嗜水气单胞菌国内分离株的毒力因子分布与致病性相关性分析

为了研究分析国内致病性嗜水气单胞菌的毒力因子的分布及与致病性的相关性及其防治,选取5种主要毒力因子气溶素(aerA)、溶血素(hlyA)、丝氨酸蛋白酶(ahpA)、抗金属蛋白酶(ast)、肠毒素(altA)设计5对特异性引物,用PCR方法进行检测,采用急性毒性实验方法测定菌株对异育银鲫的半数致死量(50%lethal dose,LD50),细菌药物敏感试验纸片扩散(K-B)法检测对抗生素的敏感性,结果显示:除菌株Hong12的LD50约为109 cfu,毒力弱外,其他毒力均较强,LD50均在106 cfu左右。aerA、ahpA、hlyA、altA、ast 5种毒力基因的携带率分别为77.8%、88.9%、100%、100%、77.8%。通过比较嗜水气单胞菌毒力基因的分布情况与其对鲫鱼致病性试验结果可以发现,aerA、ahpA为嗜水气单胞菌致病的主要毒力因子,与致病性存在相关性,ast与致病力存在一定相关性,但不是主要毒力因子,hlyA、altA与致病性不存在相关性。9株嗜水气单胞菌对青霉素、羧苄青霉素、万古霉素不敏感,对复合磺胺、头孢噻肟、利福平中毒敏感,对呋喃妥因、氯霉素、四环素、氧氟沙星高度敏感。     

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...     

翘嘴鳜病原嗜水气单胞菌分子特征及LAMP检测方法的建立

嗜水气单胞菌(Aeromonas hydrophila)是一种危害鳜鱼养殖生产的重要病原细菌, 为进一步明确该病原菌的分子特征及建立快速检测技术, 实验对引起翘嘴鳜(Siniperca chuatsi)暴发性死亡的病原嗜水气单胞菌进行了致病性、菌株毒力特征研究, 同时以嗜水气单胞菌气溶素基因aerA为分子靶标设计引物, 利用环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)建立了病原嗜水气单胞菌的快速检测方法。结果表明, 本次引起翘嘴鳜暴发性死亡的病原嗜水气单胞菌半致死浓度为1.6×106 CFU/mL, 携带aerA等14种毒力基因, 此14种毒力基因可用于其致病性分析及分子检测。以气溶素基因aerA设计引物进行的环介导恒温扩增, 结果显示可扩增出阶梯状条带, 加入SYBR Green I染色后呈现绿色的阳性反应, 而对照组均未出现任何扩增条带且反应体系呈现橙色, 表明LAMP检测方法对于嗜水气单胞菌检测具有很好的特异性; 灵敏度检测的最低检测限为4.6×101 CFU/mL; 10种经人工感染的淡水养殖鱼虾组织匀浆增菌液, 提取DNA后进行LAMP方法检测, 结果均可获得阳性扩增结果, 而对照未染菌组呈阴性, 表明该方法具有较好的应用性, 可应用于嗜水气单胞菌引起的水生动物疾病的检测。     

黄沙鳖白底板病病原菌的分离鉴定及6种毒力基因检测

用常规方法从患典型白底板病黄沙鳖的心脏、肝脏等处进行细菌的接种分离, 通过人工感染确定分离菌株的致病性, API 20NE、16S rRNA基因序列分析进行病原菌鉴定和确定其系统发育地位, K-B纸片扩散法进行药敏试验, PCR检测病原菌的6种毒力基因。试验结果, 共分离到13株病原菌, 其中嗜水气单胞菌9株, 温和气单胞菌4株。在9株嗜水气单胞菌中, 有5株与Aeromonas hydrophila ATCC 7966菌株亲源关系最近, 4株与Aeromonas hydrophila北京株QDC01的亲源关系最近; 而4株温和气单胞菌与Aeromonas sobria ATCC 43979的亲源关系最近。药敏试验结果, 仅头孢哌酮对13株病原菌都高度敏感, 来源于不同养殖区域的病原菌药敏结果相差较大。6种毒力基因的阳性率, Aer、Act和ahp均为100%, hly和Alt为92.31%, ahal为76.92%; 毒力基因型共有4种, 嗜水气单胞菌主要为hly+Aer+Alt+Act+ahal+ahp+基因型, 而温和气单胞菌主要为 hly+Aer+Alt－Act+ahal+ahp+基因型, 同时携带hly基因的菌株其致病力更强。       

嗜水气单胞菌毒力基因的研究进展

嗜水气单胞菌(Aeromonas hydrophila)隶属于气单胞菌科(Aermonadaceae)气单胞菌属(Aeromonas),为人、畜及水生动物共患的条件致病菌。该菌广泛存在于水环境中,是多种水产动物的主要致病菌。国内外学者对其进行了许多研究,现普遍认为嗜水气单胞菌的致病性与其产生的毒素密切相关。随着分子生物技术的发展,已有许多学者从分子水平上对嗜水气单胞菌进行了研究,为阐明嗜水气单胞菌的致病机理提供了一些理论依据,并建立起针对几种主要毒力因子的检测手段。本文将嗜水气单胞菌目前的研究策略、部分主要毒力因子及其检测手段作一综述,以期为嗜水气单胞菌致病机理的研究及嗜水气单胞菌病的防治提供参考和借鉴。       

嗜水气单胞菌致病性研究进展

嗜水气单胞菌(Aeromonas hydrophila,AH)是我国养殖鱼类的重要病原,对其侵袭机制和毒力因子的研究有重要的意义。本文对嗜水气单胞菌的生物学特性、致病机制、毒力因子及其防治措施进行了综述。     

湖北地区暴发病池塘中嗜水气单胞菌的遗传多样性和毒力特征研究

为了调查引起鱼类运动型气单胞菌败血症(俗称暴发病)的嗜水气单胞菌的遗传多样性和毒力特征, 阐明其流行规律, 研究于2006-2009年度从湖北省内3个不同地区的6个发病鱼塘中分离了30株嗜水气单胞菌, 其中20株为临床株(分离自血液、肝脏、肾脏或腹水), 6株为肠道株, 4株为池水株。基于所有菌株gyrB基因序列, 构建了系统发育树; 通过ERIC (Enterobacterial repetitive intergenic consensus, 肠道细菌基因间重复序列)指纹图谱进行菌株的遗传分型; 用PCR方法检测了7个毒力基因在菌株中的分布模式。这7个基因包括气溶素(aerA)、溶血素(hlyA)、热不稳定性细胞兴奋性肠毒素(alt)、热稳定性细胞兴奋性肠毒素(ast)、弹性蛋白酶(ahpB)、脂酶(lip)和鞭毛基因(fla)。此外, 以斑马鱼为感染对象, 通过腹腔注射测定了15株代表菌株的毒力。结果表明: 不同来源的20株临床株、1株肠道株和3株池水株具有相同的遗传特性, 体现为在系统树上聚为一枝, 序列相似性为100%, 具有相同的ERIC指纹图谱, 毒力基因分布模式为: aerA+hlyA+alt+ast+ahpB+lip+fla+, 且均为强毒株(LD50 9.74104cfu/尾)。与临床株相比, 其余5株肠道株和1株池水株或具有不同的ERIC指纹图谱或具有不同的毒力基因分布模式, 显示出了遗传多样性, 且毒力均弱于临床株(LD501.01106cfu/尾)。这说明在一定时间、一定区域内, 作为暴发病病原的嗜水气单胞菌为同一克隆系在流行, 不存在明显的变异或遗传多样性。此结果有助于阐明嗜水气单胞菌引起的暴发病的流行规律, 制定相应的防御措施。多种毒力基因在致病性菌株中的联合流行为发病机理的解析奠定了基础。此外, 鉴于毒力基因谱与致病性之间的相关性, 表明毒力基因可作为标记基因, 用于致病性菌株的检测。       

西伯利亚鲟源嗜水气单胞菌致病菌的分离及其全菌苗的免疫效果

从患细菌性败血症的西伯利亚鲟(Acipenser baerii)的体内分离到一株致病菌株X1,其对西伯利亚鲟的半数致死浓度(LC50)为5.62×105 cfu/ml,具有较强毒力;经ATB细菌鉴定仪生理生化鉴定和16SrDNA序列分析,菌株X1为嗜水气单胞菌(Aeromonas hydrophila);其系统发育分析表明,菌株X1与嗜水气单胞菌ATCC35654(登录号:X74676.1)的亲缘关系最近,其同源性为99%.用0.30%福尔马林灭活,将菌株X1制成灭活全菌苗,对西伯利亚鲟进行注射免疫.研究结果表明,嗜水气单胞菌X1全菌苗能够明显提高西伯利亚鲟的血清抗体水平及总蛋白、免疫球蛋白、溶菌酶含量,而且在嗜水气单胞菌X1全菌苗中加入弗氏不完全佐剂,有利于进一步增强西伯利亚鲟血清抗体水平及总蛋白、免疫球蛋白、溶菌酶含量.此外,嗜水气单胞菌X1全菌苗对西伯利亚鲟抗嗜水气单胞菌X1人工感染也具有较好的免疫保护作用,其对西伯利亚鲟的免疫保护率为50%,而且在嗜水气单胞菌X1全菌苗中加入弗氏不完全佐剂,嗜水气单胞菌X1全菌苗对西伯利亚鲟抗嗜水气单胞菌X1人工感染的免疫保护作用更好,其对西伯利亚鲟的免疫保护率为70%.因此,将嗜水气单胞菌X1全菌苗用于西伯利亚鲟细菌性败血症的防治具有广阔的发展前景.     

和厚朴酚对嗜水气单胞菌气溶素表达的抑制作用

为了研究不同浓度和厚朴酚对嗜水气单胞菌(Aeromonas hydrophila)致病力的影响, 筛选抗嗜水气单胞菌感染的天然化合物, 通过溶血试验、免疫印记试验、荧光定量PCR试验和动物试验进行了研究。结果发现, 和厚朴酚能在亚抑菌浓度下降低嗜水气单胞菌培养物上清中的溶血活性; 蛋白免疫印迹试验发现和厚朴酚能降低嗜水气单胞菌气溶素的分泌; 荧光定量PCR试验进一步表明和厚朴酚与嗜水气单胞菌共培养后降低了气溶素编码基因aerA的转录而降低气溶素的分泌。此外, 通过动物试验发现和厚朴酚治疗能显著提高斑点叉尾鮰( Ictalurus punctatus )嗜水气单胞菌感染模型的存活率。以上研究表明, 和厚朴酚能通过降低气溶素编码基因aerA的转录而降低嗜水气单胞菌的致病力, 和厚朴酚是一种潜在的新型抗嗜水气单胞菌感染的先导化合物。     

白藜芦醇抑制嗜水气单胞菌毒力作用研究

为探索白藜芦醇(Resveratrol, Res)在水产动物细菌病防控中的应用价值, 实验以淡水养殖中重要的细菌病原嗜水气单胞菌(Aeromonas hydrophila)为研究对象, 通过设置药物浓度梯度, 检测其对嗜水气单胞菌生长、生物膜形成和溶血活性的抑制作用, 和对毒力及群感调控系统相关基因表达的影响; 同时通过人工感染异育银鲫(Carassius auratus gibelio)试验检测其对鱼体保护作用和对鱼体炎症相关因子基因表达的影响。结果显示: 白藜芦醇对嗜水气单胞菌的最小抑菌浓度(MIC)>1024 μg/mL; 浓度低于64 μg/mL时, 对菌株生长影响不显著; 浓度≥32 μg/mL时, 对病原菌株生物膜形成和溶血活性具有显著抑制作用(P<0.05), 且随剂量增加而增强。荧光定量RT-PCR结果分析发现白藜芦醇能引起嗜水气单胞菌群感调控系统中luxR和luxS基因分别显著上调和下调表达; 外膜蛋白基因omp表达显著下降。人工感染试验发现攻毒前两小时腹腔注射25、50和100 mg/kg白藜芦醇处理组的异育银鲫死亡率显著下降, 鱼体炎症相关的肿瘤坏死因子(TNF-α)和Ⅱ型干扰素(IFN-γ)的mRNA表达量也显著下降。研究表明药用植物大黄、虎杖等所含白藜芦醇成分能有效抑制嗜水气单胞菌毒力, 降低鱼体炎症反应的功效; 腹腔注射25—100 mg/kg白藜芦醇对感染病原菌的异育银鲫有一定保护作用。     

Correlation between the distribution pattern of virulence genes and virulence of Aeromonas hydrophila strains

Nine strains of Aeromonas hydrophila isolated from diseased fish or soft-shelled tortoise were tested for the presence of three virulence genes including the genes encoding aerolysin,hemolysin,and extracellular serine protease (i.e.,aerA,hlyA,and ahpA,respectively).These genes were investigated using polymerase chain reaction (PCR)with specific primers for each gene.And the pathogenicities to Carrassius auratus ibebio of these strains were also assayed.PCR results demonstrated that the distribution patterns of aerA,hlyA,and ahpA were different in these strains.6/9 of A.hydrophila strains were aer A positive,8/9 of strains hly A positive,7/9 of strains ahp A positive,respectively.However,the assay for pathogenesis showed that two strains (A.hydrophila XS91-4-1 and C2)were strong virulent,two strains (A.hydrophila ST78-3-3 and 58-20-9)avirulent and the rest middle virulent was to the fish.In conclusion,there are significant correlation between the distribution pattern of the three virulence genes and the pathogenicity to Carrassius auratus ibebio.All strong virulent A.hydrophila strains were aerA+hlyA+ahpA+genotype,and all aerA+hlyA+ahpA+strains were virulent.Strains with the genotype of aerA-hlyA-ahpA+have middle pathogenicity.In the present study,we found for the first time that all A.hydrophila isolated from the ahpA positive were virulent to Carrassius auratus ibebio.Additionally,there was a positive correlation between the virulence of A.hydrophila and the presence of aerA and ahpA.     

Identification and characterization of putative virulence genes and gene clusters in Aeromonas hydrophila PPD134/91

Aeromonas hydrophila is a gram-negative opportunistic pathogen of animals and humans. The pathogenesis of A. hydrophila is multifactorial. Genomic subtraction and markers of genomic islands (GIs) were used to identify putative virulence genes in A. hydrophila PPD134/91. Two rounds of genomic subtraction led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames, which are commonly present in the eight virulence strains examined. In addition, four GIs were found, including O-antigen, capsule, phage-associated, and type III secretion system (TTSS) gene clusters. These putative virulence genes and gene clusters were positioned on a physical map of A. hydrophila PPD134/91 to determine their genetic organization in this bacterium. Further in vivo study of insertion and deletion mutants showed that the TTSS may be one of the important virulence factors in A. hydrophila pathogenesis. Furthermore, deletions of multiple virulence factors such as S-layer, serine protease, and metalloprotease also increased the 50% lethal dose to the same level as the TTSS mutation (about 1 log) in a blue gourami infection model. This observation sheds light on the multifactorial and concerted nature of pathogenicity in A. hydrophila. The large number of putative virulence genes identified in this study will form the basis for further investigation of this emerging pathogen and help to develop effective vaccines, diagnostics, and novel therapeutics.     

An attenuated plasmid-cured strain of Aeromonas hydrophila elicits protective immunity in Clarias batrachus L

Wild type Aeromonas hydrophila (Strain AO1) isolated from the lesions of ulcerative disease syndrome (UDS) affected fish bears a 21 kb virulence plasmid. With plasmid curing the isolates became attenuated and failed to induce fatal haemorrhagic ulcers in fish. The objective of the present work was to check the immunogenicity of these plasmid-cured derivatives and determine whether such strains could be used as candidate antigens for eliciting protective immunity to A. hydrophila infections in the Indian catfish Clarias batrachus L. It was observed that the plasmid-cured strains were immunogenic since infection with live plasmid-cured AO1 isolates generated effective T cell responses and led to increase in serum antibacterial agglutinin titres in C. batrachus. Plasmid-cured AO1 strains injected into C. batrachus could disseminate into head kidney (HK) and spleen but never attained the same bacterial loads obtained with wild type AO1 and were cleared rapidly from the host. Immunisation with plasmid-cured bacteria prevented systemic spread and conferred protection against lethal challenge (10 x LD(50)) with wild type A. hydrophila as well as other pathogenic strains of Aeromonas sp. These results demonstrate the potentials of plasmid-cured A. hydrophila derivatives as candidate antigens for eliciting protective immunity in fish and the possibility of using such isolates as shuttle vectors in aquaculture.     

Cold shock exoribonuclease R (VacB) is involved in Aeromonas hydrophila pathogenesis

In this study, we cloned and sequenced a virulence-associated gene (vacB) from a clinical isolate SSU of Aeromonas hydrophila. We identified this gene based on our recently annotated genome sequence of the environmental isolate ATCC 7966(T) of A. hydrophila and the vacB gene of Shigella flexneri. The A. hydrophila VacB protein contained 798 amino acid residues, had a molecular mass of 90.5 kDa, and exhibited an exoribonuclease (RNase R) activity. The RNase R of A. hydrophila was a cold-shock protein and was required for bacterial growth at low temperature. The vacB isogenic mutant, which we developed by homologous recombination using marker exchange mutagenesis, was unable to grow at 4 degrees C. In contrast, the wild-type (WT) A. hydrophila exhibited significant growth at this low temperature. Importantly, the vacB mutant was not defective in growth at 37 degrees C. The vacB mutant also exhibited reduced motility, and these growth and motility phenotype defects were restored after complementation of the vacB mutant. The A. hydrophila RNase R-lacking strain was found to be less virulent in a mouse lethality model (70% survival) when given by the intraperitoneal route at as two 50% lethal doses (LD(50)). On the other hand, the WT and complemented strains of A. hydrophila caused 80 to 90% of the mice to succumb to infection at the same LD(50) dose. Overall, this is the first report demonstrating the role of RNase R in modulating the expression of A. hydrophila virulence.     

Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.: correlation with virulence in a suckling mouse model

Previous studies have shown that two hemolytic toxins, HlyA and AerA, contribute to the virulence of Aeromonas hydrophila. A survey was performed to gauge the distribution of hlyA and aerA genes in clinical and environmental Aeromonas isolates. For A. hydrophila, A. veronii biotype sobria and A caviae, 96%, 12% and 35% of strains, respectively, were hlyA positive, whereas, 78%, 97%, 41%, respectively, were aerA positive. All virulent A. hydrophila isolates were hlyA+ aerA+. This genotype was most common in A. hydrophila (75.4%) followed by A. caviae (29.4%) and A. veronii biotype sobria (9.6%). For A. hydrophila, a two-hemolytic toxin model of virulence provides the best prediction of virulence in an animal model.     

Molecular identification and virulence of three Aeromonas hydrophila isolates cultured from infected channel catfish during a disease outbreak in west Alabama (USA) in 2009

Three isolates (AL09-71, AL09-72, and AL09-73) of Aeromonas hydrophila were cultured from infected channel catfish Ictalurus punctatus during a disease outbreak in west Alabama, USA, in August 2009. Sequence analysis of the 16S-23S rDNA intergenic spacer region (ISR), cpn60, gyrB, and rpoD genes of the 3 strains revealed that the 3 strains were closely related to each other, sharing 97 to 99% nucleotide sequence similarities. However, ISR sequences of the 3 isolates from 2009 shared only 64% nucleotide sequences with AL98-C1B, a 1998 isolate of A. hydrophila cultured from diseased fish in Alabama. Sequences of cpn60, gyrB, and rpoD from the 3 isolates from 2009 shared 91 to 95% homologies with AL98-C1B. Based on both LD50 and LD95 values of intraperitoneal injection assays, the virulences of the 3 isolates from 2009 were not significantly different from each other, but were at least 200-fold more virulent than AL98-C1B, indicating that the 3 west Alabama isolates of A. hydrophila from 2009 were highly virulent to channel catfish.     

Virulence of β-hemolytic and non-hemolytic Streptococcus mutans: lethal dose determinations in neonatal mice

The virulence of beta-hemolytic and non-hemolytic strains of Streptococcus mutans was studied in neonatal mice by LD50 determinations after intracerebral injection of the bacteria. Although the differences in LD50 values are small the results may indicate that beta-hemolytic S. mutans strains (mean LD50 of 6.3 X 10(7) c.f.u.) were more virulent than non-hemolytic S. mutans strains (mean LD50 of 47.7 X 10(7) c.f.u.). The LD50 values of other viridans streptococci varied between 4.8 and 10(7) c.f.u. Strains of S. pyogenes and S. agalactiae were highly virulent in this animal model with LD50 values lower than 10(5) c.f.u.     

Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case

AIMS: To evaluate the public health significance of representative strains of two Aeromonas spp., mainly from freshwater fish, on the basis of production of virulence-associated factors and presence of the haemolytic genes aerA and hlyA. METHODS AND RESULTS: Eleven strains of Aer. hydrophila, three strains of Aer. veronii biovar sobria (all from freshwater fish) and one strain of Aer. hydrophila from human diarrhoea were tested for potential virulence traits and for the presence of the haemolytic genes aerA and hlyA. Ten Aer. hydrophila isolates were aerA(+)hlyA(+) and two aerA(+)hlyA(-). Aeromonas veronii biovar sobria isolates were aerA(-)hlyA(-). Strains from the three genotypes showed enterotoxic activity in the suckling mouse assay. At 28 degrees C, four Aer. hydrophila fish strains could be considered as potentially virulent (possessing at least two of these characteristics: haemolytic, cytotoxic and enterotoxic). One Aer. veronii biovar sobria strain and the clinical isolate were cytotoxic on Vero cells. When grown at 4 degrees C, these six isolates fulfilled virulence criterion, but at 37 degrees C, only one fish strain, an Aer. hydrophila, did. CONCLUSIONS: The potential health risk derived from the presence of Aer. hydrophila and Aer. veronii biovar sobria in ice-stored freshwater fish should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of virulence factors is affected by temperature incubation and not always related to the presence of haemolytic genes.