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.     

Phage morphology recapitulates phylogeny: the comparative genomics of a new group of myoviruses

Among dsDNA tailed bacteriophages (Caudovirales), members of the Myoviridae family have the most sophisticated virion design that includes a complex contractile tail structure. The Myoviridae generally have larger genomes than the other phage families. Relatively few "dwarf" myoviruses, those with a genome size of less than 50 kb such as those of the Mu group, have been analyzed in extenso. Here we report on the genome sequencing and morphological characterization of a new group of such phages that infect a diverse range of Proteobacteria, namely Aeromonas salmonicida phage 56, Vibrio cholerae phages 138 and CP-T1, Bdellovibrio phage φ1422, and Pectobacterium carotovorum phage ZF40. This group of dwarf myoviruses shares an identical virion morphology, characterized by usually short contractile tails, and have genome sizes of approximately 45 kb. Although their genome sequences are variable in their lysogeny, replication, and host adaption modules, presumably reflecting differing lifestyles and hosts, their structural and morphogenesis modules have been evolutionarily constrained by their virion morphology. Comparative genomic analysis reveals that these phages, along with related prophage genomes, form a new coherent group within the Myoviridae. The results presented in this communication support the hypothesis that the diversity of phages may be more structured than generally believed and that the innumerable phages in the biosphere all belong to discrete lineages or families.     

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.     

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.     

RAPD analysis of Aeromonas salmonicida and Aeromonas hydrophila

The randomly amplified polymorphic DNA (RAPD) technique was used to analyse the genetic differentiation of 13 strains of Aeromonas salmonicida subsp. salmonicida , and seven strains of Aer. hydrophila. Reproducible profiles of genomic DNA fingerprints were generated by polymerase chain reaction (PCR) using a single randomly designed primer. The RAPD profiles of all the non-motile aeromonads, Aer. salmonicida subsp. salmonicida were identical. However, profiles of the motile aeromonads, Aer. hydrophila differed between isolates. These findings reveal genomic homogeneity in Aer. salmonicida subsp. salmonicida and genetic variety in Aer. hydrophila strains.     

Restriction endonuclease fingerprinting analysis of Canadian isolates of Aeromonas salmonicida

Restriction endonuclease fingerprinting (REF) analysis was used to examine total cellular DNA prepared from 56 independent field isolates of the fish pathogen, Aeromonas salmonicida. DNA was digested singly with the restriction enzymes EcoRI and HindIII, and the resulting fragments separated by polyacrylamide gel electrophoresis and visualized by silver staining. The REF patterns of typical isolates of A. salmonicida subsp. salmonicida were distinct from those of A. hydrophila, A. salmonicida subsp. achromogenes, A. salmonicida subsp. masoucida, and atypical isolates of A. salmonicida subsp. salmonicida. Differences between strains of typical A. salmonicida subsp. salmonicida could also be distinguished. Canadian isolates examined could be assigned to 1 of 12 different groups (REF groups), with the majority of the isolates belonging to REF groups 1 and 5. REF group 1 strains were isolated from British Columbia and New Brunswick while REF group 5 isolates were found in Ontario. None of the European strains examined had REF patterns identical to those of Canadian isolates. Based on REF analysis, there was little genetic heterogeneity detected among 23 isolates from two short-term studies of naturally occurring infections. Several different REF groups were seen among A. salmonicida collected over a 10-year period from coho salmon from the Credit River. Consistent with earlier biochemical and hybridization studies, the REF data suggest that A. salmonicida is a clonal pathogen. REF analysis can, however, permit the identification of subgroups, which may be useful in epidemiological studies.     

Characteristics of 'atypical', cytochrome oxidase-negative Aeromonas salmonicida isolated from ulcerated flounders (Platichthys flesus (L.))

'Atypical', cytochrome oxidase-negative variants of the fish pathogen Aeromonas salmonicida , isolated from ulcerated flounder ( Platichthys flesus ), were studied using different methods. Two of the strains possessed a protein that corresponded to the A-layer protein of Aer. salmonicida . The strains reacted with antibodies against the A-layer and monoclonal antibodies against the O-antigen of typical Aer. salmonicida . These tests confirm that the isolates from flounder should be classified as Aer. salmonicida . Analysis of the fatty acids showed that the isolates were rather homogenous but the values of the guanine plus cytosine content of the DNA of the bacteria varied too much for any conclusion to be drawn on their taxonomic location. The strains examined exhibited several biochemical characters that differed from those of the type strains of Aer. salmonicida subsp. salmonicida and Aer. salmonicida , subsp. achromogenes . The results suggest that these 'atypical', cytochrome oxidase-negative variants may form a new subspecies of Aer. salmonicida .     

Beyond the A‐layer: adsorption of lipopolysaccharides and characterization of bacteriophage‐insensitive mutants of Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida is a fish pathogen that causes furunculosis. Antibiotherapy used to treat furunculosis in fish has led to resistance. Virulent phages are increasingly seen as alternatives or complementary treatments against furunculosis in aquaculture environments. For phage therapy to be successful, it is essential to study the natural mechanisms of phage resistance in A. salmonicida subsp. salmonicida. Here, we generated bacteriophage‐insensitive mutants (BIMs) of A. salmonicida subsp. salmonicida, using a myophage with broad host range and characterized them. Phage plaques were different depending on whether the A‐layer surface array protein was expressed or not. The genome analysis of the BIMs helped to identify mutations in genes involved in the biogenesis of lipopolysaccharides (LPS) and on an uncharacterized gene (ASA_1998). The characterization of the LPS profile and gene complementation assays identified LPS as a phage receptor and confirmed the involvement of the uncharacterized protein ASA_1998 in phage infection. In addition, we confirmed that the presence of an A‐layer at the bacterial surface could act as protection against phages. This study brings new elements into our understanding of the phage adsorption to A. salmonicida subsp. 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.     

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.     

PCR-based assays for the fish pathogen Aeromonas salmonicida. II. Further evaluation and validation of three PCR primer sets with infected fish

Two Aeromonas salmonicida-specific polymerase chain reaction (PCR) tests and 1 A. salmonicida subsp. salmonicida-specific PCR test were used to screen salmonid populations that were either overtly or covertly infected with A. salmonicida subsp. salmonicida. It was demonstrated that these PCR assays could be used to replace the biochemical testing currently employed to confirm the identity of A. salmonicida isolates cultured from infected fish. The AP and PAAS PCR assays were also capable of direct detection of A. salmonicida in overtly infected fish, with mucus, gill and kidney samples most likely to yield a positive result. Culture was a more reliable method for the direct detection of A. salmonicida in covertly infected salmonids than was the direct PCR testing of tissue samples, with the AP and PAAS PCRs having a lower detection limit (LDL) of approximately 4 x 10(5) colony-forming units (CFU) g(-1) sample.     

Characterization of an ADP-ribosyltransferase toxin (AexT) from Aeromonas salmonicida subsp. salmonicida

An ADP-ribosylating toxin named Aeromonas salmonicida exoenzyme T (AexT) in A. salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, was characterized. Gene aexT, encoding toxin AexT, was cloned and characterized by sequence analysis. AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of Pseudomonas aeruginosa and to the YopE cytotoxin of different Yersinia species. The aexT gene was detected in all of the 12 A. salmonicida subsp. salmonicida strains tested but was absent from all other Aeromonas species. Recombinant AexT produced in Escherichia coli possesses enzymatic ADP-ribosyltransferase activity. Monospecific polyclonal antibodies directed against purified recombinant AexT detected the toxin produced by A. salmonicida subsp. salmonicida and cross-reacted with ExoS and ExoT of P. aeruginosa. AexT toxin could be detected in a wild type (wt) strain of A. salmonicida subsp. salmonicida freshly isolated from a fish with furunculosis; however, its expression required contact with RTG-2 rainbow trout gonad cells. Under these conditions, the AexT protein was found to be intracellular or tightly cell associated. No AexT was found when A. salmonicida subsp. salmonicida was incubated in cell culture medium in the absence of RTG-2 cells. Upon infection with wt A. salmonicida subsp. salmonicida, the fish gonad RTG-2 cells rapidly underwent significant morphological changes. These changes were demonstrated to constitute cell rounding, which accompanied induction of production of AexT and which led to cell lysis after extended incubation. An aexT mutant which was constructed from the wt strain with an insertionally inactivated aexT gene by allelic exchange had no toxic effect on RTG-2 cells and was devoid of AexT production. Hence AexT is directly involved in the toxicity of A. salmonicida subsp. salmonicida for RTG-2 fish cells.     

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.     

Grouping by plasmid profiles of atypical Aeromonas salmonicida isolated from fish, with special reference to salmonid fish

Plasmid profile analyses were performed for 113 strains of atypical Aeromonas salmonicida and the reference strain A. salmonicida subsp. salmonicida ATCC 14174. The atypical A. salmonicida strains comprised 98 strains obtained from fish originating from 54 farms and 2 lakes in Norway, 10 strains from Canada (2), Denmark (2), Finland (1), Iceland (1) and Sweden (4), the reference strains NCMB 1109 and ATCC 15711 (Haemophilus piscium) of A. salmonicida subsp. achromogenes, and the type cultures A. salmonicida subsp. achromogenes NCMB 1110, A. salmonicida subsp. masoucida ATCC 27013 and A. salmonicida subsp. smithia CCM 4103. A total of 95 strains of atypical A. salmonicida were separated into 7 groups (I to VII) based on the plasmid profiles. Eighteen strains of atypical A. salmonicida had no common plasmid profile. The type strain NCMB 1110 and the reference strain NCMB 1109 were included in group IV, and the type strain ATCC 27013 in group V, but the other reference and type strains had plasmid profiles different from all the other strains. An epidemiological link was documented between strains collected from different farms/localities in each of groups I, III, V and VII. Physiological and biochemical characterizations were performed for 93 of the strains to investigate phenotypic differences between the plasmid groups. Group VII strains and 3 strains with no common plasmid profile differed from the other groups in being catalase-negative. Differences in phenotypic characteristics were shown between the plasmid groups. However, significant variations in reactions for several phenotypic characteristics also occurred within each of the groups I to VII. The present study indicates that plasmid profiling may give useful epidemiological information during outbreaks of atypical A. salmonicida infections in fish. Additional comprehensive phenotypic characterisation is of limited value since the phenotypic characteristics in each plasmid group are not uniform.     

Monoclonal antibodies against AsaP1, a major exotoxin of the fish pathogen Aeromonas salmonicida subsp. achromogenes, and their application in ELISA

Two monoclonal antibodies (Mabs) binding to a toxic extracellular metallo-proteinase of Aeromonas salmonicida subsp. achromogenes, AsaP1, were produced. Both reacted with common epitopes of the native enzyme and recognized this 20 kDa antigen on Western blots. One of these Mabs had an inhibitory effect on the caseinase activity of the exotoxin. A Mab-based ELISA was set up and evaluated for serological detection of AsaP1 in bacterial culture filtrates. The exotoxin was identified serologically in the extracellular products of 11 of 26 atypical Aer. salmonicida isolates, including the type strain for subsp. achromogenes NCIMB 1110. The ELISA was approximately 100-fold more sensitive in detecting AsaP1 compared with an azocasein assay. The established serological test enables AsaP1 to be quantified reliably with a lower detection limit of about 0.12 ng ml-1 and has a potential use for the phenotypic differentiation of atypical Aer. salmonicida isolates.     

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.     

PCR-based assays for the fish pathogen Aeromonas salmonicida. I. Evaluation of three PCR primer sets for detection and identification

In an effort to develop a rapid diagnostic test for the fish pathogen Aeromonas salmonicida, the performance of 2 polymerase chain reaction (PCR) primer sets (AP and PAAS) targeting the fish pathogen A. salmonicida and 1 PCR primer set (MIY) targeting A. salmonicida subsp. salmonicida were evaluated. Initially, the PCR assays were used to screen purified DNA extracted from 308 A. salmonicida isolates. The AP and PAAS PCR tests were demonstrated to be 100% specific for the species A. salmonicida and did not cross-react with any of the non-target organisms (bacterial species other than A. salmonicida) used in this study. The combined sensitivity of the AP and PAAS tests was 99.4% and offered the best coverage in terms of identifying the target organism. The MIY PCR appeared to be 100% sensitive and specific for A. salmonicida subsp. salmonicida. Studies with tissues, spiked with known quantities of bacteria, were conducted to determine the lower detection limit of the PCR tests, and then the ability of these PCR tests to detect A. salmonicida in experimentally infected salmonids was assessed.     

长江上游网箱养殖圆口铜鱼杀鲑气单胞菌杀鲑亚种的分离与鉴定

研究首次报道了圆口铜鱼(Coreius guichenoti)疥疮病, 从患病圆口铜鱼的肝脏中分离到优势菌株YTL1, 并运用形态学观察、生理生化检测、16S rRNA和6个管家基因的系统发育分析等对分离菌株进行鉴定。基于以上实验结果, YTL1被最终鉴定为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp. salmonicida)。通过标准Kirby-Bauer纸片扩散法进行抗菌药物敏感性试验, 筛选治疗该暴发病的有效药物, 结果显示YTL1对氟苯尼考, 诺氟沙星和氨苄青霉素等13种抗生素敏感, 对6种抗生素如杆菌肽, 链霉素和卡那霉素有耐药性, 对红霉素具有中等敏感性。因此, 氟苯尼考被建议用来伴饵投喂, 并取得了较好的疾病控制效果。草鱼幼鱼(Ctenopharyngodon idella)和斑马鱼(Danio rerio)的人工感染试验结果显示, 经腹腔注射7.6×106—7.6×108 CFU/mL的YTL1菌液后, 感染鱼的症状与患病圆口铜鱼症状相似。研究证明基于6个管家基因的多序列位点分型是鉴定杀鲑气单胞菌至亚种水平的一种有效方法, 杀鲑气单胞菌是圆口铜鱼人工养殖的最大威胁之一, 并发现鲤科鱼类, 如草鱼和斑马鱼均是杀鲑气单胞菌杀鲑亚种的易感宿主。     

The development of random DNA probes specific for Aeromonas salmonicida

RAPD-PCR has been used to produce DNA probes for Aeromonas salmonicida . DNA hybridization studies showed that RAPD-PCR fragments of the same size did not necessarily hybridize to each other and therefore these sequences were not always homologous. However, a single RAPD-PCR fragment (designated 15e) was identified as being common to Aer. salmonicida . Subsequently, 15e was found to comprise five DNA fragments of similar size which differed in their nucleotide sequences. All five fragments were evaluated as DNA probes for the specific detection of Aer. salmonicida DNA: two hybridized specifically to DNA of all Aer. salmonicida isolates tested, including the four current subspecies and atypical isolates; one hybridized to subspecies salmonicida , achromogenes and masoucida , but not subspecies smithia ; one hybridized to subspecies salmonicida and achromogenes , but not subspecies masoucida or smithia ; and one hybridized to subspecies salmonicida , achromogenes and smithia , but not subspecies masoucida . It is believed that these fragments could be useful as non-radioactive probes for the safe and rapid diagnosis of these fish pathogens.