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.     

Pulsed-field gel electrophoresis in the study of mesophilic and psychrophilic Aeromonas spp.

Pulsed-field gel electrophoresis (PFGE) was used to study the genetic diversity of mesophilic Aeromonas hybridization group (HG) 1, HG 2, HG 3, HG 4, HG 5, HG 6, HG 7, HG 8/10and HG 11, psychrophilic Aeromonas salmonicida subsp. salmonicida and atypical Aerom. salmonicida strains. Xba I was chosen for restriction because it producedfragments whose numbers and size were appropriate for PFGE analysis of all studied HGs. Allmesophilic Aeromonas strains within an HG had different banding patterns. No sharedbands which could be used for identification of an HG were found. Pulsed-field gelelectrophoresis analysis further confirmed the known genetic homogeneity of Aerom.salmonicida subsp. salmonicida . Pulsed-field gel electrophoresis pattern analysissuggested that the genomic size of Aerom. salmonicida subsp. salmonicida issmaller than that of mesophilic Aeromonas spp. or atypical Aerom. salmonicida . Aeromonas salmonicida subsp. salmonicida had only one large restriction fragment (310kb) and lacked other large fragments (>160 kb). Although the PFGE patterns of atypical Aerom. salmonicida resembled the banding patterns of mesophilic Aeromonas spp.they had several small fragments (15–50 kb) shared with Aerom. salmonicida subsp. salmonicida suggesting genetic relatedness.     

Potential of the melanophore pigment response for detection of bacterial toxicity

Chromatophore cells have been investigated as potential biodetectors for function-based detection of chemically and biologically toxic substances. Oncorhynchus tshawytscha (chinook salmon) melanophores, a chromatophore cell type containing brown pigment, rapidly detect the salmonid pathogens Aeromonas salmonicida, Yersinia ruckeri, and Flavobacterium psychrophilum and the human pathogen Bacillus cereus.     

Survival of the fish pathogen Aeromonas salmonicida in seawater

Survival of Aeromonas salmonicida in natural (non-sterile) seawater, as determined from colony counts on marine agar, was found to be influenced by the presence of potentially inhibitory organisms, i.e., Acinetobacter, Aeromonas hydrophila, Chromobacterium, Escherichia coli, Flavobacterium and Pseudomonas, and their metabolites. Yet, samples, thought to be devoid of culturable A. salmonicida, were found to contain cells, which were filterable through 0.22 and 0.45 microns Millipore Millex porosity filters, and were recoverable on a specialised medium for L-forms, i.e. L-F medium.     

Association of Type III secretion genes with virulence of Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida possesses a number of potential virulence factors, including a recently identified plasmid-encoded Type III secretion system. A number of field isolates of A. salmonicida subsp. salmonicida were examined for the presence of Type III secretion genes. Using in vitro experiments, it was found that field isolates containing such genes are cytotoxic to fish cell lines, whereas those that lack these genes are not. Using a rainbow trout in vivo model, the virulence of a wild type A. salmonicida subsp. salmonicida strain (Strain JF2267), which possesses Type III secretion genes, was compared to that of a laboratory derivative of the same strain that has lost these genes. While Strain JF2267 was virulent towards rainbow trout, its derivative was not. The A. salmonicida subsp. salmonicida Type Strain ATCC 33658T, which also lacks Type III secretion genes, was also found to be avirulent by this challenge model. The findings from both the in vitro and in vivo experiments suggest that the presence of Type III secretion genes is associated with the virulence of this important fish pathogen.     

Identification and molecular characterization of two tandemly located flagellin genes from Aeromonas salmonicida A449.

Two tandemly located flagellin genes, flaA and flaB, with 79% nucleotide sequence identity were identified in Aeromonas salmonicida A449. The fla genes are conserved in typical and atypical strains of A. salmonicida, and they display significant divergence at the nucleotide level from the fla genes of the motile species Aeromonas hydrophila and Aeromonas veronii biotype sobria. flaA and flaB encode unprocessed flagellins with predicted Mrs of 32,351 and 32,056, respectively. When cloned under the control of the Ptac promoter, flaB was highly expressed when induced in Escherichia coli DH5alpha, and the FlaB protein was detectable even in the uninduced state. In flaA clones containing intact upstream sequence, FlaA was barely detectable when uninduced and poorly expressed on induction. The A. salmonicida flagellins are antigenically cross-reactive with the A. hydrophila TF7 flagellin(s) and evolutionarily closely related to the flagellins of Pseudomonas aeruginosa and Vibrio anguillarum. Electron microscopy showed that A. salmonicida A449 expresses unsheathed polar flagella at an extremely low frequency under normal laboratory growth conditions, suggesting the presence of a full complement of genes whose products are required to make flagella; e.g., immediately downstream of flaA and flaB are open reading frames encoding FlaG and FlaH homologs.     

Draft genome sequence of the virulent strain 01-B526 of the fish pathogen Aeromonas salmonicida

Aeromonas salmonicida is an important fish pathogen, mainly of salmonids. This bacterium causes a disease named furunculosis, which is particularly detrimental for the aquaculture industry. Here, we present the draft genome sequence of A. salmonicida 01-B526, a strain isolated from a brook trout that is more virulent than A. salmonicida reference strain A449, for which a genome sequence is available.     

Isolation and Characterization of a Lytic Myoviridae Bacteriophage PAS-1 with Broad Infectivity in Aeromonas salmonicida

To search for candidate control agents against Aeromonas salmonicida subsp. salmonicida infections in aquaculture, one bacteriophage (phage), designated as PAS-1, was isolated from the sediment samples of the rainbow trout (Oncorhynchus mykiss) culture farm in Korea. The PAS-1 was morphologically classified as Myoviridae and possessed approximately 48 kb of double-strand genomic DNA. The phage showed broad host ranges to other subspecies of A. salmonicida as well as A. salmonicida subsp. salmonicida including antibiotic-resistant strains. Its latent period and burst size were estimated to be approximately 40 min and 116.7 PFU/cell, respectively. Furthermore, genomic and structural proteomic analysis of PAS-1 revealed that the phage was closely related to other Myoviridae phages infecting enterobacteria or Aeromonas species. The bacteriolytic activity of phage PAS-1 was evaluated using three subspecies of A. salmonicida strain at different doses of multiplicity of infection, and the results proved to be efficient for the reduction of bacterial growth. Based on these results, PAS-1 could be considered as a novel Aeromonas phage and might have potentiality to reduce the impacts of A. salmonicida infections in aquaculture.     

Biosynthesis and Functions of a Melanoid Pigment Produced by Species of the Sporothrix Complex in the Presence of l-Tyrosine

Sporothrix schenckii is the etiological agent of sporotrichosis, the main subcutaneous mycosis in Latin America. Melanin is an important virulence factor of S. schenckii, which produces dihydroxynaphthalene melanin (DHN-melanin) in conidia and yeast cells. Additionally, l-dihydroxyphenylalanine (l-DOPA) can be used to enhance melanin production on these structures as well as on hyphae. Some fungi are able to synthesize another type of melanoid pigment, called pyomelanin, as a result of tyrosine catabolism. Since there is no information about tyrosine catabolism in Sporothrix spp., we cultured 73 strains, including representatives of newly described Sporothrix species of medical interest, such as S. brasiliensis, S. schenckii, and S. globosa, in minimal medium with tyrosine. All strains but one were able to produce a melanoid pigment with a negative charge in this culture medium after 9 days of incubation. An S. schenckii DHN-melanin mutant strain also produced pigment in the presence of tyrosine. Further analysis showed that pigment production occurs in both the filamentous and yeast phases, and pigment accumulates in supernatants during stationary-phase growth. Notably, sulcotrione inhibits pigment production. Melanin ghosts of wild-type and DHN mutant strains obtained when the fungus was cultured with tyrosine were similar to melanin ghosts yielded in the absence of the precursor, indicating that this melanin does not polymerize on the fungal cell wall. However, pyomelanin-producing fungal cells were more resistant to nitrogen-derived oxidants and to UV light. In conclusion, at least three species of the Sporothrix complex are able to produce pyomelanin in the presence of tyrosine, and this pigment might be involved in virulence.     

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.     

Complete genome sequence of bacteriophage phiAS7, a T7-like virus that infects Aeromonas salmonicida subsp. salmonicida

To date, a number of Myoviridae bacteriophages that infect Aeromonadaceae have been identified and characterized. However, the genome sequences of Aeromonas phages that not belong to the Myoviridae have not been investigated yet. Herein, we report the complete genome sequence of Aeromonas phage phiAS7, which belongs to the Podoviridae and infects Aeromonas salmonicida subsp. salmonicida.     

异育银鲫源杀鲑气单胞菌杀鲑亚种的分离鉴定

【目的】分离鉴定江苏省扬州市养殖场异育银鲫患病病原。【方法】采用常规的理化特性和分子生物学的方法,对从濒死异育银鲫肝脏处分离到的菌株YZ-1进行表型生物学、分子生物学及药敏试验的系统研究。【结果】该菌株16S r RNA基因(序列长度1 446 bp,Gen Bank登录号为JX164202)与其它杀鲑气单胞菌16S r RNA基因一致性在99%-100%之间,构建发育树确定该菌株为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp.salmonicida)。人工回感可导致异育银鲫死亡。药敏试验结果显示:对头孢呋辛、复方新诺明、恩诺沙星等23种抗生素敏感;对阿米卡星、四环素、大观霉素、头孢拉定等11种抗生素中度敏感;对青霉素G、链霉素、庆大霉素、氟苯尼考、万古霉素等10种抗生素耐药。【结论】研究结果证实引起异育银鲫死亡的病原为杀鲑气单胞菌杀鲑亚种。     

DNA:DNA reassociation analysis of Aeromonas salmonicida

DNA from 26 Aeromonas salmonicida strains, namely 11 'typical' and 15 so-called 'atypical' strains, was used to assess the taxonomic relatedness within the species. The genomes were characterized by determination of DNA base composition, DNA:DNA reassociation, calculation of sequence divergence following reassociation, and by genome size estimations. By comparison with DNA obtained from controls and the Aeromonas hydrophila group, A. salmonicida strains were determined to be correctly placed with respect to genus and species. A. salmonicida subspecies salmonicida (the 'typical' group) was an extremely homogeneous taxon. The 'atypical' strains were more diverse, but distinct biotypes were recognizable. The first biotype included several geographically diverse isolates from goldfish. The second recognizable biotype included strains isolated from European carp. Other 'atypical' isolates could not be grouped but showed enough internal homology to be retained within the species. The A. salmonicida subspecies achromogenes and masoucida were found to be closely related to the motile aeromonads. It is considered that the present classification of A. salmonicida is unsuitable and should be restructured to include A. salmonicida subspecies salmonicida, subspecies achromogenes (to include the present subspecies masoucida), and the reintroduced subspecies nova.     

Aeromonas salmonicida toxin AexT has a Rho family GTPase-activating protein domain

The N terminus of the Aeromonas salmonicida ADP-ribosylating toxin AexT displays in vitro GTPase-activating protein (GAP) activity for Rac1, CDC42, and RhoA. HeLa cells transfected with the AexT N terminus exhibit rounding and actin disordering. We propose that the Aeromonas salmonicida AexT toxin is a novel member of the growing family of bacterial RhoGAPs.     

DNA probe for Aeromonas salmonicida.

A DNA fragment that is specific to Aeromonas salmonicida has been isolated from a genomic DNA library by differential hybridization. The specificity of this fragment as a DNA probe for A. salmonicida was shown by hybridization against reference strains and clinical isolates of A. salmonicida, related aeromonads, and species from several other bacterial genera. The sensitivity of detection by a polymerase chain reaction test, based on this fragment, was approximately two A. salmonicida cells.     

DNA probe for Aeromonas salmonicida.

A DNA fragment that is specific to Aeromonas salmonicida has been isolated from a genomic DNA library by differential hybridization. The specificity of this fragment as a DNA probe for A. salmonicida was shown by hybridization against reference strains and clinical isolates of A. salmonicida, related aeromonads, and species from several other bacterial genera. The sensitivity of detection by a polymerase chain reaction test, based on this fragment, was approximately two A. salmonicida cells.     

Genetic diversity of the fish pathogen Aeromonas salmonicida demonstrated by random amplified polymorphic DNA and pulsed-field gel electrophoresis analyses

The current taxonomy of Aeromonas salmonicida includes 4 subspecies. A. salmonicida subsp. salmonicida is associated with salmonid furunculosis, and A. salmonicida subsp. achromogenes, A. salmonicida subsp. masoucida, and A. salmonicida subsp. smithia are strains that show variation in some biochemical properties. This classification does not readily encompass isolates from a wide range of fish hosts currently described as atypical A. salmonicida. This study examined 17 typical strains, 39 atypical strains and 3 type A. salmonicida subspecies strains for genetic similarity using the random amplified polymophic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) techniques. On the basis of RAPD- and PFGE-derived profiles, similarity matrices and dendrograms were constructed. The results showed that species A. salmonicida constituted a genetically heterogeneous group of strains, encompassing within an homogeneous or clonal lineage comprised solely of typical strains and the A. salmonicida subsp. salmonicida type strain.     

Identification of the major outer membrane proteins of Aeromonas salmonicida

Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the etiological agent of furunculosis, a serious infectious disease of salmonids. Aeromonas spp. are ubiquitous waterborne bacteria responsible for a wide spectrum of diseases among aquatic organisms and humans. Bacterial outer membrane proteins (OMPs) play a significant role in virulence as they comprise the outermost surface in contact with host cells and immune defense factors. To identify the major OMPs of A. salmonicida a proteomic analysis was undertaken using a carbonate OMP-enrichment protocol. The enriched OMP-extracts were separated by 2-dimensional electrophoresis (2-DE) and the spots identified using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) via an electrospray ionization source. In total, 76 unique proteins were identified from the 125 spots observed on the 2-D gel. The surface layer (S-layer) VapA protein dominated the A. salmonicida OMP 2-D profile, accounting for 60% of the protein on the 2-D gels. Among the other outer membrane proteins identified were at least 10 porins and various receptors involved in nutrient acquisition. Also identified in the carbonate insoluble fraction were phosphoglycerate kinase, enolase and others that lacked classical export sorting signals. The putative association of these proteins with the cell surface might provide new insights concerning the biological and pathogenic roles of these molecules in A. salmonicida infection. This work represents the first systematic attempt to characterize the cell surface of A. salmonicida.     

Structural studies of the core region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide

The core oligosaccharide structure of the in vivo derived rough phenotype of Aeromonas salmonicida subsp. salmonicida was investigated by a combination of compositional, methylation, CE-MS and one- and two-dimensional NMR analyses and established as the following: [carbohydrate: see text] where R=alpha-D-Galp-(1-->4)-beta-D-GalpNAc-(1--> or alpha-D-Galp-(1--> (approx. ratio 4:3). Comparative CE-MS analysis of A. salmonicida subsp. salmonicida core oligosaccharides from strains A449, 80204-1 and an in vivo rough isolate confirmed that the structure of the core oligosaccharide was conserved among different isolates of A. salmonicida.