Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp.

Aims:  The main aims of this study were to clone and express an outer membrane protein (OMP), OmpW, of Aeromonas hydrophila and to study its distribution in Aeromonas spp.
Methods and Results:  The gene encoding OmpW in A. hydrophila has been cloned and expressed in Escherichia coli . Primers were designed for amplification of full-length ompW gene and used for identification of this gene in different Aeromonas spp. Of the 42 Aeromonas strains tested, all the isolates were positive by polymerase chain reaction (PCR) except one strain of Aeromonas veronii biovar veronii (VTE338). None of the other gram-negative bacteria were positive by PCR with primers specific to ompW gene of A. hydrophila . Polyclonal antibodies were raised in rabbit against the purified recombinant protein and the reaction of these antibodies was confirmed by western blotting using the purified recombinant protein and 42 Aeromonas cultures grown at various salt concentrations.
Conclusions:  The ompW -based PCR method developed in this study was found to be 100% specific and 97% sensitive. Expression of OmpW protein of Aeromonas was found to be salt-dependant. Recombinant OmpW protein was found to be highly immunogenic in fish.
Significance and Impact of the Study:  To our knowledge, this is the first report on cloning and expression of OmpW protein of A. hydrophila . Full-length ompW gene amplification by PCR can be used for the detection of Aeromonas . Recombinant OmpW protein can be useful for vaccination of fish against Aeromonas spp.     

Phylogenetic analysis of antimicrobial lactic acid bacteria from farmed seabass Dicentrarchus labrax

The use of lactic acid bacteria (LAB) in the prevention or reduction of fish diseases is receiving increasing attention. In the present study, 47 LAB strains were isolated from farmed seabass ( Dicentrarchus labrax ) and were phenotypically and phylogenetically analysed by 16S rDNA and randomly amplified polymorphic DNA - polymerase chain reaction (RAPD-PCR). Their antimicrobial effect was tested in vitro against a wide variety of pathogenic and spoilage bacteria. Most of the strains isolated were enterococci belonging to the following species: Enterococcus faecium (59%), Enterococcus faecalis (21%), Enterococcus sanguinicola (4 strains), Enterococcus mundtii (1 strain), Enterococcus pseudoavium (1 strain), and Lactococcus lactis (1 strain). An Aerococcus viridans strain was also isolated. The survey of their antimicrobial susceptibility showed that all isolates were sensitive to vancomycin and exhibited resistance to between 4 and 10 other antibiotics relevant for therapy in human and animal medicine. Different patterns of resistance were noted for skin and intestines isolates. More than 69% (32 strains) of the isolates inhibited the growth of the majority of pathogenic and spoilage bacteria tested, including Listeria monocytogenes, Staphylococcus aureus, Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum, and Carnobacterium sp. To our knowledge, this is the first report of bioactive enterococcal species isolated from seabass that could potentially inhibit the undesirable bacteria found in food systems.     

西伯利亚鲟嗜水气单胞菌与其它3株气单胞菌间的免疫交叉反应

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

几种中药单体和抗生素对嗜水气单胞菌及温和气单胞菌的体外抑菌活性研究

考察没食子酸等6种中药单体和诺氟沙星等3种抗生素对嗜水气单胞菌及温和气单胞菌的体外抑菌活性及其联合抑菌作用。通过琼脂扩散法测定受试物的抑菌作用,用微量二倍稀释法测定受试物最小抑菌浓度（MIC）和最小杀菌浓度（MBC）,用棋盘法考察受试物的联合抑菌作用。6种中药单体中没食子酸和槲皮素对嗜水气单胞菌及温和气单胞菌抑制作用较强,抑菌圈均达到20 mm以上,没食子酸对嗜水气单胞菌及温和气单胞菌的MIC和MBC均为250 g/mL;槲皮素对嗜水气单胞菌的MIC和MBC均为500 g/mL,对温和气单胞菌的MIC和MBC分别为250和500 g/mL;而大黄素甲醚等4种中药单体无明显的抑菌作用。嗜水气单胞菌及温和气单胞菌对诺氟沙星、恩诺沙星、氟苯尼考等抗生素均极敏感。在联合药敏试验中,没食子酸与恩诺沙星或氟苯尼考、槲皮素与氟苯尼考联合用药对嗜水气单胞菌具有相加抑菌作用;没食子酸与恩诺沙星联合用药对温和气单胞菌具有协同抑菌作用,没食子酸与诺氟沙星或氟苯尼考、槲皮素与氟苯尼考联合用药对温和气单胞菌具有相加抑菌作用。没食子酸和槲皮素对嗜水气单胞菌及温和气单胞菌具有显著的抑制作用,二者与恩诺沙星、诺氟沙星、氟苯尼考等抗生素联合应用具有相加或协同作用,有助于降低抗生素的用量及残留。       

Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification

AIMS: To examine whether Aeromonas bacteria isolated from municipally treated water had virulence factor genes. METHODS AND RESULTS: A polymerase chain reaction-based genetic characterization determined the presence of six virulence factors genes, elastase (ahyB), lipase (pla/lip/lipH3/alp-1) flagella A and B (flaA and flaB), the enterotoxins, act, alt and ast, in these isolates. New primer sets were designed for all the target genes, except for act. The genes were present in 88% (ahyB), 88% (lip), 59% (fla), 43% (alt), 70% (act) and 30% (ast) of the strains, respectively. Of the 205 isolates tested only one isolate had all the virulence genes. There was a variety of combinations of virulence factors within different strains of the same species. However, a dominant strain having the same set of virulence factors, was usually isolated from any given tap in different rounds of sampling from a single tap. CONCLUSIONS: These results show that Aeromonas bacteria found in drinking water possess a wide variety of virulence-related genes and suggest the importance of examining as many isolates as possible in order to better understand the health risk these bacteria may present. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a rapid method for characterizing the virulence factors of Aeromonas bacteria and suggests that municipally treated drinking water is a source of potentially pathogenic Aeromonas bacteria.     

Population dynamics of Aeromonas spp. in an urban river watershed

Density of Aeromonas spp. at one site in the Buffalo River and at four sites on its upstream tributaries was followed from June 1992–June 1993. Membrane filtration counts of Aeromonas during the summer ranged between 18 and 4000 ml⁻¹, which were one to two logs higher than faecal coliform and faecal streptococci densities. Aeromonas spp. in the Buffalo River, and faecal coliforms, faecal streptococci, and the heterotrophic plate count throughout the watershed, increased by approximately one log during summer rainstorms. However, Aeromonas spp. increased only by a factor of two during rainstorms at the upstream sites. Aeromonas spp. showed a strong positive correlation with both indicator bacteria and total suspended solids at the upstream sites during the summer but not the winter. Correlations between Aeromonas and indicator bacteria remained strong in the Buffalo River during the winter, signifying that different conditions exist in the Buffalo River and its upstream tributaries. The strong correlation between Aeromonas spp. and indicator bacteria in the Buffalo River suggest that, in the absence of media capable of the quantitative recovery of potentially pathogenic aeromonads, standard faecal coliform analyses may adequately assess public health risks from Aeromonas spp. in an urban river used for recreational purposes.     

Preliminary selection study of potential probiotic bacteria from aquacultural area in Tunisia

In order to test the ability to produce antibacterial substances within marine bacteria, prior to select potential probiotics for use in shellfish farming, we targeted a large collection of bacterial isolates (132 strains), brought from the clamRuditapes decussatus and 37 reference strains. First, we proceeded to their biochemical identification and the screening of antibiotic resistance profiles. Else, we investigated their inhibitory activityin vitro against several fish and shellfish pathogens, using two methods: the double-layer agar and the direct simultaneous antagonism methods. The results showed high frequencies of inhibitory producing bacteria (IPB) within the isolates. These bacteria (25%) were aerobic mesophylic bacteria belonging to various bacterial groups: 33.7% oxidase-positive Gram-negative bacteria, 7.4%Enterobacteriaceae and 28% lactic acid bacteria. Besides this group, nine strains produced strong inhibition effect. These bacteria belonged to:Aeromonas hydrophila, Aeromonas sobria, Pseudomonas cepacia, Vibrio sp,Serratia liquefaciens andLactobacillus rhamnosus. They were active against pathogenic bacteria belonging to the genera:Aeromonas, Pseudomonas andVibrio. These potential probiotics were submitted to further investigations prior to their introduction in larval shellfish farming.     

从新疆哈萨克族2型糖尿病患者粪便中分离的气单胞菌对小鼠的作用研究

目的从新疆哈萨克族2型糖尿病患者粪便中分离培养气单胞菌,并回接到小鼠体内,检测其对小鼠血糖、血脂等2型糖尿病相关中间表型的作用。方法 (1)收集新鲜哈萨克族2型糖尿病患者的粪便样本,用气单胞菌专属培养基进行分离培养,微量生化反应管进行初步鉴定;提取所得菌株的DNA,使用气单胞菌属引物对此DNA进行特异性扩增,结合全自动细菌鉴定仪对菌株进行鉴定;(2)将气单胞菌菌液回接小鼠,采用羰花青素染料(CM-Dil)标记法对菌定植力情况进行检测。(3)检测气单胞菌菌液对小鼠体重、随机血糖(GLU)、空腹血糖(FBG)、总胆固醇(TC)和甘油三酯(TG)的作用,进行葡萄糖耐量(OGTT)、胰岛素耐量(ITT)试验。结果 (1)所得到的菌株经全自动细菌鉴定仪确定为气单胞菌;(2)从小鼠粪便中分离培养后的细菌能检测到荧光标记;(3)与正常饮食组小鼠相比,气单胞菌稀释液、原液、浓缩液组小鼠体重均升高;气单胞菌原液组小鼠GLU升高。小鼠FBG、TC和TG在各组间差异均无统计学意义;气单胞菌菌液组小鼠口服葡萄糖耐量试验血糖曲线下面积增加;气单胞菌原液、浓缩液组小鼠胰岛素耐量试验血糖曲线下面积增加。结论 (1)从新疆哈萨克族2型糖尿病患者粪便中分离培养得到气单胞菌;(2)从糖尿病患者粪便中分离的气单胞菌可定植于小鼠体内,并使小鼠的体重增加,GLU升高,给予气单胞菌稀释液、菌原液、菌浓缩液后第8周,小鼠出现糖耐量受损,胰岛素敏感性下降。     

Use of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside for the isolation of beta-galactosidase-positive bacteria from municipal water supplies

A new medium, mX-Gal, has been developed for the membrane filter enumeration of beta-galactosidase-positive bacteria in municipal water supplies. mX-Gal medium contains the chromogenic beta-galactosidase substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). All Aeromonas, Citrobacter, and Enterobacter strains isolated from raw water on mX-Gal medium were beta-galactosidase positive. In contrast, only 10 to 20% of these strains produced a red colony with a metallic sheen on m-Endo agar LES medium. Of 674 chlorinated water samples analyzed for total coliforms on m-Endo agar LES medium and for beta-galactosidase-positive bacteria on mX-Gal medium, 18 that were negative for coliforms on m-Endo agar LES showed beta-galactosidase-positive bacteria on mX-Gal. Of a total of 50 beta-galactosidase-positive bacteria isolated from these samples, 76% were identified as Aeromonas hydrophila.     

西伯利亚鲟致病性嗜水气单胞菌外膜蛋白及其抗原性分析

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

Broad diversity of conjugative plasmids in integron-carrying bacteria from wastewater environments

In this study we assessed the occurrence, diversity and conjugative potential of plasmids in integron-carrying Aeromonas and Enterobacteriaceae from wastewaters. Sixty-six strains were included as donors in mating assays using rifampicin-resistant Escherichia coli and Pseudomonas putida recipient strains. The diversity of plasmids from donors and transconjugants (resistant to tetracycline or streptomycin) was evaluated by restriction analysis and replicon typing targeting 19 incompatibility groups. Restriction patterns revealed a diverse plasmid pool present in these strains. Plasmids were assigned to FrepB (Aeromonas salmonicida, Aeromonas veronii, Aeromonas sp., E.?coli, Enterobacter sp.), FIC (A.?salmonicida, Aeromonas sp.), FIA (Shigella sp.), I1 (A.?veronii, Aeromonas sp., E.?coli), HI1 (E.?coli) and U (Aeromonas media) replicons. Nevertheless, 50% of the plasmids could not be assigned to any replicon type. Among integron-positive transconjugants, FrepB, I1 and HI1 replicons were detected. Results showed that wastewaters enclose a rich plasmid pool associated with integron-carrying bacteria, capable of conjugating to different bacterial hosts. Moreover, replicons detected in this study in Aeromonas strains expand our current knowledge of plasmid diversity in this genus.     

Culture-based and denaturing gradient gel electrophoresis analysis of the bacterial community structure from the intestinal tracts of earthworms(Eisenia fetida)

The bacterial communities in the intestinal tracts of earthworm were investigated by culture-dependent and - independent approaches. In total, 72 and 55 pure cultures were isolated from the intestinal tracts of earthworms under aerobic and anaerobic conditions, respectively. Aerobic bacteria were classified as Aeromonas (40%), Bacillus (37%), Photobacterium (10%), Pseudomonas (7%), and Shewanella (6%). Anaerobic bacteria were classified as Aeromonas (52%), Bacillus (27%), Shewanella (12%), Paenibacillus (5%), Clostridium (2%), and Cellulosimicrobium (2%). The dominant microorganisms were Aeromonas and Bacillus species under both aerobic and anaerobic conditions. In all, 39 DNA fragments were identified by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. Aeromonas sp. was the dominant microorganism in feeds, intestinal tracts, and casts of earthworms. The DGGE band intensity of Aeromonas from feeds, intestinal tracts, and casts of earthworms was 12.8%, 14.7%, and 15.1%, respectively. The other strains identified were Bacillus, Clostridium, Enterobacter, Photobacterium, Pseudomonas, Shewanella, Streptomyces, uncultured Chloroflexi bacterium, and uncultured bacterium. These results suggest that PCR-DGGE analysis was more efficient than the culture-dependent approach for the investigation of bacterial diversity and the identification of unculturable microorganisms.     

Effects of bacteria on the growth of an amoeba infecting the gills of turbot.

We analysed the influence of various bacteria on the in vitro growth of trophozoites of a Platyamoeba strain isolated from diseased gill tissues of cultured turbot. Little or no growth was shown by amoebae cultured in the presence of (1) the turbot-pathogenic bacteria Vibrio anguillarum, Aeromonas salmonicida or Streptococcus sp., (2) Pasteurella piscicida or Vibrio vulnificus (pathogenic for some fishes but not turbot), or (3) the non-pathogenic 'environmental' bacteria Vibrio campbelli, Vibrio fluvialis or Pseudomonas dondorofii. The only bacteria which were successfully utilized as food sources were Aeromonas hydrophila (pathogenic for some fishes but not turbot) and the non-pathogens Vibrio natriegens, Pseudomonas nautica and Escherichia coli. These results suggest that the colonization of the gills of cultured turbot by the epizoic amoeba Platyamoeba may be an indicator of faecal contamination.     

Diversity of culturable heterotrophic aerobic bacteria in pristine stream bed sediments

More than 900 culturable, heterotrophic aerobic isolates were obtained from the sediments of a forested, pristine stream and analyzed using three classical microbiological tests: API 20E, amplified ribosomal DNA restriction analysis (ARDRA), and fatty acid analysis. Gram-negative bacteria comprised most of the heterotrophic aerobic isolates (66.7%), similar to other oligotrophic environments. The isolates were assigned to the genus level as Pseudomonas, Flavobacterium, Micrococcus, Bacillus, Chromobacterium, Acinetobacter, Alcaligenes, Aeromonas, Methylobacterium, Enterobacter, Corynebacterium, and Sporolactobacillus. Genotypic analysis by ARDRA facilitated the comparison among strains within Pseudomonas, Bacillus, and Enterobacter groups. Temperature and predation may influence the survival of bacteria during seasons, as shown previously by others. Our results showed that the number of heterotrophic aerobic bacteria, especially Enterobacter, Alcaligenes, and Aeromonas, and Gram-positive bacteria, decreased in winter compared to summer conditions.     

东北七鳃鳗肠道应答气单胞菌免疫刺激相关蛋白的鉴定

动物肠道中寄生的微生物与宿主的营养、免疫及防御功能密切相关。本研究在东北七鳃鳗肠道中分离获得一株优势细菌,并鉴定为气单胞菌属（Aeromonads sp.）。建立了荧光定量PCR快速检测气单胞菌拷贝数的方法,经poly(I∶C)刺激后,气单胞菌数量下降115倍。因此,该肠道气单胞菌被认为是七鳃鳗肠道适应性免疫系统进化的关键菌群之一。为了进一步分析气单胞菌对东北七鳃鳗肠道适应性免疫影响的分子机制,将分离菌株腹腔注射七鳃鳗,利用双向电泳技术鉴定了七鳃鳗肠道应答气单胞菌免疫刺激的相关蛋白。共鉴定19个差异表达蛋白质,其中12个蛋白质表达上调,5个蛋白质表达下调,1个蛋白质刺激后消失,1个新增蛋白质,并对蛋白质功能进行分类。七鳃鳗肠道应答气单胞菌免疫刺激的差异表达蛋白分别参与了七鳃鳗适应性免疫调控、信号转导及能量代谢等。该研究可拓宽对七鳃鳗免疫研究的途径,为深入探讨适应性免疫系统发育提供理论基础和新的研究思路。     

Denaturing gradient gel electrophoresis for nonlethal detection of Aeromonas salmonicida in salmonid mucus and its potential for other bacterial fish pathogens

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA was used to nonlethally detect Aeromonas salmonicida and other bacteria in salmonid skin mucus. Mucus samples from wild spawning coho salmon (Oncorhynchus kisutch) with endemic A. salmonicida and from cultured lake trout (Salvelinus namaycush) were tested by PCR-DGGE and were compared with mucus culture on Coomassie brilliant blue agar and internal organ culture. PCR-DGGE gave a highly reproducible 4-band pattern for 9 strains of typical A. salmonicida, which was different from other Aeromonas spp. Aeromonas salmonicida presence in mucus was evident as a band that comigrated with the bottom band of the A. salmonicida 4-band pattern and was verified by sequencing. PCR-DGGE found 36 of 52 coho salmon positive for A. salmonicida, compared with 31 positive by mucus culture and 16 by organ culture. Numerous other bacteria were detected in salmonid mucus, including Pseudomonas spp., Shewanella putrefaciens, Aeromonas hydrophila and other aeromonads. However, Yersinia ruckeri was not detected in mucus from 27 lake trout, but 1 fish had a sorbitol-positive Y. ruckeri isolated from organ culture. Yersinia ruckeri seeded into a mucus sample suggested that PCR-DGGE detection of this bacterium from mucus was possible. PCR-DGGE allows nonlethal detection of A. salmonicida in mucus and differentiation of some Aeromonas spp. and has the potential to allow simultaneous detection of other pathogens present in fish mucus.     

Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus

In this study we investigated the viable but non-culturable (VBNC) state of Aeromonas hydrophila and its virulence in goldfish. Aeromonas hydrophila cultured in a 0.35% NaCl solution at pH 7.5 and at 25 degrees C for 50 days showed the VBNC state. In the VBNC state we were unable to detect viable bacteria by the plate count method but we did find 10(4) cells/ml by the direct viable count microscopical method after staining with fluorescein diacetate and ethidium bromide. The virulence comparison in goldfish showed that bacteria cultured at 25 degrees C for 1 day in a 0.35% NaCl solution were more virulent than bacteria cultured for 28 days. VBNC bacteria showed lower virulence in goldfish compared to 28-day-cultured bacteria by intraperitoneal injection. The results from the study suggest that A. hydrophila can remain in the aquatic environment for prolonged periods in the VBNC state but those cells are not pathogenic to goldfish.     

Lateral Flagella and Swarming Motility in Aeromonas Species

Swarming motility, a flagellum-dependent behavior that allows bacteria to move over solid surfaces, has been implicated in biofilm formation and bacterial virulence. In this study, light and electron microscopic analyses and genetic and functional investigations have shown that at least 50% of Aeromonas isolates from the species most commonly associated with diarrheal illness produce lateral flagella which mediate swarming motility. Aeromonas lateral flagella were optimally produced when bacteria were grown on solid medium for approximately 8 h. Transmission and thin-section electron microscopy confirmed that these flagella do not possess a sheath structure. Southern analysis of Aeromonas reference strains and strains of mesophilic species (n = 84, varied sources and geographic regions) with a probe designed to detect lateral flagellin genes (lafA1 and lafA2) showed there was no marked species association of laf distribution. Approximately 50% of these strains hybridized strongly with the probe, in good agreement with the expression studies. We established a reproducible swarming assay (0.5% Eiken agar in Difco broth, 30 degrees C) for Aeromonas spp. The laf-positive strains exhibited vigorous swarming motility, whereas laf-negative strains grew but showed no movement from the inoculation site. Light and scanning electron microscopic investigations revealed that lateral flagella formed bacterium-bacterium linkages on the agar surface. Strains of an Aeromonas caviae isolate in which lateral flagellum expression was abrogated by specific mutations in flagellar genes did not swarm, proving conclusively that lateral flagella are required for the surface movement. Whether lateral flagella and swarming motility contribute to Aeromonas intestinal colonization and virulence remains to be determined.     

Replication of Legionella pneumophila in biofilms of water distribution pipes

Biofilms similar to those present in water distribution pipes of anthropogenic aquatic systems were simulated in a rotating annular reactor using a non-Legionella community consisting of Aeromonas hydrophila, Escherichia coli, Flavobacterium breve and Pseudomonas aeruginosa. The impact of this community and Acanthamoeba castellanii on the replication of Legionella pneumophila was investigated. Despite the presence of 10(7) non-Legionella bacteria, culture and real-time polymerase chain reaction (PCR) results clearly showed that biofilm-associated Legionella bacteria only increased after intracellular replication in A. castellanii. Fluorescent in situ hybridization (FISH) staining of biofilm samples revealed that 48 h after addition of amoebae to the reactor, the amoeba population was lysing and replicated Legionella bacteria were released into the bulk water. This study demonstrated that amoebae like A. castellanii can play a crucial role in the increase and spread of L. pneumophila in anthropogenic aquatic systems and thus in the occurrence of Legionnaires' disease.     

Polymerase chain reaction (PCR)-based typing analysis of atypical isolates of the fish pathogen Aeromonas salmonicida.

Two hundred and five isolates of atypical Aeromonas salmonicida, recovered from a wide range of hosts and countries were characterized by polymerase chain reaction (PCR) targeting four genes. The chosen genes were those encoding the extracellular A-layer protein (AP), the serine protease (Sprot), the glycerophospholipid:cholestrol acetyltransferase protein (GCAT), and the 16S rRNA (16S rDNA). All the atypical A. salmonicida isolates could be assigned to 4 PCR groups. Group 1 comprised 45 strains which tested positive for PCR amplification, using the 16S rDNA, GCAT2, Sprot2, and AP primer-sets. Group 2 comprised 88 strains with produced PCR products using the 16S rDNA, GCAT2 and AP primer-sets. Group 3 comprised 21 strains which produced PCR products using 16S rDNA, GCAT2 and Sprot2 primer-sets, and group 4 comprised 51 strains which produced PCR products using the 16S rDNA and GCAT2 primer-sets only. A. salmonicida subsp. salmonicida isolates tested, belonged to group 1. The PCR primer-sets separated A. salmonicida from other reference strains of Aeromonas species and related bacteria with the exception of Aeromonas hydrophila. The results indicated that PCR typing is a useful framework for characterization of the increasing number of isolations of atypical A. salmonicida.