Isolation and Characterization of Potentially Human Pathogenic,Cytotoxin‐producing Aeromonas Strains from Retailed Seafood in Berlin,Germany

The presence of potentially human pathogenic strains of Aeromonas was investigated in 84 samples of seafood which were purchased from retail traders in Berlin, Germany in spring 2000. A total of 134 Aeromonas strains were isolated on selective [GSP agar and Aeromonas (Ryan) agar] and unselective (standard count agar and enterohaemolysin agar) media from 27 (32.1%) of the samples and were classified as Aeromonas hydrophila (67.9%), A. caviae (26.1%) and A. sobria (6.0%) by biotyping. Thirteen (48.1%) of the 27 positive samples contained more than one species of Aeromonas. Production of haemolysins on enterohaemolysin agar was found with 132 (98.5%) of the strains at 28°C and with 130 strains (97.0%) at 37°C growth temperature. Vero cytotoxins were produced by 99 (73.9%) of the strains when grown at 28°C but only by 24 of the strains (17.9%) at 37°C. The latter strains were identified as A. hydrophila (n = 22) and A. sobria (n = 2) which came from 17 (20.2%) samples of raw seafood and from ready‐to‐eat salted herring ‘Matjes’ products. Cytotoxin‐encoding genes for aerolysin (aer) and haemolysin A (hlyA) were investigated by PCR. Aer and hlyA genes were detected in both, strains which produced toxins only at 28°C and strains which produced toxins at 37°C. Our data indicate that raw seafood and ready‐to‐eat fish products can harbour potential human pathogenic, cytotoxin producing Aeromonas strains.     

Biochemical and toxigenic characteristics of Aeromonas spp. isolated from diseased mammals, moribund and healthy fish

In this study we describe biochemical, toxigenic and surface characteristics of 33 motile Aeromonas isolated from diseased mammals, 3 from moribund marine mammals, 24 from healthy fish and 4 from moribund fish. Aeromonas hydrophila, A. caviae and A. sobria were isolated from both mammals and fish but at a different incidence. Aeromonas hydrophila was the predominant species isolated from clinical specimens; it was isolated from pneumonia, wound infections, septicemia and abortion in horses, cattle and pigs. Aeromonas sobria was isolated from one mammal and 11 healthy fish. Aeromonas caviae was isolated in 2 cases from healthy fish and in 9 cases from diseased mammals. Variations in some biochemical tests including sorbitol, amylase and citrate, were observed between isolates from different sources. However, these differences did not allow the differentiation of isolates from diseased mammals and healthy fish. The majority of A. hydrophila isolates produced different extracellular products; A. sobria isolates produced less exotoxin. With A. caviae isolates no hemolysin, protease, enterotoxin or elastase were detected. There was no quantitative difference in hemolysin, protease, enterotoxin or elastase production between isolates from mammals and fish. It is suggested that A. hydrophila could be a potential pathogen for domestic animals, and fish may represent a potential reservoir of infection.     

Isolation and haemolytic activity of Aeromonas species from domestic dogs and cats.

Rectal swabs from 120 domestic dogs and 15 domestic cats were examined for Aeromonas species using alkaline peptone water (pH 8.6) as the enrichment medium and blood agar containing 15 mg/l ampicillin as the plating medium. Aeromonads were isolated from 13 (10.8%) dogs and from 1 (6.7%) cat. Of the 14 aeromonads isolated in the present study only 9 were available for speciation and testing in the haemolysin assay. Of these 5 were A. sobria (including one from a cat), 2 were A. hydrophila and 2 were A. caviae. Six were positive in the haemolysin assay; 4 A. sobria (one from a cat) and 2 A. hydrophila. The presence of haemolysin producing-Aeromonas species in the faeces of domestic dogs and cats may pose a public health problem for humans who come into contact with such animals.     

嗜温气单胞菌的鉴定及其外膜蛋白型研究

从病鳖、鳗鱼体内分离到2株嗜温气单胞菌(TAh-3、EAs-1),经形态学观察、生理生化特性等鉴定,确定TAh-3为嗜水气单胞菌,EAs-1为温和气单胞菌。动物感染试验结果表明,TAh-3株为致病性嗜水气单胞菌,对鲫鱼的致死率100%,EAs-1株为非致病性温和气单胞菌。SDS-PAGE电泳结果显示,TAh-3株外膜蛋白(OMPs)型由7条蛋白带组成,其中1条主带大小为40.0kDa;EAs-1株OMPs型由8条蛋白带组成,其中2条主带大小为38.0kDa、45.0kDa。     

两株气单胞菌的分离与鉴定

从福建省某牛蛙养殖场病死牛蛙和某鸭场病死鸭体内分离到两株细菌，检验结果表明，两株分离菌均为有运动力的革兰氏阴性杆菌。根据其临床症状、细菌培养特性、生化特性、动物回归试验以及溶血试验等一系列试验，研究证明牛蛙感染的病原为嗜水气单胞菌，鸭感染的病原为温和气单胞菌。     

Isolation and identification of motile Aeromonas species from chicken.

In this study a total of 140 broiler carcasses and carcass parts purchased at different supermarkets in Ankara including 50 whole carcass, 30 wing, 30 leg and 30 breast samples were analysed for the presence of motile Aeromonas species. According to analysis findings, motile Aeromonas spp. were isolated from 116 (82.9%) of total 140 samples. The distribution of the isolates were 94%, 86.6%, 80%, 63.3% in broiler carcass, wing, leg and breast samples, respectively. Aeromonas hydrophila was isolated the most prevalent species with 56% the range followed by Aeromonas sobria with 29.3% and Aeromonas caviae with 14.7% from all of the carcass and carcass part samples, respectively. Consequently, it was supposed that, examined broiler carcass and carcass parts have been contaminated to important level with motile Aeromonas species and it has been risk for public health.     

嗜水气单胞菌选择培养基鉴别效果的比较

本试验比较了嗜水气单胞菌（Ａｅｒｏｍｏｎａｓｈｙｄｒｏｐｈｉｌａ,Ａｈ）三种选择培养基ＡＨＭ、ＡＰＭ和ＲＳ培养基的鉴别效果,并用于１２１株细菌检测,Ａｈ的阳性检出率分别是５４．７６％、８１．３３％和８３．５４％。经过交叉比较分析,确定ＡＨＭ和ＲＳ可作为适用于生产实践的Ａｈ的选择培养基。     

嗜水气单胞菌Ⅲ型分泌系统AopN蛋白原核表达及鉴定

根据GenBank上登陆的嗜水气单胞菌AH-1株Ⅲ型分泌系统aopN基因序列,设计1对特异性引物,以J-1株的基因组为模板,PCR扩增得到aopN基因,连入pET-32a(+),转化至大肠杆菌表达,同时PCR检测aopN基因在66株嗜水气单胞菌中的分布情况。aopN基因测序分析发现,片段长度为874 bp,与嗜水气单胞菌AH-1、SSU、AH-3的同源性分别为97%、81%、82%,与杀鲑气单胞菌杀鲑亚种A449的同源性为81%,与温和气单胞菌的同源性为82%。PCR检测结果表明,57株能检测到aopN基因的目的片段。SDS-PAGE分析表明重组蛋白分子量为54.5ku,用兔抗J-1株的全菌抗血清进行Western blot分析表明,该蛋白具有较好的免疫反应性。由于多数菌株都含有aopN基因,提示AopN可能是嗜水气单胞菌的共同保护性抗原。     

温和气单胞菌RC-07-KA株溶血素基因克隆、表达及活性检测

为研究温和气单胞菌(A.sobria)溶血素基因的特性,本研究根据GenBank登录的气单胞菌属溶血素基因序列设计一对引物,经PCR扩增得到大小约1.5kb的A.sobria RC-07-KA株溶血基因片段,将该片段克隆到pGEM-T载体,测序结果显示:溶血素基因片段大小为1 467 bp,编码487个氨基酸残基,遗传进化分析结果表明该基因与气单胞菌属中的A.hydrophila菌株Sb (AY611033)、NLEPA-1607 (AF410466)、AEF (HM853019),A.sobria菌株357 (AY157998)、人源分离株(EF620533)和A.salmonicida菌株17-2 (X65048)的溶血素基因亲缘关系较近,同源性大于95％,而与其他菌株的同源性较低.通过构建溶血素重组表达质粒pET-HIy,诱导表达并通过western blot鉴定表达蛋白,结果显示重组菌能高效表达重组溶血素,而且纯化的重组溶血素具有溶解鲤鱼红细胞的活性.为该菌进一步深入研究奠定了基础.     

鱼源温和气单胞菌对氨基糖苷类和四环素类抗生素的耐药性分析

为探究温和气单胞菌对氨基糖苷类和四环素类抗生素的耐药性,试验采用PCR法检测10株来源不同的鱼源温和气单胞菌对氨基糖苷类抗生素的4种耐药基因（aph（3'）-Ⅱa、ant（3″）-Ⅰa、aac（6'）-Ⅰb、aac（3）-Ⅱa）及四环素类抗生素的3种耐药基因（tetA、tetC、tetM）的表达情况,并利用K-B纸片扩散法对6种抗生素进行耐药表型分析。结果显示,10株温和气单胞菌对氨基糖苷类耐药基因aph（3'）-Ⅱa、ant（3″）-Ⅰa、aac（6'）-Ⅰb的检出率分别为20%、30%、20%,未检测出aac（3）-Ⅱa基因;对四环素类的耐药基因tetA、tetC、tetM的检出率分别为70%、20%、60%。K-B纸片扩散法结果显示,10株菌对四环素耐药率最高,对链霉素敏感,对庆大霉素、卡那霉素、多西环素、米诺环素高度敏感。结果表明,本次分离的温和气单胞菌对氨基糖苷类和四环素类抗生素具有一定的耐药性,为深入了解温和气单胞菌的耐药机制提供参考。     

Pseudomonas aeruginosa in raw and pasteurized milk

Eighteen strains of Pseudomonas aeruginosa, i.e. 8.87%, were isolated during a year from 203 samples of raw milk. Two Pseudomonas aeruginosa strains, i.e. 4%, were isolated from 50 samples of pasteurized milk. The strains were isolated using propagation techniques in meat-peptone broth with malachite green and on selective media--on centrimide agar (CEM) and on Pseudomonas F agar. All the isolated strains produced protease, whereas lipase was produced by only five strains. The strains were devitalized when exposed to pasteurization temperatures (72 degrees C) for 20 seconds. At cold store temperatures (4 degrees C), Pseudomonas aeruginosa strain cells propagated on average by two orders, inhibitory effects of low temperatures were recorded only with one strain. Inhibitory effects of milk cultures (cream, yogurt) on Pseudomonas aeruginosa were observed; their effects were more clear-cut at the temperature of 4 degrees C. The strains were markedly susceptible to gentamycin.     

Experimental handling stress as infection-facilitating factor for the goldfish ulcerative disease

Experimental handling stress (EHS) was applied to clinically asymptomatic farmed goldfish (Carassius auratus L.). EHS affected the gills and skin integrity of the fish and was accompanied by increased levels of plasma glucose, cortisol and interleukin-10 (IL-10). EHS application was followed by highly significant enhancement of the rate of infection with a virulent Aeromonas salmonicida isolate. Cumulative ulceration at the initial phase of the ensuing goldfish ulcerative disease (GUD) evidenced a facilitating role of EHS in the onset of GUD. Host susceptibility to the pathogen increased from 40% in unstressed fish to 90% in the stressed fish. A. salmonicida could be reisolated from the early-stage skin lesions only, whereas opportunistic strains, other than A. salmonicida (A. sobria and A. hydrophila), were recovered from progressive-stage ulcers. The implication of these findings in fish aquaculture is discussed.     

五倍子对鳗鲡致病性气单胞菌的体外抗菌后效应

采用试管液体二倍稀释法测定中药五倍子对鳗鲡养殖中致病性气单胞菌(威隆气单胞菌K17、肠棕气单胞菌G03、豚鼠气单胞菌R37和嗜水气单胞菌G06)的最小抑菌浓度(MIC).然后再采用菌落计数法分别测定五倍子在0.25、0.5、1、2、4倍于各菌MIC时对鳗鲡4株致病性气单胞菌K17、G03、R37、G06的体外抗菌后效应(PAE).结果显示:五倍子在0.25、0.5、1、2、4倍MIC值时对鳗鲡4株致病性气单胞菌均具有一定的PAE,且PAE与药液浓度在一定范围内(0.25～4倍MIC)呈剂量依赖性,当药液浓度达4倍MIC时,PAE明显延长(P＜0.05);五倍子对各致病菌株K17、G03、R37、G06的PAE值在1倍MIC浓度以上差异显著(P＜0.05),在4倍MIC时对于致病菌株K17和R37的PAE之间差异较小.结论:试验结果提示,在养殖鳗鲡病害防治中设计投药方案,选用中药五倍子治疗由致病性气单胞菌感染所造成的烂鳃、肝胆肿大、败血症等疾病时,除了考虑药代动力学和MIC指标外,还应考虑PAE因素,可适当延长给药间隔时间,降低药物对鳗鲡的副作用及对其摄食量和生长的负面影响.     

鱼源气单胞菌的主要特性研究

对分离于草鱼肠炎、白鲢鱼打印病、鲢鱼白皮病、草鱼烂鳃、鲤鱼体表出血感染及甲鱼败血症等病例的80株细胞进行了主要特性研究。结果表明均为气单胞菌属（Aeromonas)的细菌，其中嗜水气单胞菌（A.hydrophila)29株（占36．25％）、豚鼠气单胞菌（A.caviae)38株（占47．5％）、温和气单胞菌A.sobria）13株（占16．25％）。在被检验的病例中，或为其中一种气单胞菌的单独感染，或为两种、三种气单胞菌的混合感染。经做人工感染试验检查，表明这些分离菌均为相应感染症的致病菌。     

致病性嗜水气单胞菌PCR快速检测方法的建立

本试验根据GenBank已登录的致病性嗜水气单胞菌保守序列16S rDNA和Aero,设计2对引物,以嗜水气单胞菌纯培养物为起始材料,建立PCR检测方法。从12株分离物中均扩增到16S rDNA片段,从3株分离物中均扩增到Aero片段,经序列测定和分析,所扩增的片段均为嗜水气单胞菌的核苷酸序列。结果表明,建立的PCR方法可用于检测致病性嗜水气单胞菌。     

Effect of Fermented Chinese Medicine Preparation by Bacillus subtilis on Growth Performance,Biochemical Indices,Antioxidant Parameters and Ability of Anti-infection of Common Carp

The objective of this study was to evaluate of the growth performance, serum biochemical indices, liver antioxidant parameters and ability of anti-infection with Aeromonas hydrophila by adding the fermented Chinese medicine by Bacillus Subtilis to the carp fish feed, and to find out the appropriate supplemental level of fermented Chinese medicine in diets. Carp fingerling with an initial body weight of (35±0.5) g were set up to 6 experimental diets supplemented with 0 (control group), 0.1‰, 0.5‰, 1‰, 2‰ and 3‰ fermented Chinese medicine for 56 days, respectively. Each group had 30 fish and set up 3 repetitions. After the feeding trail,the fish were challenged with Aeromonas hydrophila to calculate the relative percentage survival (RPS).The results showed as follows:Supplementing fermented traditional Chinese medicine by Bacillus subtilis, the weight gain rate (WGR), specific growth rate (SGR), condition factor (CF), the activities of lysozyme (LSZ), complement 3 (C3),total protein (TP),total anti-oxidation capacity (T-AOC),total superoxide dismutase (T-SOD) and catalase (CAT) in serum of common carp were enhanced at different degrees, while the feed coefficient (FCR)and malondialdehyde (MDA) in serum were declined at different degrees.When the fermented Chinese medicine supplemental levels were 2‰ and 3‰,the effects were significant (P<0.05)(except for C3, ALP and Cr).The experimental results of the virulence of common carp infected with pathogen Aeromonas hydrophila showed that,the fermented herbal could obviously improve the ability of anti-infection, the immune protective rate of the group with 2‰, 3‰ doses of the fermented Chinese medicine could reach 77.23% and 74.63%, respectively. From what had been discussed above, the group with 2‰ and 3‰ doses of the fermented Chinese medicine had significant effect on the growth performance, serum biochemical indices, liver antioxidant parameters and Aeromonas hydrophila anti-infection ability. Considering the cost of adding fermented Chinese medicine, the recommendations for the best additive quantity was 2‰.     

枯草芽孢杆菌发酵中药制剂对鲤鱼生长性能、生化指标、抗氧化指标及抗感染能力的影响

本试验旨在研究饲料中添加枯草芽孢杆菌发酵中药制剂对鲤鱼生长性能、血清生化指标、抗氧化指标及对嗜水气单胞菌抗感染能力的影响,并确定其适宜的添加水平。将枯草芽孢杆菌发酵中药制剂投喂初始体重为（35±0.5）g的鲤鱼,试验设6个组,添加水平分别为0（对照组）、0.1‰、0.5‰、1‰、2‰、3‰,每组30尾,设3个重复,试验周期56 d。养殖试验结束后,腹腔注射嗜水气单胞菌进行感染试验,计算免疫保护率（RPS）。结果显示,饲料中添加枯草芽孢杆菌发酵中药制剂,鲤鱼增重率（WGR）、特定生长率（SGR）、肥满度（CF）、溶菌酶（LSZ）、补体3（C3）、总蛋白（TP）、总抗氧化能力（T-AOC）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）均不同程度提高,饲料系数（FCR）、丙二醛（MDA）含量不同程度降低,且当饲料中发酵中药添加量为2‰、3‰时,与对照组相比各指标（C3、碱性磷酸酶（ALP）和肌酐（Cr）除外）均差异显著（P<0.05）。用嗜水气单胞菌对鲤鱼感染试验结果表明,发酵中药可显著提高鲤鱼的抗感染能力,添加发酵中药2‰、3‰组免疫保护率可达77.23%和74.63%。可见,饲料中添加枯草芽孢杆菌发酵中药2‰、3‰能显著提高鲤鱼的生长性能、血清生化指标、肝脏抗氧化能力及抗嗜水气单胞菌感染能力。基于对添加成本的考虑,建议发酵中药的最佳添加剂量为2‰。     

牛源大肠杆菌O157：H7的分离及毒力基因鉴定

从2个牛场采集新鲜粪便,增菌后,免疫磁珠富集,涂布筛选性培养基,挑取可疑菌落用rfbE/fliC二重PCR和血清学方法鉴定。设计毒力基因stx1、stx2、eae、hlyA和tccp相应引物,针对O157：H7对分离株进行PCR鉴定。口服攻毒链霉素处理的BALB/c小鼠明确分离株致病性。结果显示,成功分离到7株出血性大肠杆菌O157：H7,并且有1株迟缓性发酵山梨醇麦康凯培养基。毒力基因检测显示,其中6株毒力因子表型为stx1-stx2＋eae＋hlyA＋tccp＋,另有1株表现型为stx1＋stx2＋eae＋hlyA＋tccp＋,各分离株tccp基因均为阳性,但携带的重复片段数量有差异。所采集样品中肠出血性大肠杆菌O157：H7的检出率高达12%。1×1010 CFU同剂量口服接种经PBS洗涤的5株O157：H7分离株全菌,小鼠存活率有差异分别为40%,50%,60%,20%,50%,各分离株在小鼠体内排菌时间也有差异分别为攻毒后7,9,13,13,15d。     

嗜水气单胞菌Ⅲ型分泌系统ascV全长基因的克隆及其在不同菌株中的分布

根据已发表的嗜水气单胞菌Ⅲ型分泌系统（TTSS）的ascV基因保守区核苷酸序列设计合成1对引物,以国内疫苗菌株J-1的基因组为模板,通过PCR扩增得到331bp保守基因片段,将目的片段进行测序。在此基础上,分4段克隆J-1株的Ⅱscv基因并进一步拼接,全长为2166bp,同时PCR检测ascV基因在66株嗜水气单胞菌中的分布情况。测序分析发现,扩增出的J-1株ascV全长基因与嗜水气单胞菌AH-1、SSU、AH-3的同源性分别为97％、86％、86％,与杀鲑气单胞菌杀鲑亚种A449的同源性为86％,与温和气单胞菌的同源性为87％。PCR检测表明,64株能扩增出n5fV基因的目的片段,包括2株无毒菌株,而在2株有毒菌株中却未能检测到,说明TTSS在嗜水气单胞菌致病机制上的作用值得进一步探讨。     

Long-term cryopreservation of microbial strains relevant to food hygiene

Experiments on long-time cryopreservation at -20 degrees C were conducted, using 42 microbial strains of relevance to food hygiene. Blood slant agar was coated with 10% glycerol solution, so that all strains were in re-culturing conditions, following 12 months of storage. Re-culturing of 18 in 20 of the microbial genera used in the experiment was possible after 24 months of storage, if strains had been kept only in 10% glycerol solution. The method proved suitable particularly for strain keeping of highly sensitive genera (Lactobacillus, Pseudomonas, Aeromonas, Shigella).