溶藻弧菌噬菌体的分离

从海产品中检出29株溶藻弧菌噬菌体,从中选出4株进行鉴定。据噬菌斑特征可分为两类：一类是透明,一类是不透明。但大小各有差异,直径均在0．5—3.0mm。电镜可见形态也可分成两种类型：即头部呈长轴六角形,尾部细长,结构简单;另一类头部呈等轴六角形,但棱角不甚明显,尾部很短。这些噬菌体的增殖效价均可达108-9pfu／ml,对溶藻弧菌的综合裂解率为72.22％,单株平均裂解率在9.72—44．4％。4株噬苗体的特异性高,原液与612株属外常见菌作交叉裂解试验,均未出现交叉裂解现象;与697株属内弧菌I昊I试,也仅对副溶血弧菌有39.0％的交叉裂解,但将原液稀释至10RTD时,大部分交叉现象消失,表明两菌间有明显的亲缘关系。     

乌鳢源杀鱼爱德华菌的分离鉴定及药敏特性研究

【背景】江苏省扬州市某乌鳢养殖场发生疾病,给养殖户造成了严重的经济损失。【目的】确定病因并筛选出敏感药物,为乌鳢相关疾病的防治提供参考。【方法】从患病乌鳢体内分离致病菌,并从形态、生理生化特征、16S rRNA和gyrB基因序列及特异性PCR检测等方面对分离菌株进行鉴定,同时开展人工感染试验分析其致病性,通过纸片扩散法进行药敏特性分析。【结果】从患病乌鳢体内分离获得优势菌株SHL,经形态特征、理化特性、16SrRNA和gyrB基因序列及特异性PCR检测鉴定为杀鱼爱德华菌。进一步人工感染试验证实其对乌鳢有较强的致病性,LD50为1.6×105 CFU/g,发病症状与自然发病症状相似。药敏试验结果显示,该菌株对青霉素、氯霉素、四环素等28种抗菌药物高度敏感,对红霉素中度敏感,对苯唑西啉、克拉霉素、万古霉素等6种药物耐药。【结论】引起江苏省扬州市某养殖场的乌鳢体表溃烂及死亡的病原菌为杀鱼爱德华菌,这是我国首次从淡水鱼类中检出致病性杀鱼爱德华菌,表明该菌的感染谱在扩大,需引起水产养殖领域的重视,在养殖过程中可根据药敏实验结果选用合适的国标渔药进行防治。     

海水及海产品中溶藻弧菌的分离与鉴定

从上海东海海域随机采集海水样品50份及各市场购买海产品95份,参照国标中副溶血性弧菌的检验方法,对采集样品进行了分离与鉴定。样品增菌后选用TCBS及科玛嘉弧菌显色培养对其进行初步分离,挑取可疑单菌落进行生理生化验证和分子生物学方法鉴定,其中从9份海水、24份海产品中分离出溶藻弧菌菌株,分离率分别为18.0%与25.3%。     

两株对虾幼体弧菌病病原的分离和鉴定

从患弧菌病的凡纳滨对虾(Litopenaeus vannamei)幼体中分离到两株病原菌zouA和zouB, 常规形态和生理生化试验表明均为弧菌属菌种, 弧菌编码鉴定系统分别鉴定为溶藻弧菌(Vibrio alginolyticus)和副溶血弧菌(V. parahaemolyticus)。副溶血弧菌R72H序列检测结果进一步证实菌株zouB为副溶血弧菌。对菌株zouA的16S rRNA基因序列分析表明该菌株与溶藻弧菌、副溶血弧菌等弧菌的相似性均高于98%, 相互间不能区分; HSP60基因序列分析表明该菌株与溶藻弧菌相似性达98%以上, 而与所有其它弧菌的相似性不到92%。结合表型和分子特征的鉴定结果, 菌株zouA和zouB分别被鉴定为溶藻弧菌和副溶血弧菌。     

两株对虾幼体弧菌病病原的分离和鉴定

从患弧菌病的凡纳滨对虾（Litopenaeus vannamei）幼体中分离到两株病原菌zouA和zouB,常规形态和生理生化试验表明均为弧菌属菌种,弧菌编码鉴定系统分别鉴定为溶藻弧菌（Vibrioatginolyticus）和副溶血弧菌（V.parahaemolyticus）。副溶血弧菌R72H序列检测结果进一步证实菌株zouB为副溶血弧菌。对菌株zouA的16S rRNA基因序列分析表明该菌株与溶藻弧菌、副溶血弧菌等弧菌的相似性均高于98％,相互间不能区分;HSP60基因序列分析表明该菌株与溶藻弧菌相似性达98％以上,而与所有其它弧菌的相似性不到92％。结合表型和分子特征的鉴定结果,菌株zouA和zouB分别被鉴定为溶藻弧菌和副溶血弧菌。     

大黄鱼源溶藻弧菌的鉴定及其菌蜕制备

【背景】菌蜕是诱导Phi X174噬菌体裂解基因E(Lysis E)在革兰氏阴性菌中表达后所获得无细胞内容物的细菌空壳。菌蜕生物安全性高,能以类似活菌方式诱导机体产生良好的系统和黏膜免疫应答。【目的】对分离自患溃疡病大黄鱼肝脏中的病原菌株16-3进行种属鉴定,利用温控调节表达系统控制Phi X174噬菌体裂解基因E在该菌株中的表达来制备菌蜕,为防控鱼类溶藻弧菌感染提供有效手段。【方法】采用形态特征观察、生理生化特性测定及16S r RNA基因序列分析等方法对菌株16-3进行鉴定;构建温控裂解质粒p BV220-Lysis E,并将其电转至溶藻弧菌菌株16-3,形成重组溶藻弧菌菌株16-3(p BV220-Lysis E);将不同起始浓度的重组溶藻弧菌培养物同时进行42°C升温诱导,比较其溶菌动力曲线和裂解效率的差异;在最佳条件下制备溶藻弧菌菌株16-3菌蜕,电镜观察其形态与结构,采用倾注平板法测定冻干菌蜕中的活菌数。【结果】综合菌株16-3在形态、生理生化及16S r RNA基因系统发育等方面的特性,确定其为溶藻弧菌;构建了温控裂解质粒p BV220-Lysis E和重组溶藻弧菌菌株16-3(p BV220-Lysis E);溶藻弧菌菌株16-3菌蜕制备的最佳条件是选择起始浓度OD600为0.3的菌液进行诱导,诱导3 h后即可收获菌蜕,其裂解效率为96.9%,但经冻干处理后的菌蜕无活菌残留;电镜观察发现菌株16-3菌蜕保持原细胞的基本形态,但细胞表面有明显的溶菌孔道,且由于细胞内容流失而使细胞表面发生皱缩。【结论】制备出溶藻弧菌菌株16-3菌蜕,为其作为疫苗或疫苗递送载体奠定了基础。     

1株副溶血性弧菌Bh-06的分离、鉴定及其药敏试验

从患病的南美白对虾分离出1株副溶血性弧菌Bh-06,通过革兰染色和Biolog全自动细菌鉴定仪鉴定,并对健康南美白对虾进行攻毒试验和对该菌株进行药物敏感试验。试验结果表明:Bh-06菌株为革兰阴性弧菌,通过细菌自动鉴定仪鉴定为副溶血性弧菌;该菌在培养第5小时进入对数早期,对数期一直延续到25小时;该菌在1.0×106cfu/mL浓度时可引起南美白对虾发病,而且浓度越高,病症越严重;该菌对氧哌嗪青霉素产生高度的敏感性。     

猪源绿色气球菌的分离鉴定与药敏特性研究

目的鉴定研究2006年和2008年本实验室分别从广东清远和广西北流两个猪场发病小猪的膝关节积液中分离到2株呈四联状或成对排列,在山羊血琼脂平板上能形成明显的α-溶血圈的G+球菌。方法采用小白鼠致病性试验、细菌常规生理生化鉴定、药敏试验、耐药基因检测及16S rRNA序列分析等方法对上述分离的2株细菌进行鉴定及药敏特性研究。结果确定这2株G+球菌为正常情况下非致病性绿色气球菌(登录号分别为:GQ161096;GQ161097)。系统发育分析表明,两分离株与绿色气球菌15MS(登录号:EU075039.1)同源性分别高达99.4%和98.7%。药敏试验及耐药基因检测发现,这2株绿色气球菌除了对新霉素、氟哌酸、恩诺沙星、菌必治和环丙沙星等5种药物呈高敏外,对先锋V等13种抗菌素均不敏感。耐药基因检测结果表明,GXBL-1可检出6个耐药基因,GDQY-1则可检出多达10个耐药基因。结论从这2株分离株耐药谱检测结果可以看出,正常菌株同样携带多重耐药基因,存在着通过质粒转座子和整合子将耐药基因转移给敏感菌,导致耐药性传播的可能,应引起足够重视。     

醋酸钙不动杆菌的分离鉴定及溶藻特性

淡水微囊藻水华不仅造成水体动植物缺氧死亡,而且释放藻毒素,影响人类和其它动物的健康。利用液体感染技术,从河南省平顶山市白龟山水库分离一株能够溶解铜绿微囊藻PCC 7806的溶藻菌,命名为溶藻菌5,16S r DNA核苷酸序列测序证实该菌株为醋酸钙不动杆菌。它具有一定的溶藻特异性,只溶解PCC 7806,对FACHB-930和斜生栅藻没有影响,能够促进衣藻和红球藻的生长。最佳溶藻体积比为1∶1。溶藻菌5的菌体和无细胞培养物均具有相同的溶藻效果。显微观察藻细胞被溶解的黄化液显示细菌并未附着在藻细胞周围,也无菌胶膜形成。表明溶藻菌5可能通过释放杀藻物质和与藻竞争营养物质两种机制溶解藻细胞。     

溶藻弧菌铁载体合成及外膜蛋白表达的研究

初步研究了海洋动物病原菌溶藻弧菌的铁摄取机制。溶藻弧菌能够在高浓度铁螯合剂2-2二联吡啶的培养基中存活。在限铁环境中,溶藻弧菌生长受到抑制,补加铁可以消除这种抑制作用。通过铁载体定量检测,发现分离于发病鱼体的溶藻弧菌MVP01产铁载体量大于分离于海水的菌株No·1·1587。互补实验证明溶藻弧菌的铁载体粗提物能够被铁载体合成缺陷的大肠杆菌突变株AN93利用。在铁限制培养环境中,溶藻弧菌合成了约80kD铁调控外膜蛋白。铁摄取系统在溶藻弧菌的生存和致病性方面,都有重要的作用。     

1株虎源致病性肠球菌的分离鉴定及序列分析

从病死老虎肺脏中分离到1株肠球菌,并对该菌做了生理生化鉴定、药敏试验,致病性试验。本菌对多种抗生素高度耐药,对小白鼠有强致病性,其LD50为2.7×109.2cfu。并用PCR方法扩增分离菌株16S rDNA基因,获得1 415 bp片段,该片段核苷酸序列提交GenBank,登陆号为HM346186,将分离株的16S rDNA核苷酸序列与GenBank上其他肠球菌进行同源性分析。结果表明,分离株的16S rDNA核苷酸序列与肠球菌(EU285587)的同源性为100%,因此该分离菌株被鉴定为致病性肠球菌,命名为YN-1株(云南-1株)。     

Isolation of Vibrio parahaemolyticus and Vibrio alginolyticus from Estuarine Areas of Southeastern Alaska

The first reported isolations of halophilic vibrios, including Vibrio parahaemolyticus, from three seafood processing areas in Southeastern Alaska are described.     

Medium for Isolation and Differentiation of Vibrio parahaemolyticus and Vibrio alginolyticus

Trypticase soy agar supplemented with sucrose, sodium chloride, bile salts, and triphenyltetrazolium chloride is an improved plating medium for the isolation of Vibrio parahaemolyticus from samples of seawater, permitting better differentiation of this organism from Vibrio alginolyticus and other bacteria.     

浙江省牛犬猝死症病原菌分离鉴定及其药敏试验

对浙江省 2 0 0 3年 3～ 4月发生的牛、犬猝死综合征 (sudden deathsyndrome ,SDS)的病原体进行了分离和鉴定 ,并检测了分离菌株对不同药物的敏感性。采集 2 6头牛、2 2头猝死犬的心血 ,以及心、肝、脾、肺、肌肉组织和粪便标本 ,并同时进行尸检。采用多种培养基分离培养病原菌。根据菌落特征、革兰染色镜检和ATB半自动细菌鉴定仪检测结果鉴定病原菌的种属。用药敏试条和ATB半自动细菌鉴定仪进行上述菌株对1 0种抗生素的体外药敏试验。从 84 .6 %病牛 (2 2 / 2 6 )、72 .7%病犬 (1 6 / 2 2 )中检出魏氏梭菌 (Clostridiumwelchii) ,但未检出炭疽杆菌 ,表明魏氏梭菌是牛、犬SDS的病原体。病牛真胃、小肠和心冠状沟 ,以及病犬小肠、肝脏出现明显的点状出血 ,这可能与其死亡直接相关。所分离的魏氏梭菌对青霉素和林可霉素耐药 ,但对其它 8种抗生素敏感 ,故青霉素和林可霉素不适合用于魏氏梭菌感染引起家畜SDS的治疗     

Isolation and characterization of Vibrio alginolyticus isolated from diseased kuruma prawn, Penaeus japonicus

K.-K. LEE, S.-R. YU, T.-I. YANG, P.-C. LIU AND F.-R. CHEN. 1996. Outbreaks of serious mortality among cultured kuruma prawns ( Penaeus japonicus ) with white spotted syndrome in the carapace occurred in the summer of 1993 in I-Lan, Taiwan. A swarming bacterium, strain Swy, was isolated from the hepatopancreas of the moribund prawns using tryptic soy agar supplemented with 1% NaCl and/or thiosulphate citrate bile salt sucrose agar. This strain was characterized and identified as Vibrio alginolyticus on the basis of a number of biochemical tests. The Swy strain was virulent to both kuruma prawns ( P. japonicus ) and tiger prawns ( P. monodon ) with LD₅₀ values of 4.43 times 10⁴ and 1.57 times 10⁵ cfu g body weight^-1, respectively.     

Pathogenic analysis of Vibrio alginolyticus infection in a mouse model

Vibrio alginolyticus is a Gram-negative halophilic bacterium and has been recognized as an opportunistic pathogen to both humans and marine animals. So far, most studies have been focused on marine animals and few reports have been aimed at mammals, including human. In this study, we first established a mouse model to understand the pathogenic mechanism of V. alginolyticus infection. After infection via intraperitoneal injection, hematological and liver function indicators were evaluated and serum interleukin (IL)-1β and IL-6 expression were detected by ELISA. Furthermore, we compared the virulence of two V. alginolyticus strains, ATCC17749T and E0666. The results demonstrated that V. alginolyticus infection causes robust lung and liver damage and induces changes in IL-1β, IL-6, hematological, and liver indicators. In addition, the ATCC17749T strain appeared to be more virulent than the E0666 strain. Better understanding of the pathogenic mechanism of V. alginolyticus infection should guide effective prevention and therapy for V. alginolyticus infection.     

A Comparative Study of the Sugar Composition of O-antigenic Lipopolysaccharides Isolated from Vibrio alginolyticus and Vibrio parahaemolyticus

A comparative study of the sugar composition of O-antigenic lipopolysaccharides (LPS) isolated from Vibrio alginolyticus and those from V. parahaemolyticus was carried out. 3-Deoxy-d-mannooctulosonic acid, 2-keto-3-deoxy octonate (KDO), a regular sugar constituent of gram-negative bacterial LPS, was totally absent from LPS of all V. alginolyticus strains examined as it was from those of V. parahaemolyticus. Furthermore, a KDO-like thiobarbituric acid test-positive substance, identical with that of either V. parahaemolyticus 07 or 012, was also found in LPS from three strains, 505–78, 905–78, and 1013–79 (designated tentatively as group I), out of the five strains of V. alginolyticus tested. LPS from the members of group I contained, as component sugars, glucose, galactose, l-glycero-d-manno-heptose, glucosamine, galactosamine, the KDO-like substance, and an unidentified amino sugar P1. Thus, LPS of the members of group I possessed a similar sugar composition which is similar to that of LPS from either V. parahaemolyticus 07 or 012. LPS of strain 1027–79, one of the other two strains (designated tentatively as gorup II), contained as component sugars, glucose, l-glycero-d-mannoheptose, glucosamine, galactosamine, and the other unidentified amino sugar P2, while LPS of strain 53–79, the other member of group II, contained galactose as an additional component. The results indicate that LPS of strain 1027–79 has a sugar composition similar to that of V. parahaemolyticus 09 LPS.     

鳗弧菌胞外金属蛋白酶的化学修饰研究

用DEPC、EDC、DTNB、PMSF等8种化学修饰剂对鳗弧菌胞外金属蛋白酶进行了化学修饰。结果表明化学修饰后酶的活力发生了改变,其中组氨酸、酸性氨基酸、半胱氨酸残基的化学修饰引起酶活性的明显降低,说明组氨酸残基、酸性氨基酸、半胱氨酸残基及其二硫键在维持酶活力中发挥重要作用,是酶活力所必需;而对精氨酸、丝氨酸、ε-氨基等修饰后酶活性影响较小,表明不是酶的活性所必须的基团。     

News & Notes: Virulence of Vibrio alginolyticus Isolated from Diseased Tiger Prawn,Penaeus monodon

Outbreaks of mass mortality among cultured tiger prawns (Penaeus monodon) with white spotted syndrome (WSS) in the carapace occurred in the summer of 1994 in I-Lan, Taiwan. A swarming strain Val was isolated from hemolymph of the moribund prawns with tryptic soy agar (TSA, supplemented with 1% NaCl, Oxoid) and/or thiosulfate citrate bile salt sucrose (TCBS, Difco) agar. This strain was characterized and identified to be Vibrio alginolyticus. The strain was susceptible to antibiotics such as chloramphenicol, ciprofloxacin, doxycycline hydrochloride, nalidixic acid, oxolinic acid, and oxytetracycline while resistant to ampicillin, novobiocin, penicillin G, sulfisoxazole, and sulfonamide. The bacteria and their extracellular products (ECP) were lethal to both tiger prawns (P. monodon) and kuruma prawns (P. japonicus) with LD₅₀ values of 1.13 × 10⁵, 2.46 × 10⁵ CFU/g, and 0.23, 0.63 μg protein/g prawn body weight, respectively.