡的原因,从患病黄金鲫体内分离到致病菌JLJ-1,对该菌进行形态学观察及生理生化特性的鉴定,并对16S rDNA和管家基因gyrB、rpoD、dnaJ序列、毒力基因携带情况及耐药性进行了分析。结果表明,该菌为革兰阴性菌,结合细菌形态学特征及其生理化生化特性与维氏气单胞菌(Aerornonas veronii,A.veronii)基本相同;16S rDNA与gyrB、rpoD、dnaJ序列进化比对分析显示,该病原菌与A.veronii同属一个分支,且同源性较高。将分离菌人工感染黄金鲫,7d后黄金鲫全部死亡,且临床症状与自然病例相似。通过毒力基因检测发现,JLJ-1株可扩增出6种毒力因子:气溶素(aer)、细胞毒性肠毒素(act)、黏附素(Aha)、丝氨酸蛋白酶(ser)、核酸酶(exu)及脂肪酶(lip)。药敏试验显示,该菌对氟苯尼考、头咆哌酮及头炮曲松等药物比较敏感。本研究为黄金鲫感染A.veronii的鉴定及治疗药物的选取提供科学依据。     

3株维氏气单胞菌耐药性及毒力基因的比较

为探究不同地区的维氏气单胞菌（Aeromonas veronii）耐药性及毒力基因是否存在区别,本研究从生长形态、生化试验、药敏试验、人工感染试验、16S rDNA基因测序同源性分析及毒力基因等方面比较了草鱼源菌株GZCY2019、杂交鲟源菌株GZXY2018及斑点叉尾鮰源菌株GZBD2017的差异。结果显示,3株不同源维氏气单胞菌在鲜血平板上均为圆形、表面湿润光滑、边缘整齐半透明的灰白色菌落,菌落周围均有β溶血环;革兰氏染色镜检均为两端钝圆短小阴性杆菌;药敏试验显示,3株菌对丁胺卡那、多西环素等药物均敏感,而对复方新诺明、头孢他啶、多黏菌素B等药物均中度敏感,对其他药物则表现出不同程度的敏感性或者耐药性;人工感染试验显示,菌株GZCY2019与GZBD2017对小鼠累计死亡率为100%,菌株GZXY2018的累计死亡率为80%;16S rDNA同源性分析显示,菌株GZCY2019与菌株GZBD2017同源性为99.9%,二者与菌株GZXY2018同源性均为97.1%;毒力基因检测结果显示,3株菌均携带热不稳定性肠毒素（Alt）、溶血素（hlyA）与鞭毛（Fla）等毒力基因,而对细胞毒性肠毒素（Act）及其热稳定性肠毒素（Ast）毒力基因的携带则表现不同。本研究通过不同源维氏气单胞菌的对比发现,3株菌在生长形态、染色镜检结果、生化结果上基本相似,但耐药性和毒力基因的携带存在较大差异。     

1株鲫源维氏气单胞菌的分离鉴定

为快速分离鉴定鲫鱼出血性败血症致病因子,从病死鲫鱼肝脏分离细菌,经LB固体培养基划线分离,28℃,24 h培养后,见圆润光滑、表面微凸的菌株呈优势生长,挑取单菌落命名为AX002。观察该菌在RYAN培养基28℃,24 h后生长成深绿色有深核菌落,同条件在RS培养基培养,24 h后长成黄色菌落,后逐步由内向外变为绿色菌落,通过全自动细菌鉴定系统,检测AX002对蔗糖等23种指标呈阳性反应,对山梨醇等22种指标呈阴性反应。设计引物扩增16S r DNA和促旋酶亚基(gyr B)基因并测序。测序结果与NCBI中其他菌株序列进行比对,建立系统发育树,确定AX002为维氏气单胞菌。同时该系统显示该菌对阿米卡星、头孢唑啉、复方新诺明、环丙沙星等15种抗生素敏感。该菌对头孢唑啉、氨苄西林和氨苄西林舒巴坦钠三中抗生素药物最低抑菌浓度>16μg/m L,对四环素最低抑菌浓度>8μg/m L,结果判定AX002对头孢唑啉、氨苄西林、氨苄西林舒巴坦钠和四环素具有耐药性。该菌对体长5~15 cm,体重30~50 g鲫鱼腹腔注射,测定半致死浓度(LD50)为2.38×107CFU/m L。本研究有利于进一步研究维氏气单胞菌。     

两株草鱼源维氏气单胞菌菌株的主要表型特征及致病力比较

维氏气单胞菌(A.veronii)是严重危害草鱼的一种病原。本研究从电镜观察、Biolog微生物自动分析仪鉴定、细菌游动能力、胞外产物活性、细胞黏附性、对草鱼致病力和毒力基因等方面,比较草鱼源A.veronii菌株GZ09007和FS12001的表型特征和致病性差异。结果显示,两株细菌菌体均呈短杆状或弧形、两端钝圆,在透射电镜下可见单鞭毛。Biolog微生物自动分析仪鉴定结果显示菌株均为A.veronii,两株细菌均具有游动能力,胞外产物具有溶血活性、溶蛋白活性和脂肪酶活性,但GZ09007的游动能力、溶血活性、蛋白酶活性均显著强于FS12001 (p<0.05)。GZ09007对EPC、CIK细胞黏附性弱,FS12001的细胞黏附性强,两株细菌的细胞黏附性差异显著(p<0.05)。菌株GZ09007具有aerA、act、lip、ser、exu、fla毒力基因,但未检测到ast、alt、gcaT、ahyB等毒力基因;菌株FS12001具有aerA、act、alt、ast、lip、ser、ahyB、exu毒力基因,但未检测到gcaT、fla毒力基因。两株细菌均可以造成草鱼发病死亡,GZ09007对草鱼的LD₅₀为5.6×10~6cfu,而FS12001的LD₅₀则高于1.5×10~8cfu。表明GZ09007为强毒株,FS12001为弱毒株。推测A.veronii胞外产物的溶血活性和溶蛋白活性在其感染和致死草鱼过程中起到了关键作用。本研究结果为进一步探究A.veronii菌株的致病机理提供依据。     

1株草龟维氏气单胞菌的分离鉴定及药敏谱分析

旨在确定河北昌黎县某花鸟鱼店宠物草龟发病死亡的病因,笔者从发病草龟体内分离纯化出1株优势菌CLW-1,通过解剖观察、细菌分离培养、革兰染色镜检、生化鉴定和16S rDNA基因测序对其分析鉴定,并通过药物敏感性测试检测菌株的耐药性,通过斑马鱼接种试验和毒力基因检测其致病性。结果显示,该菌在血琼脂平板形成湿润、表面光滑、呈β溶血的灰白色菌落,在RS琼脂培养基上长出光滑湿润的黄色菌落;革兰染色镜检为阴性短小球杆菌;16S rDNA基因序列同源性分析表明,分离菌株CLW-1与维氏气单胞菌为同一族,相似性均达99%以上;斑马鱼接种试验结果显示,该菌具有较高的致病力,其LD₅₀为1.26×10⁶CFU;毒力基因扩增结果表明,分离菌株CLW-1具有aerA、Exu、Lip和Ser毒力基因;药敏试验结果显示,分离菌株CLW-1对哌拉西林、头孢唑林和恩诺沙星等敏感;对呋喃唑酮、苯唑西林和红霉素等中敏;对麦迪霉素、克林霉素和万古霉素等耐药。通过试验结果确定此分离菌株CLW-1为维氏气单胞菌,携带毒力基因,具有较高致病力,应用对应的抗生素治疗效果较好,为维氏气单胞菌病的防治奠定基础。     

中华草龟源维氏气单胞菌的分离及生物学特性鉴定

为了确定引起中华草龟(Chinemys reevesiis)死亡的病原菌并鉴定其生物学特性,从病死中华草龟病变肝脏组织分离得到1株病原菌,经生化鉴定,其为维氏气单胞菌(Aeromonas veronii),命名为CRQ1。采用斑马鱼(Brachydanio rerio)攻毒试验进行了该分离菌的致病性检验;采用PCR方法检测了该分离菌携带的毒力基因及耐药基因;采用K-B药敏纸片法检测其耐药性。结果显示:CRQ1株对斑马鱼的半数致死量(LD₅₀)为3.57×10⁷cfu/m L,携带aer A、act、aha、Ser、Exu、Lip、Hly、omp A和Lux S 9种毒力基因;分离菌对替米考星、环丙沙星和恩诺沙星等9种药物敏感,对阿米卡星、链霉素和磺胺间甲氧嘧啶等8种药物中度敏感,对新斯的明耐药;该分离菌携带四环素类耐药基因tet(A)、tet(E),氟喹诺酮类耐药基因oqx A、gyr A、gyr B、par C、par E,氨基糖苷类耐药基因aac2、aac(6)-Ib、aad B、aac4,磺胺类耐药基因sul-1,大环内酯类耐药基因er...     

牦牛源维氏气单胞菌的分离鉴定

四川省阿坝藏族羌族自治州某牧场牦牛发生腹泻和肝脏肿大为特征的疾病,本实验的目的是对病料进行细菌的分离鉴定.采集2头病死牛鼻拭子、肛拭子及肝脏样本进行细菌分离,采用16SrRNA基因和gyrB基因的序列测定鉴定细菌种,从6份病死牛样本中分离到6株维氏气单胞菌.分离菌对小鼠有较强的致病性,能导致小鼠肺脏出血和小肠肿胀出血;...     

维氏气单胞菌研究进展

维氏气单胞菌（Aeromonas veronii）渐已成为重要的人-鱼共患致病菌。文章简单介绍了其分类地位及常规和特殊的生物学性质,并进一步综述了该菌的毒力因子、鉴定方法、流行病学和疫苗研究。作为气单胞菌属的成员,维氏气单胞菌具有该属典型的毒力因子,如气溶素、肠毒素、一系列粘附因子、磷脂酶、丝氨酸蛋白酶和核酸酶等。目前,维氏气单胞菌的鉴定主要依靠生理生化方法,也有人应用分子生物学和免疫学方法鉴定。维氏气单胞菌对水产动物有相当高的致死率,患有肝胆疾病的人感染后常常产生严重的后果,需要加强针对该菌所致疾病的诊断。在疫苗研究方面,有学者研究了口服疫苗和DNA疫苗,达到了一定的免疫效果。     

杂交鲟维氏气单胞菌的分离鉴定及其耐药性分析

试验旨在对贵州省某养鱼场发病杂交鲟体内分离的优势菌株进行鉴定、致病力探究及耐药性分析。本试验通过革兰染色、生化鉴定、细菌16S rDNA基因分析、动物回归试验、药敏试验、部分毒力基因检测与耐药基因检测。结果显示:分离菌为革兰阴性短杆菌,细菌16S rDNA扩增测序与比对,得大小为1 477 bp的片段,其片段基因序列与GenBank中的维氏气单胞菌序列相似度达99.00%,药敏试验显示:分离菌对环丙沙星、氟苯尼考、头孢哌酮等药物敏感,对四环素、磺胺异唑、苯唑西林药物出现耐药,耐药基因检测显示:分离菌含有耐磺胺基因(Sul1,sul2)、Ⅰ类整合子(Intl1)3种耐药基因,毒力基因检测显示:分离菌含有气溶素(Aer)和粘附素(Aha)两种毒力基因。综合本试验结果,分离菌为维氏气单胞菌(A.veronii),含有毒力基因,并对环丙沙星、氟苯尼考、头孢哌酮等药物敏感。本试验对贵州地区鲟发病流行的病因做出准确诊断,为该地区细菌性鱼病预防与合理用药提供依据。     

Comparison of Drug Resistance and Virulence Genes of Three Strains of Aeromonas veronii

To find out whether there were differences in drug resistance and virulence genes of Aeromonas veronii (A.veronii) from different regions,this study compared morphology,biochemistry test,drug sensitive test,artificial infection test,16S rDNA gene sequencing and virulence genes homology analysis for grass carp source strain GZCY2019,hybrid sturgeon source strain GZXY2018 and Ictalurus punctatus source strain GZBD2017.The results showed that all the three strains of A.veronii from different sources presented round,moist and smooth gray and translucent colonies with neat edges on the blood plate,and the colonies were surrounded by β hemolytic rings.Gram staining showed that negative Bacillus crassus at both ends.Drug sensitivity test showed that the three strains were all sensitive to butylaminacana,doxycycline,but were all intermediary to compound xinnomin,ceftadime,polymyxin B and other drugs,while were at different degrees of sensitivity or resistance to other drugs.Artificial infection test showed that the cumulative death rates of GZCY2019 and GZBD2017 were both 100%,while the rate of GZXY2018 was 80%.The homology analysis of 16S rDNA showed that the homology between GZCY2019 and GZBD2017 was 99.9%,while the homology between the former two strains and GZXY2018 was 97.1%.Virulence gene test results showed that Alt,hlyA and Fla virulence genes were all carried,while cytotoxic enterotoxin Act and Ast virulence genes were different.In this study,through comparison of multiple experiments with different sources of A.veronii,it was found that the three strains were basically similar in growth morphology,staining microscopy and biochemical results,but there were significant differences in resistance and virulence genes.     

Isolation,Identification and Drug Sensitivity Test of a Strain of Aeromonas veronii from Chinemys reevesii

To determine the cause of death of Chinemys reevesii in a pet shop in Changli county, Hebei province, a dominant strain CLW-1 was isolated and purified from the diseased Chinemys reevesii. It was analyzed and identified through anatomical observation, bacterial isolation and culture, Gram staining and microscopic examination, biochemical identification and 16S rDNA gene sequencing, the drug resistance of the strain was detected through drug sensitivity test, and its pathogenicity was detected through zebrafish inoculation test and virulence gene experiment. The results showed that the bacteria formed moist, smooth surface, gray colony with β hemolysis on blood agar medium, and grew smooth and moist yellow colony on RS agar medium; Gram staining and microscopic examination showed negative coccidiosis; Homology analysis of 16S rDNA gene sequence showed that the isolated strain CLW-1 belongs to the same group with Aeromonas veronii, and the homology is above 99%; Inoculation test results of zebrafish showed that the strain had high pathogenicity, and its LD₅₀ was 1.26×10⁶ CFU; The virulence gene amplification results showed that the isolated strain CLW-1 had virulence genes of aerA, Exu, Lip and Ser; Drug sensitivity test results showed that the isolated strain CLW-1 was sensitive to piperacillin, cefazolin and enrofloxacin, etc; Susceptibility to furazolidone, oxacillin and erythromycin, etc; Resistance to midecamycin, clindamycin and vancomycin, etc. According to the test results, the isolated strain CLW-1 is confirmed to be Aeromonas veronii, which carries virulence genes and has high pathogenicity, it has great therapeutic effect by applying corresponding antibiotics, thus laying a foundation for the prevention and treatment of Aeromonas veronii.     

鲤源嗜水气单胞菌分离鉴定、耐药性及毒力基因检测

为查明贵州某鲤鱼养殖厂鲤鱼死亡的病因,本试验进行了细菌分离培养、菌体形态观察、细菌生理生化鉴定、16S rRNA基因测序分析、药敏试验、耐药基因检测、动物感染试验、毒力基因检测等。结果显示,从死亡鲤鱼病变组织中分离到的细菌呈圆形凸起、表面光滑的灰白色菌落,为革兰氏阴性短杆菌;生化鉴定结果显示,分离菌具有运动性,能发酵葡萄糖产酸产气,精氨酸双水解酶、赖氨酸脱羧酶、M-R和V-P试验阳性;应用细菌16S rRNA基因通用引物进行PCR扩增获得大小为1 421 bp的片段,测序发现,该分离菌与16株嗜水气单胞菌相应序列同源性达75.3%~100.0%;药敏试验结果显示,该分离菌对氟苯尼考、头孢曲松、妥布霉素、氧氟沙星、恩诺沙星5种药物呈现高度敏感,对复方新诺明、磺胺异噁唑、羧苄西林等药物呈现耐药,经耐药基因PCR检测显示,该分离菌携带Sul1、Sul2和Intl1 3种耐药基因,与药敏表型相符;人工感染试验显示,该分离菌有致病力,经毒力基因检测显示,该分离菌携带aer、hly和act 3种毒力基因。上述试验结果表明,本研究分离得到了一株耐磺胺类药物的鲤源嗜水气单胞菌,为该鲤鱼养殖厂的鲤鱼疾病防治与合理用药提供了科学依据。     

Isolation,Identification and Pathogenicity of Aeromonas veronii from Lepidorthosis Pathogenic Bacteria of Tilapia

In order to study the pathogenicity of the pathogenic bacteria in the vertical scale disease of Tilapia,one strain of pathogenic bacteria was isolated from different parts of the diseased Tilapia,named as GXKJDX01.Then the methods of 16S rDNA gene sequence analysis,bacterial morphology observation,physiological and biochemical identification,drug sensitivity test and artificial infection test were used.The results showed that the pathogenic strain GXKJDX01 was Aeromonas veronii,strain GXKJDX01 was not sensitive to 5 medicines,including penicillin G,erythromycin,vancomycin,and had a strong pathogenic effect in yellow-head catfish meat,Tilapia,loach and Procypris merus.11 kinds of drugs,including of enrofloxacin,levofloxacin,ceftriaxone,could be effective to the prevention and control.This study is the first report on the case of the pathogenic bacteria of the lepidorthosis,which was the Aeromonas veronii,and the theoretical basis was provided for the effective prevention and control.     

罗非鱼竖鳞病病原维氏气单胞菌的分离鉴定与致病性研究

为研究罗非鱼竖鳞病病原菌的致病性,本试验从出现竖鳞症状的患病罗非鱼不同部位分离得到1株病原菌,命名为GXKJDX01,采用16S rDNA基因序列分析、细菌的形态学观察、生理生化鉴定、药敏试验和人工感染试验的方法对其研究。结果显示,致病菌株GXKJDX01为维氏气单胞菌（Aeromonas veronii）;该菌株对青霉素G、红霉素、万古霉素等5种药物有高度的耐药性;对罗非鱼、黄颡鱼、泥鳅和禾花鲤均有较强的致病性。使用恩诺沙星、左氧沙星、头孢曲松等11种药物可有效对其进行防制。本试验首次报道维氏气单胞菌作为竖鳞病的案例,为其有效防控提供理论依据。     

斑点叉尾鮰源致病性维氏气单胞菌的分离与生物学特性鉴定

为鉴定一起致斑点叉尾鮰突然发病死亡的病原,本研究从发病斑点叉尾鮰中分离到1株致病菌GZTL2017,通过临床解剖观察、细菌分离培养、革兰氏染色镜检、动物回归试验、生化试验、16S rDNA序列分析、部分毒力基因检测和药敏试验进行鉴定。临床解剖观察结果显示,患病斑点叉尾鮰呈现体表溃疡、鳍根部出血、烂腮、肠管充血等症状;分离菌在培养基中呈现表面湿润凸起、边缘光滑半透明、形态均一的乳白色菌落;革兰氏染色镜检显示,分离菌为单个或成双存在、两端钝圆的短直阴性杆菌;动物回归试验显示,该分离菌有较强的致病性;生化试验结果显示,分离菌具有运动性,且氧化酶、V-P、赖氨酸脱氢酶等反应阳性,精氨酸双水解酶、硫化氢等反应阴性;16S rDNA基因序列系统进化树显示,该菌与维氏气单胞菌聚为一支,同源性均>99%;毒力基因检测结果显示,该菌能检出气溶素基因（Aer）、黏附素基因（Aha）、外膜蛋白基因（OmpA）3种毒力基因;药敏试验结果显示,该菌对氟苯尼考、强力霉素、氧氟沙星等14种药物敏感;对诺氟沙星、新霉素、万古霉素等7种药物中度敏感;对麦迪霉素、苯唑西林、头孢拉定等8种药物耐药,且对氟苯尼考、强力霉素的药物最小抑菌浓度分别为1和2 μg/mL。本试验成功分离到1株维氏气单胞菌,为斑点叉尾鮰维氏气单胞菌病防治提供参考依据。