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.     

Evidence against dormacy in the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida

Results indicate that A. salmonicida does not enter an unculturable dormant state. The resuscitation of dormant cells by nutrient broth described by previous workers appears to be due to the presence of small numbers of viable, culturable cells too few to be detected by the sampling protocol employed.     

Porphyrin binding by the surface array virulence protein of Aeromonas salmonicida.

Congo red binding by virulent A-layer-containing (A+) and avirulent A-layer-deficient (A-) strains of Aeromonas salmonicida was examined. Congo red binding to A+ cells was enhanced by salt and thus hydrophobically driven, but at low Congo red concentrations binding was salt independent. Congo red was bound by A+ cells by a kinetically distinct mechanism (Kd, 0.25 microM) which was absent in A- isogenic strains. Purified A-layer protein ("A protein") protein A also bound Congo red with similar affinity (Kd, 0.40 microM). Congo red binding was structurally specific; it was not influenced by a wide variety of compounds including amino acids and nucleotides and only weakly inhibited by structurally similar dyes. However, protoporphyrin IX and hemin were strong competitive inhibitors of Congo red binding. Protoporphyrin and hemin were bound only by A+ strains (KdS of 0.41 and 0.63 microM, respectively). Furthermore, binding of these porphyrins was strongly inhibited by Congo red but weakly inhibited by hematoporphyrin. Purified A protein also bound protoporphyrin IX and hemin with affinities similar to those of A+ cells (KdS of 0.94 and 0.41 microM, respectively.     

A stable L-form of the fish pathogen Aeromonas salmonicida

A stable L-form of Aeromonas salmonicida , which resulted from induction with benzylpenicillin, grew on brain heart infusion agar at 0–5°C. The L-form was stored successfully for 10 months in phosphate-buffered saline supplemented with 150 g l^-1 glycerol at -70°C. Reversion of the L-form to parental-type walled cells was achieved by transfer to brain heart infusion broth with incubation at 25°C.     

Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene.

A DNA-based assay was developed to detect Aeromonas salmonicida from infected fish by analyzing tissues, feces, and the tank water in which the infected fish were held. This analysis was done both by direct detection from samples and after a bacterial outgrowth step. Polymerase chain reaction (PCR) amplification of a 421-bp sequence from the 3' region of the surface array protein gene (vapA) of A. salmonicida provided a specific and sensitive method for the detection and identification of this important fish pathogen. The sensitivity of PCR detection of A. salmonicida directly from tissues was less than 10 CFU/mg. Furthermore, a detection level of 5 fg, equivalent to approximately 1 cell, was obtained by using purified chromosomal DNA as the template. This highly reproducible assay, which requires 45 min to complete, is therefore sensitive enough to be used as a noninvasive method for monitoring fish populations for the presence of carrier fish. Because the surface protein array (A-layer) is a virulence factor of A. salmonicida, PCR analysis with oligonucleotide primers directed at vapA can also be used to provide information on the potential virulence of a strain.     

Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene.

A DNA-based assay was developed to detect Aeromonas salmonicida from infected fish by analyzing tissues, feces, and the tank water in which the infected fish were held. This analysis was done both by direct detection from samples and after a bacterial outgrowth step. Polymerase chain reaction (PCR) amplification of a 421-bp sequence from the 3' region of the surface array protein gene (vapA) of A. salmonicida provided a specific and sensitive method for the detection and identification of this important fish pathogen. The sensitivity of PCR detection of A. salmonicida directly from tissues was less than 10 CFU/mg. Furthermore, a detection level of 5 fg, equivalent to approximately 1 cell, was obtained by using purified chromosomal DNA as the template. This highly reproducible assay, which requires 45 min to complete, is therefore sensitive enough to be used as a noninvasive method for monitoring fish populations for the presence of carrier fish. Because the surface protein array (A-layer) is a virulence factor of A. salmonicida, PCR analysis with oligonucleotide primers directed at vapA can also be used to provide information on the potential virulence of a strain.     

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.     

Purification and disposition of a surface protein associated with virulence of Aeromonas salmonicida.

Virulent strains of Aeromonas salmonicida observed by electron microscopy were characterized by an outer layer exhibiting a tetragonal repeat pattern. Attenuated strains had a 2.5 X 10(3)- to 5 X 10(3)-fold reduction in virulence and lost the outer layer, autoaggregating properties, and a 49-kilodalton protein (A protein) simultaneously. The A protein is the major protein component of outer membrane fractions of virulent strains. A variety of radiolabeling studies showed that this protein was surface localized and that it provided an effective barrier against iodination of other outer membrane proteins with either lactoperoxidase or diazoiodosulfanilic acid; A protein was not labeled with lactoperoxidase but was specifically labeled with diazoidosulfanilic acid. The A protein was purified by selective extraction with detergent and guanidine hydrochloride, and its amino acid composition was determined. The properties of A protein are compared with those of other bacterial surface layer proteins.     

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.     

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.     

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.     

Atypical characteristics of the salmonid pathogen Aeromonas salmonicida

Incubation of Aeromonas salmonicida at supra-optimal temperatures, i.e. 30-37 degrees C, resulted in the expression of motility by polar flagella, and changes in sugar fermentation patterns, e.g. loss of acid production from mannitol, loss of the ability to degrade complex molecules (aesculin, DNA, elastin and gelatin), and an increase in antibiotic resistance (notably co-trimoxazole). Motility was enhanced in cultures grown in brain heart infusion broth supplemented with 18% (w/v) Ficoll.     

Starvation survival of the fish pathogen Aeromonas salmonicida in seawater

Abstract Three strains of the fish pathogenic bacterium Aeromonas salmonicida were examined with respect to their ability to survive in seawater. Five to seven days after plate counts decreased below the detection limit of 10 cells/ml, the population of respiring A. salmonicida cells comprised less than 4% of the initial total bacterial population. At this stage, samples were transferred either to sterile nutrient or to control flasks containing sterile nutrient-free medium. The addition of nutrients caused reappearance of growing bacteria, detected by plating on agar medium and an increase of the population of respiring bacteria to 85–95% of the total population. In a separate experiment it was shown that after more than six months of starvation at 15°C, a few of the starved A. salmonicida cells were able to regrow in liquid media after addition of nutrients, but not on agar media. These cells evade detection by direct microscopic respiratory measurement but appear to be reactivated after addition of nutrients.     

Dormant/unculturable cells of the fish pathogen Aeromonas salmonicida

Viable cells of Aeromonas salmonicida remained in experimental marine systems after plate counts indicated an absence of culturable cells. These so-called viable but nonculturable (VBNC) cells were coccoid and smaller than their normal culturable counterparts. There was no reduction in lipopolysaccharide of the VBNC cells. There was an alteration in protein composition, however, with a decline in some (15, 70, 30, 22, and 17 kDa), but an increase in another protein (49 kDa). A significant loss of DNA occurred. The VBNC cells responded to fluorescent antibodies prepared against A. salmonicida by developing enlarged and bizarre shapes in the presence of yeast extract and nalidixic acid (the direct viable count technique), and they demonstrated respiratory activity. It was concluded that A. salmonicida survived in seawater, but major morphological changes occurred with cells retaining some viability but losing pathogenicity to Atlantic salmon (Salmo salar).     

Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida

The paracrystalline surface protein array of the pathogenic bacterium Aeromonas salmonicida is a primary virulence factor with novel binding capabilities. The species-specific structural gene (vapA) for this array protein (A-protein) was cloned into lambda gt11 but was unstable when expressed in Escherichia coli, undergoing an 816-base pair deletion due to a 21-base pair direct repeat within the gene. However, the gene was stable in cosmid pLA2917 as long as expression was poor. A-protein was located in the cytoplasmic, inner membrane and periplasmic fractions in E. coli. The DNA sequence revealed a 1,506-base pair open reading frame encoding a protein consisting of a 21-amino acid signal peptide, and a 481-residue 50,778 molecular weight protein containing considerable secondary structure. When assembled into a paracrystalline protein array on Aeromonas the cell surface A-protein was totally refractile to cleavage by trypsin, but became trypsin sensitive when disassembled. Trypsin cleavage of the isolated protein provided evidence that both the NH2- and COOH-terminal regions form distinct structural domains, consistent with three-dimensional ultrastructural evidence. The NH2-terminal 274-residue domain remained refractile to trypsin activity. This segment connects by a trypsin and CNBr-sensitive 78-residue linker region to a COOH-terminal 129-residue fragment which could apparently refold into a partially trypsin-resistant structure after cleavage at residue 323.     

Pulsed-field gel electrophoriesis analyis of Aeromonas salmonicida ssp. salmonicida

A total of 133 strains of Aeromonas salmonicida ssp. salmonicida, isolated from a wide variety of sources, were characterized by pulsed-field gel electrophoresis patterns. Sixteen profiles were demonstrated, with one profile being predominant in samples from all the countries and species of fish. Our results suggest a clonal distribution of this subspecies, with a predominant clone being responsible for most of the outbreaks worldwide.     

The outer membrane proteins of the fish pathogens Aeromonas hydrophila, Aeromonas salmonicida and Edwardsiella tarda

Abstract The profiles of the outer membrane proteins contained many major proteins and were markedly different in a number of Aeromonas hydrophila strains isolated from various origins. In contrast, the profiles of outer membrane proteins in Aeromonas salmonicida from different sources were identical and quite different from those of A. hydrophila .
In the strains of Edwardsiella tarda tested, protein components in the outer membrane were quite similar. 2 Major proteins, of 36 and approx. 46 kDa, were found in the outer membrane. Major proteins in A. hydrophila and A. salmonicida were detected in the 32–36 kDa range, as observed in many Enterobacteriaceae. In all strains tested, several 68–90 kDa polypeptides appeared in the outer membrane during iron starvation.