一株南极大磷虾共附生细菌的分离鉴定及一氧化氮靶向抑制活性筛选

从我国南极磷虾科学探捕项目所采集的南极大磷虾(Euphausia superba)样品中分离纯化到一株共附生细菌,对其进行了细菌多相分类学分析、发酵培养、代谢产物提取及HPLC分析,并对其发酵粗提物进行了针对多种重大疾病药物靶标一氧化氮(N0)抑制活性的靶向筛选.结果表明,该菌株与其进化关系最近的需盐杆菌(Salege...     

两株西施舌内脏团细菌的分离及初步鉴定

自健康西施舌内脏团中分离得到两株细菌,命名为NZT-1、NZT-2,并进行形态学观察、生理生化试验和16S rRNA基因分析.电镜观察结果显示,两株菌均为短杆状,革兰氏阴性;生化反应显示,菌株NZT-1为梅氏弧菌的概率为89.07％,NZT-2为梅氏弧菌的概率为66.21％,两菌株均产纤维素酶、蛋白质酶、右旋糖酐酶,NZT-1产淀粉酶;16S rRNA基因分析结果显示菌株NZT-1、NZT-2与弧菌属的EHP1菌株的核苷酸序列相似度分别为96％、95％,初步鉴定两株菌属弧菌属.生物学特性研究表明两株菌生长的最适pH范围为5.0～10.0,最适生长温度28℃.     

16SrDNA序列分析鉴定一株合浦珠母贝共附生乳酸菌

从合浦珠母贝（Pinctada fucata）粘液中分离纯化得到一株抗菌物质产生菌F9,利用16SrDNA方法鉴定该菌株为棒状乳杆菌（Lactobacillus coryniformis）。通过琼脂扩散法测定了该菌的抑菌谱,发现该菌发酵液上清液在排除有机酸和过氧化氢（H2O2）的影响后仍对金黄色葡萄球菌（Staphyloccocus aureus）等多种食源性致病菌有明显的抑菌作用。对该菌发酵液抑菌物质进行超滤分析及蛋白酶处理,可推断该抗菌物质为分子量在5～10kDa的蛋白类物质。     

一株产吡咯喹啉醌南极海洋细菌的分离鉴定及生物活性

从采集自南极FAO 48.1海域海水样品中分离纯化到一株产吡咯喹啉醌(PQQ)海洋细菌,对其进行了细菌分类学鉴定、摇瓶培养发酵、发酵产物提取及HPLC定量分析,并对纯化的PQQ进行了针对一氧化氮(NO)抑制活性及羟自由基(.OH)清除抗氧化活性评价。结果表明,该菌株与副球菌属(Paracoccus sp.)内菌株的16S rRNA基因序列同源性较高。综合多个分类学指标的分析结果,将其鉴定为属于副球菌属,命名为Paracoccus sp.PQ-08。生物活性评价结果表明,PQQ可显著抑制人脐静脉内皮(ECV-304)模式细胞内诱导型一氧化氮合成酶(iNOS)在mRNA转录水平及蛋白水平的表达量,明显降低胞内的NO释放量。当其作用浓度为100μM时,对iNOS蛋白表达的最高抑制率为67.9%(P<0.05)。此外,实验还证实PQQ具有较强的清除.OH的抗氧化活性。     

一株黄颡鱼源维氏气单胞菌的分离鉴定及其对10种中草药的敏感性研究

从湖北省某养殖场患病黄颡鱼组织中分离到1株细菌性病原,经形态学观察、16S rRNA基因序列测定及系统发育学分析,鉴定为维氏气单胞菌(Aeromonas veronii)。为确定10种中草药(诃子、五倍子、穿心莲、乌梅、连翘、五味子、秦皮、鱼腥草、牡丹皮、青蒿)对黄颡鱼源维氏气单胞菌的抑菌效果,分别制备这10种中草药的水提物,使用不同浓度梯度的水提物进行了抑菌试验,并以盐酸多西环素和氟苯尼考作阳性对照。试验结果表明：诃子、五倍子、穿心莲有较好的抑菌效果,其中诃子抑菌效果最好;乌梅、连翘、五味子、秦皮、鱼腥草、牡丹皮、青蒿的抑菌效果较差,其中五味子的抑菌效果最差。     

光合细菌Ⅰ的分离及其培养条件的优化

从虾塘淤泥中分离出1株能降解亚硝酸盐的光合细菌I,初步鉴定其为球形红假单胞菌。利用Plackett-Barman设计法,对影响光合细菌I生长量的11个因素进行了筛选,确定影响该菌生长的培养基的主要因素为NH_4Cl、NaHCO_3、温度和接种量;采用响应面法对其中的4个显著因子的最佳水平进行研究,结果表明,当NH_4Cl为0.99 g/L、NaHCO_3为1.68 g/L、温度30.18℃、接种量8.90%时,经过5 d培养,菌体密度显著提高,培养液OD_(660)可以从0.972提高至1.343。     

一株海洋过氧化氢酶高产菌的鉴定及产酶条件优化

对来自青岛近海海域底泥的一株产过氧化氢酶菌株YS0810进行形态学观察、16S rDNA序列同源性分析及生理生化特性的鉴定,在250 ml摇瓶中进行发酵产酶条件优化。初步确定该菌属于不动杆菌属Acinetobacter。发酵培养的最佳碳、氮源分别为蔗糖20 g/L和蛋白胨15 g/L,无机盐MgSO4·7H2O、NaCl、KH2PO4最佳浓度分别为0.9、5.0和1.0 g/L;菌株在培养基起始pH=7.0、4%接种量、50 ml装液量和25℃的条件下发酵24 h获得较高的酶产量。在最佳培养条件下酶产量为2469 U/ml,是优化前的5倍。     

一株草鱼源荧光假单胞菌的分离鉴定及其耐药特性分析

为探究临沂市某草鱼养殖场发生病害的原因,从患病的草鱼(Ctenopharyngodon idellus)肝脏和肾脏混合组织中分离得到一株优势致病菌G-13,通过细菌形态学观察、革兰氏染色、生理生化特征、16S rRNA基因测序,以及系统发育分析等完成菌株鉴定,并测定其耐药特性。形态特征显示G-13是一种短杆状的革兰氏阴性菌,在LB固体平板上形成乳白色、圆形、整体呈隆起状、边缘较为整齐、表面光滑湿润的菌落;16S rRNA系统发育进化分析发现G-13与荧光假单胞菌(Pseudomonas fluorescens)聚在一支,序列同源性高达100%;生理生化试验显示葡萄糖、精氨酸双水解酶、鸟氨酸脱羧酶、赖氨酸脱羧酶、氧化酶、无盐和3%NaCl胰胨水等7项指标为阳性,与假单胞菌属特征相近,确定分离株G-13为P.fluorescens。药敏试验结果显示菌株G-13对丁胺卡那、多西环素、四环素、新霉素、环丙沙星、氧氟沙星等13种抗生素敏感,对氨苄西林、头孢拉定、克林霉素、呋喃唑酮等7种抗生素耐药。     

一株病原发光杆菌的分离与鉴定

从三疣梭子蟹育苗场发病且大量死亡的大眼幼体中分离到优势生长的菌株SX1,人工感染试验证明,SX1对健康三疣梭子蟹大眼幼体及Ⅲ期幼蟹有较强的致病性;对菌株SX1进行了形态特征、理化特性等表型生物学特性检验,并进行了菌株SX1的16S rRNA、gyrB和rpoA 3种基因的同源性检索与系统发育学分析。结果显示,菌株SX1具有发光杆菌属的特征,且16S rRNA、gyrB和rpoA 3种基因序列均与发光杆菌具有较高的同源性,在系统发育树中与Photobacteriumganghwense聚为一个分支,自举数据值达100%。综合菌株SX1的形态特征、理化特性及3种基因的同源性检索与系统发育学分析,研究表明菌株SX1与P.ganghwense亲缘关系更近,鉴定SX1为P.ganghwense且为本次引起三疣梭子蟹大眼幼体大量死亡的病原菌。     

Characterization and distribution of Staphylococcus sp. implicated for improvement of fish sauce odor

ABSTRACT:   The two Staphylococcus strains that had been isolated from fish sauce mush (moromi) made from frigate mackerel in Japan and proved to improve fish sauce odor, were examined for their taxonomic positions. The sequence analysis based on 16S rRNA and rpoB showed that the two strains, R4Nu and R5G, had an identical sequence with sequence identities of 99.5% and 99.0% to the above two genes from the closest species of S. nepalensis , respectively. A DNA hybridization test of the two strains showed more than 80% DNA similarity with S. nepalensis , thus confirming the above-mentioned species identification. Polymerase chain reaction primers specific to the strain isolated from fish sauce mush were designed from rpoB and examined for the distribution of this species to various fish sauces made in Asian countries as well as to fish sauce starter (malt) made from soy beans and barley in Toyama Prefecture, Japan. The amplified DNA fragment bearing the S. nepalensis gene was detected in the enriched culture of the malt, although no positive reaction was shown with fish sauce samples. These results suggest that S. nepalensis indebted to improve fish sauce odor was originated from the fish sauce starter malt.     

凡纳滨对虾养殖池塘硝化细菌的分离鉴定及脱氮效果研究

利用平板稀释涂布法和平板划线分离纯化法,自凡纳滨对虾养殖池塘中分离得到5株细菌,经过形态观察、生理生化特性以及16S rDNA序列分析,对分离菌株进行鉴定,同时对其硝化功能和氨氮去除特性进行研究。试验结果显示,分离得到的5株细菌分别为门多萨假单胞菌、嗜胺甲基杆菌、放射根瘤菌、施氏假单胞菌和氧化微杆菌。对其净化效果进行验证可知,门多萨假单胞菌和施氏假单胞菌对氨氮的去除率超过85%,分别为88.83%和91.73%;对亚硝态氮的去除率超过90%,分别为95.08%和97.39%;对硝态氮的去除率超过90%,分别为90.49%和91.22%。以上两种菌株有较高的氨氮和亚硝态氮去除效果,具有潜在的应用价值。     

乌江网箱养殖中患病花鲈的细菌分离与分子鉴定

利用微生物学常用的细菌分离、纯化方法从患病花鲈(Lateolabrax japonicus)分离出9个菌株,编号分别为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ、Ⅶ、Ⅷ、Ⅸ。对9个菌株的16SrRNA基因序列进行PCR扩增,均得到约600bp的片段。将扩增结果进行测序,结果输入到NCBI(http://www.ncbi.nlm.nih.gov/)中,用BLAST工具对序列进行同源性比对分析。结果表明,Ⅰ为杀鲑气单胞菌,同源性98%;Ⅱ为不动菌属,同源性97%;Ⅲ为肺炎克雷伯氏杆菌,同源性98%;Ⅳ为嗜水气单胞菌,同源性99%;Ⅴ为希瓦氏菌,同源性99%;Ⅵ为腐生葡萄球菌,同源性99%;Ⅶ为枯草芽孢杆菌,同源性99%;Ⅷ为沙雷氏菌属,同源性95%;Ⅸ为杀鲑气单胞菌,同源性97%。     

养殖澳洲宝石鱼迟缓爱德华氏菌的分离鉴定及致病基因的检测

从患病澳洲宝石鱼体内分离到一株致病菌(编号Et4),对该菌的生化特性及致病基因进行检测,并进行了药敏和人工感染实验。结果显示:菌株Et4的生化鉴定结果与迟缓爱德华氏菌(Edwardsiella tarda)标准菌株(AY77513)一致;其16S rRNA序列,经同源性比对与迟缓爱德华氏菌核苷酸相似度最高,达99%;根据迟缓爱德华氏菌的致病基因Ⅲ型分泌系统装置蛋白esaV基因序列设计引物,进行PCR扩增,得到708 bp序列,该序列与迟缓爱德华氏菌的esaV基因序列相似性达99.3%,说明菌株Et4具有esaV基因。菌株Et4对环丙沙星等较敏感,对其它药物中度或不敏感,具有较强的致病力(LD50=3.74×104CFU/mL),从病鱼中可以重新分离出此菌。综合形态学、生化特性、16S rDNA序列及致病基因序列鉴定其为迟缓爱德华氏菌。     

Identification and partial characterization of potential probiotic lactic acid bacteria in freshwater Labeo rohita and Cirrhinus mrigala

Carps are the most diversified freshwater fish belonging to family Cyprinidae. Numerous probiotic and pathogenic lactic acid bacteria (LAB) have been characterized from carps. However, the diversity of these ecologically important bacteria is entirely unknown in freshwater fish of Pakistan. The present study aimed to characterize and identify the lactic acid bacteria from two carps viz. Laboe rohita and Cirrhinus mrigala and determine their antagonistic activity. Seventeen bacterial isolates were purified from the gastrointestinal tract and gills of these fish and characterized morphologically. Initially, seven isolates were screened as LAB using agar supplemented with CaCO₃. Subsequently, only two isolates CILB2 and RIL10 were selected as LAB after high‐performance liquid chromatography analysis for lactic acid production. Isolates CILB2 and RIL10 were genetically identified as Enterococcus faecalis and Weissella sp., respectively after 16S rRNA gene sequence analysis. Both strains exhibited significant antagonistic activity against common fish pathogens Streptococcus agalactiae, Staphylococcus aureus and Escherichia coli. Enterococcus faecalis CILB2 and Weissella sp. RIL10 were also found negative for haemolysis and gelatinase activities and were sensitive to ampicillin, amoxicillin, doxycycline, erythromycin, chloramphenicol and co‐trimoxazole antibiotics. The identified LAB strains may further be investigated for their potential probiotic application in fish feed and food preservation techniques.     

一株芽孢杆菌的分离鉴定及在生物絮团对虾养殖中的应用

从对虾养殖池中分离到1株细菌(编号2013042402,简称菌株02),分别用16S rDNA序列比对法和细菌全细胞脂肪酸气相色谱法对该菌进行鉴定.结果显示,菌株02为芽孢杆菌(Bacillus sp.).为探讨该芽孢杆菌在生物絮团对虾养殖中的使用效果,实验分别设置加菌加糖组(菌株02的量为2.0× 104 CFU/ml,蔗糖量为饵料的70％)、加菌组、加糖组(生物絮团组)及空白对照组,研究了菌株02对养殖水质(温度、盐度、溶氧、pH、氨氮及亚硝酸氮)、对虾存活率及水体中主要菌群组成等指标的影响.结果显示,加菌加糖组能显著降低养殖水体中的氨氮和亚硝酸氮浓度,提高对虾存活率.生物絮团对虾养殖系统中添加菌株02,能够改善菌群结构,抑制弧菌生长.研究结果可为生物絮团对虾养殖中定向培养有益微生物提供技术支持.     

First report of Shewanella sp. and Listonella sp. infection in freshwater cultured loach,Misgurnus anguillicaudatus

A novel disease outbreak occurred among cultured loach, Misgurnus anguillicaudatus in China. The diseased fish displayed lethargy, loss of appetite, black discolouration, anasarca and ventral skin hyperaemia. Clinical signs were observed in most of the fish examined, which consisted of discoloured liver, swollen spleen and petechial haemorrhage in the intestine and peritoneal membranes. The causative agent was found to be two species of bacteria by experimental infection, identified as Shewanella sp. and Listonella sp. The result showed that these were likely to be Shewanella putrefaciens and Listonella anguillarum respectively. Bacterial identification consisted of physiological and biochemical tests as well as 16S rRNA sequence analysis. In addition, antibiotic susceptibility testing was performed, revealing that Shewanella putrefaciens and Listonella anguillarum were susceptible to enrofloxacin, ciprofloxacin, levofloxacin, pipemidicacid and norfloxacin. This is the first report of S. putrefaciens and L. anguillarum from cultured loach.     

患病草鱼肠道病原菌的分离鉴定及毒力研究

自环洞庭湖区精养鱼池患病草鱼肠道中分离获得4种病原菌,进行16S rRNA基因和gyrB基因序列测定和在线比对,并对草鱼进行回归感染。随后进行4种病原菌的药敏特征、溶血活性试验,并利用PCR方法检测了6种毒力因子的携带情况。试验结果表明,病原菌分别为温和气单胞菌、异常嗜糖气单胞菌、类志贺邻单胞菌和格氏乳球菌;4种病原菌回归感染草鱼,均能使草鱼患病致死,自死亡的草鱼体内均能分离得到相应感染的病原菌;4种病原菌对多种抗生素具有耐药性,且在脱纤维绵羊血血平板上检测到了温和气单胞菌、异常嗜糖气单胞菌AaB007的溶血活性;PCR结果表明,4种病原菌携带多种毒力因子。研究结果可为环洞庭湖区草鱼精养鱼池健康养殖及病害防治提供科学依据。