异育银鲫源杀鲑气单胞菌杀鲑亚种的分离鉴定

【目的】分离鉴定江苏省扬州市养殖场异育银鲫患病病原。【方法】采用常规的理化特性和分子生物学的方法,对从濒死异育银鲫肝脏处分离到的菌株YZ-1进行表型生物学、分子生物学及药敏试验的系统研究。【结果】该菌株16S r RNA基因(序列长度1 446 bp,Gen Bank登录号为JX164202)与其它杀鲑气单胞菌16S r RNA基因一致性在99%-100%之间,构建发育树确定该菌株为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp.salmonicida)。人工回感可导致异育银鲫死亡。药敏试验结果显示:对头孢呋辛、复方新诺明、恩诺沙星等23种抗生素敏感;对阿米卡星、四环素、大观霉素、头孢拉定等11种抗生素中度敏感;对青霉素G、链霉素、庆大霉素、氟苯尼考、万古霉素等10种抗生素耐药。【结论】研究结果证实引起异育银鲫死亡的病原为杀鲑气单胞菌杀鲑亚种。     

Uptake of the fish pathogen, Aeromonas salmonicida, by rainbow trout (Salmo gairdneri L.)

Aeromonas salmonicida was detected in the blood, kidney and spleen of rainbow trout within 2 min of immersion in a suspension containing 10⁴ cells/ml. Uptake into fish was enhanced by culturing the pathogen in low levels of nutrient, i.e., 0.1% (w/v) brain heart infusion (BHI) broth and by the addition of latex particles to the bacterial suspensions. However, there was no apparent difference in the uptake of pathogenic or non-pathogenic isolates. Moreover, the fish did not succumb to clinical signs of disease.     

长江江豚杀鲑气单胞菌的分离鉴定及生物学特性分析

本文通过细菌的分离培养,首次从临床症状表现为溃疡、腐烂的患病江豚表皮分离到一株杀鲑气单胞菌XJ-JT株。通过细菌理化性质鉴定、遗传进化分析、药敏试验、致病性试验对其生物学特性进行分析,结果表明：菌株XJ-JT为革兰氏阴性短杆菌,两端钝圆,无芽孢;细菌分离鉴定的结果显示分离菌株为杀鲑气单胞菌;系统进化分析揭示其基因序列与银鲫源性杀鲑气单胞菌分离株高度同源;20种抗生素的药敏试验结果表明分离菌株对阿米卡星、克拉霉素、克林霉素等8种药物敏感,对头孢噻肟、头孢曲松、卡那霉素中介,对阿莫西林、氨苄西林、四环素等9种药物耐药;人工感染试验,结果显示分离菌对鲫鱼有较强的致病性。     

Partial purification and properties of a haemolytic activity (T-lysin) from Aeromonas salmonicida

Abstract An extracellular haemolytic activity, produced by the fish pathogen Aeromonas salmonicida , against trout erythrocytes (T-lysin) was partially purified by ammonium sulphate precipitation and gel chromatography. Lysis of trout erythrocytes was found to be due to the combined activity of a caseinase and another factor, apparently membrane-associated, which when separated caused incomplete lysis. Incomplete lysis was also observed when caseinase production was suppressed by the incorporation of ammonium sulphate in the growth medium, or in caseinase-negative mutants. Inhibition of caseinase activity by phenyl methyl sulphonyl fluoride also resulted in the loss of full lytic potential from culture supernates containing T-lysin.     

Application of an immunoaffinity-based preconcentration method for mass spectrometric analysis of the O-chain polysaccharide of Aeromonas salmonicida from in vitro- and in vivo-grown cells

In this study, application of magnetic beads (Dynabeads) coated with Aeromonas salmonicida lipopolysaccharide-specific polyclonal antisera to MS-based characterization of bacterial lipopolysaccharides has been evaluated. The results showed that the affinity-based preconcentration strategy resulted in at least a 100-fold increase in the detection of sensitivity, affording direct capillary electrophoresis (CE)-MS analysis of A. salmonicida lipopolysaccharide O-chain polysaccharide from in vitro- cultured cells. Subsequent CE-MS analysis of in vivo -grown cells of A. salmonicida confirmed significant changes in the structure of the lipopolysaccharide O-chain polysaccharide as a result of in vivo cultivation.     

The quantitative relationship of lethality between extracellular protease and extracellular haemolysin of Aeromonas salmonicida in Atlantic salmon (Salmo salar L.)

An additive relationship of lethality between purified protease and haemolysin of the extracellular products (ECP) of Aeromonas salmonicida was demonstrated by i.p. injection in Atlantic salmon (Salmo salar L.). The lethal toxicity of the combinations of protease and haemolysin follow a linear regression line y = -54.54x + 2400. The LD50 of protease and haemolysin when injected separately was 2400 ng/g fish and 44 ng protein/g fish, respectively.     

Characterization of conjugative transposon Tn5251 of Streptococcus pneumoniae

Abstract Tn5251 belongs to the Tn916-Tn1545 family of conjugative transposons (CT) and was found integrated into CT Tn5252 , to form the composite element Tn5253 of Streptococcus pneumoniae . We show that Tn5251 is identical in structure and size to Tn916 . DNA sequence analysis of a 4,419-bp segment containing the tet(M) gene showed that only 73 nucleotides out of 4,419 were different in the the two CT. Essentially all differences (66 / 73) were clustered in a 688-bp segment of tet(M) , which was 90% identical to Tn916 and 100% identical to the tet(M) genes of Tn1545 from S. pneumoniae and pOZ101 from Neisseria gonorrhoeae . DNA sequence analysis of the Tn5251/Tn5252 junction fragments allowed us (i) to determine Tn5251 termini, (ii) to define the 6-bp coupling sequences flanking the CT, and (iii) to infer the structure of the integration site ( attB ) of Tn5251 into Tn5252 . Conjugal transfer of Tn5251 independent from Tn5253 could not be detected, even if we could show excision and formation of Tn5251 circular intermediates at a level of 5.4 copies per 10⁶ chromosomes.     

Prevalence of tet gene and complete genome sequencing of tet gene-encoded plasmid (pAHH01) isolated from Aeromonas species in South Korea

Aims: To investigate the tetracycline resistance related to tet genes in Aeromonas isolates collected from water and diseased fish in South Korea. Methods and Results: A total of 34 Aeromonas strains were examined for their susceptibility to tetracycline using the minimum inhibitory concentration (MIC) assay, and the genetic determinants (tetA to E) were analysed. Among these strains, the tetA and tetE genes were predominant (tetA was found in six strains, and tetE was found in nine strains), and 15 strains were tetracycline‐resistant by the MIC assay. Additionally, the 8979‐bp plasmid that contains the tetE gene was fully sequenced. Conclusions: These data may be important with regard to the spread and persistence of tetracycline resistance genes in the bacterial populations that are present in aquaculture systems. Significance and Impact of the Study: Interestingly, no isolate has previously been shown to harbour three tet genes that are mediated by efflux systems, but the tetA, tetD and tetE genes were all isolated from one strain, which had the highest MIC value for tetracycline among the strains analysed in this study. We also investigated the full‐length plasmid that encoded the tetE gene from a tetracycline‐resistant strain.     

Characterization of tetracycline resistance lactobacilli isolated from swine intestines at western area of Taiwan

To investigate the frequency of tetracycline resistance (Tet-R) lactobacilli in pig intestines, a total of 256 pig colons were analyzed and found to contain typical colonies of Tet-R lactic acid bacteria in every sample, ranging from 3.2 × 10³ to 6.6 × 10⁵ CFU/cm². From these samples, a total of 159 isolates of Tet-R lactobacilli were obtained and identified as belonging to 11 species, including Lactobacillus reuteri, Lactobacillus amylovorus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus ruminis, Lactobacillus kefiri, Lactobacillus fermentum, Lactobacillus sakei, Lactobacillus coryniformis, Lactobacillus parabuchneri and Lactobacillus letivazi. Based on the EFSA (2008) breakpoints, all isolates, after MIC analysis, were qualified as Tet-R, from which the significant high Tet-R MIC₅₀ and MIC₉₀ values indicated an ecological distribution of Tet-R lactobacilli mostly with high resistance potency in pig colons. PCR-detection identified 5 tet genes in these isolates, the most predominant one being tet (W), followed by tet (M), (L), (K), and (Q). Their detection rates were 82.0%, 22.5%, 14.4%, 8.1% and 0.9%, respectively. Noteworthily, isolates of the same species carrying identical tet gene(s) usually had a wide different MIC values. Furthermore, strain-subtyping of these isolates by REP-PCR demonstrated a notable genotypic biodiversity % (average = 62%).     

Sphingobacterium sp. strain PM2-P1-29 harbours a functional tet(X) gene encoding for the degradation of tetracycline

Aims:  The tet (X) gene has previously been found in obligate anaerobic Bacteroides spp., which is curious because tet (X) encodes for a NADP-dependent monooxygenase that requires oxygen to degrade tetracycline. In this study, we characterized a tetracycline resistant, aerobic, Gram-negative Sphingobacterium sp. strain PM2-P1-29 that harbours a tet (X) gene.
Methods and Results:  Sphingobacterium sp. PM2-P1-29 demonstrated the ability to transform tetracycline compared with killed controls. The presence of the tet (X) gene was verified by PCR and nucleotide sequence analysis. Additional nucleotide sequence analysis of regions flanking the tet (X) gene revealed a mobilizable transposon-like element (Tn 6031 ) that shared organizational features and genes with the previously described Bacteroides conjugative transposon CTnDOT. A circular transposition intermediate of the tet (X) region, characteristic of mobilizable transposons, was detected. However, we could not demonstrate the conjugal transfer of the tet (X) gene using three different recipient strains and numerous experimental conditions.
Conclusions:  This study suggests that Sphingobacterium sp. PM2-P1-29 or a related bacterium may be an ancestral source of the tet (X) gene.
Significance and Impact of the Study:  This study demonstrates the importance of environmental bacteria and lateral gene transfer in the dissemination and proliferation of antibiotic resistance among bacteria.     

嗜水气单胞菌J-1株弹性蛋白酶的表达、纯化及特性分析

摘要：【目的】表达、纯化嗜水气单胞菌J-1株弹性蛋白酶,并对弹性蛋白酶的性质进行分析。【方法】以pET-32a为表达载体将弹性蛋白酶基因ahyB转化至大肠杆菌BL21菌株中进行诱导表达,表达重组酶用His TaqNi2+亲和层析柱纯化并用6 mol/L盐酸胍进行复性;利用硫酸铵分级沉淀、阴离子交换层析和分子筛层析对嗜水气单胞菌培养上清液中的弹性蛋白酶进行纯化。将【结果】从嗜水气单胞菌培养上清液中获得的弹性蛋白酶原酶的最适pH 为8.5,而表达重组酶为 10.0;对热的稳定性,原酶高于表达酶。两种形式酶的性     

Oseltamivir-resistant influenza A(H1N1)pdm09 virus in southern Brazil

The neuraminidase (NA) genes of A(H1N1)pdm09 influenza virus isolates from 306 infected patients were analysed. The circulation of oseltamivir-resistant viruses in Brazil has not been reported previously. Clinical samples were collected in the state of Rio Grande do Sul (RS) from 2009-2011 and two NA inhibitor-resistant mutants were identified, one in 2009 (H275Y) and the other in 2011 (S247N). This study revealed a low prevalence of resistant viruses (0.8%) with no spread of the resistant mutants throughout RS.     

Characterization of adjacent E-box and nuclear factor 1-like DNA binding sequence in the human CYP1A2 promoter

To better understand the molecular mechanisms of cytochrome P450 1A2 (CYP1A2) regulation, we have characterized a region of the promoter (+3 to -176) that contains a single E-box and an adjacent nuclear factor 1 (NF1)-like DNA binding site. The E-box was shown to specifically bind nuclear proteins that were recognized by antibodies against upstream stimulatory factor (USF) 1 and 2. Comparison of NF1 binding proteins in HepG2 cells and primary cultures of rat hepatocytes revealed different patterns of DNA-protein complexes, all of which were recognized by a general NF1 antibody. Mutations of the E-box resulted in substantial reduction of promoter activity in either primary hepatocytes or HepG2 cells regardless of the presence in the reporter constructs of other CYP1A2 regulatory elements, such as the hepatic nuclear factor 1 (HNF-1) binding site. In contrast, reporter gene activity of the promoter construct harboring the mutated NF1-like binding site was affected by upstream sequences when transfected into HepG2 cells, but not in primary hepatocytes. We conclude that both USF proteins and different isoforms of NF1 contribute to the constitutive expression of CYP1A2.     

Molecular findings from influenza A(H1N1)pdm09 detected in patients from a Brazilian equatorial region during the pandemic period

After the World Health Organization officially declared the end of the first pandemic of the XXI century in August 2010, the influenza A(H1N1)pdm09 virus has been disseminated in the human population. In spite of its sustained circulation, very little on phylogenetic data or oseltamivir (OST) resistance is available for the virus in equatorial regions of South America. In order to shed more light on this topic, we analysed the haemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09 positive samples collected during the pandemic period in the Pernambuco (PE), a northeastern Brazilian state. Complete HA sequences were compared and amino acid changes were related to clinical outcome. In addition, the H275Y substitution in NA, associated with OST resistance, was investigated by pyrosequencing. Samples from PE were grouped in phylogenetic clades 6 and 7, being clustered together with sequences from South and Southeast Brazil. The D222N/G HA gene mutation, associated with severity, was found in one deceased patient that was pregnant. Additionally, the HA mutation K308E, which appeared in Brazil in 2010 and was only detected worldwide the following year, was identified in samples from hospitalised cases. The resistance marker H275Y was not identified in samples tested. However, broader studies are needed to establish the real frequency of resistance in this Brazilian region.     

Expressed sequence tags analysis of Atlantic halibut (Hippoglossus hippoglossus) liver, kidney and spleen tissues following vaccination against Vibrio anguillarum and Aeromonas salmonicida

To investigate the response of Atlantic halibut to vaccination and pathogen exposure, a cDNA library was constructed from liver, kidney and spleen mRNA collected following vaccination against Vibrio anguillarum and Aeromonas salmonicida. After sequencing 1114 clones 1072 (96.23%) readable sequences were obtained of which 106 sequences are the first reported from the fish. Of these, 182 clones (16.98%) contained cell/organism defence genes including immunoglobulin light chain, MHC class I and II, interferon consensus sequence binding protein, B-cell receptor-associated protein, early B-cell factor, 10 complement components, heat shock protein 70 and 90, antimicrobial peptides hepcidin type 1 and 2, and CC chemokine (macrophage inflammatory protein-1 beta-like chemokine, MIP-1beta). Expression of MIP-1beta-like was elevated in the kidney and spleen at 1, 2, 7 and 14 days post vaccination. Functional genes involved in cellular processes of hematopoietic tissues were also identified. These results indicate that this cDNA library contains many important genes involved in the immune response, making it an important resource for studying the response of Atlantic halibut to vaccination or pathogen exposure.     

紫茎泽兰花蕾发育过程中类Beclin1基因的克隆表达分析

以紫茎泽兰不同发育时期花蕾为实验材料,通过光学显微观察、DNA ladder检测表明紫茎泽兰花蕾发育过程中绒毡层有PCD现象发生,自紫茎泽兰花蕾中克隆长为679 bp的Beclin1 cDNA基因部分片段,同烟草叶片的Beclin1基因序列（AY701316）同源性为98%,通过Northern blotting分析,在紫茎泽兰花蕾发育过程中细胞程序性死亡（PCD）相关Beclin1基因表达在花蕾发育第Ⅱ期和Ⅲ期较为旺盛,且在花蕾发育第Ⅱ期最强烈。本研究首次克隆并证实紫茎泽兰花蕾Beclin1基因与PCD有一定的相关性,为分析紫茎泽兰入侵机理提供新的思路和手段。     

Characterization of electrochemical activity of a strain ISO2‐3 phylogenetically related to Aeromonas sp. isolated from a glucose‐fed microbial fuel cell

The microbial communities associated with electrodes in closed and open circuit microbial fuel cells (MFCs) fed with glucose were analyzed by 16S rRNA approach and compared. The comparison revealed that bacteria affiliated with the Aeromonas sp. within the Gammaproteobacteria constituted the major population in the closed circuit MFC (harvesting electricity) and considered to play important roles in current generation. We, therefore, attempted to isolate the dominant bacteria from the anode biofilm, successfully isolated a Fe (III)‐reducing bacterium phylogenetically related to Aeromonas sp. and designated as strain ISO2‐3. The isolated strain ISO2‐3 could grow and concomitantly produce current (max. 0.24 A/m²) via oxidation of glucose or hydrogen with an electrode serving as the sole electron acceptor. The strain could ferment glucose, but generate less electrical current. Cyclic voltammetry supported the strain ISO2‐3 was electrically active and likely to transfer electrons to the electrode though membrane‐associated compounds (most likely c‐type cytochrome). This mechanism requires intimate contact with the anode surface. Scanning electron microscopy revealed that the strain ISO2‐3 developed multiplayer biofilms on the anode surface and also produced anchor‐like filamentous appendages (most likely pili) that may promote long‐range electron transport across the thick biofilm. Biotechnol. Bioeng. 2009; 104: 901–910. © 2009 Wiley Periodicals, Inc.