2011年通州区腹泻源性嗜水气单胞菌的分子分型特征

目的了解北京市通州区2011年腹泻患者粪便中分离到的27株嗜水气单胞菌（Aeromonas hydrophila）的生物学和分子分型特征,为该菌引发疾病的防控提供参考依据。方法对27株腹泻源性嗜水气单胞菌进行Aer毒素检测和PFGE分型,并进行同源性比较。结果27株腹泻源性嗜水气单胞菌中7株菌的Aer毒素为阳性,占总数的25．93％;PFGE图谱分为27个带型。结论在通州区腹泻患者粪便中检出的菌株部分携带Aer毒力因子,目前无优势流行菌株。建议相关部门加强对该菌的监测,避免该菌引发的各类疾患的发生。     

西伯利亚鲟嗜水气单胞菌与其它3株气单胞菌间的免疫交叉反应

采用间接免疫荧光技术分析了西伯利亚鲟细菌性败血症致病菌嗜水气单胞菌(Aeromonas hydrophlia)X1菌株、豚鼠气单胞菌(Aeromonas caviae)XL2-T菌株、致病性温和气单胞菌(Aeromonas sobria)W1菌株与无致病性嗜水气单胞菌(Aeromonas hydrophlia)M3菌株等水产养殖主要病原菌与抗血清之间的免疫交叉反应。结果显示具有致病性的同属菌株X1菌株、XL2-T菌株、W1菌株交叉反应程度较大,说明这3株菌表面存在较多相同抗原决定簇。而无致病性菌株M3与其他3株致病性菌株免疫交叉反应程度较小。     

嗜水气单胞菌毒力基因在传代过程中的稳定性研究

为了解嗜水气单胞菌毒力基因在传代过程中的稳定性,对传代前后嗜水气单胞菌(Aeromonas hydrophila,A.hydrophila)的毒力和毒力基因进行测定和检测.采用急性毒性试验方法测定10株嗜水气单胞菌原代、第10代及第20代的菌株对异育银鲫(Carassais auratus gibebio)的半数致死量,通过聚合酶链式反应(PCR)检测原代、第10代及第20代的菌株中5种毒力基因aerA、hlyA、ahpA、altA及ast的变化情况.试验结果表明,嗜水气单胞菌在传代过程中毒力基因分布相对较稳定.但是ahpA基因极易发生变异,在培养基上连续多次传代后,毒力逐渐减弱甚至消失.可推知,ahpA基因与毒力相关性最大,并易受传代影响.     

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。     

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.     

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...     

西伯利亚鲟致病性嗜水气单胞菌外膜蛋白及其抗原性分析

采用十二烷基肌氨酸钠(Sarkosyl)法提取西伯利亚鲟嗜水气单胞菌(Aeromonas hydrophila)外膜蛋白,电泳显示所提取的主要外膜蛋白分子量为26～120 kDa;为比较该菌株与气单胞菌菌属其他细菌外膜蛋白组分及抗原性异同,以致病性豚鼠气单胞菌(A.caviae)、温和气单胞菌(A.sobria)和无致病力的嗜水气单胞菌为对照,电泳图谱显示4种气单胞菌外膜蛋白的分子量主要集中在26～120 kDa之间;利用抗西伯利亚鲟嗜水气单胞菌血清的免疫印迹试验表明该菌株外膜蛋白中分子量为75 kDa、52 kDa、43 kDa、40 kDa、34 kDa、28 kDa的蛋白条带呈现阳性反应,其他3种气单胞菌外膜蛋白中均有与该抗血清反应的条带,且分子量为28 kDa、34 kDa的反应条带为4株菌共有;43 kDa与75 kDa反应条带为部分菌株共有.为进一步筛选和研究致病性气单胞菌的共同保护抗原提供参考.     

嗜水气单胞菌侵袭力与宿主细胞信号转导和骨架的关系

嗜水气单胞菌(Aeromonas hydrophila,Ah)是淡水鱼暴发性败血症的主要病原,该菌能够引致淡水鱼等的败血症和人的腹泻等^[1]。嗜水气单胞菌有多种致病因子,如毒素、蛋白酶、S层蛋白等^[2],还发现它具有侵袭作用,有报道嗜水气单胞菌粪分离株能侵袭HEp-2细胞^[3],但对于鱼源菌株的侵袭特性知之甚少。仅有一些报道认为嗜水气单胞菌能引致细胞病变^[4,5]。     

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。     

Occurrence, diversity and pathogenicity of mesophilic Aeromonas in estuarine waters of the Italian coast of the Adriatic Sea

A total of 208 strains of Aeromonas were isolated by monthly sampling from two estuaries (one provided with, and the other devoid of a waste-water treatment system) on the Italian coast of the Adriatic sea between September 1994 and August 1995. Biotyping at the species level allowed the identification of 96 strains (46%) as Aer. caviae , 46 (22%) as Aer. sobria , 33 (16%) as Aer. hydrophila and 25 (12%) as Aer. veronii . Eight strains (4%) were regarded as unnamed aeromonads. Aeromonas caviae was the most prevalent species in water with a high degree of pollution, while Aer. hydrophila strains were more commonly isolated from cleaner water. Aeromonas sobria and Aer. veronii were equally distributed in both estuaries. There was no correlation between temperature and numbers of aeromonads in either estuary. Using a biochemical fingerprinting method, strains were divided into similarity groups (PhP-types) based on their biochemical phenotypes. Several different PhP-types were found in each estuary, yielding a high diversity for these strains. However, some identical PhP-types were also found in both estuaries and at different times of the year, indicating that certain Aeromonas strains can survive more widely varying physico-chemical conditions. The production of toxins capable of causing cytoskeletal-dependent changes in the morphology of Chinese hamster ovary (CHO) cells was detected in 14 strains and appeared to be dependent on the season.     

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.     

Survival of Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria in soil

The survival of mesophilic Aeromonas spp. in soil in the presence or absence of indigenous microflora was evaluated in a laboratory study. Two cytotoxic ( Aer. hydrophila and Aer. caviae ) and one invasive ( Aer. sobria ) clinical isolate strains were selected for this study. After contamination of sterile or unsterilized soil with the three strains of Aeromonas , the number of living cells was determined over at least 5 months. For all strains the survival curves were characterized by an initial re-growth followed by a slow inactivation of bacteria, with significant differences due to the presence of indigenous microflora. The times necessary to achieve a 95% reduction of the initial population were > 140, 113 and 62 d in sterilized soil respectively for Aer. caviae, Aer. hydrophila and Aer. sobria , while the corresponding times in unsterilized soil were 42, 38 and 11 d. All strains preserved the virulence factors for the entire period of the study. These results suggest that the soil may be an important reservoir for Aeromonas spp. and, thus, may play an important role in the epidemiology of Aeromonas -associated human infections.     

Production of a monoclonal antibody against Aeromonas hydrophila and its application to bacterial identification

AIMS: to develop a monoclonal antibody (MAb) for the rapid detection of Aeromonas hydrophila in human faeces. METHODS AND RESULTS: A monoclonal antibody with strong specificity against Aer. hydrophila was obtained by the fusion of myeloma cells and splenocytes of a mouse immunized with vegetative cells of Aer. hydrophila ATCC 7966, followed by a two-step selection against other species of the genera. ELISA analyses revealed that MAb 5F3 strongly reacts with all the Aer. hydrophila strains evaluated, showing a just basal reactivity against other species of the genera, especially Aer. sobria and Aer. caviae. CONCLUSIONS: MAb 5F3 was characterized as an IgG that recognized a polypeptide of approximately 110 kDa. SIGNIFICANCE AND IMPACT OF THE STUDY: This MAb could be used to detect Aer. hydrophila in human stool samples.     

A method for the enumeration of Aeromonas bacteriophage in aquatic environments

K.P. FLINT. 1996. Bacteriophage were isolated against type strains and environmental isolates of Aeromonas hydrophila, Aeromonas sobria and Aeromonas caviae . A most probable number method for estimating the number of bacteriophage in a water sample was devised and tested using some of these isolates. The maximum number of bacteriophage against all three type strains were found in water from below a sewage effluent outfall. This corresponds to the increased numbers of each species of bacterium also found in this water sample. High numbers of bacteriophage against Aer. hydrophila were also found in the lake sample examined. Bacteriophage against Aer. caviae were rare in water samples other than those contaminated with sewage effluent.     

Rapid Aeromonas hydrophila identification by TaqMan PCR assay: comparison with a phenotypic method

Aims:  Aeromonas hydrophila is recognized as a human pathogen following wound exposure or ingestion of contaminated water and food. For rapid identification of this bacterium, a TaqMan-based real-time PCR assay has been developed.
Methods and Results:  Primers and probes that target specific sequences of the 16S rRNA gene and cytolytic enterotoxin gene ( aerA ) were combined in a duplex assay. Presence and size of PCR products were confirmed with microchannel fluidics electrophoresis analysis. After validation, using type strain CIP7614T DNA, the PCR assay was tested on 12 positive and negative controls. Twenty-one Aeromonas strains were isolated from environmental samples and were identified with biochemical tests as Aer. sobria , Aer. caviae and Aer. hydrophila . Only Aer. hydrophila strains tested positive by PCR assay.
Conclusions:  The PCR developed here was successfully applied for the identification of Aer. hydrophila from reference, clinical and environmental samples and showed a high discrimination between Aer. hydrophila and other Aeromonas species.
Significance and Impact of the Study:  This molecular method is convenient, rapid (2·5 h vs 24 h), specific to identify Aer. hydrophila and usable for diagnosis in medical and veterinary laboratories.     

Growth of Aeromonas species on increasing concentrations of sodium chloride

The growth of 16 strains of Aeromonas, representing 12 species of the genera, were examined at different salt levels (0-1.71 M NaCl). All the strains grew on media with 0.34 M NaCl, and nine on media with 0.68 M. Two strains, Aer. enteropelogenes and Aer. trota, were able to grow on media with 0.85 M and 1.02 M NaCl, respectively. Comparison of the growth curves of Aer. hydrophila ATCC7966 and Aer. trota ATCC 49657 on four concentrations of NaCl (0.08, 0.34, 0.68 and 1.02 M) confirm the high tolerance of Aer. trota, and indicate that high concentrations of salt increase the lag time and decrease the maximum growth rate. However, both strains were able to grow, slowly, in at least 0.68 M NaCl, a sodium chloride concentration currently used as food preservative.     

Incidence and identification of mesophilic Aeromonas spp. from retail foods

Sixty-eight food samples were examined for the presence of mesophilic Aeromonas species both qualitatively and quantitatively. Aeromonads were isolated from 26% of the vegetable samples, 70% of the meat and poultry samples and 72% of the fish and shrimps. Numbers of motile aeromonads present in the food samples varied from <10(2) cfu g(-1) to >10(5) cfu g(-1). GLC analysis of FAMEs was used to identify a selection of presumptive Aeromonas colonies to fenospecies or genomic species level. Aeromonas strains belonging to the Aer. caviae complex, which also includes the potentially pathogenic genospecies HG4, were mostly isolated from vegetables but were also found in meat, poultry and fish. In addition, three strains of the virulent taxon Aer. veronii biovar sobria HG8 were isolated from poultry and minced meat. All members of the Aer. hydrophila complex, predominant in the fish, meat and poultry samples, were classified in the non-virulent taxon HG3. Although the significance of Aeromonas in foods remains undefined, the isolation of Aeromonas HG4 and HG8 strains from a variety of retail foods may indicate that these products can act as possible vehicles for the dissemination of food-borne Aeromonas gastroenteritis.     

Phenotypic study by numerical taxonomy of strains belonging to the genus Aeromonas

AIMS: This study was undertaken to cluster and identify a large collection of Aeromonas strains. METHODS AND RESULTS: Numerical taxonomy was used to analyse phenotypic data obtained on 54 new isolates taken from water, fish, snails, sputum and 99 type and reference strains. Each strain was tested for 121 characters but only the data for 71 were analysed using the 'SSM' and 'SJ' coefficients, and the UPGMA clustering algorithm. At SJ values of > or = 81.6% the strains clustered into 22 phenons which were identified as Aer. jandaei, Aer. hydrophila, Aer. encheleia, Aer. veronii biogroup veronii, Aer. trota, Aer. caviae, Aer. eucrenophila, Aer. ichthiosmia, Aer. sobria, Aer. allosaccharophila, Aer. media, Aer. schubertii and Aer. salmonicida. The species Aer. veronii biogroup sobria was represented by several clusters which formed two phenotypic cores, the first related to reference strain CECT 4246 and the second related to CECT 4835. A good correlation was generally observed among this phenotypic clustering and previous genomic and phylogenetic data. In addition, three new phenotypic groups were found, which may represent new Aeromonas species. CONCLUSIONS: The phenetic approach was found to be a necessary tool to delimitate and identify the Aeromonas species. SIGNIFICANCE AND IMPACT OF THE STUDY: Valuable traits for identifying Aeromonas as well as the possible existence of new Aeromonas species or biotypes are indicated.     

Genetic diversity and antimicrobial susceptibility of motile aeromonads isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) farms

A total of 22 motile Aeromonas strains were detected in 48 (18.53%) of 259 fish and 6 (10.71%) of 56 water samples obtained from seven commercial rainbow trout ( Oncorhynchus mykiss , Walbaum) farms in the province of Mersin, Turkey. These strains were identified by conventional microbiological techniques and by using an ID32GN system. Of these isolates 20 (91.3%) were identified as Aeromonas hydrophila and 2 (8.7%) as Aeromonas sobria . While 8 of the A. hydrophila strains were isolated from water samples, 12 isolates were from fish samples. Whereas A. hydrophila strains were found in all farms, A. sobria was detected in only two farms. Genetic diversity by arbitrarily primed polymerase chain reaction (AP-PCR) and antimicrobial sensitivity tests were carried out on eight A. hydrophila isolates obtained from water samples, and isolates from seven A. hydrophila and one A. sobria from fish samples. The AP-PCR band patterns of motile aeromonads demonstrated weak similarity to the A. hydrophila reference strain ATCC 7966. Five A. hydrophila strains in the water samples displayed genetic similarity, but three others were different. Aeromonas hydrophila isolates from fish samples possessed slight similarities, and A. sobria was genetically distant to all A. hydrophila strains. An antimicrobial sensitivity test of 16 isolates revealed that 100% were sensitive to gentamicin, 87.5% to sulphamethoxazole–trimethoprim, 62.5% to enrofloxacin, 43.8% to oxytetracycline, 37.5% to neomycin, 18.75% to streptomycin and 6.25% to erythromycin. All isolates were resistant to novobiocin.