嗜水气单胞菌浮游态和生物被膜状态比较蛋白质组学及相关蛋白特性分析
|
摘要
嗜水气单胞菌(Aeromonas hydrophila)是一种重要的人-兽-鱼共患病病原。在自然环境中该茵大多以生物被膜形式存在,该存在形式作为一种同浮游细菌相对应的生长方式,在细菌的致病过程中发挥重要作用。现有的研究主要集中在嗜水气单胞菌生物被膜耐药性及疫苗方面,而毒力及生物被膜形成能力的关系以及生物被膜状态下与浮游态细菌蛋白表达差异的研究,国内外尚属空白。本研究观察了嗜水气单胞茵体外生物被膜的结构特征及形成过程,比较了生物被膜状态同浮游态细菌蛋白表达的差异,研究了生物被膜形成相关蛋白OmpAⅡ、FlaA、Omp38及LuxS勺生物学功能。
1.嗜水气单胞菌生物被膜形成能力分析及其与毒力关系
结合FITC-conA、FDA/PI荧光染色技术,对生物被膜的不同成分进行染色,利用荧光显微镜、扫描电镜及激光共聚焦显微镜观察不同时间生物被膜的结构特征及形成过程；同时将绿色荧光蛋白(GFP)表达载体导入嗜水气单胞菌,利用GFP标记技术示踪嗜水气单胞茵体外生物被膜的形成。结果显示,嗜水气单胞菌培养8h后聚集形成微菌落团,24h后生物被膜具有一定的厚度,且随时间延长,生物被膜厚度增加,死菌比例增加。气单胞茵不同分离株生物被膜的形成能力分析显示,86.7%(100/113)的气单胞茵菌株可形成生物被膜。通过选取生物被膜形成能力不同的菌株进行斑马鱼攻毒试验,结果表明生物被膜形成能力与菌株的致病性呈明显正相关。
2.嗜水气单胞菌浮游态和生物被膜状态比较蛋白质组学研究
利用比较蛋白质组学的方法,研究了嗜水气单胞菌J-1浮游态与生物被膜状态全菌蛋白及外膜蛋白的表达差异。结果表明,生物被膜状态下全菌蛋白中,17种蛋白表达上调,另有17种蛋白表达下调；而外膜蛋白中,11种蛋白表达上调,1种蛋白表达下调。采用嗜水气单胞菌鲫鱼感染血清与上述两种状态细菌的外膜蛋白进行免疫蛋白质组学分析,发现7种蛋白在两种状态均具有较强的免疫反应性,其中6种蛋白在生物被膜状态下表达升高,1种蛋白仅在生物被膜状态下具有免疫反应性。结果表明生物被膜状态下的细菌与浮游态相比,蛋白表达存在一定的差异,所鉴定的具有免疫反应性的蛋白作为疫苗侯选抗原,在抵抗嗜水气单胞菌持续性感染中具有较好的应用前景。
3.嗜水气单胞菌主要外膜蛋白OmpAⅡ的生物学功能
分析了嗜水气单胞菌生物被膜相关基因ompAII序列及其在252株不同气单胞菌中的分布,同时利用同源重组技术构建了ompAⅡ基因缺失株,通过比较野生株、缺失株的生物学特性,阐述主要外膜蛋白OmpAⅡ在嗜水气单胞菌致病过程中所发挥的作用。序列分析结果表明,基因ompAⅡ开放阅读框(ORF)为1002bp,编码333aa的主要外膜蛋白OmpAⅡ,该蛋白具有微孔蛋白超家族及OmpA蛋白家族的特性。基因ompAⅡ在嗜水气单胞菌中的分布率为83.3%(60/72)。ompAⅡ基因缺失株胞外产物溶血活性、溶蛋白活性及细胞毒性均有明显降低,生物被膜的形成能力也有所下降。此外,缺失株的黏附能力及抵抗巨噬细胞RAW264.7的吞噬能力与亲本目比,分别降低了25%和33%,据此可以推测嗜水气单胞菌的OmpAⅡ可能与该菌的黏附有关。以上研究结果揭示主要外膜蛋白OmpAⅡ是嗜水气单胞菌重要的毒力因子,在嗜水气单胞茵对宿主细胞的黏附过程中发挥作用。
4.嗜水气单胞菌鞭毛蛋白FlaA的生物学功能
分析了嗜水气单胞茵生物被膜相关基因flaA序列及其在252株不同气单胞茵中的分布,同时利用同源重组技术构建了flaA基因缺失株,通过比较野生株、缺失株的生物学特性,阐述鞭毛蛋白FlaA在嗜水气单胞茵致病过程中发挥的作用。序列分析结果表明,基因flaA ORF为909bp,编码302aa的鞭毛蛋白FlaA.从FlaA进化途径上看,嗜水气单胞菌J-1株FlaA位于气单胞茵属FlaA第2群中。flaA基因在嗜水气单胞菌中的分布率为55.6%(40/72),与其他气单胞菌属细菌的鞭毛蛋白的同源性在88%-99%之间。flaA基因缺失株、互补株及野生株的生物学特性比较结果表明,毒力基因aer和vgrG3的表达下调为野生株的0.44和0.45倍,该菌株的溶血活性显著下降。此外,缺失株生物被膜的形成能力降低,为野生株的85%。flaA基因缺失株对HEp-2细胞的黏附能力降低了48%,其茵体和上清液对巨噬细胞RAW264.7的毒性作用分别降低了35%和55%。研究结果表明,鞭毛蛋白FlaA与细菌的黏附作用密切相关,且在生物被膜形成过程中发挥重要作用。
5.嗜水气单胞茵外膜蛋白Omp38的生物学功能
分析了嗜水气单胞菌生物被膜相关基因omp38及其在252株不同气单胞菌中的分布,同时利用同源重组技术构建了omp38基因缺失株,通过比较野生株、缺失株的生物学特性,阐述外膜蛋白Omp38在嗜水气单胞菌致病过程中发挥的作用。序列分析结果表明,基因omp38ORF为1074bp,编码357aa的微孔蛋白为Omp38,属于微孔蛋白超家族。omp38基因在嗜水气单胞菌中的分布率为,73.6%(53/72)。通过结晶紫染色、激光共聚焦显微镜观察发现缺失株生物被膜的形成能力显著增强,FITC-conA标记后荧光显微镜观察发现生物被膜多糖的产生能力显著增强。噬菌体敏感试验表明,omp38基因缺失株使噬菌体的增殖能力降低,推测Omp38可能是细菌表面噬菌体受体。omp38基因缺失后细菌的溶血活性及溶蛋白活性降低,可能与aer、ahe2、 emp的表达下调有关。omp38基因缺失株对HEp-2细胞的黏附能力、抵抗血清杀菌能力及抗巨噬细胞的吞噬能力均显著增强,分别为野生株的11.45、10和1.73倍,这可能与多糖的保护作用有关。同时缺失株细菌及上清液对巨噬细胞RAW264.7的毒性分别增加19%和10%。此外,缺失株Ⅵ型分泌系统(T6SS)的vgrG2基因表达上调为亲本株的3.4倍。上述结果表明omp38基因缺失可引起嗜水气单胞菌毒力增强。
6.嗜水气单胞菌J-1株ompAⅡ和omp38双基因缺失株的构建及生物学特性分析
利用接合转移技术在omp38基因缺失株的基础上构建了ompAⅡ-omp38双基因缺失株,并对缺失株与野生株的生物学特性进行了比较。结果显示,缺失株具有较好的遗传稳定性,生长能力显著增强。荧光定量PCR结果显示,缺失株毒力基因aer的表达量下调为亲本株的0.83,而ahe2、emp、hcp2、vgrG2的表达量上调为亲本株的1.60、1.49、1.71、4.46倍。缺失株溶血活性的降低及溶蛋白活性的增强,可能与其毒力基因aer表达量下调及ahe2、emp表达量上调有关。此外,ompAⅡ与omp38双基因的缺失使得细菌抵抗血清杀菌能力及细胞毒性增强,而对HEp-2细胞的黏附能力及对巨噬细胞RAW264.7抗吞噬能力则表现为降低。以上结果表明,双基因缺失株的特性并非是ompAⅡ及omp38单基因缺失株特性的简单加合。
7.嗜水气单胞茵J-1株luxS基因缺失株的生物学特性分析
群体感应(QS)系统luxS基因缺失后,细菌的生物被膜形成能力显著降低,这与QS系统在生物被膜形成过程中发挥的作用密切相关。荧光定量PCR结果显示,缺失株所有毒力基因表达均下调,其中aer、ahe2、emp、hcp2的表达量显著下调为亲本株的0.14、0.21、0.06、0.24倍,这与细菌溶血活性及溶蛋白活性显著降低相一致。缺失株对细胞的黏附能力及菌体、上清对细胞的毒性显著降低,分别为野生株的84%、85%、48%。以上结果表明,LuxS参与调控细菌的毒力,与细菌生物被膜的形成有关。
8.嗜水气单胞菌Omp38重组蛋白的动物免疫效果评价
嗜水气单胞菌重组蛋白Omp38抗血清可与76%(47/62)的不同血清型嗜水气单胞菌外膜蛋白反应,表明其是一种潜在的共同保护性抗原。将重组蛋白Omp38分别注射免疫小鼠、鳊鱼,评价其免疫效果。结果表明,小鼠免疫后灭活疫苗组(I组)及蛋白免疫组(II组)IgG抗体水平显著高于对照组(III组),第35d达到最高。腹腔巨噬细胞吞噬活性及脾淋巴细胞转化能力均呈增加趋势,与血清IgG抗体产生水平基本一致。免疫后42d脾脏淋巴细胞中CD3+、CD3+CD4+CD8所占比率最高,I组为65.17%、52.08%,II组为53.22%、39.58%。荧光定量PCR分析结果表明免疫后21d开始,免疫组淋巴细胞IFN-y及IL-4mRNA表达水平上调,35d达到最高。鳊鱼免疫后,灭活疫苗组(I组)及蛋白免疫组(II组)IgM抗体水平显著高于对照组(III组),21d达到最高,此时Ⅱ组抗体水平高于I组,之后一直维持在较高水平。各免疫组血清超氧化物歧化酶(SOD)活性在免疫初期变化较为明显,14d II组SOD活性显著高于Ⅲ组(P<0.05),21d时SOD活性达到最高。各免疫组血清溶茵酶(LSZ)活性在14d呈现较高水平,且Ⅰ组高于Ⅱ组(P<0.05),之后呈现波动且高于同时间点III组LSZ活性(P<0.05)。流式细胞仪及平板计数结果表明各免疫组鳊鱼头肾细胞吞噬活性显著高于Ⅲ组(P<0.05)。免疫保护试验显示,Ⅰ组、Ⅱ组小鼠分别获得了82.00%、58.82%的相对保护率；Ⅰ组、Ⅱ组鳊鱼分别获得了50.00%、57.14%的相对保护率.结果表明,重组蛋白Omp38能够有效地刺激机体产生体液免疫应答及细胞免疫应答,对小鼠及鳊鱼的嗜水气单胞茵感染具有较好的免疫保护效果.
Aeromonas hydrophila is an important bacterial pathogen of aquatic animals and human. The bacterium lives in biofilms in the natural environment, as another life way corresponding to planktonic cells, which plays a key role in the pathogenesis of many bacterial infections. Current research of biofilms is focused on the antimicrobial resistance and vaccine development, however, the relationship between virulence and biofilm formation ability and the differences in protein expression between A. hydrophila planktonic cells and biofilms have not been investigated. In this study, structure characteristics and forming progress of biofilm in vitro were observed, protein expression between biofilms and planktonic cells was compared, and the biological functions of proteins including OmpAII, FlaA, Omp38and LuxS related to biofilm formation were studied.
1. Analysis of biofilm formation ability of A. hydrophila and its relationship with virulence
A. hydrophila can form biofilm, which causes the persistent infection of aquatic animals. In this study, using FITC-conA and FDA/PI staining techniques, the different components of biofilm were stained, and the characteristics of biofilm at different times were observed by fluorescence microscope, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and green fluorescent protein (GFP)-labled technology. The results showed that the bacteria began to cluster and form microcolony at8h, the biofilm was with certain thickness at24h, and the thickness and proportion of dead bacteria increased with the duration of culture time. In addition, the results of biofilm formation ability of Aeromonas strains showed that86.7%(100/113) could form biofilm. Additionally, the virulence of representative strains with different biofilm formation ability to zebrafish was investigated, and the positive correlation between biofilm formation ability and pathogenicity of strains was demonstrated.
2. Comparative proteomics analysis between biofilm and planktonic cells of A. hydrophila
The differences in expression profiles of whole bacterial proteins and outer membrane proteins (OMPs) from A. hydrophila J-1between planktonic cells and biofilm were investigated by comparative proteomics analysis. The results showed that compared with the planktonic controls,17proteins were unregulated and other17proteins were downregulated in the whole bacterial proteins from A. hydrophila biofilms, and11proteins were unregulated and one protein were downregulated in the OMPs from A. hydrophila biofilms. In addition, an immunoproteomics assay was developed to identify candidate antigens in OMPs of planktonic cells and biofilm with convalescent serum from Crucian carp. Seven immunoreactive proteins were identified under both planktonic and biofilm growth conditions, among which six proteins were upregulated in biofilm, and one protein was immunoreactive only in the biofilm growth conditions.
3. Biological functions of major outer membrane protein OmpAⅡ in A. hydrophila
To explore the function of OmpAⅡ, the gene sequence of ompAII was analyzed and its distribution in252different Aeromonas strains was investigated, and the ompAⅡ mutant was constructed by homologous recombination method. The open reading frame (ORF) of ompAⅡ gene was1002bp at length, which encoded333aa major outer membrane protein OmpAⅡ. OmpAⅡ comprises the structural motifs characterized by members of the family of porin and OmpA. The distribution of ompAⅡ in A. hydrophila was83.3%(60/72). The hemolytic activity, protease activity and cytotoxicity of extracellular products in the ompAⅡ mutant were significantly decreased. As well, the biofilm formation ability of the mutant strain was decreased. Moreover, the adhesion capacity to HEp-2cell and antiphagocytosis capacity to RAW264.7of the mutant strain were decreased by25%and33%compared to wide type strain. It suggested that OmpAⅡ was an important virulence factor of A. hydrophila, which might be involved in the adhesion of A. hydrophila to host cells.
4. Biological functions of FlaA in A. hydrophila
The gene sequence of flaA was analyzed and its distribution in252different Aeromonas strains was investigated. The flaA mutant was constructed by homologous recombination method. The ORF of flaA gene was909bp, which encoded302aa FlaA. According to the evolutionary pathway of FlaA, the homolog in A. hydrophila J-1located in the second group of Aeromonas FlaA. The distribution of gene flaA in A. hydrophila was 55.6%(40/72), and the deduced amino acid was88%-99%identical to other Aeromonas strains. The expression of virulence genes aer and vgrG3in the flaA mutant was decreased to0.44and0.45, respectively, which correlated with the significant decrease of hemolytic activity. Moreover, the biofilm formation ability of the mutant strain was reduced to85%compared to wide type strain. Furthermore, the adhesion capacity to the HEp-2cell decreased by48%, and the cytotoxic activities of bacteria and supernantant to RAW264.7cell were reduced by35%and55%, respectively. It indicated that FlaA was related to the adhesion of bacterium and contributed to biofilm formation, and thus played an important role in pathogenesis of A. hydrophila.
5. Biological functions of outer membrane protein Omp38in A. hydrophila
The gene sequence of omp38was analyzed and its distribution in252different Aeromonas strains was investigated. The omp38mutant was constructed by homologous recombination method. The ORF of omp38gene was1074bp, which encoded357aa porin Omp38. Omp38belongs to the porin superfamily. PCR and Western blot analysis showed that the gene of Omp38was widespread in A. hydrophila, with the73.6%(53/72) distribution percentage, and this protein was the potential common protective antigen, which could be used as candidate antigen of genetic engineering subunit vaccine. The omp38mutant showed increased biofilm formation with crystal violet staining and CLSM, and increased polysaccharide production by fluorescence microscope with FITC-conA labled. The bacteriophage sensitivity results showed that the omp38mutant reduced the reproductive activity of bacteriophage, which revealed that Omp38maybe phage receptor. The expressions of virulence genes aer, ahe2and emp in the omp38mutant were decreased, which may associated with the reduce of hemolytic activity and protease activity. In addition, adhesion capacity to HEp-2cell, antiserum and antiphagocytosis capacity was significantly increased to11.45,10and1.73fold compared to wide type strain, which may associated with the protection of increased polysaccharide. As well, the cytotoxic activities of bacteria and supernantant to RAW264.7cell were notably increased by19%and10%, respectively. Furthermore, virulence of the mutant to zebrafish was increased, which associated with the increased expression of virulence gene vgrG2in T6SS of A. hydrophila. It is presumed that the increased polysaccharide resulted in other characteristic changes of the omp38mutant. It suggested that A. hydrophila virulence increased after omp38was deleted.
6. Construction and characterization of ompAⅡ and omp38double gene mutant strain in A. hydrophila J-1
The ompAⅡ-omp38double gene mutant strain was constructed on the basis of the omp38mutant by homologous recombination method and biological characteristics were analyzed. The mutant strain showed well genetic stability and enhanced growth ability. Real time PCR analysis showed that expression of the virulence genes aer was decreased by0.83and the expressions of ahe2, emp, hcp2and vgrG2were increased by1.60,1.49,1.71and4.46compared to wide type strain, respectively. The decreased hemolytic activity and increased protease activity in the mutant strain, which corelated with the downregulation of aer and upregulation of ahe and emp. Moreover, antiserum ability and cytotoxicity to RAW264.7cell were increased, whereas, adhesion capacity to the HEp-2cell and antiphagocytosis to RAW264.7cell were decreased compared to wide type strain. It indicated that characteristics of the double gene mutant strain were not a simple adduct of characteristics of ompAⅡ and omp38single gene mutant strains.
7. Characterization of luxS gene mutant strain in A. hydrophila J-1
After the deletion of luxS gene in quorum sensing (QS), the biofilm formation capability of A. hydrophila J-1was significantly decreased. Real time PCR analysis showed that expressions of all virulence genes were decreased, among which expressions of aer, ahe2, emp and hcp2decreased by0.14,0.21,0.06and0.24, respectively, which might contrbute to the notably decreased hemolytic activity and protease activity.. Moreover, adhesion capacity to the HEp-2cell, cytotoxic activities of bacteria and supernantant to RAW264.7cell were significantly reduced to84%,85%and48%. It suggested that LuxS might be involved in biofilm formation and contribute to the virulence of the bacterium.
8. Animal immune efficacy of recombinant protein Omp38of A. hydrophila
To evaluate the immune efficacy of recombinant protein Omp38of A. hydrophila, mice and Parabramis pekinensis were immunized by injection, respectively. The results of vaccination in mice showed that IgG level of inactivation vaccine (group Ⅰ) and protein immunization (group Ⅱ) groups were significantly higher than that of control group (group Ⅲ), and reached the highest level at35d. Peritoneal macrophage phagocytosis activity and spleen lymphocyte proliferation activity were increased at the same trend as well as the production of antibody level. The percentages of CD3+and CD3+CD4+CD8-in spleen lymphocyte reached the highest level at42d after vaccination. They were65.17%and 52.08%in Group I, and53.22%and39.58%in group II. Real time PCR analysis showed that mRNA expression levels in IFN-y and IL-4began to increase at21d and reached the highest level at35d after vaccination in both immunization groups. The results of vaccination in Parabramis pekinensis showed that IgM level of inactivation vaccine (group I) and protein immunization (group Ⅱ) groups were significantly higher than that of control group (group Ⅲ), and reached the highest level at21d, at which time antibody titer of group Ⅱ was higher than that of group I, and maintained at a high level since this time. The activities of lysozyme (LSZ) and superoxide dismutases (SOD) in the sera of immunization groups had obvious changes at the early stage after immunization. The SOD activity of group II was higher than that of group Ⅲ at14d (P<0.05), and reached the highest level at21d. Likewise, the LSZ activity of immunization groups was at a high level at14d, and the activity of group I was higher than that of group II (P<0.05). Since this time the LSZ activity was fluctuated and higher than that of group III at the same day after immunization (P<0.05). The flow cytometer and plate count analysis showed that phagocytisis activity of lymphocytes in head kidney of immunization groups were significantly higher than that of group Ⅲ(P<0.05). The challenge test showed that the relative protection rates (RPS) of immunized mice in group I and group II were82.00%and58.82%, respectively, and the RPS of immunized Parabramis pekinensis in group I and group II was50.00%and57.14%, respectively. It suggested that the recombinant protein Omp38could stimulate the humoral and cellular immune response effectively, and provide better protection efficacy in mice and Parabramis pekinensis against infections caused by A. hydrophila.
1.嗜水气单胞菌生物被膜形成能力分析及其与毒力关系
结合FITC-conA、FDA/PI荧光染色技术,对生物被膜的不同成分进行染色,利用荧光显微镜、扫描电镜及激光共聚焦显微镜观察不同时间生物被膜的结构特征及形成过程；同时将绿色荧光蛋白(GFP)表达载体导入嗜水气单胞菌,利用GFP标记技术示踪嗜水气单胞茵体外生物被膜的形成。结果显示,嗜水气单胞菌培养8h后聚集形成微菌落团,24h后生物被膜具有一定的厚度,且随时间延长,生物被膜厚度增加,死菌比例增加。气单胞茵不同分离株生物被膜的形成能力分析显示,86.7%(100/113)的气单胞茵菌株可形成生物被膜。通过选取生物被膜形成能力不同的菌株进行斑马鱼攻毒试验,结果表明生物被膜形成能力与菌株的致病性呈明显正相关。
2.嗜水气单胞菌浮游态和生物被膜状态比较蛋白质组学研究
利用比较蛋白质组学的方法,研究了嗜水气单胞菌J-1浮游态与生物被膜状态全菌蛋白及外膜蛋白的表达差异。结果表明,生物被膜状态下全菌蛋白中,17种蛋白表达上调,另有17种蛋白表达下调；而外膜蛋白中,11种蛋白表达上调,1种蛋白表达下调。采用嗜水气单胞菌鲫鱼感染血清与上述两种状态细菌的外膜蛋白进行免疫蛋白质组学分析,发现7种蛋白在两种状态均具有较强的免疫反应性,其中6种蛋白在生物被膜状态下表达升高,1种蛋白仅在生物被膜状态下具有免疫反应性。结果表明生物被膜状态下的细菌与浮游态相比,蛋白表达存在一定的差异,所鉴定的具有免疫反应性的蛋白作为疫苗侯选抗原,在抵抗嗜水气单胞菌持续性感染中具有较好的应用前景。
3.嗜水气单胞菌主要外膜蛋白OmpAⅡ的生物学功能
分析了嗜水气单胞菌生物被膜相关基因ompAII序列及其在252株不同气单胞菌中的分布,同时利用同源重组技术构建了ompAⅡ基因缺失株,通过比较野生株、缺失株的生物学特性,阐述主要外膜蛋白OmpAⅡ在嗜水气单胞菌致病过程中所发挥的作用。序列分析结果表明,基因ompAⅡ开放阅读框(ORF)为1002bp,编码333aa的主要外膜蛋白OmpAⅡ,该蛋白具有微孔蛋白超家族及OmpA蛋白家族的特性。基因ompAⅡ在嗜水气单胞菌中的分布率为83.3%(60/72)。ompAⅡ基因缺失株胞外产物溶血活性、溶蛋白活性及细胞毒性均有明显降低,生物被膜的形成能力也有所下降。此外,缺失株的黏附能力及抵抗巨噬细胞RAW264.7的吞噬能力与亲本目比,分别降低了25%和33%,据此可以推测嗜水气单胞菌的OmpAⅡ可能与该菌的黏附有关。以上研究结果揭示主要外膜蛋白OmpAⅡ是嗜水气单胞菌重要的毒力因子,在嗜水气单胞茵对宿主细胞的黏附过程中发挥作用。
4.嗜水气单胞菌鞭毛蛋白FlaA的生物学功能
分析了嗜水气单胞茵生物被膜相关基因flaA序列及其在252株不同气单胞茵中的分布,同时利用同源重组技术构建了flaA基因缺失株,通过比较野生株、缺失株的生物学特性,阐述鞭毛蛋白FlaA在嗜水气单胞茵致病过程中发挥的作用。序列分析结果表明,基因flaA ORF为909bp,编码302aa的鞭毛蛋白FlaA.从FlaA进化途径上看,嗜水气单胞菌J-1株FlaA位于气单胞茵属FlaA第2群中。flaA基因在嗜水气单胞菌中的分布率为55.6%(40/72),与其他气单胞菌属细菌的鞭毛蛋白的同源性在88%-99%之间。flaA基因缺失株、互补株及野生株的生物学特性比较结果表明,毒力基因aer和vgrG3的表达下调为野生株的0.44和0.45倍,该菌株的溶血活性显著下降。此外,缺失株生物被膜的形成能力降低,为野生株的85%。flaA基因缺失株对HEp-2细胞的黏附能力降低了48%,其茵体和上清液对巨噬细胞RAW264.7的毒性作用分别降低了35%和55%。研究结果表明,鞭毛蛋白FlaA与细菌的黏附作用密切相关,且在生物被膜形成过程中发挥重要作用。
5.嗜水气单胞茵外膜蛋白Omp38的生物学功能
分析了嗜水气单胞菌生物被膜相关基因omp38及其在252株不同气单胞菌中的分布,同时利用同源重组技术构建了omp38基因缺失株,通过比较野生株、缺失株的生物学特性,阐述外膜蛋白Omp38在嗜水气单胞菌致病过程中发挥的作用。序列分析结果表明,基因omp38ORF为1074bp,编码357aa的微孔蛋白为Omp38,属于微孔蛋白超家族。omp38基因在嗜水气单胞菌中的分布率为,73.6%(53/72)。通过结晶紫染色、激光共聚焦显微镜观察发现缺失株生物被膜的形成能力显著增强,FITC-conA标记后荧光显微镜观察发现生物被膜多糖的产生能力显著增强。噬菌体敏感试验表明,omp38基因缺失株使噬菌体的增殖能力降低,推测Omp38可能是细菌表面噬菌体受体。omp38基因缺失后细菌的溶血活性及溶蛋白活性降低,可能与aer、ahe2、 emp的表达下调有关。omp38基因缺失株对HEp-2细胞的黏附能力、抵抗血清杀菌能力及抗巨噬细胞的吞噬能力均显著增强,分别为野生株的11.45、10和1.73倍,这可能与多糖的保护作用有关。同时缺失株细菌及上清液对巨噬细胞RAW264.7的毒性分别增加19%和10%。此外,缺失株Ⅵ型分泌系统(T6SS)的vgrG2基因表达上调为亲本株的3.4倍。上述结果表明omp38基因缺失可引起嗜水气单胞菌毒力增强。
6.嗜水气单胞菌J-1株ompAⅡ和omp38双基因缺失株的构建及生物学特性分析
利用接合转移技术在omp38基因缺失株的基础上构建了ompAⅡ-omp38双基因缺失株,并对缺失株与野生株的生物学特性进行了比较。结果显示,缺失株具有较好的遗传稳定性,生长能力显著增强。荧光定量PCR结果显示,缺失株毒力基因aer的表达量下调为亲本株的0.83,而ahe2、emp、hcp2、vgrG2的表达量上调为亲本株的1.60、1.49、1.71、4.46倍。缺失株溶血活性的降低及溶蛋白活性的增强,可能与其毒力基因aer表达量下调及ahe2、emp表达量上调有关。此外,ompAⅡ与omp38双基因的缺失使得细菌抵抗血清杀菌能力及细胞毒性增强,而对HEp-2细胞的黏附能力及对巨噬细胞RAW264.7抗吞噬能力则表现为降低。以上结果表明,双基因缺失株的特性并非是ompAⅡ及omp38单基因缺失株特性的简单加合。
7.嗜水气单胞茵J-1株luxS基因缺失株的生物学特性分析
群体感应(QS)系统luxS基因缺失后,细菌的生物被膜形成能力显著降低,这与QS系统在生物被膜形成过程中发挥的作用密切相关。荧光定量PCR结果显示,缺失株所有毒力基因表达均下调,其中aer、ahe2、emp、hcp2的表达量显著下调为亲本株的0.14、0.21、0.06、0.24倍,这与细菌溶血活性及溶蛋白活性显著降低相一致。缺失株对细胞的黏附能力及菌体、上清对细胞的毒性显著降低,分别为野生株的84%、85%、48%。以上结果表明,LuxS参与调控细菌的毒力,与细菌生物被膜的形成有关。
8.嗜水气单胞菌Omp38重组蛋白的动物免疫效果评价
嗜水气单胞菌重组蛋白Omp38抗血清可与76%(47/62)的不同血清型嗜水气单胞菌外膜蛋白反应,表明其是一种潜在的共同保护性抗原。将重组蛋白Omp38分别注射免疫小鼠、鳊鱼,评价其免疫效果。结果表明,小鼠免疫后灭活疫苗组(I组)及蛋白免疫组(II组)IgG抗体水平显著高于对照组(III组),第35d达到最高。腹腔巨噬细胞吞噬活性及脾淋巴细胞转化能力均呈增加趋势,与血清IgG抗体产生水平基本一致。免疫后42d脾脏淋巴细胞中CD3+、CD3+CD4+CD8所占比率最高,I组为65.17%、52.08%,II组为53.22%、39.58%。荧光定量PCR分析结果表明免疫后21d开始,免疫组淋巴细胞IFN-y及IL-4mRNA表达水平上调,35d达到最高。鳊鱼免疫后,灭活疫苗组(I组)及蛋白免疫组(II组)IgM抗体水平显著高于对照组(III组),21d达到最高,此时Ⅱ组抗体水平高于I组,之后一直维持在较高水平。各免疫组血清超氧化物歧化酶(SOD)活性在免疫初期变化较为明显,14d II组SOD活性显著高于Ⅲ组(P<0.05),21d时SOD活性达到最高。各免疫组血清溶茵酶(LSZ)活性在14d呈现较高水平,且Ⅰ组高于Ⅱ组(P<0.05),之后呈现波动且高于同时间点III组LSZ活性(P<0.05)。流式细胞仪及平板计数结果表明各免疫组鳊鱼头肾细胞吞噬活性显著高于Ⅲ组(P<0.05)。免疫保护试验显示,Ⅰ组、Ⅱ组小鼠分别获得了82.00%、58.82%的相对保护率；Ⅰ组、Ⅱ组鳊鱼分别获得了50.00%、57.14%的相对保护率.结果表明,重组蛋白Omp38能够有效地刺激机体产生体液免疫应答及细胞免疫应答,对小鼠及鳊鱼的嗜水气单胞茵感染具有较好的免疫保护效果.
Aeromonas hydrophila is an important bacterial pathogen of aquatic animals and human. The bacterium lives in biofilms in the natural environment, as another life way corresponding to planktonic cells, which plays a key role in the pathogenesis of many bacterial infections. Current research of biofilms is focused on the antimicrobial resistance and vaccine development, however, the relationship between virulence and biofilm formation ability and the differences in protein expression between A. hydrophila planktonic cells and biofilms have not been investigated. In this study, structure characteristics and forming progress of biofilm in vitro were observed, protein expression between biofilms and planktonic cells was compared, and the biological functions of proteins including OmpAII, FlaA, Omp38and LuxS related to biofilm formation were studied.
1. Analysis of biofilm formation ability of A. hydrophila and its relationship with virulence
A. hydrophila can form biofilm, which causes the persistent infection of aquatic animals. In this study, using FITC-conA and FDA/PI staining techniques, the different components of biofilm were stained, and the characteristics of biofilm at different times were observed by fluorescence microscope, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and green fluorescent protein (GFP)-labled technology. The results showed that the bacteria began to cluster and form microcolony at8h, the biofilm was with certain thickness at24h, and the thickness and proportion of dead bacteria increased with the duration of culture time. In addition, the results of biofilm formation ability of Aeromonas strains showed that86.7%(100/113) could form biofilm. Additionally, the virulence of representative strains with different biofilm formation ability to zebrafish was investigated, and the positive correlation between biofilm formation ability and pathogenicity of strains was demonstrated.
2. Comparative proteomics analysis between biofilm and planktonic cells of A. hydrophila
The differences in expression profiles of whole bacterial proteins and outer membrane proteins (OMPs) from A. hydrophila J-1between planktonic cells and biofilm were investigated by comparative proteomics analysis. The results showed that compared with the planktonic controls,17proteins were unregulated and other17proteins were downregulated in the whole bacterial proteins from A. hydrophila biofilms, and11proteins were unregulated and one protein were downregulated in the OMPs from A. hydrophila biofilms. In addition, an immunoproteomics assay was developed to identify candidate antigens in OMPs of planktonic cells and biofilm with convalescent serum from Crucian carp. Seven immunoreactive proteins were identified under both planktonic and biofilm growth conditions, among which six proteins were upregulated in biofilm, and one protein was immunoreactive only in the biofilm growth conditions.
3. Biological functions of major outer membrane protein OmpAⅡ in A. hydrophila
To explore the function of OmpAⅡ, the gene sequence of ompAII was analyzed and its distribution in252different Aeromonas strains was investigated, and the ompAⅡ mutant was constructed by homologous recombination method. The open reading frame (ORF) of ompAⅡ gene was1002bp at length, which encoded333aa major outer membrane protein OmpAⅡ. OmpAⅡ comprises the structural motifs characterized by members of the family of porin and OmpA. The distribution of ompAⅡ in A. hydrophila was83.3%(60/72). The hemolytic activity, protease activity and cytotoxicity of extracellular products in the ompAⅡ mutant were significantly decreased. As well, the biofilm formation ability of the mutant strain was decreased. Moreover, the adhesion capacity to HEp-2cell and antiphagocytosis capacity to RAW264.7of the mutant strain were decreased by25%and33%compared to wide type strain. It suggested that OmpAⅡ was an important virulence factor of A. hydrophila, which might be involved in the adhesion of A. hydrophila to host cells.
4. Biological functions of FlaA in A. hydrophila
The gene sequence of flaA was analyzed and its distribution in252different Aeromonas strains was investigated. The flaA mutant was constructed by homologous recombination method. The ORF of flaA gene was909bp, which encoded302aa FlaA. According to the evolutionary pathway of FlaA, the homolog in A. hydrophila J-1located in the second group of Aeromonas FlaA. The distribution of gene flaA in A. hydrophila was 55.6%(40/72), and the deduced amino acid was88%-99%identical to other Aeromonas strains. The expression of virulence genes aer and vgrG3in the flaA mutant was decreased to0.44and0.45, respectively, which correlated with the significant decrease of hemolytic activity. Moreover, the biofilm formation ability of the mutant strain was reduced to85%compared to wide type strain. Furthermore, the adhesion capacity to the HEp-2cell decreased by48%, and the cytotoxic activities of bacteria and supernantant to RAW264.7cell were reduced by35%and55%, respectively. It indicated that FlaA was related to the adhesion of bacterium and contributed to biofilm formation, and thus played an important role in pathogenesis of A. hydrophila.
5. Biological functions of outer membrane protein Omp38in A. hydrophila
The gene sequence of omp38was analyzed and its distribution in252different Aeromonas strains was investigated. The omp38mutant was constructed by homologous recombination method. The ORF of omp38gene was1074bp, which encoded357aa porin Omp38. Omp38belongs to the porin superfamily. PCR and Western blot analysis showed that the gene of Omp38was widespread in A. hydrophila, with the73.6%(53/72) distribution percentage, and this protein was the potential common protective antigen, which could be used as candidate antigen of genetic engineering subunit vaccine. The omp38mutant showed increased biofilm formation with crystal violet staining and CLSM, and increased polysaccharide production by fluorescence microscope with FITC-conA labled. The bacteriophage sensitivity results showed that the omp38mutant reduced the reproductive activity of bacteriophage, which revealed that Omp38maybe phage receptor. The expressions of virulence genes aer, ahe2and emp in the omp38mutant were decreased, which may associated with the reduce of hemolytic activity and protease activity. In addition, adhesion capacity to HEp-2cell, antiserum and antiphagocytosis capacity was significantly increased to11.45,10and1.73fold compared to wide type strain, which may associated with the protection of increased polysaccharide. As well, the cytotoxic activities of bacteria and supernantant to RAW264.7cell were notably increased by19%and10%, respectively. Furthermore, virulence of the mutant to zebrafish was increased, which associated with the increased expression of virulence gene vgrG2in T6SS of A. hydrophila. It is presumed that the increased polysaccharide resulted in other characteristic changes of the omp38mutant. It suggested that A. hydrophila virulence increased after omp38was deleted.
6. Construction and characterization of ompAⅡ and omp38double gene mutant strain in A. hydrophila J-1
The ompAⅡ-omp38double gene mutant strain was constructed on the basis of the omp38mutant by homologous recombination method and biological characteristics were analyzed. The mutant strain showed well genetic stability and enhanced growth ability. Real time PCR analysis showed that expression of the virulence genes aer was decreased by0.83and the expressions of ahe2, emp, hcp2and vgrG2were increased by1.60,1.49,1.71and4.46compared to wide type strain, respectively. The decreased hemolytic activity and increased protease activity in the mutant strain, which corelated with the downregulation of aer and upregulation of ahe and emp. Moreover, antiserum ability and cytotoxicity to RAW264.7cell were increased, whereas, adhesion capacity to the HEp-2cell and antiphagocytosis to RAW264.7cell were decreased compared to wide type strain. It indicated that characteristics of the double gene mutant strain were not a simple adduct of characteristics of ompAⅡ and omp38single gene mutant strains.
7. Characterization of luxS gene mutant strain in A. hydrophila J-1
After the deletion of luxS gene in quorum sensing (QS), the biofilm formation capability of A. hydrophila J-1was significantly decreased. Real time PCR analysis showed that expressions of all virulence genes were decreased, among which expressions of aer, ahe2, emp and hcp2decreased by0.14,0.21,0.06and0.24, respectively, which might contrbute to the notably decreased hemolytic activity and protease activity.. Moreover, adhesion capacity to the HEp-2cell, cytotoxic activities of bacteria and supernantant to RAW264.7cell were significantly reduced to84%,85%and48%. It suggested that LuxS might be involved in biofilm formation and contribute to the virulence of the bacterium.
8. Animal immune efficacy of recombinant protein Omp38of A. hydrophila
To evaluate the immune efficacy of recombinant protein Omp38of A. hydrophila, mice and Parabramis pekinensis were immunized by injection, respectively. The results of vaccination in mice showed that IgG level of inactivation vaccine (group Ⅰ) and protein immunization (group Ⅱ) groups were significantly higher than that of control group (group Ⅲ), and reached the highest level at35d. Peritoneal macrophage phagocytosis activity and spleen lymphocyte proliferation activity were increased at the same trend as well as the production of antibody level. The percentages of CD3+and CD3+CD4+CD8-in spleen lymphocyte reached the highest level at42d after vaccination. They were65.17%and 52.08%in Group I, and53.22%and39.58%in group II. Real time PCR analysis showed that mRNA expression levels in IFN-y and IL-4began to increase at21d and reached the highest level at35d after vaccination in both immunization groups. The results of vaccination in Parabramis pekinensis showed that IgM level of inactivation vaccine (group I) and protein immunization (group Ⅱ) groups were significantly higher than that of control group (group Ⅲ), and reached the highest level at21d, at which time antibody titer of group Ⅱ was higher than that of group I, and maintained at a high level since this time. The activities of lysozyme (LSZ) and superoxide dismutases (SOD) in the sera of immunization groups had obvious changes at the early stage after immunization. The SOD activity of group II was higher than that of group Ⅲ at14d (P<0.05), and reached the highest level at21d. Likewise, the LSZ activity of immunization groups was at a high level at14d, and the activity of group I was higher than that of group II (P<0.05). Since this time the LSZ activity was fluctuated and higher than that of group III at the same day after immunization (P<0.05). The flow cytometer and plate count analysis showed that phagocytisis activity of lymphocytes in head kidney of immunization groups were significantly higher than that of group Ⅲ(P<0.05). The challenge test showed that the relative protection rates (RPS) of immunized mice in group I and group II were82.00%and58.82%, respectively, and the RPS of immunized Parabramis pekinensis in group I and group II was50.00%and57.14%, respectively. It suggested that the recombinant protein Omp38could stimulate the humoral and cellular immune response effectively, and provide better protection efficacy in mice and Parabramis pekinensis against infections caused by A. hydrophila.
引文
[1]McCoy W, Bryers J, Robbins J, Costerton J. Observations of fouling biofilm formation. Can J Microbiol,1981,27:910-7.
[2]Milev-Milovanovic I, Long S, Wilson M, Bengten E, Miller NW, Chinchar VG. Identification and expression analysis of interferon gamma genes in channel catfish. Immunogenetics, 2006,58:70-80.
[3]Costerton JW, Stewart PS, Greenberg EP. Bacterial biofihlms:a common cause of persistent infections. Science,1999,284:1318-27.
[4]Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms:from the natural environment to infectious diseases. Nat Rev Microbiol,2004,2:95-108.
[5]Donlan RM, Costerton JW. Biofilms:survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev,2002,15:167-75.
[6]Monds RD, O'Toole GA. The developmental model of microbial biofilms:ten years of a paradigm up for review. Trends Microbiol,2009,17:73-87.
[7]Van Wey AS, Cookson AL, Soboleva TK, Roy NC, McNabb WC, Bridier A, Briandet R, Shorten PR. Anisotropic nutrient transport in three-dimensional single species bacterial biofilms. Biotechnol Bioeng,2011,12:156-63.
[8]Costerton JW. Introduction to biofilm. Int J Antimicrob Agents,1999,11:217-21.
[9]Costerton JW, Stewart PS. Battling biofilms. Sci Am,2001,285:74-81.
[10]Branda SS, Vik S, Friedman L, Kolter R. Biofilms:the matrix revisited. Trends Microbiol, 2005,13:20-6.
[11]Habash M, Reid G Microbial biofihns:their development and significance for medical device-related infections. JClin Pharmacol,1999,39:887-98.
[12]Hoiby N, Ciofu O, Johansen HK, Song ZJ, Moser C, Jensen PO, Molin S, Givskov M, Tolker-Nielsen T, Bjarnsholt T. The clinical impact of bacterial biofilms. Int J Oral Sci, 2011,3:55-65.
[13]Costerton JW. Overview of microbial biofilms. JInd Microbiol,1995,15:137-40.
[14]Kilty SJ, Duval M, Chan FT, Ferris W, Slinger R. Methylglyoxal:(active agent of manuka honey) in vitro activity against bacterial biofihns. Int Forum Allergy Rhinol,2011,1:348-50.
[15]Jones WL, Sutton MP, McKittrick L, Stewart PS. Chemical and antimicrobial treatments change the viscoelastic properties of bacterial biofilms. Biofouling,2011,27:207-15.
[16]Mah TF, O'Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol,2001,9:34-9.
[17]Simoes M, Simoes LC, Vieira MJ. A review of current and emergent biofilm control strategies. LWT-Food Sci Technol,2010,43:573-83.
[18]Bordi C, de Bentzmann S. Hacking into bacterial biofilms:a new therapeutic challenge. Ann Intensive Care,2011,1:19-26.
[19]Perez-Mendoza D, Coulthurst SJ, Sanjuan J, Salmond GP. N-Acetylglucosamine-dependent biofilm formation in Pectobacterium atrosepticum is cryptic and activated by elevated c-di-GMP levels. Microbiology,2011,157:3340-8.
[20]Liu H, Wang Q, Liu Q, Cao X, Shi C, Zhang Y. Roles of Hfq in the stress adaptation and virulence in fish pathogen Vibrio alginolyticus and its potential application as a target for live attenuated vaccine. Appl Microbiol Biot,2011:1-12.
[21]Fong JCN, Yildiz FH. The rbmBCDEF gene cluster modulates development of rugose colony morphology and biofilm formation in Vibrio cholerae. JBacteriol,2007,189:2319-25.
[22]Jin H, Zhou R, Kang M, Luo R, Cai X, Chen H. Biofilm formation by field isolates and reference strains of Haemophilus parasuis. Vet Microbiol,2006,118:117-23.
[23]Drenkard E, Ausubel FM. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature,2002,416:740-3.
[24]Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science,1994,263:802-11.
[25]Whiteley M, Bangera MG, Bumgarner RE, Parsek MR, Teitzel GM, Lory S, Greenberg EP. Gene expression in Pseudomonas aeruginosa biofilms. Nature,2001,413:860-4.
[26]Shemesh M, Tam A, Steinberg D. Differential gene expression profiling of Streptococcus mutans cultured under biofilm and planktonic conditions. Microbiology,2007,153:1307-17.
[27]Schembri MA, Kjaergaard K, Klemm P. Global gene expression in Escherichia coli biofilms. Mol Microbiol,2003,48:253-67.
[28]Ren D, Bedzyk LA, Thomas SM, Ye RW, Wood TK. Gene expression in Escherichia coli biofilms. Appl Microbiol Biotechnol,2004,64:515-24.
[29]Ren D, Bedzyk LA, Setlow P, Thomas SM, Ye RW, Wood TK. Gene expression in Bacillus subtilis surface biofilms with and without sporulation and the importance of yveR for biofilm maintenance. Biotechnol Bioeng,2004,86:344-64.
[30]Domka J, Lee J, Bansal T, Wood TK. Temporal gene-expression in Escherichia coli K-12 biofilms. Environ Microbiol,2007,9:332-46.
[31]Waite RD, Papakonstantinopoulou A, Littler E, Curtis MA. Transcriptome analysis of Pseudomonas aeruginosa growth:comparison of gene expression in planktonic cultures and developing and mature biofilms. JBacterial,2005,187:6571-6.
[32]Yamamoto R, Noiri Y, Yamaguchi M, Asahi Y, Maezono H, Ebisu S. Time course of gene expression during Porphyromonas gingivalis strain ATCC 33277 biofilm formation. Appl Environ Microbiol,2011,77:6733-6.
[33]He X, Ahn J. Differential gene expression in planktonic and biofilm cells of multiple antibiotic-resistant Salmonella Typhimurium and Staphylococcus aureus. FEMS Microbiol Lett, 2011,325:180-8.
[34]Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. JBacteriol,2002,184:1140-54.
[35]Khan AU, Islam B, Khan SN, Akram M. A proteomic approach for exploring biofilm in Streptococcus mutans. Bioinformation,2011,5:440-5.
[36]Tremoulet F, Duche O, Namane A, Martinie B, Labadie JC. A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth mode. FEMS Microbiol Lett,2002,215:7-14.
[37]Kalmokoff M, Lanthier P, Tremblay TL, Foss M, Lau PC, Sanders G, Austin J, Kelly J, Szymanski CM. Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation. JBacteriol,2006,188:4312-20.
[38]Koerdt A, Orell A, Pham TK, Mukherjee J, Wlodkowski A, Karunakaran E, Biggs CA, Wright PC, Albers SV. Macromolecular fingerprinting of sulfolobus species in biofilm:a transcriptomic and proteomic approach combined with spectroscopic analysis. J Proteome Res, 2011,10:4105-19.
[39]Pham TK, Roy S, Noirel J, Douglas I, Wright PC, Stafford GP. A quantitative proteomic analysis of biofilm adaptation by the periodontal pathogen Tannerella forsythia. Proteomics, 2010,10:3130-41.
[40]Shin JH, Lee HW, Kim SM, Kim J. Proteomic analysis of Acinetobacter baumannii in biofilm and planktonic growth mode. J Microbiol,2009,47:728-35.
[41]Espinal P, Marti S, Vila J. Effect of biofilm formation on the survival of Acinetobacter baumannii on dry surfaces. JHosp Infect,2012,80:56-60.
[42]Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cell Microbiol, 2009,11:1034-43.
[43]Cucarella C, Tormo MA, Ubeda C, Trotonda MP, Monzon M, Peris C, Amorena B, Lasa I, Penades JR. Role of biofilm-associated protein bap in the pathogenesis of bovine Staphylococcus aureus. Infect Immun,2004,72:2177-85.
[44]Norouzi F, Mansouri S, Moradi M, Razavi M. Comparison of cell surface hydrophobicity and biofilm formation among ESBL-and non-ESBL-producing Pseudomonas aeruginosa clinical isolates. Afr JMicrobiol Res,2010,4:1143-7.
[45]Jain A, Agarwal A. Biofilm production, a marker of pathogenic potential of colonizing and commensal staphylococci. J Microbiol Meth,2009,76:88-92.
[46]Holmberg A, Lood R, Morgelin M, Soderquist B, Holst E, Collin M, Christensson B, Rasmussen M. Biofilm formation by Propionibacterium acnes is a characteristic of invasive isolates. Clin MicrobiolInfec,2009,15:787-95.
[47]Deighton M, Borland R, Capstick J. Virulence of Staphylococcus epidermidis in a mouse model: significance of extracellular slime. Epidemiol Infect,1996,117:267-80.
[48]Yamanaka T, Furukawa T, Matsumoto-Mashimo C, Yamane K, Sugimori C, Nambu T, Mori N, Nishikawa H, Walker C, Leung KP. Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17. BMC Microbiol,2009,9:11-19.
[49]Zhang L, Hinz AJ, Nadeau JP, Mah TF. Pseudomonas aeruginosa tssCl links type VI secretion and biofilm-specific antibiotic resistance. JBacteriol,2011,193:5510-3.
[50]Turnock LL, Somers EB, Faith NG, Czuprynski CJ, Lee WA. The effects of prior growth as a biofilm on the virulence of Salmonella typhimurium for mice. Comp Immunol Microbiol Infect Dis,2002,25:43-8.
[51]Puttamreddy S, Minion FC. Linkage between cellular adherence and biofilm formation in Escherichia coli O157-H7 EDL933. FEMS Microbiol Lett,2011,315:46-53.
[52]Klausen M, Heydorn A, Ragas P, Lambertsen L, Aaes-Jorgensen A, Molin S, Tolker-Nielsen T. Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol,2003,48:1511-24.
[53]Naves P, del Prado G, Huelves L, Gracia M, Ruiz V, Blanco J, Dahbi G, Blanco M, Ponte Mdel C, Soriano F. Correlation between virulence factors and in vitro biofilm formation by Escherichia coli strains. Microb Pathog,2008,45:86-91.
[54]Simoes M. Antimicrobial strategies effective against infectious bacterial biofilms. Curr Med Chem,2011,18:2129-45.
[55]Goodman SD, Obergfell KP, Jurcisek JA, Novotny LA, Downey JS, Ayala EA, Tjokro N, Li B, Justice SS, Bakaletz LO. Biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid-associated proteins. Mucosal Immunol,2011,4:625-37.
[56]Brackman G, Cos P, Maes L, Nelis HJ, Coenye T. Quorum sensing inhibitors increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivo. Antimicrob Agents Chemother,2011,55:2655-61.
[57]Ma Q, Yang Z, Pu M, Peti W, Wood TK. Engineering a novel c-di-GMP-binding protein for biofilm dispersal. Environ Microbiol,2011,13:631-42.
[58]Lynch MJ, Swift S, Kirke DF, Keevil CW, Dodd CE, Williams P. The regulation of biofilm development by quorum sensing in Aeromonas hydrophila. Environ Microbiol,2002,4:18-28.
[59]Khajanchi BK, Sha J, Kozlova EV, Erova TE, Suarez G, Sierra JC, Popov VL, Horneman AJ, Chopra AK. N-acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila. Microbiology,2009,155:3518-31.
[60]Kozlova EV, Popov VL, Sha J, Foltz SM, Erova TE, Agar SL, Horneman AJ, Chopra AK. Mutation in the S-ribosylhomocysteinase (luxS) gene involved in quorum sensing affects biofilm formation and virulence in a clinical isolate of Aeromonas hydrophila. Microb Pathogenesis,2008,45:343-54.
[61]Azad IS, Shankar KM, Mohan CV, Kalita B. Uptake and processing of biofilm and free-cell vaccines of Aeromonas hydrophila in indian major carps and common carp following oral vaccination Dis Aquat Organ,2000,43:103-8.
[62]Nayak DK, Asha A, Shankar KM, Mohan CV. Evaluation of biofilm of Aeromonas hydrophila for oral vaccination of Clarias batrachus--a carnivore model. Fish Shellfish Immunol, 2004,16:613-9.
[63]Asha A, Nayak DK, Shankar KM, Mohan CV. Antigen expression in biofilm cells of Aeromonas hydrophila employed in oral vaccination of fish. Fish Shellfish Immunol,2004,16:429-36.
[1]Parker JL, Shaw JG. Aeromonas spp. clinical microbiology and disease. J Infection, 2011,62:109-18..
[2]Agger WA, McCormick J, Gurwith MJ. Clinical and microbiological features of Aeromonas hydrophila-associated diarrhea. J Clin Mcrobiol,1985,21:909.
[3]Bogdanovi R, obelji M, Markovi M, Nikoli V, Ognjanovi M, Sarjanovi L, Maki D. Haemolytic-uraemic syndrome associated with Aeromonas hydrophila enterocolitis. Pediatr Nephrol,1991,5:293-5.
[4]Kao HT, Huang YC, Lin TY. Fatal bacteremic pneumonia caused by Aeromonas hydrophila in a previously healthy child. JMicrobiol Immunol,2003,36:209-11.
[5]Mukhopadhyay C, Bhargava A, Ayyagari A. Aeromonas hydrophila and aspiration pneumonia:a diverse presentation. Yonsei Med J,2003,44:1087-90.
[6]Vally H, Whittle A, Cameron S, Dowse GK, Watson T. Outbreak of Aeromonas hydrophila wound infections associated with mud football. Clin Infect Dis,2004,38:1084.
[7]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin Mcrobiol Rev 2010,23:35.
[8]Dooley JS, Trust TJ. Surface protein composition of Aeromonas hydrophila strains virulent for fish:identification of a surface array protein. JBacteriol,1988,170:499-506.
[9]孙建和,严亚贤,陈怀青,陆承平.温度对嗜水气单胞菌外膜蛋白表达的影响.中国兽医学报,1999：555-8.
[10]Fang HM, Ge R, Sin YM. Cloning, characterisation and expression of Aeromonas hydrophila major adhesin. Fish Shellfish Immunol,2004,16:645-58.
[11]Lee S, Yin Z, Ge R, Sin Y. Isolation and characterization of fish Aeromonas hydrophila adhesins important for in vitro epithelial cell invasion. JFish Dis,1997,20:169-75.
[12]Bogaerts A, Temmerman L, Boerjan B, Husson S, Schoofs L, Verleyen P. A differential proteomics study of Caenorhabditis elegans infected with Aeromonas hydrophila. Dev Comp Immunol,2010,10:69-74..
[13]Upadhyaya T, Singh RK, Dixit A. Molecular cloning and sequence analysis of lamB encoding outer membrane maltose-inducible porin of Aeromonas hydrophila. DNA Seq,2007,18:302-6.
[14]Vazquez- Juarez R, Romero M, Ascencio F. Adhesive properties of a LamB - like outer - membrane protein and its contribution to Aeromonas veronii adhesion. JAppl Microbiol, 2004,96:700-8.
[15]Jiang B, Howard SP. The Aeromonas hydrophila exeE gene, required both for protein secretion and normal outer membrane biogenesis, is a member of a general secretion pathway. Mol Microbiol,1992,6:1351-61.
[16]Pillai L, Sha J, Erova TE, Fadl AA, Khajanchi BK, Chopra AK. Molecular and functional characterization of a ToxR-regulated lipoprotein from a clinical isolate of Aeromonas hydrophila. Infect Immun,2006,74:3742-55.
[17]Maji S, Mali P, Joardar S. Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.). Fish Shellfish Immunol,2006,20:462-73.
[18]Rahman MH, Kawai K. Outer membrane proteins of Aeromonas hydrophila induce protective immunity in goldfish. Fish Shellfish Immunol,2000,10:379-82.
[19]Maiti B, Raghunath P, Karunasagar I. Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp. J Appl Microbiol,2009,107:1157-67.
[20]Khushiramani R, Girisha SK, Karunasagar I. Protective efficacy of recombinant OmpTS protein of Aeromonas hydrophila in Indian major carp. Vaccine,2007,25:1157-8.
[21]Khushiramani RM, Maiti B, Shekar M, Girisha S, Akash N, Deepanjali A, Karunasagar I. Recombinant Aeromonas hydrophila outer membrane protein 48 (Omp48) induces a protective immune response against Aeromonas hydrophila and Edwardsiella tarda. Res Microbiol,2012.
[22]Merino S, Vilches S, Canals R, Ramirez S, Tomas JM. A Clq-binding 40kDa porin from Aeromonas salmonicida:Cloning, sequencing, role in serum susceptibility and fish immunoprotection. Microb Pathogenesis,2005,38:227-37.
[23]Guan R, Xiong J, Huang W, Guo S. Enhancement of protective immunity in European eel (Anguilla anguilla) against Aeromonas hydrophila and Aeromonas sobria by a recombinant Aeromonas outer membrane protein. Acta Biochim Biophys Sin (Shanghai),2011,43:79-88.
[24]Stintzi A, Raymond KN. Amonabactin-mediated iron acquisition from transferrin and lactoferrin by Aeromonas hydrophila:direct measurement of individual microscopic rate constants. J Bio Inorg Chem,2000,5:57-66.
[25]Jeanteur D, Gletsu N, Pattus F, Buckley JT. Purification of Aeromonas hydrophila major outer-membrane proteins:N-terminal sequence analysis and channel-forming properties. Mol Microbiol,1992,6:3355-63.
[26]Merino S, Nogueras MM, Aguilar A, Rubires X, Alberti S, Benedi VJ, Tomas JM. Activation of the complement classical pathway (Clq binding) by mesophilic Aeromonas hydrophila outer membrane protein. Infect Immun,1998,66:3825-31.
[27]Nogueras MM, Merino S, Aguilar A, Benedi VJ, Tomas JM. Cloning, sequencing, and role in serum susceptibility of porin II from mesophilic Aeromonas hydrophila. Infect Immun, 2000,68:1849.
[28]吕爱军,李槿年,余为一,屈艾,温洪宇.8株嗜温气单胞菌的外膜蛋白型及其聚类分析.中国兽医学报,2005：157-9.
[29]董传甫,林天龙,俞伏松,龚晖.嗜水气单胞菌细胞外膜蛋白及S层蛋白分析.中国水产科学,2003：201-5.
[30]Chhabra G, Upadhyaya T, Dixit A. Molecular cloning, sequence analysis and structure modeling of OmpR, the response regulator of Aeromonas hydrophila. Mol Biol Rep,2012,39:41-50.
[1]Parker JL, Shaw JG. Aeromonas spp. clinical microbiology and disease. J Infection, 2011,62:109-18.
[2]Agger WA, McCormick J, Gurwith MJ. Clinical and microbiological features of Aeromonas hydrophila-associated diarrhea. JClin Microbiol,1985,21:909-18.
[3]Bogdanovi R, obelji M, Markovi M, Nikoli V, Ognjanovi M, Sarjanovi L, Maki D. Haemolytic-uraemic syndrome associated with Aeromonas hydrophila enterocolitis. Pediatr Nephrol,1991,5:293-5.
[4]Kao HT, Huang YC, Lin TY. Fatal bacteremic pneumonia caused by Aeromonas hydrophila in a previously healthy child. J Microbiol Immunol,2003,36:209-11.
[5]Mukhopadhyay C, Bhargava A, Ayyagari A. Aeromonas hydrophila and aspiration pneumonia:a diverse presentation. Yonsei Med J,2003,44:1087-90.
[6]Vally H, Whittle A, Cameron S, Dowse GK, Watson T. Outbreak of Aeromonas hydrophila wound infections associated with mud football. Clin Infect Dis,2004,38:1084-92.
[7]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin Microbiol Rev,2010,23:35-42.
[8]Chauret C, Volk C, Creason R, Jarosh J, Robinson J, Warnes C. Detection of Aeromonas hydrophila in a drinking-water distribution system:a field and pilot study. Can J Microbiol, 2001,47:782-6.
[9]Monds RD, O'Toole GA. The developmental model of microbial biofilms:ten years of a paradigm up for review. Trends Microbiol,2009,17:73-87.
[10]Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms:from the natural environment to infectious diseases. Nat Rev Microbiol,2004,2:95-108.
[11]Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms:a common cause of persistent infections. Science,1999,284:1318-25.
[12]Sim es M, Sim es LC, Vieira MJ. A review of current and emergent biofilm control strategies. LWT-Food Sci Technol,2010,43:573-83.
[13]Stepanovic S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Meth,2000,40:175-9.
[14]Watnick PI, Kolter R. Steps in the development of a Vibrio cholerae El Tor biofilm. Mol Microbiol,1999,34:586-95.
[15]Fengqing H, Song Y. Electroporation-mediated transformation of Aeromonas hydrophila. Plasmid,2005,54:283-7.
[16]Neely M, Pfeifer J, Caparon M. Streptococcus-zebrafish model of bacterial pathogenesis. Infect Immun,2002,70:3904.
[17]Reed L, Muench H. A simple method of estimating fifty per cent endpoints. Am J Epidemiol, 1938,27:493-7.
[18]Potera C. Forging a link between biofilms and disease. Science,1999,283:1837-43.
[19]Davey ME, OToole G A. Microbial biofilms:from ecology to molecular genetics. Microbiol Mol Biol Rev,2000,64:847-67.
[20]Drenkard E, Ausubel FM. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature,2002,416:740-3.
[21]Trevors JT. Viable but non-culturable (VBNC) bacteria:Gene expression in planktonic and biofilm cells. J Microbiol Meth,2011,86:266-73.
[22]Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science,1994,263:802-11.
[23]Tsien RY. The green fluorescent protein. Annu Rev Biochem,1998,67:509-44.
[24]van der Sar AM, Appelmelk BJ, Vandenbroucke-Grauls CMJE, Bitter W. A star with stripes: zebrafish as an infection model. Trends Microbiol,2004,12:451-7.
[25]Wu Z, Zhang W, Lu Y, Lu C. Transcriptome profiling of zebrafish infected with Streptococcus suis. Microb pathogenesis,2010,48:178-87.
[26]Li J, Ni X, Liu Y, Lu C. Detection of three virulence genes alt, ahp and aerA in Aeromonas hydraphila and their relationship with actual virulence to zebrafish. J Appl Microbiol, 2011,316:160-8.
[27]Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cell Microbiol, 2009,11:1034-43.
[28]Cucarella C, Tormo MA, Ubeda C, Trotonda MP, Monzon M, Peris C, Amorena B, Lasa I, Penades JR. Role of biofilm-associated protein bap in the pathogenesis of bovine Staphylococcus aureus. Infect Immun,2004,72:2177-85.
[29]Norouzi F, Mansouri S, Moradi M, Razavi M. Comparison of cell surface hydrophobicity and biofilm formation among ESBL-and non-ESBL-producing Pseudomonas aeruginosa clinical isolates. Afr J Microbiol Res,2010,4:1143-7.
[30]Jain A, Agarwal A. Biofilm production, a marker of pathogenic potential of colonizing and commensal staphylococci. J Microbiol Meth,2009,76:88-92.
[31]Holmberg A, Lood R, Morgelin M, S6derquist B, Hoist E, Collin M, Christensson B, Rasmussen M. Biofilm fonnation by Propionibacterium acnes is a characteristic of invasive isolates. Clin Microbiol Infec,2009,15:787-95.
[32]Deighton M, Borland R, Capsiick J. Virulence of Staphylococcus epidermidis in a mouse model: significance of extracellular slime. Epidemiol Infect,1996,117:267-80.
[33]Yamanaka T, Furukawa T, Matsumoto-Mashimo C, Yamane K, Sugimori C, Nambu T, Mori N, Nishikawa H, Walker C, Leung KP. Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17. BMC Microbiol,2009,9:11-19.
[34]Rahman MH, Suzuki S, Kawai K. Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus. Microbiol Res, 2001,156:103-6.
[1]Parker JL, Shaw JG. Aeromonas spp. clinical microbiology and disease. J Infection,2011, 62:109-18.
[2]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin Microbiol Rev,2010,23:35-73.
[3]Monds RD, O'Toole GA. The developmental model of microbial biofilms:ten years of a paradigm up for review. Trends Microbiol,2009,17:73-87.
[4]Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cell Microbiol, 2009,11:1034-43.
[5]Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms:a common cause of persistent infections. Science,1999,284:1318-25.
[6]Fux C, Costerton J, Stewart P, Stoodley P. Survival strategies of infectious biofilms. Trends Microbiol,2005,13:34-40.
[7]Yu HB, Zhang YL, Lau YL, Yao F, Vilches S, Merino S, Tomas JM, Howard SP, Leung KY. Identification and characterization of putative virulence genes and gene clusters in Aeromonas hydrophila PPD134/91. Appl Environ Microbiol,2005,71:4469-77.
[8]Dooley JS, Trust TJ. Surface protein composition of Aeromonas hydrophila strains virulent for fish:identification of a surface array protein. JBacteriol,1988,170:499-506.
[9]Esteve C, Alcaide E, Canals R, Merino S, Blasco D, Figueras MJ, Tomas JM. Pathogenic Aeromonas hydrophila serogroup 0:14 and 0:81 strains with an S layer. Appl Environ Microbiol,2004,70:5898-904.
[10]Schulz GE. The structure of bacterial outer membrane proteins. BBA-Biomembranes, 2002,1565:308-17.
[11]Lee S, Yin Z, Ge R, Sin Y. Isolation and characterization of fish Aeromonas hydrophila adhesins important for in vitro epithelial cell invasion. JFish Dis,1997,20:169-75.
[12]Buckley JT, Peter S, Chopra A, Houston C. The cytotoxic enterotoxin of Aeromonas hydrophila is aerolysin. Infect Immun,1999,67:466-75.
[13]Upadhyaya T, Singh RK, Dixit A. Molecular cloning and sequence analysis of lamB encoding outer membrane maltose-inducible porin of Aeromonas hydrophila. Mitochondr DNA, 2007,18:302-6.
[14]Maiti B, Raghunath P, Karunasagar I. Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp. J Appl Microbiol,2009,107:1157-67.
[15]Maji S, Mali P, Joardar SN. Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.). Fish Shellfish Immunol,2006,20:462-73.
[16]Khushiramani R, Girisha SK, Karunasagar I. Cloning and expression of an outer membrane protein ompTS of Aeromonas hydrophila and study of immunogenicity in fish. Protein Expr Purif,2007,51:303-7.
[17]Guan R, Xiong J, Huang W, Guo S. Enhancement of protective immunity in European eel (Anguilla anguilla) against Aeromonas hydrophila and Aeromonas sobria by a recombinant Aeromonas outer membrane protein. Acta Biochim Biophys Sin (Shanghai),2011,43:79-88.
[18]Filip C, Fletcher G, Wulff JL, Earhart CF. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. JBacteriol,1973,115:717-22.
[19]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-[Delta][Delta] CT method. Methods,2001,25:402-8.
[20]van Alen T, Claus H, Zahedi RP, Groh J, Blazyca H, Lappann M, Sickmann A, Vogel U. Comparative proteomic analysis of biofilm and planktonic cells of Neisseria meningitidis. Proteomics,2010,8:60-9.
[21]Benard L, Litzler PY, Cosette P, Lemeland JF, Jouenne T, Junter GA. Proteomic analysis of Staphylococcus aureus biofilms grown in vitro on mechanical heart valve leaflets. J Biomed Mater Res A,2009,88:1069-78.
[22]Tremoulet F, Duche O, Namane A, Martinie B, Labadie JC. A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth mode. FEMS Microbiol Lett,2002,215:7-14.
[23]Shin JH, Lee HW, Kim SM, Kim J. Proteomic analysis of Acinetobacter baumannii in biofilm and planktonic growth mode. J Microbiol,2009,47:728-35.
[24]Souza AA, Takita MA, Coletta-Filho HD, Caldana C, Yanai GM, Muto NH, Oliveira RC, Nunes LR, Machado MA. Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro. FEMS Microbiol Lett,2004,237:341-53.
[25]Kim DG, Kim YR, Kim EY, Cho HM, Ann SH, Kong IS. Isolation of the groESL cluster from Vibrio anguillarum and PCR detection targeting groEL gene. Fisheries Sci,2010,76:803-10.
[26]Kalmokoff M, Lanthier P, Tremblay TL, Foss M, Lau PC, Sanders G, Austin J, Kelly J, Szymanski CM. Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation. JBacteriol,2006,188:4312-20.
[27]Hefford MA, D'Aoust S, Cyr TD, Austin JW, Sanders G, Kheradpir E, Kalmokoff ML. Proteomic and microscopic analysis of biofilms formed by Listeria monocytogenes 568. Can J Microbiol,2005,51:197-208.
[28]Zhang D, Jiang B, Xiang Z, Wang S. Functional characterisation of altered outer membrane proteins for tetracycline resistance in Escherichia coli. Int JAntimicrob Ag,2008,32:315-9.
[29]Garcia-Sureda L, Juan C, Domenech-Sanchez A, Alberti S. Role of Klebsiella pneumoniae LamB porin in antimicrobial resistance. Antimicrob Agents Ch,2011:AAC.01441-10v1.
[30]Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature,2000,405:914-9.
[31]Galindo CL, Sha J, Ribardo DA, Fadl AA, Pillai L, Chopra AK. Identification of Aeromonas hydrophila cytotoxic enterotoxin-induced genes in macrophages using microarrays. J Biol Chem, 2003,278:40198-212.
[32]Chopra A, Xu XJ, Ribardo D, Gonzalez M, Kuhl K, Peterson J, Houston C. The cytotoxic enterotoxin of Aeromonas hydrophila induces proinflammatory cytokine production and activates arachidonic acid metabolism in macrophages. Infect Immun,2000,68:2808.
[33]Villamon E, Villalba V, Nogueras MM, Tomas JM, Gozalbo D, Gil ML. Glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme present in the periplasm of Aeromonas hydrophila. Anton Leeuw,2003,84:31-8.
[34]Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG. Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol,2002,184:1140-9.
[35]Ang CS, Veith PD, Dashper SG, Reynolds EC. Application of 16O/18O reverse proteolytic labeling to determine the effect of biofilm culture on the cell envelope proteome of Porphyromonas gingivalis W50. Proteomics,2008,8:1645-60.
[36]Planchon S, Desvaux M, Chafsey I, Chambon C, Leroy S, Hebraud M, Talon R. Comparative subproteome analyses of planktonic and sessile Staphylococcus xylosus C2a:new insight in cell physiology of a coagulase-negative Staphylococcus in biofilm. J Proteome Res, 2009,8:1797-809.
[37]Wang D, Calla B, Vimolmangkang S, Wu X, Korban SS, Huber SC, Clough SJ, Zhao Y. The orphan gene ybjN conveys pleiotropic effects on multicellular behavior and survival of Escherichia coli. PLoS One,2011,6:e25293.
[38]Wang D. Regulation of amylovoran biosynthesis in Erwinia amylovora.2011.
[39]Zhao Y, Wang D, Nakka S, Sundin G, Korban S. Systems level analysis of two-component signal transduction systems in Erwinia amylovora:Role in virulence, regulation of amylovoran biosynthesis and swarming motility. BMC genomics,2009,10:245-53.
[40]Suarez G, Sierra JC, Sha J, Wang S, Erova TE, Fadl AA, Foltz SM, Horneman AJ, Chopra AK. Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila. Microb Pathogenesis,2008,44:344-61.
[41]Trevors JT. Viable but non-culturable (VBNC) bacteria:Gene expression in planktonic and biofilm cells. J Microbiol Meth,2011,86:266-73.
[42]Rahman MH, Suzuki S, Kawai K. Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus. Microbiol Res, 2001,156:103-6.
[43]Hanning I, Donoghue DJ, Jarquin R, Kumar GS, Aguiar VF, Metcalf JH, Reyes-Herrera I, Slavik M. Campylobacter biofilm phenotype exhibits reduced colonization potential in young chickens and altered in vitro virulence. Poult Sci,2009,88:1102-7.
[44]Pham TK, Roy S, Noirel J, Douglas I, Wright PC, Stafford GP. A quantitative proteomic analysis of biofilm adaptation by the periodontal pathogen Tannerella forsythia. Proteomics, 2010,10:3130-41.
[45]Tenorio E, Saeki T, Fujita K, Kitakawa M, Baba T, Mori H, Isono K. Systematic characterization of Escherichia coli genes/ORFs affecting biofilm formation. FEMS Microbiol Lett,2003,225:107-14.
[46]Kirov SM, Castrisios M, Shaw JG. Aeromonas flagella (polar and lateral) are enterocyte adhesins that contribute to biofilm formation on surfaces. Infect Immun,2004,72:1939-47.
[47]Santos P, Santos P, Bello A, Freitas Almeida A. Association of Aeromonas caviae polar and lateral flagella with biofilm formation. Lett Appl Microbiol,2010,9:10-9..
[48]Lemon KP, Higgins DE, Kolter R. Flagellar motility is critical for Listeria monocytogenes biofilm formation. J Bacteriol,2007,189:4418-26.
[49]Watnick PI, Lauriano CM, Klose KE, Croal L, Kolter R. The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139. Mol Microbiol,2001,39:223-35.
[50]Wolfe AJ, Millikan DS, Campbell JM, Visick KL. Vibrio fischeri 54 controls motility, biofilm formation, luminescence, and colonization. Appl Environ Microbiol,2004,70:2520-9.
[51]Ho HT, Lipman LJ, Wosten MM, van Asten AJ, Gaastra W. Arcobacter spp. possess two very short flagellins of which FlaA is essential for motility. FEMS Immunol Med Microbiol, 2008,53:85-95.
[52]Costello GM, Vipond R, MacIntyre S. Aeromonas salmonicida possesses two genes encoding homologs of the major outer membrane protein, OmpA. J Bacteriol,1996,178:1623-31.
[53]Orme R, Douglas CW, Rimmer S, Webb M. Proteomic analysis of Escherichia coli biofilms reveals the overexpression of the outer membrane protein OmpA. Proteomics,2006,6:4269-77.
[54]Namba A, Mano N, Takano H, Beppu T, Ueda K, Hirose H. OmpA is an adhesion factor of Aeromonas veronii, an optimistic pathogen that habituates in carp intestinal tract. J Appl Mcrobiol,2008,105:1441-51.
[55]Bordi C, de Bentzmann S. Hacking into bacterial biofilms:a new therapeutic challenge. Ann Intensive Care,2011,1:19-27.
[56]Brady RA, O'May GA, Leid JG, Prior ML, Costerton JW, Shirtliff ME. Resolution of Staphylococcus aureus biofilm infection using vaccination and antibiotic treatment. Infect Immun,2011,79:1797-803.
[57]Chen Z, Peng B, Wang S, Peng X. Rapid screening of highly efficient vaccine candidates by immunoproteomics. Proteomics,2004,4:3203-13.
[1]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin Microbiol Rev,2010,23:35-73.
[2]Parker JL, Shaw JG. Aeromonas spp. clinical microbiology and disease. J Infection, 2011,62:109-18.
[3]Agger WA, McCormick J, Gurwith MJ. Clinical and microbiological features of Aeromonas hydrophila-associated diarrhea. J Clin Microbiol,1985,21:909-18.
[4]Kao HT, Huang YC, Lin TY. Fatal bacteremic pneumonia caused by Aeromonas hydrophila in a previously healthy child. J Microbiol Immunol,2003,36:209-11.
[5]Vally H, Whittle A, Cameron S, Dowse GK, Watson T. Outbreak of Aeromonas hydrophila wound infections associated with mud football. Clin Infect Dis,2004,38:1084-95.
[6]Ni XD, Wang N, Liu YJ, Lu CP. Immunoproteomics of extracellular proteins of the Aeromonas hydrophila China vaccine strain J-1 reveal a highly immunoreactive outer membrane protein. FEMS Immunol Med Microbiol,2010,58:363-73.
[7]Kirov SM, Tassell BC, Semmler ABT, ODonovan LA, Rabaan AA, Shaw JG. Lateral flagella and swarming motility in Aeromonas species. JBacteriol,2002,184:547-56.
[8]Sambrook J, Fritsch E, Maniatis T. Molecular cloning:a laboratory manual, Spring Harbor Laboratory Press. Cold Spring Harbor, NY; 1989.
[9]Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem,1976,72:248-54.
[10]Farfan M, Minana-Galbis D, Fuste MC, Loren JG. Divergent evolution and purifying selection of the flaA gene sequences in Aeromonas. Biol Direct,2009,4:23-31.
[11]Umelo E, Trust TJ. Identification and molecular characterization of two tandemly located flagellin genes from Aeromonas salmonicida A449. JBacteriol,1997,179:5292-9.
[12]Yu HB, Zhang YL, Lau YL, Yao F, Vilches S, Merino S, Tomas JM, Howard SP, Leung KY. Identification and characterization of putative virulence genes and gene clusters in Aeromonas hydrophila PPD134/91. Appl Environ Microbiol,2005,71:4469-77.
[13]Esteve C, Alcaide E, Canals R, Merino S, Blasco D, Figueras MJ, Tomas JM. Pathogenic Aeromonas hydrophila serogroup 0:14 and 0:81 strains with an S layer. Appl Environ Microbiol,2004,70:5898-904.
[14]Maiti B, Raghunath P, Karunasagar I. Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp. J Appl Microbiol,2009,107:1157-67.
[15]Weiser JN, Gotschlich EC. Outer membrane protein A (OmpA) contributes to serum resistance andpathogenicity of Escherichia coli K-1. Infect Immun,1991,59:2252-61.
[16]Fito-Boncompte L, Chapalain A, Bouffartigues E, Chaker H, Lesouhaitier O, Gicquel G, Bazire A, Madi A, Connil N, Veron W. Full virulence of Pseudomonas aeruginosa requires OprF. Infect Immun,2011,79:1176-84.
[17]Bartra SS, Gong X, Lorica CD, Jain C, Nair MKM, Schifferli D, Qian L, Li Z, Plano GV, Schesser K. The outer membrane protein A (OmpA) of Yersinia pestis promotes intracellular survival and virulence in mice. Microbl Pathogenesis,2011,9:167-74.
[18]Duperthuy M, Binesse J, Le Roux F, Romestand B, Caro A, Got P, Givaudan A, Mazel D, Bachere E, Destoumieux-Garz6n D. The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas. Environ Microbiol,2010,12:951-63.
[19]Xu'ai L, Aihua S, Ping R, Zhe Z, Jie Y. Characterization of conserved combined T and B cell epitopes in Leptospira interrogans major outer membrane proteins OmpL1 and LipL41. BMC Microbiol,2011,11:21-9.
[20]Danese PN, Pratt LA, Dove SL, Kolter R. The outer membrane protein, Antigen 43, mediates cell to cell interactions within Escherichia coli biofilms. Mol Microbiol,2000,37:424-32.
[21]MacIntyre S, Trust T, Buckley J. Identification and characterization of outer membrane fragments released by Aeromonas sp. CanJBiochem,1980,58:1018-25.
[22]Vazquez - Juarez R, Barrera-Saldafia H, Hernandez - Saavedra N, Gomez - Chiarri M, Ascencio F. Molecular cloning, sequencing and characterization of omp48, the gene encoding for an antigenic outer membrane protein from Aeromonas veronii. J Appl Microbiol, 2003,94:908-18.
[23]Loeb MR, Kilner J. Release of a special fraction of the outer membrane from both growing and phage T4-infected Escherichia coli B. BBA-Biomembranes,1978,514:117-27..
[25]Merino S, Vilches S, Canals R, Ramirez S, Tomas JM. A C1q-binding 40kDa porin from Aeromonas salmonicida:cloning, sequencing, role in serum susceptibility and fish immunoprotection. Microb Pathogenesis,2005,38:227-37.
[1]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin MicrobiolRev,2010,23:35-73.
[2]Parker JL., Shaw JG Aeromonas spp. clinical microbiology and disease. J Infection, 20101,62:109-18.
[3]Agger WA, McCormick J, Gurwith MJ. Clinical and microbiological features of Aeromonas hydrophila-associated diarrhea. J Clin Microbiol,1985,21:909-18.
[4]Kao HT, Huang YC, Lin TY. Fatal bacteremic pneumonia caused by Aeromonas hydrophila in a previously healthy child. J Microbiol Immunol,2003,36:209-11.
[5]Vally H, Whittle A, Cameron S, Dowse GK, Watson T. Outbreak of Aeromonas hydrophila wound infections associated with mud football. Clin Infect Dis,2004,38:1084-95.
[6]Ni XD, Wang N, Liu YJ, Lu CP. Immunoproteomics of extracellular proteins of the Aeromonas hydrophila China vaccine strain J-1 reveal a highly immunoreactive outer membrane protein. FEMS Immunol Med Microbiol,2010,58:363-73.
[7]Lin J, Huang S, Zhang Q. Outer membrane proteins:key players for bacterial adaptation in host niches. Microbes Infect,2002,4:325-31.
[8]Maiti B, Raghunath P, Karunasagar I. Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp. J Appl Microbiol,2009,107:1157-67.
[9]Maji S, Mali P, Joardar SN. Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.). Fish Shellfish Immunol,2006,20:462-73.
[10]Khushiramani R, Girisha SK, Karunasagar I. Cloning and expression of an outer membrane protein ompTS of Aeromonas hydrophila and study of immunogenicity in fish. Protein Expr Purif,2007,51:303-7.
[11]Guan R, Xiong J, Huang W, Guo S. Enhancement of protective immunity in European eel (Anguilla anguilla) against Aeromonas hydrophila and Aeromonas sobria by a recombinant Aeromonas outer membrane protein. Acta Biochim Biophys Sin (Shanghai),2011,43:79-88.
[12]Khushiramani R, Girisha SK, Karunasagar I. Protective efficacy of recombinant OmpTS protein of Aeromonas hydrophila in Indian major carp. Vaccine,2007,25:1157-8.
[13]Merino S, Vilches S, Canals R, Ramirez S, Tomas JM. A Clq-binding 40kDa porin from Aeromonas salmonicida:Cloning, sequencing, role in serum susceptibility and fish immunoprotection. Microbl Pathogenesis,2005,38:227-37.
[14]Daly J, Moore A, Olivier G. A colorimetric assay for the quantification of brook trout (Salvelinus fontinalis) lymphocyte mitogenesis. Fish Shellfish Immunol,1995,5:265-73.
[15]Maji S, Mali P, Joardar S. Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.). Fish Shellfish Immunol,2006,20:462-73.
[16]Murray JS. How the MHC selects Thl/Th2 immunity. Immunol Today,1998,19:157-62.
[17]McGuirk P, Mills KHG. Pathogen-specific regulatory T cells provoke a shift in the Thl/Th2 paradigm in immunity to infectious diseases. Trends Immunol,2002,23:450-5.
[18]Parronchi P, De Carli M, Manetti R, Simonelli C, Sampognaro S, Piccinni M, Macchia D, Maggi E, Del Prete G, Romagnani S. IL-4 and IFN (alpha and gamma) exert opposite regulatory effects on the development of cytolytic potential by Thl or Th2 human T cell clones. J Immunol, 1992,149:2977-83.
[19]Manetti R, Parronchi P, Giudizi MQ Piccinni M, Maggi E, Trinchieri G, Romagnani S. Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Thl)-specific immune responses and inhibits the development of IL-4-producing Th cells. J Exp Med, 1993,177:1199-204.
[20]Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-y:an overview of signals, mechanisms and functions. J Leukocyte biol,2004,75:163-89.
[21]Schoenborn JR, Wilson CB. Regulation of Interferon-y During Innate and Adaptive Immune Responses. Adv Immunol,2007,96:41-101.
[22]Walton CB, Inos ABH, Andres OA, Jube S, Couet HG Douglas JT, Patek PQ, Borthakur D. Immunization with hybrid recombinant Mycobacterium tuberculosis H37Rv proteins increases the TH1 cytokine response in mice following a pulmonary instillation of irradiated mycobacteria. Vaccine,2008,26:4396-402.
[23]Hamdy S, Molavi O, Ma Z, Haddadi A, Alshamsan A, Gobti Z, Elhasi S, Samuel J, Lavasanifar A. Co-delivery of cancer-associated antigen and Toll-like receptor 4 ligand in PLGA nanoparticles induces potent CD8+T cell-mediated anti-tumor immunity. Vaccine, 2008,26:5046-57.
[24]Beckman JS, Carson M, Smith CD, Koppenol WH. ALS, SOD and peroxynitrite. Nature, 1993,364:584-92.
[25]Wilhelm FD. Fish antioxidant defenses--a comparative approach. Braz J Med Biol Res, 1996,29:1735-43.
[26]Pascual P, Pedrajas J, Toribio F, Lopez-Barea J, Peinado J. Effect of food deprivation on oxidative stress biomarkers in fish(Sparus aurata). Chem Biol Interact,2003,145:191-9.
[27]Sharp G, Secombes C. The role of reactive oxygen species in the killing of the bacterial fish pathogen Aeromonas salmonicida by rainbow trout macrophages. Fish Shellfish Immunol, 1993,3:119-29.
[1]Parker JL, Shaw JG Aeromonas spp. clinical microbiology and disease. J Infection, 2011,62:109-18.
[2]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin Microbiol Rev,2010,23:35-73.
[3]Asha A, Nayak DK, Shankar KM, Mohan CV. Antigen expression in biofilm cells of Aeromonas hydrophila employed in oral vaccination of fish. Fish Shellfish Immunol,2004,16:429-36.
[4]Azad IS, Shankar KM, Mohan CV, Kalita B. Uptake and processing of biofilm and free-cell vaccines of Aeromonas hydrophila in indian major carps and common carp following oral vaccination Dis Aquat Organ,2000,43:103-8.
[5]Simoes M, Simoes LC, Vieira MJ. A review of current and emergent biofilm control strategies. LWT-Food Sci Technol,2010,43:573-83.
[6]Esteve C, Alcaide E, Canals R, Merino S, Blasco D, Figueras MJ, Tomas JM. Pathogenic Aeromonas hydrophila serogroup O:14 and O:81 strains with an S layer. Appl Environ Microbiol, 2004,70:5898-904.
[7]Weiser JN, Gotschlich EC. Outer membrane protein A (OmpA) contributes to serum resistance and pathogenicity of Escherichia coli K-1. Infect Immun,1991,59:2252-64.
[8]Fito-Boncompte L, Chapalain A, Bouffartigues E, Chaker H, Lesouhaitier O, Gicquel G, Bazire A, Madi A, Connil N, Veron W. Full virulence of Pseudomonas aeruginosa requires OprF. Infect Immun,2011,79:1176-85.
[9]Bartra SS, Gong X, Lorica CD, Jain C, Nair MKM, Schifferli D, Qian L, Li Z, Piano GV, Schesser K. The outer membrane protein A (OmpA) of Yersinia pestis promotes intracellular survival and virulence in mice. Microb Pathogenesis,2011,12:53-65.
[10]Duperthuy M, Binesse J, Le Roux F, Romestand B, Caro A, Got P, Givaudan A, Mazel D, Bachere E, Destoumieux-Garzon D. The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas. Environ Microbiol,2010,12:951-63.
[11]Xu'ai L, Aihua S, Ping R, Zhe Z, Jie Y. Characterization of conservedcombined T and B cell epitopes in Leptospira interrogans major outer membrane proteins OmpLl and LipL41. BMC Microb iol,2011,11:21-9.
[12]Kirov SM, Castrisios M, Shaw JG Aeromonas flagella (polar and lateral) are enterocyte adhesins that contribute to biofilm formation on surfaces. Infect Immun,2004,72:1939-50.
[13]Ho HT, Lipman LJ, Wosten MM, van Asten AJ, Gaastra W. Arcobacter spp. possess two very short flagellins of which FlaA is essential for motility. FEMS Immunol Med Microbiol, 2008,53:85-95.
[14]Lemon KP, Higgins DE, Kolter R. Flagellar motility is critical for Listeria monocytogenes biofilm formation. JBacteriol,,2007,189:4418-28.
[15]Watnick PI, Lauriano CM, Klose KE, Croal L, Kolter R. The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139. Mol Microbiol,2001,39:223-35.
[16]Wolfe AJ, Millikan DS, Campbell JM, Visick KL. Vibrio fischeri 54 controls motility, biofilm formation, luminescence, and colonization. Appl Environ Microbiol,,2004,70:2520-29.
[17]Zhu J, Chai Y, Zhong Z, Li S, Winans SC. Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules:detection of autoinducers in Mesorhizobium huakuii. Appl Environ Microbiol,2003,69:6949-53.
[18]Wang RF, Kushner SR. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene,1991,100:195-9.
[19]陆承平.兽医微生物学.第四版：中国农业出版社,2007,p.31-2.
[20]Quandt J, Hynes MF. Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. Gene,1993,127:15-21.
[1]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin Mcrobiol Rev,2010,23:35-73.
[2]Parker JL, Shaw JG. Aeromonas spp. clinical microbiology and disease. J Infection, 2011,62:109-18.
[3]陈怀青,陆承平.家养鲤科鱼暴发性传染病的病原研究.南京农业大学学报,1991：87－91.
[4]Lee S, Yin Z, Ge R, Sin Y. Isolation and characterization of fish Aeromonas hydrophila adhesins important for in vitro epithelial cell invasion. J Fish Dis,1997,20:169-75.
[5]Buckley JT, Peter S, Chopra A, Houston C. The cytotoxic enterotoxin of Aeromonas hydrophila is aerolysin. Infect Immun,1999,67:466-78.
[6]Upadhyaya T, Singh RK, Dixit A. Molecular cloning and sequence analysis of lamB encoding outer membrane maltose-inducible porin of Aeromonas hydrophila. Mitochondr DNA, 2007,18:302-6.
[7]Simoes M, Simoes LC, Vieira MJ. A review of current and emergent biofilm control strategies. LWT-Food Sci Technol,2010,43:573-83.
[8]Ling H, Kang A, Tan MH, Qi X, Chang MW. The absence of the luxS gene increases swimming motility and flagella synthesis in Escherichia coli K12. Biochem Bioph Res Co,2010,401:521-6.
[9]Huang Z, Meric G, Liu Z, Ma R, Tang Z, Lejeune P.luxS-based quorum-sensing signaling affects Biofilm formation in Streptococcus mutans. JMol Mcrobiol Biotechnol,2009,17:12-9.
[10]Weiser JN, Gotschlich EC. Outer membrane protein A (OmpA) contributes to serum resistance and pathogenicity of Escherichia coli K-1. Infect Immun,1991,59:2252-63.
[11]Rabaan AA, Gryllos I, Tomas JM, Shaw JG. Motility and the polar flagellum are required for Aeromonas caviae adherence to HEp-2 cells. Infect Immun,2001,69:4257-68.
[12]Kirov SM, Castrisios M, Shaw JG. Aeromonas flagella (polar and lateral) are enterocyte adhesins that contribute to biofilm formation on surfaces. Infect Immun,2004,72:1939-48.
[13]Bartra SS, Gong X, Lorica CD, Jain C, Nair MKM, Schifferli D, Qian L, Li Z, Plano GV, Schesser K. The outer membrane protein A (OmpA) of Yersinia pestis promotes intracellular survival and virulence in mice. Microb Pathogenesis,2011,12:53-65.
[14]Duperthuy M, Binesse J, Le Roux F, Romestand B, Caro A, Got P, Givaudan A, Mazel D, Bachere E, Destoumieux-Garzon D. The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas. Environ Microbiol,2010,12:951-63.
[15]Lemon KP, Higgins DE, Kolter R. Flagellar motility is critical for Listeria monocytogenes biofilm formation. J Bacteriol,2007,189:4418-27.
[16]Watnick PI, Lauriano CM, Klose KE, Croal L, Kolter R. The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae 0139. Mol Microbiol,2001,39:223-35.
[17]Wolfe AJ, Millikan DS, Campbell JM, Visick KL. Vibrio fischeri controls motility, biofilm formation, luminescence, and colonization. Appl Environ Microbiol,,2004,70:2520-8.
[18]Aeckersberg F, Lupp C, Feliciano B, Ruby E. Vibrio fischeri outer membrane protein OmpU plays a role in normal symbiotic colonization. JBacteriol,2001,183:6590-9.
[19]Fito-Boncompte L, Chapalain A, Bouffartigues E, Chaker H, Lesouhaitier O, Gicquel G, Bazire A, Madi A, Connil N, Veron W. Full virulence of Pseudomonas aeruginosa requires OprF. Infect Immun,2011,79:1176-85.
[20]张小军.嗜水气单胞菌J-1株luxS基因缺失株的构建及特性分析：南京农业大学；2009.
[21]Bassler BL, Wright M, Silverman MR. Multiple signalling systems controlling expression of luminescence in Vibrio harveyi:sequence and function of genes encoding a second sensory pathway. Mol Microbiol,1994,13:273-86.
[22]Bumann D, Holland P, Siejak F, Koesling J, Sabarth N, Lamer S, Zimny-Arndt U, Jungblut PR, Meyer TF. A comparison of murine and human immunoproteomes of Helicobacter pylori validates the preclinical murine infection model for antigen screening. Infect Immun, 2002,70:6494-8.
[23]Ju Z, Karsi A, Kocabas A, Patterson A, Li P, Cao D, Dunham R, Liu Z. Transcriptome analysis of channel catfish (Ictalurus punctatus):genes and expression profile from the brain. Gene, 2000,261:373-82.
[24]Erova TE, Pillai L, Fadl AA, Sha J, Wang S, Galindo CL, Chopra AK. DNA adenine methyltransferase influences the virulence of Aeromonas hydrophila. Infect Immun, 2006,74:410-24.
[25]Swift S, Lynch MJ, Fish L, Kirke DF, Tomas JM, Stewart GS, Williams P. Quorum sensing-dependent regulation and blockade of exoprotease production in Aeromonas hydrophila. Infect Immun,1999,67:5192-9.
[26]Kozlova EV, Popov VL, Sha J, Foltz SM, Erova TE, Agar SL, Horneman AJ, Chopra AK. Mutation in the S-ribosylhomocysteinase (luxS) gene involved in quorum sensing affects biofilm formation and virulence in a clinical isolate of Aeromonas hydrophila. Microb Pathogenesis,2008,45:343-54.
[27]Kirov SM, Tassell BC, Semmler ABT, O'Donovan LA, Rabaan AA, Shaw JG. Lateral flagella and swarming motility in Aeromonas species. JBacteriol,2002,184:547-58.
[28]Sha J, Pillai L, Fadl AA, Galindo CL, Erova TE, Chopra AK. The type Ⅲ secretion system and cytotoxic enterotoxin alter the virulence of Aeromonas hydrophila. Infect Immun, 2005,73:6446-57.
[29]Costello GM, Vipond R, Maclntyre S. Aeromonas salmonicida possesses two genes encoding homologs of the major outer membrane protein, OmpA. JBacteriol,1996,178:1623-32.
[30]Namba A, Mano N, Takano H, Beppu T, Ueda K, Hirose H. OmpA is an adhesion factor of Aeromonas veronii, an optimistic pathogen that habituates in carp intestinal tract. J Appl Microbiol,2008,105:1441-51.
[31]Orme R, Douglas CW, Rimmer S, Webb M. Proteomic analysis of Escherichia coli biofilms reveals the overexpression of the outer membrane protein OmpA. Proteomics,2006,6:4269-77.
[32]Santos P, Santos P, Bello A, Freitas Almeida A. Association of Aeromonas caviae polar and lateral flagella with biofilm formation. Lett Appl Microbiol,2010,9:10-9.
[33]Wolfe AJ, Millikan DS, Campbell JM, Visick KL. Vibrio fischeri controls motility, biofilm formation, luminescence, and colonization. Appl Environ Microbiol,2004,70:2520-32.
[34]Ho HT, Lipman LJ, Wosten MM, van Asten AJ, Gaastra W. Arcobacter spp. possess two very short flagellins of which FlaA is essential for motility. FEMS Immunol Med Microbiol, 2008,53:85-95.
[35]Pagel M, Simonet V, Li J, Lallemand M, Lauman B, Delcour AH. Phenotypic characterization of pore mutants of the Vibrio cholerae porin OmpU. JBacteriol,2007,189:8593-600.
[36]Tsai YK, Fung CP, Lin JC, Chen JH, Chang FY, Chen TL, Siu LK. The Klebsiella pneumoniae outer membrane porins OmpK35 and OmpK36 play roles in both antimicrobial resistance and virulence. JAntimicrob Chemother,2011:AAC.01275-10v1.
[37]Chen JH, Siu LK, Fung CP, Lin JC, Yeh KM, Chen TL, Tsai YK, Chang FY. Contribution of outer membrane protein K36 to antimicrobial resistance and virulence in Klebsiella pneumoniae. JAntimicrob Chemother,2010,65:986-90.
[38]Dooley JS, Trust TJ. Surface protein composition of Aeromonas hydrophila strains virulent for fish:identification of a surface array protein. JBacteriol,1988,170:499-506.
[39]Belland RJ, Trust TJ. Synthesis, export, and assembly of Aeromonas salmonicida A-layer analyzed by transposon mutagenesis. JBacteriol,1985,163:877-81.
[40]Merino S, Nogueras MM, Aguilar A, Rubires X, Alberti S, Benedi VJ, Tomas JM. Activation of the complement classical pathway (Clq binding) by mesophilic Aeromonas hydrophila outer membrane protein. Infect Immun,1998,66:3825-31.
[41]Nogueras MM, Merino S, Aguilar A, Benedi VJ, Tomas JM. Cloning, sequencing, and role in serum susceptibility of porin II from mesophilic Aeromonas hydrophila. Infect Immun, 2000,68:1849-62.
[42]Brackman G, Cos P, Maes L, Nelis HJ, Coenye T. Quorum sensing inhibitors increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivo. Antimicrob Agents Chemother,2011,55:2655-61.
[43]Revel A T NDM, et al. Expression of a luxS gene is not required for Borreliaburgdorferi infection of mice via needle inoculation. Infect Immun,2003,71-8.
[44]Winzer K, Sun YH, Green A, Delory M, Blackley D, Hardie KR, Baldwin TJ, Tang CM. Role of Neisseria meningitidis luxS in cell-to-cell signaling and bacteremic infection. Infect Immun, 2002,70:2245-8.
[45]Merritt J, Qi F, Goodman SD, Anderson MH, Shi W. Mutation of luxS affects biofilm formation in Streptococcus mutans. Infect Immun,2003,71:1972-9.
[46]Huang Z, Meric G, Liu Z, Ma R, Tang Z, Lejeune P.luxS-based quorum-sensing signaling affects biofilm formation in Streptococcus mutans. J Mol Microbiol Biotechnol,2008,5:55-61.
[47]Yoshida A, Ansai T, Takehara T, Kuramitsu HK. LuxS-based signaling affects Streptococcus mutans biofihn formation. Appl Environ Microbiol,2005,71:2372-80.
[48]Zhang M, Sun K, Sun L. Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain. Microbiology,2008,154:2060-9.
[2]Milev-Milovanovic I, Long S, Wilson M, Bengten E, Miller NW, Chinchar VG. Identification and expression analysis of interferon gamma genes in channel catfish. Immunogenetics, 2006,58:70-80.
[3]Costerton JW, Stewart PS, Greenberg EP. Bacterial biofihlms:a common cause of persistent infections. Science,1999,284:1318-27.
[4]Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms:from the natural environment to infectious diseases. Nat Rev Microbiol,2004,2:95-108.
[5]Donlan RM, Costerton JW. Biofilms:survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev,2002,15:167-75.
[6]Monds RD, O'Toole GA. The developmental model of microbial biofilms:ten years of a paradigm up for review. Trends Microbiol,2009,17:73-87.
[7]Van Wey AS, Cookson AL, Soboleva TK, Roy NC, McNabb WC, Bridier A, Briandet R, Shorten PR. Anisotropic nutrient transport in three-dimensional single species bacterial biofilms. Biotechnol Bioeng,2011,12:156-63.
[8]Costerton JW. Introduction to biofilm. Int J Antimicrob Agents,1999,11:217-21.
[9]Costerton JW, Stewart PS. Battling biofilms. Sci Am,2001,285:74-81.
[10]Branda SS, Vik S, Friedman L, Kolter R. Biofilms:the matrix revisited. Trends Microbiol, 2005,13:20-6.
[11]Habash M, Reid G Microbial biofihns:their development and significance for medical device-related infections. JClin Pharmacol,1999,39:887-98.
[12]Hoiby N, Ciofu O, Johansen HK, Song ZJ, Moser C, Jensen PO, Molin S, Givskov M, Tolker-Nielsen T, Bjarnsholt T. The clinical impact of bacterial biofilms. Int J Oral Sci, 2011,3:55-65.
[13]Costerton JW. Overview of microbial biofilms. JInd Microbiol,1995,15:137-40.
[14]Kilty SJ, Duval M, Chan FT, Ferris W, Slinger R. Methylglyoxal:(active agent of manuka honey) in vitro activity against bacterial biofihns. Int Forum Allergy Rhinol,2011,1:348-50.
[15]Jones WL, Sutton MP, McKittrick L, Stewart PS. Chemical and antimicrobial treatments change the viscoelastic properties of bacterial biofilms. Biofouling,2011,27:207-15.
[16]Mah TF, O'Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol,2001,9:34-9.
[17]Simoes M, Simoes LC, Vieira MJ. A review of current and emergent biofilm control strategies. LWT-Food Sci Technol,2010,43:573-83.
[18]Bordi C, de Bentzmann S. Hacking into bacterial biofilms:a new therapeutic challenge. Ann Intensive Care,2011,1:19-26.
[19]Perez-Mendoza D, Coulthurst SJ, Sanjuan J, Salmond GP. N-Acetylglucosamine-dependent biofilm formation in Pectobacterium atrosepticum is cryptic and activated by elevated c-di-GMP levels. Microbiology,2011,157:3340-8.
[20]Liu H, Wang Q, Liu Q, Cao X, Shi C, Zhang Y. Roles of Hfq in the stress adaptation and virulence in fish pathogen Vibrio alginolyticus and its potential application as a target for live attenuated vaccine. Appl Microbiol Biot,2011:1-12.
[21]Fong JCN, Yildiz FH. The rbmBCDEF gene cluster modulates development of rugose colony morphology and biofilm formation in Vibrio cholerae. JBacteriol,2007,189:2319-25.
[22]Jin H, Zhou R, Kang M, Luo R, Cai X, Chen H. Biofilm formation by field isolates and reference strains of Haemophilus parasuis. Vet Microbiol,2006,118:117-23.
[23]Drenkard E, Ausubel FM. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature,2002,416:740-3.
[24]Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science,1994,263:802-11.
[25]Whiteley M, Bangera MG, Bumgarner RE, Parsek MR, Teitzel GM, Lory S, Greenberg EP. Gene expression in Pseudomonas aeruginosa biofilms. Nature,2001,413:860-4.
[26]Shemesh M, Tam A, Steinberg D. Differential gene expression profiling of Streptococcus mutans cultured under biofilm and planktonic conditions. Microbiology,2007,153:1307-17.
[27]Schembri MA, Kjaergaard K, Klemm P. Global gene expression in Escherichia coli biofilms. Mol Microbiol,2003,48:253-67.
[28]Ren D, Bedzyk LA, Thomas SM, Ye RW, Wood TK. Gene expression in Escherichia coli biofilms. Appl Microbiol Biotechnol,2004,64:515-24.
[29]Ren D, Bedzyk LA, Setlow P, Thomas SM, Ye RW, Wood TK. Gene expression in Bacillus subtilis surface biofilms with and without sporulation and the importance of yveR for biofilm maintenance. Biotechnol Bioeng,2004,86:344-64.
[30]Domka J, Lee J, Bansal T, Wood TK. Temporal gene-expression in Escherichia coli K-12 biofilms. Environ Microbiol,2007,9:332-46.
[31]Waite RD, Papakonstantinopoulou A, Littler E, Curtis MA. Transcriptome analysis of Pseudomonas aeruginosa growth:comparison of gene expression in planktonic cultures and developing and mature biofilms. JBacterial,2005,187:6571-6.
[32]Yamamoto R, Noiri Y, Yamaguchi M, Asahi Y, Maezono H, Ebisu S. Time course of gene expression during Porphyromonas gingivalis strain ATCC 33277 biofilm formation. Appl Environ Microbiol,2011,77:6733-6.
[33]He X, Ahn J. Differential gene expression in planktonic and biofilm cells of multiple antibiotic-resistant Salmonella Typhimurium and Staphylococcus aureus. FEMS Microbiol Lett, 2011,325:180-8.
[34]Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. JBacteriol,2002,184:1140-54.
[35]Khan AU, Islam B, Khan SN, Akram M. A proteomic approach for exploring biofilm in Streptococcus mutans. Bioinformation,2011,5:440-5.
[36]Tremoulet F, Duche O, Namane A, Martinie B, Labadie JC. A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth mode. FEMS Microbiol Lett,2002,215:7-14.
[37]Kalmokoff M, Lanthier P, Tremblay TL, Foss M, Lau PC, Sanders G, Austin J, Kelly J, Szymanski CM. Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation. JBacteriol,2006,188:4312-20.
[38]Koerdt A, Orell A, Pham TK, Mukherjee J, Wlodkowski A, Karunakaran E, Biggs CA, Wright PC, Albers SV. Macromolecular fingerprinting of sulfolobus species in biofilm:a transcriptomic and proteomic approach combined with spectroscopic analysis. J Proteome Res, 2011,10:4105-19.
[39]Pham TK, Roy S, Noirel J, Douglas I, Wright PC, Stafford GP. A quantitative proteomic analysis of biofilm adaptation by the periodontal pathogen Tannerella forsythia. Proteomics, 2010,10:3130-41.
[40]Shin JH, Lee HW, Kim SM, Kim J. Proteomic analysis of Acinetobacter baumannii in biofilm and planktonic growth mode. J Microbiol,2009,47:728-35.
[41]Espinal P, Marti S, Vila J. Effect of biofilm formation on the survival of Acinetobacter baumannii on dry surfaces. JHosp Infect,2012,80:56-60.
[42]Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cell Microbiol, 2009,11:1034-43.
[43]Cucarella C, Tormo MA, Ubeda C, Trotonda MP, Monzon M, Peris C, Amorena B, Lasa I, Penades JR. Role of biofilm-associated protein bap in the pathogenesis of bovine Staphylococcus aureus. Infect Immun,2004,72:2177-85.
[44]Norouzi F, Mansouri S, Moradi M, Razavi M. Comparison of cell surface hydrophobicity and biofilm formation among ESBL-and non-ESBL-producing Pseudomonas aeruginosa clinical isolates. Afr JMicrobiol Res,2010,4:1143-7.
[45]Jain A, Agarwal A. Biofilm production, a marker of pathogenic potential of colonizing and commensal staphylococci. J Microbiol Meth,2009,76:88-92.
[46]Holmberg A, Lood R, Morgelin M, Soderquist B, Holst E, Collin M, Christensson B, Rasmussen M. Biofilm formation by Propionibacterium acnes is a characteristic of invasive isolates. Clin MicrobiolInfec,2009,15:787-95.
[47]Deighton M, Borland R, Capstick J. Virulence of Staphylococcus epidermidis in a mouse model: significance of extracellular slime. Epidemiol Infect,1996,117:267-80.
[48]Yamanaka T, Furukawa T, Matsumoto-Mashimo C, Yamane K, Sugimori C, Nambu T, Mori N, Nishikawa H, Walker C, Leung KP. Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17. BMC Microbiol,2009,9:11-19.
[49]Zhang L, Hinz AJ, Nadeau JP, Mah TF. Pseudomonas aeruginosa tssCl links type VI secretion and biofilm-specific antibiotic resistance. JBacteriol,2011,193:5510-3.
[50]Turnock LL, Somers EB, Faith NG, Czuprynski CJ, Lee WA. The effects of prior growth as a biofilm on the virulence of Salmonella typhimurium for mice. Comp Immunol Microbiol Infect Dis,2002,25:43-8.
[51]Puttamreddy S, Minion FC. Linkage between cellular adherence and biofilm formation in Escherichia coli O157-H7 EDL933. FEMS Microbiol Lett,2011,315:46-53.
[52]Klausen M, Heydorn A, Ragas P, Lambertsen L, Aaes-Jorgensen A, Molin S, Tolker-Nielsen T. Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol,2003,48:1511-24.
[53]Naves P, del Prado G, Huelves L, Gracia M, Ruiz V, Blanco J, Dahbi G, Blanco M, Ponte Mdel C, Soriano F. Correlation between virulence factors and in vitro biofilm formation by Escherichia coli strains. Microb Pathog,2008,45:86-91.
[54]Simoes M. Antimicrobial strategies effective against infectious bacterial biofilms. Curr Med Chem,2011,18:2129-45.
[55]Goodman SD, Obergfell KP, Jurcisek JA, Novotny LA, Downey JS, Ayala EA, Tjokro N, Li B, Justice SS, Bakaletz LO. Biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid-associated proteins. Mucosal Immunol,2011,4:625-37.
[56]Brackman G, Cos P, Maes L, Nelis HJ, Coenye T. Quorum sensing inhibitors increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivo. Antimicrob Agents Chemother,2011,55:2655-61.
[57]Ma Q, Yang Z, Pu M, Peti W, Wood TK. Engineering a novel c-di-GMP-binding protein for biofilm dispersal. Environ Microbiol,2011,13:631-42.
[58]Lynch MJ, Swift S, Kirke DF, Keevil CW, Dodd CE, Williams P. The regulation of biofilm development by quorum sensing in Aeromonas hydrophila. Environ Microbiol,2002,4:18-28.
[59]Khajanchi BK, Sha J, Kozlova EV, Erova TE, Suarez G, Sierra JC, Popov VL, Horneman AJ, Chopra AK. N-acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila. Microbiology,2009,155:3518-31.
[60]Kozlova EV, Popov VL, Sha J, Foltz SM, Erova TE, Agar SL, Horneman AJ, Chopra AK. Mutation in the S-ribosylhomocysteinase (luxS) gene involved in quorum sensing affects biofilm formation and virulence in a clinical isolate of Aeromonas hydrophila. Microb Pathogenesis,2008,45:343-54.
[61]Azad IS, Shankar KM, Mohan CV, Kalita B. Uptake and processing of biofilm and free-cell vaccines of Aeromonas hydrophila in indian major carps and common carp following oral vaccination Dis Aquat Organ,2000,43:103-8.
[62]Nayak DK, Asha A, Shankar KM, Mohan CV. Evaluation of biofilm of Aeromonas hydrophila for oral vaccination of Clarias batrachus--a carnivore model. Fish Shellfish Immunol, 2004,16:613-9.
[63]Asha A, Nayak DK, Shankar KM, Mohan CV. Antigen expression in biofilm cells of Aeromonas hydrophila employed in oral vaccination of fish. Fish Shellfish Immunol,2004,16:429-36.
[1]Parker JL, Shaw JG. Aeromonas spp. clinical microbiology and disease. J Infection, 2011,62:109-18..
[2]Agger WA, McCormick J, Gurwith MJ. Clinical and microbiological features of Aeromonas hydrophila-associated diarrhea. J Clin Mcrobiol,1985,21:909.
[3]Bogdanovi R, obelji M, Markovi M, Nikoli V, Ognjanovi M, Sarjanovi L, Maki D. Haemolytic-uraemic syndrome associated with Aeromonas hydrophila enterocolitis. Pediatr Nephrol,1991,5:293-5.
[4]Kao HT, Huang YC, Lin TY. Fatal bacteremic pneumonia caused by Aeromonas hydrophila in a previously healthy child. JMicrobiol Immunol,2003,36:209-11.
[5]Mukhopadhyay C, Bhargava A, Ayyagari A. Aeromonas hydrophila and aspiration pneumonia:a diverse presentation. Yonsei Med J,2003,44:1087-90.
[6]Vally H, Whittle A, Cameron S, Dowse GK, Watson T. Outbreak of Aeromonas hydrophila wound infections associated with mud football. Clin Infect Dis,2004,38:1084.
[7]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin Mcrobiol Rev 2010,23:35.
[8]Dooley JS, Trust TJ. Surface protein composition of Aeromonas hydrophila strains virulent for fish:identification of a surface array protein. JBacteriol,1988,170:499-506.
[9]孙建和,严亚贤,陈怀青,陆承平.温度对嗜水气单胞菌外膜蛋白表达的影响.中国兽医学报,1999：555-8.
[10]Fang HM, Ge R, Sin YM. Cloning, characterisation and expression of Aeromonas hydrophila major adhesin. Fish Shellfish Immunol,2004,16:645-58.
[11]Lee S, Yin Z, Ge R, Sin Y. Isolation and characterization of fish Aeromonas hydrophila adhesins important for in vitro epithelial cell invasion. JFish Dis,1997,20:169-75.
[12]Bogaerts A, Temmerman L, Boerjan B, Husson S, Schoofs L, Verleyen P. A differential proteomics study of Caenorhabditis elegans infected with Aeromonas hydrophila. Dev Comp Immunol,2010,10:69-74..
[13]Upadhyaya T, Singh RK, Dixit A. Molecular cloning and sequence analysis of lamB encoding outer membrane maltose-inducible porin of Aeromonas hydrophila. DNA Seq,2007,18:302-6.
[14]Vazquez- Juarez R, Romero M, Ascencio F. Adhesive properties of a LamB - like outer - membrane protein and its contribution to Aeromonas veronii adhesion. JAppl Microbiol, 2004,96:700-8.
[15]Jiang B, Howard SP. The Aeromonas hydrophila exeE gene, required both for protein secretion and normal outer membrane biogenesis, is a member of a general secretion pathway. Mol Microbiol,1992,6:1351-61.
[16]Pillai L, Sha J, Erova TE, Fadl AA, Khajanchi BK, Chopra AK. Molecular and functional characterization of a ToxR-regulated lipoprotein from a clinical isolate of Aeromonas hydrophila. Infect Immun,2006,74:3742-55.
[17]Maji S, Mali P, Joardar S. Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.). Fish Shellfish Immunol,2006,20:462-73.
[18]Rahman MH, Kawai K. Outer membrane proteins of Aeromonas hydrophila induce protective immunity in goldfish. Fish Shellfish Immunol,2000,10:379-82.
[19]Maiti B, Raghunath P, Karunasagar I. Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp. J Appl Microbiol,2009,107:1157-67.
[20]Khushiramani R, Girisha SK, Karunasagar I. Protective efficacy of recombinant OmpTS protein of Aeromonas hydrophila in Indian major carp. Vaccine,2007,25:1157-8.
[21]Khushiramani RM, Maiti B, Shekar M, Girisha S, Akash N, Deepanjali A, Karunasagar I. Recombinant Aeromonas hydrophila outer membrane protein 48 (Omp48) induces a protective immune response against Aeromonas hydrophila and Edwardsiella tarda. Res Microbiol,2012.
[22]Merino S, Vilches S, Canals R, Ramirez S, Tomas JM. A Clq-binding 40kDa porin from Aeromonas salmonicida:Cloning, sequencing, role in serum susceptibility and fish immunoprotection. Microb Pathogenesis,2005,38:227-37.
[23]Guan R, Xiong J, Huang W, Guo S. Enhancement of protective immunity in European eel (Anguilla anguilla) against Aeromonas hydrophila and Aeromonas sobria by a recombinant Aeromonas outer membrane protein. Acta Biochim Biophys Sin (Shanghai),2011,43:79-88.
[24]Stintzi A, Raymond KN. Amonabactin-mediated iron acquisition from transferrin and lactoferrin by Aeromonas hydrophila:direct measurement of individual microscopic rate constants. J Bio Inorg Chem,2000,5:57-66.
[25]Jeanteur D, Gletsu N, Pattus F, Buckley JT. Purification of Aeromonas hydrophila major outer-membrane proteins:N-terminal sequence analysis and channel-forming properties. Mol Microbiol,1992,6:3355-63.
[26]Merino S, Nogueras MM, Aguilar A, Rubires X, Alberti S, Benedi VJ, Tomas JM. Activation of the complement classical pathway (Clq binding) by mesophilic Aeromonas hydrophila outer membrane protein. Infect Immun,1998,66:3825-31.
[27]Nogueras MM, Merino S, Aguilar A, Benedi VJ, Tomas JM. Cloning, sequencing, and role in serum susceptibility of porin II from mesophilic Aeromonas hydrophila. Infect Immun, 2000,68:1849.
[28]吕爱军,李槿年,余为一,屈艾,温洪宇.8株嗜温气单胞菌的外膜蛋白型及其聚类分析.中国兽医学报,2005：157-9.
[29]董传甫,林天龙,俞伏松,龚晖.嗜水气单胞菌细胞外膜蛋白及S层蛋白分析.中国水产科学,2003：201-5.
[30]Chhabra G, Upadhyaya T, Dixit A. Molecular cloning, sequence analysis and structure modeling of OmpR, the response regulator of Aeromonas hydrophila. Mol Biol Rep,2012,39:41-50.
[1]Parker JL, Shaw JG. Aeromonas spp. clinical microbiology and disease. J Infection, 2011,62:109-18.
[2]Agger WA, McCormick J, Gurwith MJ. Clinical and microbiological features of Aeromonas hydrophila-associated diarrhea. JClin Microbiol,1985,21:909-18.
[3]Bogdanovi R, obelji M, Markovi M, Nikoli V, Ognjanovi M, Sarjanovi L, Maki D. Haemolytic-uraemic syndrome associated with Aeromonas hydrophila enterocolitis. Pediatr Nephrol,1991,5:293-5.
[4]Kao HT, Huang YC, Lin TY. Fatal bacteremic pneumonia caused by Aeromonas hydrophila in a previously healthy child. J Microbiol Immunol,2003,36:209-11.
[5]Mukhopadhyay C, Bhargava A, Ayyagari A. Aeromonas hydrophila and aspiration pneumonia:a diverse presentation. Yonsei Med J,2003,44:1087-90.
[6]Vally H, Whittle A, Cameron S, Dowse GK, Watson T. Outbreak of Aeromonas hydrophila wound infections associated with mud football. Clin Infect Dis,2004,38:1084-92.
[7]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin Microbiol Rev,2010,23:35-42.
[8]Chauret C, Volk C, Creason R, Jarosh J, Robinson J, Warnes C. Detection of Aeromonas hydrophila in a drinking-water distribution system:a field and pilot study. Can J Microbiol, 2001,47:782-6.
[9]Monds RD, O'Toole GA. The developmental model of microbial biofilms:ten years of a paradigm up for review. Trends Microbiol,2009,17:73-87.
[10]Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms:from the natural environment to infectious diseases. Nat Rev Microbiol,2004,2:95-108.
[11]Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms:a common cause of persistent infections. Science,1999,284:1318-25.
[12]Sim es M, Sim es LC, Vieira MJ. A review of current and emergent biofilm control strategies. LWT-Food Sci Technol,2010,43:573-83.
[13]Stepanovic S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Meth,2000,40:175-9.
[14]Watnick PI, Kolter R. Steps in the development of a Vibrio cholerae El Tor biofilm. Mol Microbiol,1999,34:586-95.
[15]Fengqing H, Song Y. Electroporation-mediated transformation of Aeromonas hydrophila. Plasmid,2005,54:283-7.
[16]Neely M, Pfeifer J, Caparon M. Streptococcus-zebrafish model of bacterial pathogenesis. Infect Immun,2002,70:3904.
[17]Reed L, Muench H. A simple method of estimating fifty per cent endpoints. Am J Epidemiol, 1938,27:493-7.
[18]Potera C. Forging a link between biofilms and disease. Science,1999,283:1837-43.
[19]Davey ME, OToole G A. Microbial biofilms:from ecology to molecular genetics. Microbiol Mol Biol Rev,2000,64:847-67.
[20]Drenkard E, Ausubel FM. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature,2002,416:740-3.
[21]Trevors JT. Viable but non-culturable (VBNC) bacteria:Gene expression in planktonic and biofilm cells. J Microbiol Meth,2011,86:266-73.
[22]Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science,1994,263:802-11.
[23]Tsien RY. The green fluorescent protein. Annu Rev Biochem,1998,67:509-44.
[24]van der Sar AM, Appelmelk BJ, Vandenbroucke-Grauls CMJE, Bitter W. A star with stripes: zebrafish as an infection model. Trends Microbiol,2004,12:451-7.
[25]Wu Z, Zhang W, Lu Y, Lu C. Transcriptome profiling of zebrafish infected with Streptococcus suis. Microb pathogenesis,2010,48:178-87.
[26]Li J, Ni X, Liu Y, Lu C. Detection of three virulence genes alt, ahp and aerA in Aeromonas hydraphila and their relationship with actual virulence to zebrafish. J Appl Microbiol, 2011,316:160-8.
[27]Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cell Microbiol, 2009,11:1034-43.
[28]Cucarella C, Tormo MA, Ubeda C, Trotonda MP, Monzon M, Peris C, Amorena B, Lasa I, Penades JR. Role of biofilm-associated protein bap in the pathogenesis of bovine Staphylococcus aureus. Infect Immun,2004,72:2177-85.
[29]Norouzi F, Mansouri S, Moradi M, Razavi M. Comparison of cell surface hydrophobicity and biofilm formation among ESBL-and non-ESBL-producing Pseudomonas aeruginosa clinical isolates. Afr J Microbiol Res,2010,4:1143-7.
[30]Jain A, Agarwal A. Biofilm production, a marker of pathogenic potential of colonizing and commensal staphylococci. J Microbiol Meth,2009,76:88-92.
[31]Holmberg A, Lood R, Morgelin M, S6derquist B, Hoist E, Collin M, Christensson B, Rasmussen M. Biofilm fonnation by Propionibacterium acnes is a characteristic of invasive isolates. Clin Microbiol Infec,2009,15:787-95.
[32]Deighton M, Borland R, Capsiick J. Virulence of Staphylococcus epidermidis in a mouse model: significance of extracellular slime. Epidemiol Infect,1996,117:267-80.
[33]Yamanaka T, Furukawa T, Matsumoto-Mashimo C, Yamane K, Sugimori C, Nambu T, Mori N, Nishikawa H, Walker C, Leung KP. Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17. BMC Microbiol,2009,9:11-19.
[34]Rahman MH, Suzuki S, Kawai K. Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus. Microbiol Res, 2001,156:103-6.
[1]Parker JL, Shaw JG. Aeromonas spp. clinical microbiology and disease. J Infection,2011, 62:109-18.
[2]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin Microbiol Rev,2010,23:35-73.
[3]Monds RD, O'Toole GA. The developmental model of microbial biofilms:ten years of a paradigm up for review. Trends Microbiol,2009,17:73-87.
[4]Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cell Microbiol, 2009,11:1034-43.
[5]Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms:a common cause of persistent infections. Science,1999,284:1318-25.
[6]Fux C, Costerton J, Stewart P, Stoodley P. Survival strategies of infectious biofilms. Trends Microbiol,2005,13:34-40.
[7]Yu HB, Zhang YL, Lau YL, Yao F, Vilches S, Merino S, Tomas JM, Howard SP, Leung KY. Identification and characterization of putative virulence genes and gene clusters in Aeromonas hydrophila PPD134/91. Appl Environ Microbiol,2005,71:4469-77.
[8]Dooley JS, Trust TJ. Surface protein composition of Aeromonas hydrophila strains virulent for fish:identification of a surface array protein. JBacteriol,1988,170:499-506.
[9]Esteve C, Alcaide E, Canals R, Merino S, Blasco D, Figueras MJ, Tomas JM. Pathogenic Aeromonas hydrophila serogroup 0:14 and 0:81 strains with an S layer. Appl Environ Microbiol,2004,70:5898-904.
[10]Schulz GE. The structure of bacterial outer membrane proteins. BBA-Biomembranes, 2002,1565:308-17.
[11]Lee S, Yin Z, Ge R, Sin Y. Isolation and characterization of fish Aeromonas hydrophila adhesins important for in vitro epithelial cell invasion. JFish Dis,1997,20:169-75.
[12]Buckley JT, Peter S, Chopra A, Houston C. The cytotoxic enterotoxin of Aeromonas hydrophila is aerolysin. Infect Immun,1999,67:466-75.
[13]Upadhyaya T, Singh RK, Dixit A. Molecular cloning and sequence analysis of lamB encoding outer membrane maltose-inducible porin of Aeromonas hydrophila. Mitochondr DNA, 2007,18:302-6.
[14]Maiti B, Raghunath P, Karunasagar I. Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp. J Appl Microbiol,2009,107:1157-67.
[15]Maji S, Mali P, Joardar SN. Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.). Fish Shellfish Immunol,2006,20:462-73.
[16]Khushiramani R, Girisha SK, Karunasagar I. Cloning and expression of an outer membrane protein ompTS of Aeromonas hydrophila and study of immunogenicity in fish. Protein Expr Purif,2007,51:303-7.
[17]Guan R, Xiong J, Huang W, Guo S. Enhancement of protective immunity in European eel (Anguilla anguilla) against Aeromonas hydrophila and Aeromonas sobria by a recombinant Aeromonas outer membrane protein. Acta Biochim Biophys Sin (Shanghai),2011,43:79-88.
[18]Filip C, Fletcher G, Wulff JL, Earhart CF. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. JBacteriol,1973,115:717-22.
[19]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-[Delta][Delta] CT method. Methods,2001,25:402-8.
[20]van Alen T, Claus H, Zahedi RP, Groh J, Blazyca H, Lappann M, Sickmann A, Vogel U. Comparative proteomic analysis of biofilm and planktonic cells of Neisseria meningitidis. Proteomics,2010,8:60-9.
[21]Benard L, Litzler PY, Cosette P, Lemeland JF, Jouenne T, Junter GA. Proteomic analysis of Staphylococcus aureus biofilms grown in vitro on mechanical heart valve leaflets. J Biomed Mater Res A,2009,88:1069-78.
[22]Tremoulet F, Duche O, Namane A, Martinie B, Labadie JC. A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth mode. FEMS Microbiol Lett,2002,215:7-14.
[23]Shin JH, Lee HW, Kim SM, Kim J. Proteomic analysis of Acinetobacter baumannii in biofilm and planktonic growth mode. J Microbiol,2009,47:728-35.
[24]Souza AA, Takita MA, Coletta-Filho HD, Caldana C, Yanai GM, Muto NH, Oliveira RC, Nunes LR, Machado MA. Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro. FEMS Microbiol Lett,2004,237:341-53.
[25]Kim DG, Kim YR, Kim EY, Cho HM, Ann SH, Kong IS. Isolation of the groESL cluster from Vibrio anguillarum and PCR detection targeting groEL gene. Fisheries Sci,2010,76:803-10.
[26]Kalmokoff M, Lanthier P, Tremblay TL, Foss M, Lau PC, Sanders G, Austin J, Kelly J, Szymanski CM. Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation. JBacteriol,2006,188:4312-20.
[27]Hefford MA, D'Aoust S, Cyr TD, Austin JW, Sanders G, Kheradpir E, Kalmokoff ML. Proteomic and microscopic analysis of biofilms formed by Listeria monocytogenes 568. Can J Microbiol,2005,51:197-208.
[28]Zhang D, Jiang B, Xiang Z, Wang S. Functional characterisation of altered outer membrane proteins for tetracycline resistance in Escherichia coli. Int JAntimicrob Ag,2008,32:315-9.
[29]Garcia-Sureda L, Juan C, Domenech-Sanchez A, Alberti S. Role of Klebsiella pneumoniae LamB porin in antimicrobial resistance. Antimicrob Agents Ch,2011:AAC.01441-10v1.
[30]Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature,2000,405:914-9.
[31]Galindo CL, Sha J, Ribardo DA, Fadl AA, Pillai L, Chopra AK. Identification of Aeromonas hydrophila cytotoxic enterotoxin-induced genes in macrophages using microarrays. J Biol Chem, 2003,278:40198-212.
[32]Chopra A, Xu XJ, Ribardo D, Gonzalez M, Kuhl K, Peterson J, Houston C. The cytotoxic enterotoxin of Aeromonas hydrophila induces proinflammatory cytokine production and activates arachidonic acid metabolism in macrophages. Infect Immun,2000,68:2808.
[33]Villamon E, Villalba V, Nogueras MM, Tomas JM, Gozalbo D, Gil ML. Glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme present in the periplasm of Aeromonas hydrophila. Anton Leeuw,2003,84:31-8.
[34]Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG. Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol,2002,184:1140-9.
[35]Ang CS, Veith PD, Dashper SG, Reynolds EC. Application of 16O/18O reverse proteolytic labeling to determine the effect of biofilm culture on the cell envelope proteome of Porphyromonas gingivalis W50. Proteomics,2008,8:1645-60.
[36]Planchon S, Desvaux M, Chafsey I, Chambon C, Leroy S, Hebraud M, Talon R. Comparative subproteome analyses of planktonic and sessile Staphylococcus xylosus C2a:new insight in cell physiology of a coagulase-negative Staphylococcus in biofilm. J Proteome Res, 2009,8:1797-809.
[37]Wang D, Calla B, Vimolmangkang S, Wu X, Korban SS, Huber SC, Clough SJ, Zhao Y. The orphan gene ybjN conveys pleiotropic effects on multicellular behavior and survival of Escherichia coli. PLoS One,2011,6:e25293.
[38]Wang D. Regulation of amylovoran biosynthesis in Erwinia amylovora.2011.
[39]Zhao Y, Wang D, Nakka S, Sundin G, Korban S. Systems level analysis of two-component signal transduction systems in Erwinia amylovora:Role in virulence, regulation of amylovoran biosynthesis and swarming motility. BMC genomics,2009,10:245-53.
[40]Suarez G, Sierra JC, Sha J, Wang S, Erova TE, Fadl AA, Foltz SM, Horneman AJ, Chopra AK. Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila. Microb Pathogenesis,2008,44:344-61.
[41]Trevors JT. Viable but non-culturable (VBNC) bacteria:Gene expression in planktonic and biofilm cells. J Microbiol Meth,2011,86:266-73.
[42]Rahman MH, Suzuki S, Kawai K. Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus. Microbiol Res, 2001,156:103-6.
[43]Hanning I, Donoghue DJ, Jarquin R, Kumar GS, Aguiar VF, Metcalf JH, Reyes-Herrera I, Slavik M. Campylobacter biofilm phenotype exhibits reduced colonization potential in young chickens and altered in vitro virulence. Poult Sci,2009,88:1102-7.
[44]Pham TK, Roy S, Noirel J, Douglas I, Wright PC, Stafford GP. A quantitative proteomic analysis of biofilm adaptation by the periodontal pathogen Tannerella forsythia. Proteomics, 2010,10:3130-41.
[45]Tenorio E, Saeki T, Fujita K, Kitakawa M, Baba T, Mori H, Isono K. Systematic characterization of Escherichia coli genes/ORFs affecting biofilm formation. FEMS Microbiol Lett,2003,225:107-14.
[46]Kirov SM, Castrisios M, Shaw JG. Aeromonas flagella (polar and lateral) are enterocyte adhesins that contribute to biofilm formation on surfaces. Infect Immun,2004,72:1939-47.
[47]Santos P, Santos P, Bello A, Freitas Almeida A. Association of Aeromonas caviae polar and lateral flagella with biofilm formation. Lett Appl Microbiol,2010,9:10-9..
[48]Lemon KP, Higgins DE, Kolter R. Flagellar motility is critical for Listeria monocytogenes biofilm formation. J Bacteriol,2007,189:4418-26.
[49]Watnick PI, Lauriano CM, Klose KE, Croal L, Kolter R. The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139. Mol Microbiol,2001,39:223-35.
[50]Wolfe AJ, Millikan DS, Campbell JM, Visick KL. Vibrio fischeri 54 controls motility, biofilm formation, luminescence, and colonization. Appl Environ Microbiol,2004,70:2520-9.
[51]Ho HT, Lipman LJ, Wosten MM, van Asten AJ, Gaastra W. Arcobacter spp. possess two very short flagellins of which FlaA is essential for motility. FEMS Immunol Med Microbiol, 2008,53:85-95.
[52]Costello GM, Vipond R, MacIntyre S. Aeromonas salmonicida possesses two genes encoding homologs of the major outer membrane protein, OmpA. J Bacteriol,1996,178:1623-31.
[53]Orme R, Douglas CW, Rimmer S, Webb M. Proteomic analysis of Escherichia coli biofilms reveals the overexpression of the outer membrane protein OmpA. Proteomics,2006,6:4269-77.
[54]Namba A, Mano N, Takano H, Beppu T, Ueda K, Hirose H. OmpA is an adhesion factor of Aeromonas veronii, an optimistic pathogen that habituates in carp intestinal tract. J Appl Mcrobiol,2008,105:1441-51.
[55]Bordi C, de Bentzmann S. Hacking into bacterial biofilms:a new therapeutic challenge. Ann Intensive Care,2011,1:19-27.
[56]Brady RA, O'May GA, Leid JG, Prior ML, Costerton JW, Shirtliff ME. Resolution of Staphylococcus aureus biofilm infection using vaccination and antibiotic treatment. Infect Immun,2011,79:1797-803.
[57]Chen Z, Peng B, Wang S, Peng X. Rapid screening of highly efficient vaccine candidates by immunoproteomics. Proteomics,2004,4:3203-13.
[1]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin Microbiol Rev,2010,23:35-73.
[2]Parker JL, Shaw JG. Aeromonas spp. clinical microbiology and disease. J Infection, 2011,62:109-18.
[3]Agger WA, McCormick J, Gurwith MJ. Clinical and microbiological features of Aeromonas hydrophila-associated diarrhea. J Clin Microbiol,1985,21:909-18.
[4]Kao HT, Huang YC, Lin TY. Fatal bacteremic pneumonia caused by Aeromonas hydrophila in a previously healthy child. J Microbiol Immunol,2003,36:209-11.
[5]Vally H, Whittle A, Cameron S, Dowse GK, Watson T. Outbreak of Aeromonas hydrophila wound infections associated with mud football. Clin Infect Dis,2004,38:1084-95.
[6]Ni XD, Wang N, Liu YJ, Lu CP. Immunoproteomics of extracellular proteins of the Aeromonas hydrophila China vaccine strain J-1 reveal a highly immunoreactive outer membrane protein. FEMS Immunol Med Microbiol,2010,58:363-73.
[7]Kirov SM, Tassell BC, Semmler ABT, ODonovan LA, Rabaan AA, Shaw JG. Lateral flagella and swarming motility in Aeromonas species. JBacteriol,2002,184:547-56.
[8]Sambrook J, Fritsch E, Maniatis T. Molecular cloning:a laboratory manual, Spring Harbor Laboratory Press. Cold Spring Harbor, NY; 1989.
[9]Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem,1976,72:248-54.
[10]Farfan M, Minana-Galbis D, Fuste MC, Loren JG. Divergent evolution and purifying selection of the flaA gene sequences in Aeromonas. Biol Direct,2009,4:23-31.
[11]Umelo E, Trust TJ. Identification and molecular characterization of two tandemly located flagellin genes from Aeromonas salmonicida A449. JBacteriol,1997,179:5292-9.
[12]Yu HB, Zhang YL, Lau YL, Yao F, Vilches S, Merino S, Tomas JM, Howard SP, Leung KY. Identification and characterization of putative virulence genes and gene clusters in Aeromonas hydrophila PPD134/91. Appl Environ Microbiol,2005,71:4469-77.
[13]Esteve C, Alcaide E, Canals R, Merino S, Blasco D, Figueras MJ, Tomas JM. Pathogenic Aeromonas hydrophila serogroup 0:14 and 0:81 strains with an S layer. Appl Environ Microbiol,2004,70:5898-904.
[14]Maiti B, Raghunath P, Karunasagar I. Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp. J Appl Microbiol,2009,107:1157-67.
[15]Weiser JN, Gotschlich EC. Outer membrane protein A (OmpA) contributes to serum resistance andpathogenicity of Escherichia coli K-1. Infect Immun,1991,59:2252-61.
[16]Fito-Boncompte L, Chapalain A, Bouffartigues E, Chaker H, Lesouhaitier O, Gicquel G, Bazire A, Madi A, Connil N, Veron W. Full virulence of Pseudomonas aeruginosa requires OprF. Infect Immun,2011,79:1176-84.
[17]Bartra SS, Gong X, Lorica CD, Jain C, Nair MKM, Schifferli D, Qian L, Li Z, Plano GV, Schesser K. The outer membrane protein A (OmpA) of Yersinia pestis promotes intracellular survival and virulence in mice. Microbl Pathogenesis,2011,9:167-74.
[18]Duperthuy M, Binesse J, Le Roux F, Romestand B, Caro A, Got P, Givaudan A, Mazel D, Bachere E, Destoumieux-Garz6n D. The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas. Environ Microbiol,2010,12:951-63.
[19]Xu'ai L, Aihua S, Ping R, Zhe Z, Jie Y. Characterization of conserved combined T and B cell epitopes in Leptospira interrogans major outer membrane proteins OmpL1 and LipL41. BMC Microbiol,2011,11:21-9.
[20]Danese PN, Pratt LA, Dove SL, Kolter R. The outer membrane protein, Antigen 43, mediates cell to cell interactions within Escherichia coli biofilms. Mol Microbiol,2000,37:424-32.
[21]MacIntyre S, Trust T, Buckley J. Identification and characterization of outer membrane fragments released by Aeromonas sp. CanJBiochem,1980,58:1018-25.
[22]Vazquez - Juarez R, Barrera-Saldafia H, Hernandez - Saavedra N, Gomez - Chiarri M, Ascencio F. Molecular cloning, sequencing and characterization of omp48, the gene encoding for an antigenic outer membrane protein from Aeromonas veronii. J Appl Microbiol, 2003,94:908-18.
[23]Loeb MR, Kilner J. Release of a special fraction of the outer membrane from both growing and phage T4-infected Escherichia coli B. BBA-Biomembranes,1978,514:117-27..
[25]Merino S, Vilches S, Canals R, Ramirez S, Tomas JM. A C1q-binding 40kDa porin from Aeromonas salmonicida:cloning, sequencing, role in serum susceptibility and fish immunoprotection. Microb Pathogenesis,2005,38:227-37.
[1]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin MicrobiolRev,2010,23:35-73.
[2]Parker JL., Shaw JG Aeromonas spp. clinical microbiology and disease. J Infection, 20101,62:109-18.
[3]Agger WA, McCormick J, Gurwith MJ. Clinical and microbiological features of Aeromonas hydrophila-associated diarrhea. J Clin Microbiol,1985,21:909-18.
[4]Kao HT, Huang YC, Lin TY. Fatal bacteremic pneumonia caused by Aeromonas hydrophila in a previously healthy child. J Microbiol Immunol,2003,36:209-11.
[5]Vally H, Whittle A, Cameron S, Dowse GK, Watson T. Outbreak of Aeromonas hydrophila wound infections associated with mud football. Clin Infect Dis,2004,38:1084-95.
[6]Ni XD, Wang N, Liu YJ, Lu CP. Immunoproteomics of extracellular proteins of the Aeromonas hydrophila China vaccine strain J-1 reveal a highly immunoreactive outer membrane protein. FEMS Immunol Med Microbiol,2010,58:363-73.
[7]Lin J, Huang S, Zhang Q. Outer membrane proteins:key players for bacterial adaptation in host niches. Microbes Infect,2002,4:325-31.
[8]Maiti B, Raghunath P, Karunasagar I. Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp. J Appl Microbiol,2009,107:1157-67.
[9]Maji S, Mali P, Joardar SN. Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.). Fish Shellfish Immunol,2006,20:462-73.
[10]Khushiramani R, Girisha SK, Karunasagar I. Cloning and expression of an outer membrane protein ompTS of Aeromonas hydrophila and study of immunogenicity in fish. Protein Expr Purif,2007,51:303-7.
[11]Guan R, Xiong J, Huang W, Guo S. Enhancement of protective immunity in European eel (Anguilla anguilla) against Aeromonas hydrophila and Aeromonas sobria by a recombinant Aeromonas outer membrane protein. Acta Biochim Biophys Sin (Shanghai),2011,43:79-88.
[12]Khushiramani R, Girisha SK, Karunasagar I. Protective efficacy of recombinant OmpTS protein of Aeromonas hydrophila in Indian major carp. Vaccine,2007,25:1157-8.
[13]Merino S, Vilches S, Canals R, Ramirez S, Tomas JM. A Clq-binding 40kDa porin from Aeromonas salmonicida:Cloning, sequencing, role in serum susceptibility and fish immunoprotection. Microbl Pathogenesis,2005,38:227-37.
[14]Daly J, Moore A, Olivier G. A colorimetric assay for the quantification of brook trout (Salvelinus fontinalis) lymphocyte mitogenesis. Fish Shellfish Immunol,1995,5:265-73.
[15]Maji S, Mali P, Joardar S. Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.). Fish Shellfish Immunol,2006,20:462-73.
[16]Murray JS. How the MHC selects Thl/Th2 immunity. Immunol Today,1998,19:157-62.
[17]McGuirk P, Mills KHG. Pathogen-specific regulatory T cells provoke a shift in the Thl/Th2 paradigm in immunity to infectious diseases. Trends Immunol,2002,23:450-5.
[18]Parronchi P, De Carli M, Manetti R, Simonelli C, Sampognaro S, Piccinni M, Macchia D, Maggi E, Del Prete G, Romagnani S. IL-4 and IFN (alpha and gamma) exert opposite regulatory effects on the development of cytolytic potential by Thl or Th2 human T cell clones. J Immunol, 1992,149:2977-83.
[19]Manetti R, Parronchi P, Giudizi MQ Piccinni M, Maggi E, Trinchieri G, Romagnani S. Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Thl)-specific immune responses and inhibits the development of IL-4-producing Th cells. J Exp Med, 1993,177:1199-204.
[20]Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-y:an overview of signals, mechanisms and functions. J Leukocyte biol,2004,75:163-89.
[21]Schoenborn JR, Wilson CB. Regulation of Interferon-y During Innate and Adaptive Immune Responses. Adv Immunol,2007,96:41-101.
[22]Walton CB, Inos ABH, Andres OA, Jube S, Couet HG Douglas JT, Patek PQ, Borthakur D. Immunization with hybrid recombinant Mycobacterium tuberculosis H37Rv proteins increases the TH1 cytokine response in mice following a pulmonary instillation of irradiated mycobacteria. Vaccine,2008,26:4396-402.
[23]Hamdy S, Molavi O, Ma Z, Haddadi A, Alshamsan A, Gobti Z, Elhasi S, Samuel J, Lavasanifar A. Co-delivery of cancer-associated antigen and Toll-like receptor 4 ligand in PLGA nanoparticles induces potent CD8+T cell-mediated anti-tumor immunity. Vaccine, 2008,26:5046-57.
[24]Beckman JS, Carson M, Smith CD, Koppenol WH. ALS, SOD and peroxynitrite. Nature, 1993,364:584-92.
[25]Wilhelm FD. Fish antioxidant defenses--a comparative approach. Braz J Med Biol Res, 1996,29:1735-43.
[26]Pascual P, Pedrajas J, Toribio F, Lopez-Barea J, Peinado J. Effect of food deprivation on oxidative stress biomarkers in fish(Sparus aurata). Chem Biol Interact,2003,145:191-9.
[27]Sharp G, Secombes C. The role of reactive oxygen species in the killing of the bacterial fish pathogen Aeromonas salmonicida by rainbow trout macrophages. Fish Shellfish Immunol, 1993,3:119-29.
[1]Parker JL, Shaw JG Aeromonas spp. clinical microbiology and disease. J Infection, 2011,62:109-18.
[2]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin Microbiol Rev,2010,23:35-73.
[3]Asha A, Nayak DK, Shankar KM, Mohan CV. Antigen expression in biofilm cells of Aeromonas hydrophila employed in oral vaccination of fish. Fish Shellfish Immunol,2004,16:429-36.
[4]Azad IS, Shankar KM, Mohan CV, Kalita B. Uptake and processing of biofilm and free-cell vaccines of Aeromonas hydrophila in indian major carps and common carp following oral vaccination Dis Aquat Organ,2000,43:103-8.
[5]Simoes M, Simoes LC, Vieira MJ. A review of current and emergent biofilm control strategies. LWT-Food Sci Technol,2010,43:573-83.
[6]Esteve C, Alcaide E, Canals R, Merino S, Blasco D, Figueras MJ, Tomas JM. Pathogenic Aeromonas hydrophila serogroup O:14 and O:81 strains with an S layer. Appl Environ Microbiol, 2004,70:5898-904.
[7]Weiser JN, Gotschlich EC. Outer membrane protein A (OmpA) contributes to serum resistance and pathogenicity of Escherichia coli K-1. Infect Immun,1991,59:2252-64.
[8]Fito-Boncompte L, Chapalain A, Bouffartigues E, Chaker H, Lesouhaitier O, Gicquel G, Bazire A, Madi A, Connil N, Veron W. Full virulence of Pseudomonas aeruginosa requires OprF. Infect Immun,2011,79:1176-85.
[9]Bartra SS, Gong X, Lorica CD, Jain C, Nair MKM, Schifferli D, Qian L, Li Z, Piano GV, Schesser K. The outer membrane protein A (OmpA) of Yersinia pestis promotes intracellular survival and virulence in mice. Microb Pathogenesis,2011,12:53-65.
[10]Duperthuy M, Binesse J, Le Roux F, Romestand B, Caro A, Got P, Givaudan A, Mazel D, Bachere E, Destoumieux-Garzon D. The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas. Environ Microbiol,2010,12:951-63.
[11]Xu'ai L, Aihua S, Ping R, Zhe Z, Jie Y. Characterization of conservedcombined T and B cell epitopes in Leptospira interrogans major outer membrane proteins OmpLl and LipL41. BMC Microb iol,2011,11:21-9.
[12]Kirov SM, Castrisios M, Shaw JG Aeromonas flagella (polar and lateral) are enterocyte adhesins that contribute to biofilm formation on surfaces. Infect Immun,2004,72:1939-50.
[13]Ho HT, Lipman LJ, Wosten MM, van Asten AJ, Gaastra W. Arcobacter spp. possess two very short flagellins of which FlaA is essential for motility. FEMS Immunol Med Microbiol, 2008,53:85-95.
[14]Lemon KP, Higgins DE, Kolter R. Flagellar motility is critical for Listeria monocytogenes biofilm formation. JBacteriol,,2007,189:4418-28.
[15]Watnick PI, Lauriano CM, Klose KE, Croal L, Kolter R. The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139. Mol Microbiol,2001,39:223-35.
[16]Wolfe AJ, Millikan DS, Campbell JM, Visick KL. Vibrio fischeri 54 controls motility, biofilm formation, luminescence, and colonization. Appl Environ Microbiol,,2004,70:2520-29.
[17]Zhu J, Chai Y, Zhong Z, Li S, Winans SC. Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules:detection of autoinducers in Mesorhizobium huakuii. Appl Environ Microbiol,2003,69:6949-53.
[18]Wang RF, Kushner SR. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene,1991,100:195-9.
[19]陆承平.兽医微生物学.第四版：中国农业出版社,2007,p.31-2.
[20]Quandt J, Hynes MF. Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. Gene,1993,127:15-21.
[1]Janda JM, Abbott SL. The genus Aeromonas:taxonomy, pathogenicity, and infection. Clin Mcrobiol Rev,2010,23:35-73.
[2]Parker JL, Shaw JG. Aeromonas spp. clinical microbiology and disease. J Infection, 2011,62:109-18.
[3]陈怀青,陆承平.家养鲤科鱼暴发性传染病的病原研究.南京农业大学学报,1991：87－91.
[4]Lee S, Yin Z, Ge R, Sin Y. Isolation and characterization of fish Aeromonas hydrophila adhesins important for in vitro epithelial cell invasion. J Fish Dis,1997,20:169-75.
[5]Buckley JT, Peter S, Chopra A, Houston C. The cytotoxic enterotoxin of Aeromonas hydrophila is aerolysin. Infect Immun,1999,67:466-78.
[6]Upadhyaya T, Singh RK, Dixit A. Molecular cloning and sequence analysis of lamB encoding outer membrane maltose-inducible porin of Aeromonas hydrophila. Mitochondr DNA, 2007,18:302-6.
[7]Simoes M, Simoes LC, Vieira MJ. A review of current and emergent biofilm control strategies. LWT-Food Sci Technol,2010,43:573-83.
[8]Ling H, Kang A, Tan MH, Qi X, Chang MW. The absence of the luxS gene increases swimming motility and flagella synthesis in Escherichia coli K12. Biochem Bioph Res Co,2010,401:521-6.
[9]Huang Z, Meric G, Liu Z, Ma R, Tang Z, Lejeune P.luxS-based quorum-sensing signaling affects Biofilm formation in Streptococcus mutans. JMol Mcrobiol Biotechnol,2009,17:12-9.
[10]Weiser JN, Gotschlich EC. Outer membrane protein A (OmpA) contributes to serum resistance and pathogenicity of Escherichia coli K-1. Infect Immun,1991,59:2252-63.
[11]Rabaan AA, Gryllos I, Tomas JM, Shaw JG. Motility and the polar flagellum are required for Aeromonas caviae adherence to HEp-2 cells. Infect Immun,2001,69:4257-68.
[12]Kirov SM, Castrisios M, Shaw JG. Aeromonas flagella (polar and lateral) are enterocyte adhesins that contribute to biofilm formation on surfaces. Infect Immun,2004,72:1939-48.
[13]Bartra SS, Gong X, Lorica CD, Jain C, Nair MKM, Schifferli D, Qian L, Li Z, Plano GV, Schesser K. The outer membrane protein A (OmpA) of Yersinia pestis promotes intracellular survival and virulence in mice. Microb Pathogenesis,2011,12:53-65.
[14]Duperthuy M, Binesse J, Le Roux F, Romestand B, Caro A, Got P, Givaudan A, Mazel D, Bachere E, Destoumieux-Garzon D. The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas. Environ Microbiol,2010,12:951-63.
[15]Lemon KP, Higgins DE, Kolter R. Flagellar motility is critical for Listeria monocytogenes biofilm formation. J Bacteriol,2007,189:4418-27.
[16]Watnick PI, Lauriano CM, Klose KE, Croal L, Kolter R. The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae 0139. Mol Microbiol,2001,39:223-35.
[17]Wolfe AJ, Millikan DS, Campbell JM, Visick KL. Vibrio fischeri controls motility, biofilm formation, luminescence, and colonization. Appl Environ Microbiol,,2004,70:2520-8.
[18]Aeckersberg F, Lupp C, Feliciano B, Ruby E. Vibrio fischeri outer membrane protein OmpU plays a role in normal symbiotic colonization. JBacteriol,2001,183:6590-9.
[19]Fito-Boncompte L, Chapalain A, Bouffartigues E, Chaker H, Lesouhaitier O, Gicquel G, Bazire A, Madi A, Connil N, Veron W. Full virulence of Pseudomonas aeruginosa requires OprF. Infect Immun,2011,79:1176-85.
[20]张小军.嗜水气单胞菌J-1株luxS基因缺失株的构建及特性分析：南京农业大学；2009.
[21]Bassler BL, Wright M, Silverman MR. Multiple signalling systems controlling expression of luminescence in Vibrio harveyi:sequence and function of genes encoding a second sensory pathway. Mol Microbiol,1994,13:273-86.
[22]Bumann D, Holland P, Siejak F, Koesling J, Sabarth N, Lamer S, Zimny-Arndt U, Jungblut PR, Meyer TF. A comparison of murine and human immunoproteomes of Helicobacter pylori validates the preclinical murine infection model for antigen screening. Infect Immun, 2002,70:6494-8.
[23]Ju Z, Karsi A, Kocabas A, Patterson A, Li P, Cao D, Dunham R, Liu Z. Transcriptome analysis of channel catfish (Ictalurus punctatus):genes and expression profile from the brain. Gene, 2000,261:373-82.
[24]Erova TE, Pillai L, Fadl AA, Sha J, Wang S, Galindo CL, Chopra AK. DNA adenine methyltransferase influences the virulence of Aeromonas hydrophila. Infect Immun, 2006,74:410-24.
[25]Swift S, Lynch MJ, Fish L, Kirke DF, Tomas JM, Stewart GS, Williams P. Quorum sensing-dependent regulation and blockade of exoprotease production in Aeromonas hydrophila. Infect Immun,1999,67:5192-9.
[26]Kozlova EV, Popov VL, Sha J, Foltz SM, Erova TE, Agar SL, Horneman AJ, Chopra AK. Mutation in the S-ribosylhomocysteinase (luxS) gene involved in quorum sensing affects biofilm formation and virulence in a clinical isolate of Aeromonas hydrophila. Microb Pathogenesis,2008,45:343-54.
[27]Kirov SM, Tassell BC, Semmler ABT, O'Donovan LA, Rabaan AA, Shaw JG. Lateral flagella and swarming motility in Aeromonas species. JBacteriol,2002,184:547-58.
[28]Sha J, Pillai L, Fadl AA, Galindo CL, Erova TE, Chopra AK. The type Ⅲ secretion system and cytotoxic enterotoxin alter the virulence of Aeromonas hydrophila. Infect Immun, 2005,73:6446-57.
[29]Costello GM, Vipond R, Maclntyre S. Aeromonas salmonicida possesses two genes encoding homologs of the major outer membrane protein, OmpA. JBacteriol,1996,178:1623-32.
[30]Namba A, Mano N, Takano H, Beppu T, Ueda K, Hirose H. OmpA is an adhesion factor of Aeromonas veronii, an optimistic pathogen that habituates in carp intestinal tract. J Appl Microbiol,2008,105:1441-51.
[31]Orme R, Douglas CW, Rimmer S, Webb M. Proteomic analysis of Escherichia coli biofilms reveals the overexpression of the outer membrane protein OmpA. Proteomics,2006,6:4269-77.
[32]Santos P, Santos P, Bello A, Freitas Almeida A. Association of Aeromonas caviae polar and lateral flagella with biofilm formation. Lett Appl Microbiol,2010,9:10-9.
[33]Wolfe AJ, Millikan DS, Campbell JM, Visick KL. Vibrio fischeri controls motility, biofilm formation, luminescence, and colonization. Appl Environ Microbiol,2004,70:2520-32.
[34]Ho HT, Lipman LJ, Wosten MM, van Asten AJ, Gaastra W. Arcobacter spp. possess two very short flagellins of which FlaA is essential for motility. FEMS Immunol Med Microbiol, 2008,53:85-95.
[35]Pagel M, Simonet V, Li J, Lallemand M, Lauman B, Delcour AH. Phenotypic characterization of pore mutants of the Vibrio cholerae porin OmpU. JBacteriol,2007,189:8593-600.
[36]Tsai YK, Fung CP, Lin JC, Chen JH, Chang FY, Chen TL, Siu LK. The Klebsiella pneumoniae outer membrane porins OmpK35 and OmpK36 play roles in both antimicrobial resistance and virulence. JAntimicrob Chemother,2011:AAC.01275-10v1.
[37]Chen JH, Siu LK, Fung CP, Lin JC, Yeh KM, Chen TL, Tsai YK, Chang FY. Contribution of outer membrane protein K36 to antimicrobial resistance and virulence in Klebsiella pneumoniae. JAntimicrob Chemother,2010,65:986-90.
[38]Dooley JS, Trust TJ. Surface protein composition of Aeromonas hydrophila strains virulent for fish:identification of a surface array protein. JBacteriol,1988,170:499-506.
[39]Belland RJ, Trust TJ. Synthesis, export, and assembly of Aeromonas salmonicida A-layer analyzed by transposon mutagenesis. JBacteriol,1985,163:877-81.
[40]Merino S, Nogueras MM, Aguilar A, Rubires X, Alberti S, Benedi VJ, Tomas JM. Activation of the complement classical pathway (Clq binding) by mesophilic Aeromonas hydrophila outer membrane protein. Infect Immun,1998,66:3825-31.
[41]Nogueras MM, Merino S, Aguilar A, Benedi VJ, Tomas JM. Cloning, sequencing, and role in serum susceptibility of porin II from mesophilic Aeromonas hydrophila. Infect Immun, 2000,68:1849-62.
[42]Brackman G, Cos P, Maes L, Nelis HJ, Coenye T. Quorum sensing inhibitors increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivo. Antimicrob Agents Chemother,2011,55:2655-61.
[43]Revel A T NDM, et al. Expression of a luxS gene is not required for Borreliaburgdorferi infection of mice via needle inoculation. Infect Immun,2003,71-8.
[44]Winzer K, Sun YH, Green A, Delory M, Blackley D, Hardie KR, Baldwin TJ, Tang CM. Role of Neisseria meningitidis luxS in cell-to-cell signaling and bacteremic infection. Infect Immun, 2002,70:2245-8.
[45]Merritt J, Qi F, Goodman SD, Anderson MH, Shi W. Mutation of luxS affects biofilm formation in Streptococcus mutans. Infect Immun,2003,71:1972-9.
[46]Huang Z, Meric G, Liu Z, Ma R, Tang Z, Lejeune P.luxS-based quorum-sensing signaling affects biofilm formation in Streptococcus mutans. J Mol Microbiol Biotechnol,2008,5:55-61.
[47]Yoshida A, Ansai T, Takehara T, Kuramitsu HK. LuxS-based signaling affects Streptococcus mutans biofihn formation. Appl Environ Microbiol,2005,71:2372-80.
[48]Zhang M, Sun K, Sun L. Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain. Microbiology,2008,154:2060-9.