香港养殖海鲷弧菌致病菌药物敏感性及耐药质粒研究

从发病海鲷(Sparus sarba)中共分离到51株弧菌(\%Vibrio)\%,经API20E细菌快速鉴定系统及Alsina和Blanch关键生理生化特性分析鉴定为7个种,它们分别是：溶藻胶弧菌(\%V.alginolyticus)(24株),创伤弧菌(V.vulnificus)(12株)和副溶血弧菌(V.parahaemolyticus)(7株),火神弧菌(V.logei)(4株),远洋弧菌Ⅱ菌(V.pelagius Ⅱ)(2株),河弧菌(V.fluvialis)(1株)和地中海弧菌(V.mediterranei)(1株)\%。其中3种优势菌溶藻胶弧菌创伤弧菌和副溶血弧菌证实对海鲷有致病性。另外采用平板稀释法检测了51株菌对16种抗菌素的敏感性。发现所有菌株对ceftriaxone,链霉素,萘啶酮酸和利福霉素敏感,几乎所有菌株对ceftazidime, netilimicin,氯霉素和sulfamethoxazole敏感.大部分菌株对氨苄青霉素 (60.8%),cefuroxime(667%),丁胺卡那霉素(55%),卡那霉素(588%)和三甲氧苄氨嘧啶(765%)等具有较强的耐药性。通过对菌株中所含有的耐药质粒进行分析,发现15株菌株含有1～4个质粒,分子量范围为9～123kb之间,对12株既含有较大分子量质粒又具有耐药性的菌株进行了质粒转化试验,结果其中9株菌的质粒具有转化能力,转化率为１０－１１～１０－９,表明所分离的菌株的抗药性是由于细菌染色体相关突变造成的。     

宁海地区香鱼弧菌病病原菌鉴定

摘要：【目的】香鱼弧菌病对中国沿海地区的香鱼养殖业造成了巨大的危害,然而,病原不明导致了防治上的许多问题。本文鉴定了引起宁海地区香鱼爆发性弧菌病的病原。【方法】采用TCBS平板分离优势菌;采用回归感染试验确认病原菌,采用改进的寇氏法计算LD50;采用形态学观察、生理生化特征测定、细菌特异性引物PCR扩增检测及细菌16S rRNA和金属蛋白酶（MP）基因序列分析鉴定细菌;采用药敏实验测定它对部分抗生素的敏感性。【结果】分离并鉴定优势菌株ayu-H080701为宁海地区香鱼弧菌病的病原菌,它对香鱼的半致死量为1.2×104 CFU。形态学观察和生理生化特征测定表明,ayu-H080701与鳗利斯顿氏菌最为接近。PCR扩增检测表明,细菌16S rRNA 基因通用引物和鳗利斯顿氏菌MP基因特异引物均能扩增到预期大小的特异性条带。ayu-H080701与鳗利斯顿氏菌16S rRNA基因核苷酸序列同源性最高,为99.4%～99.5%,与同属的海弧菌和美人鱼发光杆菌分别为94.3%和91.9%;ayu-H080701与鳗利斯顿氏菌MP氨基酸序列同源性高达97.6%～98.8 %,与其它弧菌科成员则低于75.6 %,系统进化树分析也揭示ayu-H080701与鳗利斯顿氏菌进化相关性最高。【结论】引起宁海地区香鱼弧菌病的菌株ayu-H080701被鉴定为鳗利斯顿氏菌。     

鳗鲡弧菌病病原菌的分离与鉴定

从患弧菌病的鳗鲡的肝脏分离到编号为E-3-11、E-10-1和E-11-1的三株菌。用此菌人工感染鳗鲡能出现相似的病症,并从肝、肾又重新分离到这种菌,证实为该病的病原菌。这些菌株的特性基本一致,经鉴定为鳗弧菌(Vibrio anguillarum)。     

一株茶碱降解菌的分离和鉴定

从制药废水生物处理系统的活性污泥中经富集分离到一株能以茶碱作为唯一碳、氮源生长的茶碱降解菌Tcn3,该菌株可以利用茶碱的最高浓度为3 000 mg/L。当茶碱浓度为1000 mg/L时被彻底降解的时间仅需48 h。Tcn3菌株降解茶碱的最适pH为80。K＋是该菌株降解茶碱的必需元素。采用16S rDNA序列分析法及传统的生理生化特征鉴定法对该菌株进行鉴定,结果表明,Tcn3的16S rDNA的核苷酸序列与善变副球菌(Paracoccus versutus) ATCC 25364的同源性为997%,在细菌系统发育分类学上属于变形菌α亚类,Rhodobacter组：副球菌属,善变副球菌。     

鞘氨醇单胞菌PY3菲降解基因的克隆及序列分析

将菲降解菌鞘氨醇单胞菌(Sphingomanas sp.)PY3的DNA片段与pUC119质粒连接后,转化大肠杆菌JM109,经筛选得到两个质粒,分别命名为pUp1(带有23kb外源DNA片段)和pUp2(带有39kb外源DNA片段)。pUp 1的DNA含有2个ORF。ORF 1由275个氨基酸组成,与恶臭假单胞菌(Pseudomonas putida)F1的甲苯水解酶及菌株Pseudomonas CF600的甲苯水解酶在氨基酸水平上有47%的同源性。ORF 2由327个氨基酸组成,与嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)的邻苯二酚双加氧酶(phe B)及紫红红球菌(Rhodococcus rhodochrous)CTM的邻苯二酚双加氧酶(C23O)在氨基酸水平上分别有57%和44%的同源性。     

编码1,3-丙二醇氧化还原酶基因的克隆和表达

采用PCR法克隆了巴氏梭菌（Clostridium pasteurianum）CpN86菌株编码1,3丙二醇氧化还原酶基因（dhaT基因）;完成了dhaT基因测序、表达载体构建和在大肠杆菌中表达;分离和纯化了dhaT基因表达的重组蛋白。实验结果：（1）PCR法克隆的dhaT基因和肺炎克雷伯氏菌Klebsiella pneumoniae菌株dhaT基因的序列同源性为829%;（2）dhaT基因表达蛋白的酶活为108U/mg;（3）dhaT基因表达的蛋白分子量为43kD;（4）Western blot确定了dhaT基因表达的蛋白和 CpN86菌株天然蛋白有相同的抗原反应。     

钝齿棒杆菌天冬氨酸激酶基因的克隆和序列分析

运用PCR方法,从野生型钝齿棒杆菌株(Corynebacterium crenatum)AS1542及具有AEC抗性的突变株CD945染色体上分别扩增出天冬氨酸激酶(AK)基因(ask),构建了重组质粒。核苷酸序列分析表明,C.crenatum AS1542AK基因与C.crenatum CD945相比,第1199位的碱基由T变为C,引起酶蛋白β亚基第80位氨基酸从亮氨酸变成脯氨酸。该氨基酸的突变在蛋白结构上位于ACT结构域内,该区受赖氨酸调控。C.crenatum AS1542的AK基因的编码区核苷酸序列与C.glutamicum\,C.flavum及B.lactofermentum相比,同源性分别为97.23%、97.55%和97.62%,酶蛋白氨基酸序列的同源性分别为99.76%、99.52%和99.76%。但在AK基因的启动子上游序列部分与其它棒杆菌相比有较大差异。     

分离自鸡眼草和木蓝的根瘤菌分类研究

采用数值分类方法对分离自鸡眼草(Kummerowia)和木蓝(Indigofera)的根瘤菌及已知参比菌株进行聚类分析,发现在83%的相似性水平上形成2个不同与已知菌种的新类群。以SDS全细胞蛋白电泳技术快速聚类分群扩大菌株数,在86%的相似性水平上,分离自鸡眼草的24株菌形成第1类群,分离自木蓝的20株菌形成第2类群。DNA同源性测定结果表明,这2个类群中心菌株SH713和SHL042与13个已知根瘤菌种的DNA同源性均小于61%。因此,分离自鸡眼草和木蓝的根瘤菌分别构成2个独立的根瘤菌新种群。     

斑点叉尾(鱼回)一株致病菌的分离鉴定及系统发育分析

从发生急性流行性传染病的斑点叉尾NFDA5肝、肾分离到一高致病性的菌株（CCF00024）,经人工感染实验证实其为该病的病原菌。对该菌的形态、生理生化及16S rDNA序列分析结果表明,其为非发酵型,严格需氧,革兰氏阴性杆菌,极生多鞭毛,对除麦芽糖和甘露糖以外的多种糖类不能利用产酸,氧化酶阴性,DNA酶、蛋白酶、脲酶、赖氨酸脱羧酶阳性,MR阴性。在以该菌16S rDNA序列（GenBank登录号AY970826）和GenBank及RDP数据库内同源性较高的细菌16S rDNA序列构建的系统发育树中,分离菌CCF00024与嗜麦芽寡养单胞菌（Stenotrophomonas maltophilia）聚在一簇,特别是与S. maltophilia M51的同源性最高,其序列相似性达99.6%,结合形态和生理生化特点将其鉴定为嗜麦芽寡养单胞菌（Stenotrophomonas maltophilia）。     

一株高毒力致病杆菌CB6的鉴定

从北京郊区果园采集的小卷蛾斯氏线虫（Steinernema carpocapsae）肠道内分离到一株具有较强杀虫和抑菌活性的致病杆菌菌株CB6。形态特征及生理生化特征测定结果表明,CB6菌株与致病杆菌属（Xenorhabdus）中的嗜线虫致病杆菌（X. nematophila）种的特征基本一致。测定了该菌株的16S rRNA序列并根据16S rRNA序列构建了系统发育树;在系统发育树中,CB6菌株与嗜线虫致病杆菌其他4个菌株形成一个类群,序列同源性大于99%。但CB6菌株的酪氨酸酶、脂酶（蛋黄）的产生、核糖产酸等生化特征与嗜线虫致病杆菌种内的其他菌株存在一定的差异,且具有更强的杀虫和抑菌活性。因此认为CB6菌株是嗜线虫致病杆菌的一个变种,命名为嗜线虫致病杆菌北京变种（X. nematophila var. pekingensis）。     

Ribotypes and plasmid contents of Vibrio anguillarum strains in relation to serovar.

Eighty-six strains of the 10 major agglutination types of Vibrio anguillarum (serovars O1 to O10) and 6 nontypeable strains of V. anguillarum have been characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by plasmid profile analysis. Forty-four different ribotypes were observed with the restriction enzyme HindIII. Ribotype similarity was compared by using the Dice coefficient (Sd), and three significantly different levels of homogeneity within the V. anguillarum serovars were observed (serovars O1, O3A, O7, and O9, Sds of > 90%; serovars O2B, O4, and O10, Sds of 80 to 90%; serovars O2A, O3B, O5, and O8, Sds between 46 and 70%). None of the ribotype patterns of V. anguillarum strains were observed among 20 other Vibrio strains typed for comparison. By cluster analysis, the V. anguillarum strains were divided into a main cluster containing 83 strains, while all strains of serovar O3B, one strain (each) of serovars O2A, O5, and O8, and a nontypeable strain were separated from this cluster by at least 15% difference in similarity coefficients. Plasmids were demonstrated in only six strains other than serovar O1. In serovar O1, a 67- to 70-kilobase-pair (kb) plasmid molecule was present in 17 of 19 strains tested; of the two remaining strains, one strain harbored two plasmids (45 and 6.5 kb) and one strain had no plasmids.     

Vibrio anguillarum serogroup O3 and V. anguillarum-like serogroup O3 cross-reactive species--comparison and characterization

Forty-five Vibrio anguillarum-like isolates reacting with V. anguillarum serogroup O3 antiserum were examined in 30 characters to clarify their phenotypical properties, while their genotype was examined by ribotyping. The strains were isolated from diseased and dead fish or from environmental sources such as water, sediment, plankton, and faeces and gills of healthy fish. Phenotypically, the similarity of all the strains was more than 90%. However, significant differences between the fish-associated and environmental strains were detected. Biochemically, deviations were found in the Voges-Proskauer test and lysine decarboxylase reaction. Clustering analysis of the ribotypes showed two distinct clusters with a similarity of only 32%. Two strains representing each of these groups were used in a LD50 study, which showed some difference also in the pathogenicity between environmental and fish strains. It is suggested that the environmental strains belong to another species than V. anguillarum, but serologically cross-reacting with the V. anguillarum serogroup O3. The ribotyping as well as biochemical results indicated that the environmental strains possibly belong to Vibrio aestuarianus. The bona fide V. anguillarum serogroup O3 strains proved to be very homogeneous both phenotypically and genotypically, and the similarity of ribotypes was more than 96%. The V. anguillarum-like, serogroup O3-reactive strains from the environment were more heterogeneous in their biochemical behaviour, and showed an approximately 70% similarity in ribotypes.     

Identification and characterization of Epp, the secreted processing protease for the Vibrio anguillarum EmpA metalloprotease

The zinc metalloprotease EmpA is a virulence factor for the fish pathogen Vibrio anguillarum. Previous studies demonstrated that EmpA is secreted as a 46-kDa proenzyme that is activated extracellularly by the removal of an approximately 10-kDa propeptide. We hypothesized that a specific protease is responsible for processing secreted pro-EmpA into mature EmpA. To identify the protease responsible for processing pro-EmpA, a minitransposon mutagenesis (using mini-Tn10Km) clone bank of V. anguillarum was screened for reduced protease activity due to insertions in undescribed genes. One mutant with reduced protease activity was identified. The region containing the mini-Tn10Km was cloned, sequenced, and found to contain epp, an open reading frame encoding a putative protease. Further characterization of epp was done using strain M101, created by single-crossover insertional mutagenesis. Protease activity was absent in M101 cultures even when empA protease activity was induced by salmon gastrointestinal mucus. When the epp mutation was complemented with a wild-type copy of epp (M102), protease activity was restored. Western blot analysis of sterile filtered culture supernatants from wild-type (M93Sm) cells, M101 cells, and M102 cells revealed that only pro-EmpA was present in M101supernatants; both pro-EmpA and mature EmpA were detected in M93Sm and M102 supernatants. When sterile filtered culture supernatants from the empA mutant strain (M99) and M101 were mixed, protease activity was restored. Western blot analysis revealed that pro-EmpA in M101 culture supernatant was processed to mature EmpA only after mixing with M99 culture supernatant. These data show that Epp is the EmpA-processing protease.     

The Chemotactic Response of Vibrio anguillarum to Fish Intestinal Mucus Is Mediated by a Combination of Multiple Mucus Components

Chemotactic motility has previously been shown to be essential for the virulence of Vibrio anguillarum in waterborne infections of fish. To investigate the mechanisms by which chemotaxis may function during infection, mucus was isolated from the intestinal and skin epithelial surfaces of rainbow trout. Chemotaxis assays revealed that V. anguillarum swims towards both types of mucus, with a higher chemotactic response being observed for intestinal mucus. Work was performed to examine the basis, in terms of mucus composition, of this chemotactic response. Intestinal mucus was analyzed by using chromatographic and mass spectrometric techniques, and the compounds identified were tested in a chemotaxis assay to determine the attractants present. A number of mucus-associated components, in particular, amino acids and carbohydrates, acted as chemoattractants for V. anguillarum. Importantly, only upon combination of these attractants into a single mixture were levels of chemotactic activity similar to those of intestinal mucus generated. A comparative analysis of skin mucus revealed its free amino acid and carbohydrate content to be considerably lower than that of the more chemotactically active intestinal mucus. To study whether host specificity exists in relation to vibrio chemotaxis towards mucus, comparisons with a human Vibrio pathogen were made. A cheR mutant of a Vibrio cholerae El Tor strain was constructed, and it was found that V. cholerae and V. anguillarum exhibit a chemotactic response to mucus from several animal sources in addition to that from the human jejunum and fish epithelium, respectively.     

Cloning and nucleotide sequence analysis of a chloramphenicol acetyltransferase gene from Vibrio anguillarum.

The chloramphenicol resistant gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a transferable R plasmid (pJA7324) isolated from the fish pathogen Vibrio anguillarum strain PT24 was cloned into the plasmid vector pUC19. The nucleotide sequence analysis of 1,348 base pair DNA identified an open reading frame encoding a protein of 216 amino acid residues with a calculated molecular mass of 25,471 daltons. The predicted amino acid sequences for this cat gene are 37-69% homologous with other CAT proteins of both Gram-negative and -positive bacteria. Colony hybridization performed with a PvuII-BamHI fragment including this cat gene as a probe, revealed that the same or similar chloramphenicol resistance genes existed among V. anguillarum isolates.     

Comparative Proteomics of two Vibrio anguillarum Serotype O1 Strains with Different Virulence Phenotypes

Vibrio anguillarum is part of the natural flora in the aquatic habitat, but under certain circumstances it can cause terminal hemorrhagic septicemia in marine fish due to the action of virulence-associated proteins. In our study, V. anguillarum MN and 3010 were identified as serotype O1 by AFLP analysis, and the virulence of V. anguillarum MN was shown 50-fold higher than that of the strain 3101 by LD(50) tests with Japanese flounder Paralichthys olivaceus. Nine spots were noted as differentially expressed proteins by comparing the cellular and extracellular protein profiles of V. anguillarum MN and 3101. Mass spectrometry results showed OmpU and PrtV were highly expressed in the virulent strain MN but lowly expressed in the less virulent strain 3101. Expression level confirmed by semiquantitative RT-PCR showed that ompU and prtV were indeed highly expressed in the virulent strain MN. Together with similar amino acid sequences of both OmpU and PrtV in V. anguillarum MN and 3101, our study indicated that the expression level of OmpU and PrtV may be associated with the virulence of V. anguillarum.     

Plasmids mediating iron uptake in Vibrio anguillarum strains isolated from turbot in Spain

Vibrio strains isolated from diseased turbot in an experimental fish farm on the Atlantic coast of northwest Spain were identified as Vibrio anguillarum. The isolates shared many biochemical characteristics with V. anguillarum strains obtained from other sources, and harboured a plasmid species that showed extensive homology with plasmid pJM1, carried by V. anguillarum strain 775 isolated from an epizootic in North America. Restriction endonuclease analysis showed that the two plasmids were very similar albeit not identical. The presence of the plasmid in the turbot isolates was associated with their ability to cause disease in fish. Plasmid-carrying bacteria could also grow under conditions of iron limitation. Two outer membrane proteins, of 86 and 79 kDal, were induced, and a similar siderophore activity to that produced by V. anguillarum 775 was also detected under these conditions. The 86 kDal outer membrane protein cross-reacted immunologically with antiserum raised against the outer membrane protein OM2 produced by strain 775. Nonvirulent plasmidless derivatives were unable to grow under iron-limiting conditions, and were also unable to produce either siderophore activity or the 86 kDal outer membrane protein, suggesting the plasmid-mediated nature of these components.     

环丙沙星与新氟康杀灭鳗弧菌及其影响因素研究

对环丙沙星与新氟康杀灭鳗弧菌及其影响因素进行了研究,结果表明：环丙沙星（Ciproflozacin）和新氟康（Florfenicol）杀灭鳗弧菌的最高稀释度分别为10^-5和10^-4。在20℃～50℃的范围里,环丙沙星与新氟康杀灭鳗弧菌的效果随着温度升高而下降;杀菌最佳pH值分别为3和5;两种药品杀灭鳗弧菌的效果均随着可溶性淀粉浓度的增加而减弱。综合各最佳因素,环丙沙星在20℃和pH3时,杀灭鳗弧菌效果提高了19．13％;新氟康在20℃和pH5时,杀灭鳗弧菌效果提高了59．47％。     

A medium for presumptive identification of Vibrio anguillarum.

A medium (VAM) for differentiation of Vibrio anguillarum is described. The presence of bile salts, the high pH, and the high NaCl concentration select mainly for Vibrio species. The high salinity and the ampicillin select for a fraction of Vibrio species, and sorbitol fermentation differentiates among those vibrios still able to grow. One hundred ninety-seven of 227 strains of V. anguillarum were identified with this medium. Only 3 of 66 strains of Vibrio that were not V. anguillarum or V. anguillarum-like were recognized with this medium, and any of 7 non-Vibrio strains related to fish diseases or Escherichia coli grew on the medium. It is our contention that the medium described here constitutes an efficient instrument for presumptive detection of V. anguillarum in pathological and environmental samples.