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 .     

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.     

Comparison of extracellular proteases produced by Aeromonas salmonicida strains, isolated from various fish species

Extracellular products (ECPs) of five typical and 25 atypical Aeromonas salmonicida isolates from various fish species and geographical locations were analysed by substrate specificity, inhibition of proteolytic activity and substrate SDS-PAGE. The type strains of Aer. salmonicida subsp. salmonicida and Aer. salmonicida subsp. achromogenes were included for comparison. The results indicated that the strains formed six protease groups. The proteases produced by the two type strains were of a different nature. All the typical strains belonged to one group and showed proteolytic activities comparable to P1 and P2 proteases. Three atypical (oxidase-negative) strains secreted a protease comparable to P1. With the exception of these three, all strains produced metallo-gelatinases. A metallo-caseinase (AsaP1) was detected in the ECP of subsp. achromogenes type strain and 10 of the atypical strains. A number of proteolytic components with different apparent molecular weights (AMWs) were identified. These include caseinases with AMWs of > 100, 80, 60 and 30 kDa and gelatinolytic components with different AMWs, including some with AMW higher than P1 and lower than P2. The protease production of the isolates was not found to be host specific.     

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.     

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.     

Characterization of atypical Aeromonas salmonicida isolates by ribotyping and plasmid profiling

A total of 38 strains of atypical Aeromonas salmonicida , three oxidase-negative but otherwise typical Aer. salmonicida , three typical Aer. salmonicida , and two reference strains, isolated from several countries and fish species were examined with respect to rRNA gene restriction patterns (ribotypes) and plasmid profiles. Most epidemiologically unrelated strains had different ribotypes, whereas isolates from the same outbreak were identical. All strains, except one, carried one or more large plasmids (> 55 kbp) and all strains, except two, additionally carried one or more smaller plasmids. Many strains isolated from the same outbreak showed different plasmid profiles although some plasmids were identical. The results suggest the existence of several atypical Aer. salmonicida. It also seems that ribotypes are stable properties for these bacteria while the plasmids are more labile.     

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.     

Assessment of genetic variability and relatedness among atypical Aeromonas salmonicida from marine fishes,using AFLP-fingerprinting

Atypical strains of Aeromonas salmonicida are the causal agent of atypical furunculosis or ulcer disease in various fish species, including spotted wolffish Anarhichas minor, which is a promising species in the Norwegian fish-farming industry. Isolates of atypical A. salmonicida comprise a very heterogenous group showing large variety in biochemical, molecular and virulence characteristics. The genetic variability among atypical isolates from wolffish was characterised using amplified fragment length polymorphism analysis: AFLP-fingerprinting. Additional isolates from halibut Hippoglossus hippoglossus, turbot Scophthalmus maximus, cod Gadus morhua and several salmonid fishes were included for assessment of variability and relatedness among a total of 56 atypical isolates of A. salmonicida. They were compared to reference strains of A. salmonicida subspecies and to other Aeromonas species pathogenic in fishes. AFLP-fingerprints subjected to similarity analysis yielded a grouping of the isolates into several clusters, revealing genetic heterogeneity among the isolates. There seems to be a correlation between genetic similarity among isolates and the fish host. The Icelandic isolates, mainly from cod, formed a very homogeneous subcluster, which was closely related to the wolffish isolates. All atypical isolates from spotted and common wolffish grouped together in a large cluster and appear to be very homogeneous, even though they had been isolated over a period of 8 yr at different locations in Norway. On the other hand, most of the isolates from turbot and halibut grouped together into 2 different clusters, while the 9 atypical isolates from salmonids appeared in 4 different clusters. Thus, the atypical isolates of A. salmonicida from halibut, turbot and salmonid fishes seem to be more genetically diverse than those from wolffish and cod.     

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.     

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.     

Characterization of Aeromonas encheleia strains isolated from aquatic environments in the Czech Republic

Aims:  Characterization and identification of Aeromonas strains isolated from surface and underground waters using phenotypic and genotyping methods.
Methods and Results:  Biotyping using the ENTEROtest 24 kit and conventional biochemical and physiological tests assigned four strains to Aeromonas encheleia , whereas three isolates were identified as ambiguous Aeromonas bestiarum/Aeromonas caviae and one strain as Aeromonas eucrenophila/Aeromonas encheleia . Further characterization grouped the analysed strains together with Aer. encheleia CCM 4582^T and assigned the analysed group as members of Aer. encheleia species using ribotyping, whole-cell protein analysis and ERIC-PCR fingerprinting. The results obtained were verified by DNA gyrase A subunit gene sequencing. All analysed isolates showed unique molecular patterns, except for isolates P 1769 and CCM 7407, which revealed the same Eco RI ribotype profile and proved to be identical strains.
Conclusions:  Our results imply that Aer. encheleia strains occur in unpolluted surface as well as in underground waters and demonstrate applied methods as suitable for their identification.
Significance and Impact of the Study:  To our best knowledge, this is the first report of the isolation and identification of Aer. encheleia in the Czech Republic.     

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.     

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.     

The interaction of trimethoprim and quinolones against Gram-negative fish pathogens

The effect of combination of trimethoprim with other non-sulphonamide antibacterial agents, in particular oxolinic acid and nalidixic acid, was evaluated against Gram-negative fish pathogens. The species included Aeromonas salmonicida, Yersinia ruckeri , some Vibrio spp. and Escherichia coli as a reference. The extent of synergy found by other workers with these substances against human Gram-negative bacteria was not apparent here. Some positive interaction between trimethoprim and oxolinic acid was found with Aer. salmonicida, Y. ruckeri and E. coli and between trimethoprim and nalidixic acid with Y. ruckeri in double disc diffusion tests but was not supported by fractional inhibitory concentration indices. The combinations were not effective in preventing emergence of resistance in passage on a drug gradient. Trimethoprim-resistant isolates of Aer. salmonicida were inhibited by low levels of oxolinic acid but the converse did not apply.     

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

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

Automated biochemical identification of bacterial fish pathogens using the Abbott Quantum II

The Quantum II, originally designed by Abbott Diagnostics for automated rapid identification of members of Enterobacteriaceae, was adapted for the identification of bacterial fish pathogens. The instrument operates as a spectrophotometer at a wavelength of 492.600 nm. A sample cartridge containing 20 inoculated biochemical chambers is inserted in the path of the analyzing beam. Reactions are converted into a 7-digit octal biocode, relayed via a sensor to the memory module, and compared to biocodes preprogrammed in the memory. An identification is then printed. Presently, the Quantum II is capable of identifying human strains of Aeromonas hydrophila and Edwardsiella tarda. This study was initiated to determine the feasibility of expanding the use of the Quantum II to include identification of bacterial fish pathogens. Ten to 50 isolates of Edwardsiella ictaluri, Serratia liquefaciens, Yersinia ruckeri, Aeromonas hydrophila, typical Aeromonas salmonicida, and atypical Aeromonas salmonicida were utilized to determine optimal incubation conditions, relative stability of the biochemicals, and ability to obtain consistent biocode numbers. After sorting the octal biocodes from the 169 isolates into groupings using a cluster analysis technique, it was shown by a Chi-square goodness of fit test that isolates of a given species were sorted into the same cluster group at a frequency of at least 99%. Results of this study illustrate the usefulness of the Quantum II BID system for the identification of bacterial fish pathogens not contained within the system's memory module.     

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.     

Characterization of Aeromonas salmonicida subsp. salmonicida: a comparative study of strains of different geographic origin

A total of 130 strains of the fish pathogen Aeromonas salmonicida isolated in Denmark, Norway, Scotland, Canada and the USA were examined. The strains originated from farmed salmonid fish. The biochemical, physiological and serological characteristics, antibiotic resistance patterns and cell surface-related properties were compared. Aeromonas salmonicida was found to be remarkably consistent in general cultural and biochemical characteristics. It is noteworthy that the strains were positive in the fermentation of L-arabinose and were negative in the fermentation of D-arabinose. All the strains were highly proteolytic. It was observed, however, that 5% of the strains did not digest calf and trout serum and the production of haemolysin and degradation of casein by the same strains were delayed compared with the other strains. Common to all of the rough strains were auto-aggregation and ability to bind the dyes Coomassie brilliant blue and Congo red and the majority of these strains were highly hydrophobic. The strains were tested for their susceptibility to 22 antibacterial agents. Antibiotic resistance profiles of Aer. salmonicida indicated that resistance to the quinolones and oxytetracycline was increasing and that multi-resistant strains were found in several countries. The variation found in antibiograms could have potential as epidemiological markers in certain geographic areas.