Reclassification of Aeromonas hydrophila subsp. dhakensis Huys et al. 2002 and Aeromonas aquariorum Martínez-Murcia et al. 2008 as Aeromonas dhakensis sp. nov. comb nov. and emendation of the species Aeromonas hydrophila

Previous studies indicate that Aeromonas aquariorum and Aeromonas hydrophila subsp. dhakensis are the same taxon and suggest that they should be synonymized. Using a polyphasic approach, the phenotypic and phylogenetic relationship of A. aquariorum with the 3 defined A. hydrophila subspecies (i.e. dhakensis, hydrophila, ranae) was investigated. Phylogenetic trees derived from the 16S rRNA, rpoD or gyrB genes and a multilocus phylogenetic analysis (with the concatenated sequences of gyrB, rpoD, recA, dnaJ and gyrA) confirmed that both A. aquariorum and A. hydrophila subsp. dhakensis are a unique taxon, different from the other A. hydrophila subspecies, corroborating the phenotypic and DNA–DNA hybridization (DDH) results. A formal synonymization of A. aquariorum and A. hydrophila subsp. dhakensis and a reclassification of both as Aeromonas dhakensis sp. nov. comb nov. is therefore proposed.     

双氟沙星在人工感染嗜水气单胞菌的异育银鲫体内的药代动力学

为了阐明双氟沙星在健康与处于患病状态下的异育银鲫(Carassius aurutus gibelio)体内的药代动力学特征差异,为双氟沙星的正确合理用药提供参考,本研究通过人工创伤感染的方式采用最佳浓度的嗜水气单胞菌(Aeromonas hydrophila)感染异育银鲫,在此基础上进一步以双氟沙星在健康异育银鲫体内的药代动力学特征为对照,采用反相高效液相色谱法测定双氟沙星在感染嗜水气单胞菌的异育银鲫体内的药代动力学特征。实验结果表明,以鱼体重的20 mg/kg口灌给药后,双氟沙星在人工感染嗜水气单胞菌的异育银鲫与健康异育银鲫体内的总药时曲线均符合一级吸收开放性二室模型,其药动学方程分别为C=6.227e^-0.109t-8.074e^-2.752t+1.847e^-0.006t和C=110.295e^-0.331t+1.533e^-0.01t-111.828e^-0.412t,但与双氟沙星在健康异育银鲫体内的药代动力学参数相比,双氟沙星在人工感染嗜水气单胞菌的异育银鲫体内的吸收、分布、消除速度减慢,其在人工感染嗜水气单胞菌的异育银鲫体内的分 布半衰期、消除半衰期、吸收速率常数、曲线下面积分别增加了4.25 h、36.17 h、2.34/h和74.52 mg·h/L,达峰时间延长了5.75 h,峰浓度降低了61.16%,且未出现重吸收现象。本研究证实嗜水气单胞菌感染能够导致异育银鲫肝肾功能损伤,因而双氟沙星在人工感染嗜水气单胞菌的异育银鲫体内的吸收、分布、代谢和消除均会减慢。     

Analysis of the interaction of Aeromonas caviae, A. hydrophila and A. sobria with mucins

Aeromonas species are known to be involved in human gastrointestinal diseases. These organisms colonize the gastrointestinal tract. Aeromonas hydrophila, A. caviae, and A. sobria have been demonstrated microscopically to adhere to animal cell lines that express mucous receptors, but quantitative studies of adherence to mucosal components such as mucin have not been published to date. Purified bovine submaxillary gland, hog gastric mucin, and fish skin mucin were used as a model to study mucin-binding activity among A. caviae, A. hydrophila, and A. sobria strains. Our findings revealed that binding of radiolabeled and enzyme-conjugated mucins to Aeromonas cells varied depending on the labeling procedure. The highest binding was observed when the three mucin preparations were labeled with horseradish peroxidase. Binding of the various horseradish peroxidase-labeled mucins by A. caviae, A. hydrophila, and A. sobria cells is a common property among Aeromonas species isolated from human infections, diseased fish, and from environmental sources. The proportion of Aeromonas strains which bind the various horseradish peroxidase-labeled mucins was significantly higher for A. hydrophila than for A. caviae and A. sobria. Bacterial cell-surface extracts containing active mucin-binding components recognized the horseradish peroxidase-labeled mucins. The molecular masses of the mucin-binding proteins were estimated by SDS-PAGE and Western blot as follows: A. caviae strain A4812 (95 and 44 kDa); A. hydrophila strain 48748 (97, 45, 33 and 22 kDa); and A. sobria strain 48739 (95 and 43 kDa). Mucin interaction with Aeromonas cells was also studied in terms of growth in mucin-rich media. The culture conditions greatly influence the expression of A. hydrophila mucin-binding activity.     

Presenting a foreign antigen on live attenuated Edwardsiella tarda using twin-arginine translocation signal peptide as a multivalent vaccine

The twin-arginine translocation (Tat) system is a major pathway for transmembrane translocation of fully folded proteins. In this study, a multivalent vaccine to present foreign antigens on live attenuated vaccine Edwardsiella tarda WED using screened Tat signal peptide was constructed. Because the Tat system increases the yields of folded antigens in periplasmic space or extracellular milieu, it is expected to contribute to the production of conformational epitope-derived specific antibodies. E. tarda Tat signal peptides fused with the green fluorescent protein (GFP) was constructed under the control of an in vivo inducible dps promoter. The resulting plasmids were electroporated into WED and the subcellular localizations of GFP were analyzed with Western blotting. Eight signal peptides with optimized GFP translocation efficiency were further fused to a protective antigen glyceraldehyde-3-phosphate dehydrogenase (GapA) from a fish pathogen Aeromonas hydrophila. Signal peptides of DmsA, NapA, and SufI displayed high efficiency for GapA translocation. The relative percent survival (RPS) of turbot was measured with a co-infection of E. tarda and A. hydrophila, and the strain with DmsA signal peptide showed the maximal protection. This study demonstrated a new platform to construct multivalent vaccines using optimized Tat signal peptide in E. tarda.     

Metabolic engineering of Aeromonas hydrophila 4AK4 for production of copolymers of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoate

A mutant termed Aeromonas hydrophila AKLF was constructed by deleting acetic acid pathway related genes pta and ackA in A. hydrophila 4AK4. Accumulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) in A. hydrophila AKLF was increased by 47% from 2.11 to 3.10 g/L associated with a reduction on acetic acid formation compared with A. hydrophila 4AK4 when lauric acid was used as carbon resource. A. hydrophila AKLF harboring pVGAB encoding Vitreoscilla hemoglobin, β-ketothiolase and acetoacetyl-CoA reductase was found to produce 85% more PHBHHx compared to its wild type. Expression of plasmid pD_EcL_Pp harboring genes related to fatty acid metabolism in A. hydrophila AKLF led to 63% more PHBHHx production than A. hydrophila 4AK4. Replacing phaC in A. hydrophila AKLF with a mutant phaC2 from Pseudomonas stutzeri 1317 resulted in enhanced production of copolymers of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates compared to A. hydrophila 4AK4 harboring the mutant phaC2 in the chromosome as control.     

Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli

Potassium tellurite (K₂TeO₃) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.     

嗜水气单胞菌ntrC调控的生理功能及其机制研究

[目的] NtrC是一种与DNA结合的转录调控因子,在激活氮同化基因的转录和维持氮源供应中具有重要作用,本研究拟探究其对嗜水气单胞菌生理功能的影响及其作用机理。[方法] 本研究采用同源重组方法构建了嗜水气单胞菌ATCC 7966 ntrC的缺失株,并以野生株为对照,对缺失株的生理表型进行测定和分析,利用定量蛋白质组学技术比较野生株和ntrC缺失株的蛋白表达差异。[结果] 发现敲除ntrC基因后,嗜水气单胞菌在缺氮、渗透压、重金属离子、氧化以及不同抗生素胁迫下的耐受性都发生显著变化,且这些表型在其补救菌株中均能得到恢复。定量蛋白质组学分析发现,野生株和ntrC缺失株的差异表达蛋白可能参与氨基酸生物合成、抗坏血酸和醛糖酸盐等代谢通路的调控。[结论] 本研究阐明了ntrC在嗜水气单胞菌中的重要作用及其对细菌生物学功能的影响,探讨了ntrC直接或间接调控的蛋白与生理表型之间的联系,研究结果可为未来水产致病菌的防治提供理论支持。     

Production and characterization of homopolymer poly(3-hydroxyvalerate) (PHV) accumulated by wild type and recombinant Aeromonas hydrophila strain 4AK4

Aeromonas hydrophila 4AK4 normally produces copolyesters (PHBHHx) consisting of 3-hydroxybutyrate (C4) and 3-hydroxyhexanoate (C6). Wild type and recombinant A. hydrophila 4AK4 (pSXW02) expressing vgb and fadD genes encoding Vitreoscilla haemoglobin and Escherichia coli acyl-CoA synthase respectively, were found able to produce homopolyester poly(3-hydroxyvalerate) (PHV) (C5) on undecanoic acid as a single carbon source. The recombinant grew to 5.59 g/L cell dry weight (CDW) containing 47.74 wt% PHV in shake flasks when growth was conducted in LB medium and PHV production in undecanoic acid. The cells grew to 47.12 g/L CDW containing 60.08 wt% PHV in a 6 L fermentor study. Physical characterization of PHV produced by recombinant A. hydrophila 4AK4 (pSXW02) in fermentor showed a weight average molecular weight (M_w) of 230,000 Da, a polydispersity of 3.52, a melting temperature of 103 °C and a glass transition temperature of −15.8 °C. The degradation temperature at 5% weight loss of the PHV was around 258 °C.     

黄芩苷对嗜水气单胞菌生物膜形成的影响及体内外的抑菌作用

[目的]探讨中药单体黄芩苷对嗜水气单胞菌在体内外生长及生物膜形成的影响.[方法]体外实验中,利用牛津杯法检测抑菌圈直径,结晶紫法检测生物膜的形成,通过泳动实验检测黄芩苷对嗜水气单胞菌运动性的影响,紫外吸收法检测细胞膜完整性,用透射电镜技术观察黄芩苷对细菌形态的影响.体内实验利用草鱼为对象检测黄芩苷对嗜水气单胞菌增殖的影...     

Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus

In this study we investigated the viable but non-culturable (VBNC) state of Aeromonas hydrophila and its virulence in goldfish. Aeromonas hydrophila cultured in a 0.35% NaCl solution at pH 7.5 and at 25 °C for 50 days showed the VBNC state. In the VBNC state we were unable to detect viable bacteria by the plate count method but we did find 10⁴ cells/ml by the direct viable count microscopical method after staining with fluorescein diacetate and ethidium bromide. The virulence comparison in goldfish showed that bacteria cultured at 25 °C for 1 day in a 0.35% NaCl solution were more virulent than bacteria cultured for 28 days. VBNC bacteria showed lower virulence in goldfish compared to 28-day-cultured bacteria by intraperitoneal injection.The results from the study suggest that A. hydrophila can remain in the aquatic environment for prolonged periods in the VBNC state but those cells are not pathogenic to goldfish.     

广东中山地区气单胞菌环境株的分离、鉴定及系统发生关系

从广东省中山市的池塘水样、底泥、健康鱼、肠道及稻田土样中用Aeromonas的选择培养基分离到10株气单胞菌。通过生理生化测试、16S rDNA序列测定、与气单胞菌典型菌株的16S rDNA序列进行比对和聚类分析,对它们进行了鉴定,并研究了它们之间的系统发生关系。结果显示该地区环境中气单胞菌的优势种除A. hydrophila（HG1组）外, 还有A. caviae（HG4组）、A. jandaei(HG9组)和A. veronii(HG10组),其中后两种是国内新记录。这是国内首次对环境中气单胞菌多样性进行研究。     

Immunomodulatory effect of Withania somnifera supplementation diet in the giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila

The effect of Withania somnifera extract supplementation diets on innate immune response in giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila was investigated. The bacterial clearance efficiency significantly increased in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet against pathogen from weeks 1-4 as compared to the control. The innate immune parameters such as, phenoloxidase activity, superoxide anion level, superoxide dismutase activity, nitrate, and nitrite concentrations were significantly enhanced in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet from weeks 1-4 against pathogen. The total hemocyte counts (THC) significantly increased in prawn fed with 0.1% and 1.0% doses diet from weeks 1-4 against pathogen as compared to the control. These results strongly suggested that administration of W. somnifera through supplementation diet positively enhances the innate immune system and enhanced survival rate in M. rosenbergii against A. hydrophila infection.     

Phytoextracts-Synthesized Silver Nanoparticles Inhibit Bacterial Fish Pathogen Aeromonas hydrophila

Fish disease is a major stumbling block towards sustainable growth of the fisheries sector. Aeromonas hydrophila, which is a major infectious aquatic pathogen is reportedly the causative agent of ulcers, fin-rot, tail-rot, hemorrhagic septicemia in fish, and has reportedly developed resistance against many of the available antibiotics. In this context, the inhibitory function of silver nanoparticles (AgNPs) against A. hydrophila was studied to evaluate its possible application in aquaculture as alternative to antibiotics. AgNPs were synthesized using the leaf extracts of subtropical plants Mangifera indica (Mango), Eucalyptus terticornis (Eucalyptus), Carica papaya (Papaya) and Musa paradisiaca (Banana). The absorbance maxima, size range and shape of the AgNPs as characterized by the UV–Vis spectroscopy, high resolution transmission electron microscopy (HR-TEM), and energy dispersive X-ray spectroscopy (EDX) were, Mangifera—442, 50–65 nm, ovular; Eucalyptus—465, 60–150 nm, oval; Carica—442, 25–40 nm, round, irregular; and Musa—454, 10–50 nm, round, irregular, respectively. Well-diffusion of these AgNPs for their antimicrobial characteristics exhibited that, the papaya leaf extract synthesized AgNPs had maximum antimicrobial activity at 153.6 μg/ml concentrations, and that from the eucalyptus leaves was least effective. As observed, the potency of the nanoparticles enhanced with the decrease in particle size, from 60–150 nm in eucalyptus to 25–40 nm in papaya. Due to its purely natural sourcing, phytosynthesized AgNPs can be applied as alternative to antibiotics and other biocides as a cost-effective and eco-friendly therapeutic agent against A. hydrophila stimulated diseases in aquatic animals.     

Production of N-acyl Homoserine Lactones and Virulence Factors of Waterborne Aeromonas hydrophila

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. The purpose of this study was to investigate the production of N-acyl-homoserine lactone (AHL) signal molecules and some virulence factors, including hemolysins, proteases, extracellular nucleases production and cytotoxicity by waterborne Aeromonas hydrophila. A total of 24 strains isolated from fresh-water or diseased fish were used in the study. The majority A.hydrophila strains produce two AHL molecules (21/24), one is N-butanoyl homoserine lactone (BHL), and the other is N-hexanoyl homoserine lactone (HHL) according to thin-layer chromatography analysis. Among the virulence factors tested, more than 83 % of the isolates produced β haemolysin when inoculated on sheep blood agar, only 50 % of the isolates displayed DNase activity, 75 % of the isolates shown proteolytic activity on skimmed milk plate, and cytotoxic activity was detected in 20 of 24 of the isolates. The strains producing AHLs possessed one or more virulence factors. In conclusion, the production of quorum sensing signal molecules is common among the strains that we examined, and there seems to some relationships between quorum sensing signal production and virulence factors in A. hydrophila.     

嗜水气单胞菌菌落多重PCR方法的建立及应用

[背景]嗜水气单胞菌(Aeromonas hydrophila)对水产动物、畜禽和人类均有致病性.基因表达的溶血素、气溶素和肠毒素是重要毒力因子,在致病性嗜水气单胞菌早期检测及防治中尤为重要.目前采用菌落直接提取DNA用于多重PCR研究的相关报道较少.[目的]基于菌落PCR方法建立针对嗜水气单胞菌溶血性基因、肠毒素基因...     

Comparison of phenotypical and genetic identification of Aeromonas strains isolated from diseased fish

Phenotypicaly identified Aeromonas strains (n=119) recovered mainly from diseased fish were genetically re-identified and the concordance between the results was analysed. Molecular characterization based on the GCAT genus specific gene showed that only 90 (75.6%) strains belonged to the genus Aeromonas. The 16S rDNA-RFLP method identified correctly most of the strains with the exception of a few that belonged to A. bestiarum, A. salmonicida or A. piscicola. Separation of these 3 species was correctly assessed with the rpoD gene sequences, which revealed that 5 strains with the RFLP pattern of A. salmonicida belonged to A. piscicola, as did 1 strain with the pattern of A. bestiarum. Correct phenotypic identification occurred in only 32 (35.5%) of the 90 strains. Only 14 (21.8%) of the 64 phenotypically identified A. hydrophila strains belonged to this species. However, coincident results were obtained in 88% (15/17) of the genetically identified A. salmonicida strains. Phenotypic tests were re-evaluated on the 90 genetically characterized Aeromonas strains and there were contradictions in the species A. sobria for a number of previously published species-specific traits. After genetic identification, the prevailing species were A. sobria, A. salmonicida, A. bestiarum, A. hydrophila, A. piscicola and A. media but we could also identify a new isolate of the recently described species A. tecta. This work emphasizes the need to rely on the 16S rDNA-RFLP method and sequencing of housekeeping genes such as rpoD for the correct identification of Aeromonas strains.     

Diversity of Babesia and Theileria species in symptomatic and asymptomatic dogs in Croatia

Babesiosis, the disease caused by tick-borne hematozoan parasites of the genus Babesia, is particularly common in dogs, and is caused by several “large” species of Babesia, as well as by an increasing number of “small” species of Babesia, some of which appear to be more closely related to members of the genus Theileria. In this work, blood samples were collected from 848 randomly selected, asymptomatic dogs and from 81 symptomatic dogs, microscopically positive for Babesia, and characterised by PCR and sequence analysis of a fragment of the ssrRNA gene. A prevalence of 3.42% (29 of 848) was found in asymptomatic dogs and sequence analysis revealed the presence of Babesia canis canis in 20 dogs (69%), Babesia gibsoni in six dogs (21%), Babesia canis vogeli in two dogs (7%) and Theileria annae in one dog (3%). In the group of symptomatic dogs, which were all positive by PCR, B. canis canis was the predominant species (78 dogs, or 96%), followed by single infections with B. canis vogeli, Babesia caballi and Theileria equi. Our study has confirmed that dogs are infected with a wide range of both large and small piroplasm species and subspecies, including B. caballi and T. equi, two parasites usually found in horses. The detection of the pathogenic species B. canis canis and B. gibsoni in asymptomatic dogs indicates that the relationship between parasite species/subspecies and clinical signs of infection in dogs deserves further investigation. Finally, the identities of the tick vectors transmitting T. annae and B. caballi remain to be elucidated.     

Toxicity of Crude Extracellular Products of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> on Rohu, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.)

In the present study the haemolytic and proteolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila (CAHH14 strain) were studied with respect to temperature and different time of incubation as well as its lethal toxicity on rohu, Labeo rohita. The strain was isolated from Catla catla (showing abdominal dropsy symptom) collected from the pond of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India and was characterized on the basis of biochemical tests. The highest production of haemolysin was achieved when the bacteria was grown at 35°C for 30 h. The proteolytic activity was found to be highest when the bacterium was grown at 30°C for 36 h. The haemolytic and proteolytic toxin produced by Aeromonas hydrophila was found to be lethal to rohu (LD₅₀ 1.7 × 10⁴ cfu/ml). The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min. This indicates that protease activity and haemolytic activity of A. hydrophila ECP was temperature dependant.     

Innate immune response and disease resistance in Carassius auratus by triherbal solvent extracts

This study reports the effect of aqueous, ethanol and methanol triherbal solvent extract from Azadirachta indica, Ocimum sanctum and Curcuma longa on innate immune mechanisms such as phagocytosis activity, respiratory burst activity, alternative complement activity and lysozyme activity and disease resistance in goldfish (Carassius auratus) against Aeromonas hydrophila. Fish were intraperitoneally injected with different doses of 0, 5, 50 and 100 mg kg⁻¹ body weight of each triherbal solvent extracts. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) and innate immune response was assessed on week 1, 2 and 4 by challenging with live A. hydrophila (1 × 10⁷ cells ml⁻¹). All the chosen innate immune parameters were enhanced in the ethanol and methanol triherbal solvent extract treatment after week 2. However, the aqueous triherbal extract was enhanced only after week 4. The ethanol and methanol triherbal solvent extracts administration preceding the challenge with live A. hydrophila decreased the percentage mortality in the experimental groups with the consequence increase in RPS values. The study indicates that all the doses of ethanol or methanol triberbal solvent extracts could be positively influence the immune response and protect the heath status of goldfish against A. hydrophila infection.     

嗜水气单胞菌中转录因子AHA_1581对细菌生理功能调控机制的研究

[目的] LuxR家族转录因子能够抑制或刺激不同功能类型基因的表达,来维持细胞功能的稳定性。嗜水气单胞菌是水产养殖中重要的致病菌之一,目前对该菌中的LuxR家族转录因子功能的研究还较少。[方法] 本研究利用含有sacB标记的自杀载体pRE112和同源重组技术敲除LuxR家族转录因子AHA_1581基因。[结果] 生理表型测定结果发现,ΔAHA_1581的运动与胞外蛋白的酶活增强、生物被膜形成能力降低,且耐受低温、卡那霉素、庆大霉素胁迫,但是对K₂Cr₂O₇更加敏感。进一步对野生型Ah和ΔAHA_1581的定量蛋白质组学分析,共鉴定到2654个蛋白,其中59个蛋白下调表达,142个蛋白上调表达。生物信息学分析表明AHA_1581参与调控双组分调节系统、丙酮酸代谢、碳代谢、TCA循环等细菌重要生理过程,以及细菌耐药基因和毒力因子的差异表达。[结论] 了解AHA_1581基因在调控细菌毒力以及生物过程中所起的重要作用,对预防和控制嗜水气单胞菌引起疾病的发生和传播可能具有重要的科学意义。