泥鳅(Misgurnus anguillicaudatus)病原温和气单胞菌(Aeromonas sobria)分子鉴定及耐药性研究

采用常规生理生化特性测定及16SrRNA、gyrB及rpoD基因同源性检索与系统发育学分析等分子生物学方法,对引起养殖泥鳅大量死亡的病原细菌进行了综合鉴定。生理生化特性结果表明,分离菌株NQ090701与气单胞菌属的温和气单胞菌相近;16SrRNA、gyrB及rpoD基因Blast序列同源性检索与系统发育学分析均与温和气单胞菌相似性最高;综合形态与生理生化特征及16SrRNA、gyrB与rpoD基因的分子特征,确认分离菌株NQ090701为温和气单胞菌(Aeromonas sobria)。同时,采用试管稀释法测定了养殖生产中常用的10种抗菌药物对病原温和气单胞菌的体外最小杀菌浓度(Minimal Bactericidal Concentration,MBC),结果表明氟哌酸杀菌作用最好,其次为盐酸沙拉沙星;甲砜霉素和复方磺胺甲恶唑在所试验的药物浓度范围对温和气单胞菌无杀菌效果。

Mortality of therapeutic fish Garra rufa caused by Aeromonas sobria

Objective:To investigate a case of mass mortality of Garra rufa(G.rufa)from a fish hatchery farm in Slovakia.Methods:Causative bacterial agent was swabbing out of affected fish skin area and subsequently identified using commercial test system.Antibiotic susceptibility was determined by the disk diffusion method.Results:Infected G.rufa was characterized by abnormal swimming behaviour,bleeding of skin lesions and local haemorrhages.Despite of using recommended aquatic antibiotic treatment no improvement was achieved and Aeromonas sobria{A.sobria)was identified as a causative agent of fish mortality.Due to massive fish mortality,antibiotic susceptibility of pure isolated culture of A.sobria was evaluated employing eight antibiotics against human infections.A.sobria was resistant only against one antibiotic,namely ampicilin.Conclusions:These results indicate that A.sobria can act as a primary pathogen of G.rufa and may be a potential risk factor for immunodeficient or immunoincompetent patients during the ichthyotherapy.

海洋菌株Aeromonas sp.YS-54来源几丁质酶的制备与性质研究

为实现甲壳类原料的高效降解与N-乙酰氨基寡糖的绿色制备,本研究以青岛近海土壤中筛选的菌株YS-54为材料,使用60%硫酸铵沉淀、Sephadex G-100凝胶过滤层析制备几丁质酶;对几丁质酶的最适温度、最适pH、底物偏好性等性质进行表征;使用质谱技术确认了酶解产物的聚合度。结果表明,菌株YS-54为Aeromonas属,该菌株来源几丁质酶比活力为23.44 U/mg,分子量为63与75 kDa。几丁质酶在40℃和pH为5.0条件下具有良好的稳定性,Ba^(2+)、Co^(2+)、Mg^(2+)与TritonX-100显著促进了几丁质酶活力(P<0.05),而Fe^(3+)、Cu^(2+)、SDS对酶活力起抑制作用。几丁质酶对α-几丁质的比活力为7.99 U/mg,为水解胶体几丁质比活力的34.09%。几丁质酶对胶体几丁质与α-几丁质的酶解产物聚合度分别为1~4与1~3。Aeromonas sp.YS-54来源几丁质酶在较宽的温度与pH范围内保持稳定,同时对α-几丁质具有良好的水解能力,可为甲壳类废弃物的高效综合利用提供技术支持。

Model prediction of inactivation of Aeromonas salmonicida grown on poultry in situ by intense pulsed light

The aim of this study was to evaluate the factors influencing the inactivation effect of intense pulsed light(IPL)on Aeromonas salmonicida grown on chicken meat and skin,and to further develop prediction models of inactivation.In this work,chicken meat and skin inoculated with meat-borne A.salmonicida isolates were subjected to IPL treatments under different conditions.The results showed that IPL had obvious bactericidal effect in the chicken skin and thickness groups when the treatment voltage and time were 7 V combined with 5 s.In addition,the lethality curves of A.salmonicida were fitted under IPL conditions of 3.5-7.5 V.The comparison of statistical parameters revealed that the Weibull model could best fit the mortality curves and could accurately predict the mortality dynamic of A.salmonicida grown on chicken skin.And further a secondary model between the scale factor b and the treatment voltage in Weibull model was established using linear equations,which determined that the secondary model could accurately predict the inactivation of A.salmonicida.This study provides a theoretical basis for future prediction models of Aeromonas,and also provides new ideas for sterilization approaches of meat-borne Aeromonas.

Isolation,identification,and virulence gene analysis of pathogenic Aeromonas dhakensis in Macrobrachium rosenbergii and histopathological observation

To identify the cause of mass mortality of adult Macrobrachium rosenbergii in a farm in Gaoyou City,Jiangsu Province,China,a dominant strain named DKQ-1 was isolated from the hepatopancreas of dying M.rosenbergii and identified as Aeromonas dhakensis by purification culture,biochemical characterization,and 16S rRNA and gyrB gene sequence analysis.The results of the challenge test revealed that the strain was highly pathogenic and the 50%lethal dose(LD_(50))in 72 h to M.rosenbergii was 1.54×10^(5)CFU/mL.The amplification results of virulence genes show that strain DKQ-1 carried 9 virulence genes,including ascV,aexT,aer,act,lip,ompAI,gcaT,acg,and exu,supporting the strong virulence of strain DKQ-1 to M.rosenbergii.Histopathological observation of the hepatopancreas,gills,and intestines indicated that DKQ-1 injection into M.rosenbergii could cause serious tissue damage,which further supported the strong virulence of this strain.In addition,a drug susceptibility test revealed that strain DKQ-1 was sensitive to 16 kinds of antibiotics,resistant to 9 kinds of antibiotics,and had intermediate resistance to spectinomycin and kanamycin.This study is the first report of A.dhakensis isolated from M.rosenbergii and provided a reference for the pathogen identification of bacterial diseases in M.rosenbergii,and for the prevention and treatment caused by A.dhakensis.

Cloning and Bioinformatics Analysis of hcp Gene in Aeromonas hydrophila

[Objectives]To explore the function of hcp gene in Aeromonas hydrophila.[Methods]A pair of specific primers was designed referring to the hcp gene sequence of A.hydrophila.The hcp gene was amplified by PCR,and performed bioinformatics analysis.[Results]The hcp gene had a total length of 1650 bp and encoded 549 amino acids.The theoretical molecular weight of the protein predicted was about 59476.44 kDa.After predicting the N-terminal signal peptide structure of the amino acid sequence,neither obvious signal peptide cleavage site nor signal peptide was found,and the protein had no transmembrane region.The amino acid sequence had a N-glycosylation site,4 protein kinase C phosphorylation sites,7 casein kinase II phosphorylation sites,9 N-myristoylation sites,4 isoprene binding sites,10 microbody C-terminal target signal sites,and an ATP/GTP binding site motif A(P-ring).The amino acid sequence of hcp gene of A.hydrophila was performed homology analysis with other Aeromonas strains,and it showed higher homology with A.veronii.In the secondary structure,theα-helix,β-sheet,random coil and extended strand accounted for 45.36%,6.01%,37.52%and 11.11%,respectively.The tertiary structure model consisted of 18α-helix and 22β-sheet.Analysis of protein-protein network interaction demonstrated that the proteins interacting with Hcp protein were AHA_3407,nrfA,nirB-1,nirB-2 and AHA_1112.[Conclusions]Through the bioinformatics prediction results,the basic information of hcp gene of A.hydrophila is preliminarily understood,and the possible function of this protein is predicted,in order to provide guidance for subsequent vaccine research.

Antimicrobial Resistance of Aeromonas spp.and Vibrio spp.Isolated from Fresh Pangasius Fish in Cambodia

The study was conducted to identify Aeromonas spp.and Vibrio spp.from fresh Pangasius fish(n=153)in Cambodia and test their antimicrobial susceptibility to antibiotics.The samples were collected from different wet markets of Phnom Penh city and Kampong Thom,and Siem Reap provinces.The bacteria were isolated by using selective medium and their AMR(Antimicrobial Resistance)profile was tested by API 20E technique,respectively.Susceptibility profile was determined for seven antibiotics commonly used.The Vibrio spp.(34.64%,n=53)was found to be higher than Aeromonas spp.(24.83%,n=38).Four Vibrio and four Aeromonas species were identified where V.parahaemolyticus(57%,n=30)was the highest,followed by V.cholerae(38%,n=20),V.fluvialis(3.8%,n=2)and V.aglinolyticus(1.9%,n=1),whereas A.hydrophila(47%,n=18)was the highest,followed by A.hydrophila/caviae(45%,n=17),A.sobria(5%,n=2),A.caviae(2.6%,n=1).All the species presented high multi-resistance to the tested antibiotics.The antibiotic susceptibility profile to ampicillin(74%-100%),ciprofloxacin(7%-100%),sulfamethoxazole/trimethoprim(14%-100%),florfenicol(14%-100%),oxytetracycline(7%-100%),erythromycin(10%-100%)and colistin sulphate(33%-100%)was revealed resistance level in Aeromonas spp.whereas few species of Vibrio spp.resistant to ampicillin(43%-100%),ciprofloxacin(14%-100%),sulfamethoxazole/trimethoprim(14%-100%),florfenicol(14%-100%),oxytetracycline(20%-100%),erythromycin(29%-100%),colistin sulphate(33%-100%)were also identified.The results revealed these Aeromonas spp.and Vibrio spp.are potentially reservoirs of antibiotic resistance genes.MDR(Multidrug Resistance)was widespread among the samples isolated.That is a high-risk source of contamination since those pathogens and antimicrobials are often used.Our findings highlight that the aquatic environment and fresh Pangasius fish act as reservoirs of AMR Aeromonas spp.and Vibrio spp.which underline the need for a judicious use of antimicrobials and timely surveillance of AMR in aquaculture.Overall,the findings of our study indicated the presence of A.hydrophila,A.hydrophila/caviae,A.caviae,A.sobria,V.parahaemolyticus,V.cholerae,V.alginolyticus and V.fluvialis and high MDR.This result will allow us to identify the potential risk over circulating isolates in animal health and public health and the spread through the food chain offering supports for appropriate sanitary actions.

斑点叉尾(Ictalunes punctatus)源中间气单胞菌(Aeromonas media)分离鉴定及药敏特性

从湖南常德某养殖场死亡的斑点叉尾（Ictalunes punctatus）肝脏、肾脏、腹水及血水中分离到一株致病性菌株zy02。对该菌进行了形态特征观察、理化特性测定及分子生物学方法鉴定,用PCR方法同时扩增其16S rDNA和gyrB基因,分析了16S rDNA和gyrB两种基因序列的同源性,并构建了系统发生树。经生理生化测定和16S rDNA与gyrB基因序列分析,zy02株为中间气单胞菌。人工感染该菌后发病鱼表现为与自然发病类似症状,且从组织中再分离的细菌特性与原感染菌相同。腹腔注射后该菌株对斑点叉尾的半致死浓度为3.89×10~6CFU/mL。菌株zy02对多西环素、庆大霉素及左氧氟沙星等高度敏感;对红霉素中度敏感;对阿莫西林、磺胺异噁唑、克林霉素及利福平等5种药物耐药。本研究为斑点叉尾该病防控提供了理论依据。

Effects of Compound Probiotics on Intestine and Intestinal Aeromonas hydrophila of Fish

[Objective] This study was conducted to develop compound probiotics that can be used as the alternatives of chemical drugs and antibiotics in aquaculture. [Method] Different concentrations of EM （effective microorganisms） and Bacillus amyloliquefaciens were mixed at a ratio of 1：1, and sprayed on fish feed. The growth of intestinal villi of the fishes that had been fed with the feed mixed with compound probiotics for three months was observed; meanwhile, the content of in- testinal Aeromonas hydrophila was detected by real-time quantitative PCR. ｜Result] The compound probiotics promoted the development of intestinal villi, and inhibited the growth of A. hydrophila, and the effects were also concentration dependent. However, the compound probiotics did not increase the thickness of the intestinal serous layer, muscular layer and submucosal layer. [Conclusion] The compound probiotics we prepared can be used and popularized in aquiculture as it can inhibit the growth of A. hydrophila.

罗氏沼虾(Macrobrachium rosenbergii)致病性豚鼠气单胞菌(Aeromonas caviae)的分离鉴定及其特性分析

近年来细菌性疾病因病原菌种类繁多、覆盖区域广而影响了其养殖业的健康发展,造成较大的经济损失。从体表具有黑斑、肢体残损症状的罗氏沼虾(Macrobrachium rosenbergii)肝胰腺组织中分离得到1株病原菌,通过形态学观察、生理生化特性鉴定、16S rRNA序列和特定毒力基因检测鉴定,以及人工回归感染试验和药敏试验分析其致病性和耐药性。结果表明,该菌为豚鼠气单胞菌(Aeromonas caviae),含有aer、lip、gcat、ser和OMPA I等毒力基因;药敏试验显示其对头孢曲松、氯霉素等7种抗生素高度敏感,对恩诺沙星、阿米卡星及复方新诺明3种抗生素中度敏感,对四环素、环丙沙星等10种抗生素具有耐药性;人工回归感染实验表明该菌具有较高的致病性,对罗氏沼虾的半致死浓度为6.54×10~5 CFU/mL。豚鼠气单胞菌作为水产养殖中的条件性致病菌,研究结果揭示了该菌株的部分生物学特性,有利于丰富豚鼠气单胞菌属的基础数据,同时也为罗氏沼虾养殖过程中的病害防控提供了一定参考依据。

SELEX筛选嗜水气单胞菌(Aeromonas hydrophila)适体方法的建立

嗜水气单胞菌是水产养殖中一种比较常见的致病病原菌。本研究以嗜水气单胞菌为研究对象,利用SELEX指数富集系统进化技术(Systematic Evolution of Ligands by Exponential Enrichment,SELEX),经过12轮的筛选,通过生物素-抗地高辛碱性磷酸酶显色系统检测DNA适配子与嗜水气单胞菌的亲和力,克隆、测序后,用DNAMAN软件分析其一级序列与二级结构。研究结果表明,通过12轮筛选后,亲和力由最初的0.32升到第9轮的0.72,亲和力提高了2.25倍。一级结构主要由GT组成,说明同源性适体有了一定程度的富集。二级结构模拟图以茎环状结构为主,说明这些茎环状结构可能是适体与靶分子进行结合作用的基础,也是其进一步形成三维空间结构的基础。

四株鳖源致病性嗜水气单胞菌(Aeromonas hydrophila)的表型、分子鉴定及其毒力基因检测

采用生态毒理学、表观分类学及分子生物学方法,对具白底板病典型症状濒死中华鳖内脏中分离获得的4株病原菌开展了以致病性、表型分析、分子鉴定及毒力基因检测为内容的实验研究,结果表明:(1)4株病原菌均具致病性,致死力由大到小依次为ZHYYZ-2、ZHYYZ-4、ZHYYZ-1、ZHYYZ-3;(2)4株病原菌均为呈短杆状、具溶血活性的革兰氏阴性细菌,VITEK2型全自动细菌鉴定与药敏系统和ATBExpression型细菌鉴定与药敏智能系统均显示为嗜水气单胞菌;(3)ZHYYZ-1、ZHYYZ-2、ZHYYZ-3、ZHYYZ-4的16SrDNA序列长度分别为1460、1464、1466、1461,经Blast同源性检索表明它们所扩增的16SrDNA序列与GenBank数据库中登记的71株嗜水气单胞菌的相似性均为99%;(4)经PCR特异性检测,各实验菌均含有Aha、AHH、AerA和OMP。根据4株实验菌的表型和分子生物学特征,判定它们均为气单胞菌属的致病性嗜水气单胞菌。

Isolation,Identification and in vitro Antimicrobial Susceptibility of Pathogenic Aeromonas veronii from Soft-shelled Turtles

In order to effectively control diseases caused by Aeromonas veronii,pathogenic bacteria were isolated and incubated from infected soft-shelled turtles with traditional bacterial isolation method. Four strains of pathogenic bacteria were isolated and identified with traditional biochemical identification method and modern molecular biological identification techniques. According to the results, four strains of pathogenic bacteria were identified as A. veronii biovar sobria. Drug sensitivity test and in vitro antimicrobial test against Bacillus amyloliquefaciens were performed with agar diffusion method. The results showed that B. amyloliquefaciens and several antibiotics such as ofloxacin and gentamicin exerted strong antimicrobial effects on four isolates. B. amyloliquefaciens could be used for the prevention and control of diseases caused by A. veronii in aquaculture.

重组嗜水气单胞菌Aeromonas hydrophila 4AK4中3-羟基丁酸3-羟基己酸共聚酯PHBHHx的发酵生产

3 羟基丁酸和 3 羟基己酸共聚酯 (PHBHHx)是一种性能优良的新型生物可降解材料 ,其机械和加工性能与3 羟基己酸 (3HHx)在共聚物中的含量密切相关。在嗜水气单孢菌Aeromonashydrophila 4AK4中引入了编码 β 酮基硫解酶 (β ketothiolase)的phbA基因和编码乙酰乙酰辅酶A还原酶 (Acetoacetyl CoAreductase)的phbB基因 ,使重组菌增加了一条利用乙酰辅酶A合成 3 羟基丁酸 CoA的代谢途径 ,这使得利用非相关性碳源调控PHBHHx的单体组成比例成为可能。利用葡萄糖酸钠和月桂酸作为碳源 ,对重组Aeromonashydrophila 4AK4进行了摇瓶培养及 5L发酵罐培养的研究。在摇瓶实验中 ,通过改变碳源中两种组分的比例 ,可以使A .hydrophila 4AK4合成的PHBHHx中的3HHx摩尔含量由原来的 15 %左右降低到 3%～ 12 % ,成功地实现了对PHBHHx单体组成的调控 ;当以月桂酸为唯一碳源时 ,在 5L发酵罐中 ,经过 5 6h的培养 ,获得了 5 1.5g L的细胞干重 (CDW) ,其中 6 2 %为PHBHHx,3HHx在PHBHHx中的摩尔含量为 9.7% ;当以 1:1的葡萄糖酸钠和月桂酸为碳源时 ,4 8h的 5L发酵罐培养获得了 32 .8g L的CDW和 5 2 %的PHBHHx含量 ,其中 3HHx在PHBHHx中的摩尔含量为 6 .7%。

海洋细菌Aeromonas hydrophila和Vibrio vulnificus对单萜柠檬烯的生物转化产物分析

研究了南海海洋细菌嗜水性气单胞菌Aerom onas hydrophila(5-213)和创伤弧菌Vibrio vulnificus(13-203)在培养基中添加单萜(D)-柠檬烯后提取产物的组成变化情况。经GC-MS分析发现该两细菌对(D)-柠檬烯有显著的生物催化转化作用,能对底物进行不同程度的氧化、还原、水解、酯化、开环等反应,并产生丰富的系列结构不同的其它萜类化合物:包括单萜、倍半萜、二萜、以及三萜等。通过单萜微生物转化和生物合成必将获得新的活性萜类产品,也为丰富的天然单萜资源的开发与利用提供新的途径和方法。

一株嗜水气单胞菌Aeromonas hydrophila的16S-23S rDNA间区序列分析及应用

根据细菌的16SrDNA3′端和23SrDNA5′端的高度保守区设计引物,扩增了一株中华鳖Trionyxsinensis"红板病"病原菌嗜水气单胞菌的16S-23SrDNA间区,克隆到pGEM＿T载体上,测序。用BLAST和DNAstar软件对16S-23SrDNA间区序列以及其内tRNA基因与已发表的嗜水气单胞菌和近缘种的IGSG序列进行比较分析,设计出对嗜水气单胞菌特异的PCR引物,建立了检测嗜水气单胞菌的PCR方法,进行了特异性、灵敏性检测,并用此方法检测了不同的水样。

Research on Bacteriostatic and Bactericidal Efficacy of Povidone Iodine on Aeromonas

[Objective] The aim of this study is to investigate the bacteriostatic and bactericidal efficacy of povidone iodine as one kind of disinfectants for aquaculture on Aeromonas.[Method] With the standard strain of Aeromonas ATCC7966 and the Aeromonas strains isolated from diseased fish or aquatic environment as the tested strains,the bacteriostatic and bactericidal efficacy of povidone iodine was studied by double broth dilution method.[Result] The povidone iodine could inhibit the growth of Aeromonas strains at lower concentration,and killed Aeromonas strains at higher concentration.The minimum bacteriostatic concentration（MIC） of povidone iodine on Aeromonas sp.T1,Aeromonas sp T2 and Aeromonas sp.T4 was 125.00 mg/L,and the minimum bactericidal concentration（MBC） was 4.00 g/L.Whereas,the minimum bacteriostatic concentration（MIC） of povidone iodine on ATCC7966,Aeromonas sp.T3,Aeromonas sp T5 and Aeromonas sp.T6 was 250.00 mg/L,and the minimum bactericidal concentration（MBC） was 8.00 g/L.[Conclusion] This study provides certain basis for reasonable application of povidone iodine.

Separation and Identification of Pathogens Causing Soft-shelled Turtle Fulminant Infectious Disease and Preparation of Anti-Aeromonas Serum

[Objective] This study aimed to isolate and identify the pathogens of soft-shelled turtle fulminant infectious disease,to provide the basis for the prevention and control of aquatic animal diseases.[Method] Commercially available sick soft-shelled turtles were used as experimental materials,to isolate and identify the causative pathogens;pathogenicity test and toxin determination of the isolates were carried out;in addition,hyperimmune serum was prepared and its therapeutic effect was detected.[Result] Nine bacteria were isolated from the livers of sick soft-shelled turtles,including seven Aeromonas sobria strains（JA-1） and two A.caviae strains（JA-2）.The median lethal dose（LD50） of JA-1 and JA-2 was 4.3×10^5/ml and 5.5×10^7/ml,respectively;the broth culture supernatant of the two bacteria contains a type of toxin with hemolytic activity,indicating that the soft-shelled turtle fulminant infectious disease is caused by A.sobria and A.caviae.Vaccine was prepared by using the two bacteria and rabbit immunization with immunopotentiator,showing a titer of 1：512 and a cure rate of 100%.[Conclusion] This study laid foundation for the prevention and control of aquatic animal diseases.

pH和NaCl质量浓度对发酵鱼糜中腐败菌Aeromonas veronii bv. veronii群体感应的影响

细菌通过群体感应(Quorum sensing,QS)系统来调控自身一些代谢行为,如生物膜的形成、鞭毛运动等,可引起食品腐败和食源性疾病,分析环境因素对细菌QS的影响对控制水产品质量安全具有重要意义。该研究以从发酵鱼糜中分离的腐败菌Aeromonas veronii bv.veronii为研究对象,利用生物报告菌根癌农杆菌及激光共聚焦显微镜等技术分析了pH(6.0、7.0、8.0)和Na Cl(5、10、20 g/L)对细菌N-酰基高丝氨酸内酯(N-acyl homoserine lactone,AHL)类信号分子介导的QS及代谢行为的影响。研究发现,培养基初始pH 8.0或NaCl质量浓度为20g/L时能显著降低细菌产生的AHLs活性、生物膜形成能力及泳动能力(p<0.05)。由此认为,通过控制环境中的pH和Na Cl质量浓度能影响细菌合成AHLs及其QS系统的表达,并调控细菌的代谢行为。