Purification and characterization of Aeromonas sobria pili, a possible colonization factor.

Pili of Aeromonas sobria Ae1 were purified and characterized. The molecular mass of the pilin was estimated to be about 23 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Ae1 pili were electrophoretically and immunologically distinguishable from the W pili of A. hydrophila Ae6, although the two pili were morphologically indistinguishable. The N-terminal amino acid sequences of the two pilins were identical in the first 10 residues. Strain Ae1 and its purified pili adhered to human and rabbit intestines and agglutinated human and rabbit erythrocytes. Hemagglutination was inhibited by D-galactose and D-mannose, but not by L-fucose. Organisms pretreated with the Fab fraction of the antipilus antibody failed to adhere to the intestines. Organisms did not adhere to intestines pretreated with the purified pili. These findings suggest that the pili are a colonization factor of A. sobria Ae1.

Survival of Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.

Cellular fatty acid composition of Plesiomonas shigelloides.

The cellular fatty acid compositions of 29 strains of Plesiomonas shigelloides and 5 strains of Aeromonas hydrophila were studied. The cellular fatty acid compositions of all the Plesiomonas strains were identical and characterized by the presence of hexadecanoate (16:0) (33%), hexadecenoate (16:1) (28%), octadecenoate (18:1) (9%), and octadecanoate (18:0) (6%). The cellular fatty acid composition of A. hydrophila was similar to that of the Plesiomonas strains, except that the former contained an average of 25% 16:0, 29% 16:1, 12% 18:1, and 2% 18:0 acids compared with 33, 28, 9, and 6%, respectively, for the latter. The percentage ratios of 16:1 to 16:0 and 18:1 to 18:0 could be used to differentiate P. shigelloides from A. hydrophila. These ratios were 0.8 and 1.5 for the former and 1.2 and 6.0 for the latter.

Comparison of different media for the identification and quantification of Aeromonas spp. in water.

In order to assess the suitability of the Starch glutamate ampicillin penicillin-10C agar for the isolation of Aeromonas spp. from waters it was necessary to compare the properties of this medium with those of three others, Starch ampicillin agar, Ampicillin dextrin agar and m-Aeromonas medium, and to monitor different kinds of waters. A selection of forty eight samples were taken from moderately polluted river water, highly polluted river water, polluted sea water (littoral) and treatment & distribution water and monitored using these media. The results were similar with Ampicillin dextrin agar, m-Aeromonas medium and Starch glutamate ampicillin penicillin-10C, but the simplicity of composition and use and its selectivity recommends the last medium as the most adequate for the isolation of Aeromonas spp.

A new and rapid method for the assay of bacterial fatty acids using high resolution capillary gas chromatography and trimethylsulfonium hydroxide.

Gas-liquid chromatography of cellular fatty acids is a useful tool for the identification of bacteria. Derivatization of bacterial fatty acids to methyl esters by conventional techniques is usually time-consuming and complicated. A new one-step technique using trimethyl-sulfonium hydroxide allows the direct formation of fatty acid methyl esters within 1-2 min. Some random examples of profiles demonstrate that straight, branched, saturated, unsaturated, hydroxy and cyclopropyl fatty acids match conventional preparations well. The method is a very sensitive one, since only a few colonies are sufficient for preparation of fatty acid methyl esters.

Electrophoretic analysis of the surface components of autoagglutinating surface array protein-positive and surface array protein-negative Aeromonas hydrophila and Aeromonas sobria.

The protein and lipopolysaccharide (LPS) compositions of 10 autoagglutinating Aeromonas hydrophila and Aeromonas sobria strains were studied; one group consisted of five serogroup O:11 strains that contained an S layer, while a second group was composed of diverse serogroups that were S layer negative by transmission electron microscopy. All serogroup O:11 strains were found to contain a predominant 52,000- to 54,000-molecular-weight protein that was present on both whole-cell and outer membrane protein profiles; this protein was found to be glycine extractable under low-pH (pH 4) conditions and was identified as the surface array protein. LPS analysis revealed that all O:11 strains exhibited homogeneous-length O-polysaccharide side chains characterized primarily by two or three major bands. In contrast, S-layer-negative autoagglutinating strains of other serogroups lacked this predominant surface array protein, and silver stain analysis of LPS indicated that such profiles mainly consisted of core antigens and were deficient in or devoid of O-polysaccharide side chains. These collective results offer potential explanations for observed differences between these two groups in virulence, disease spectrum, and pathogenic properties.

Dynamics of Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae in a sewage treatment pond.

The spatiotemporal dynamics of Aeromonas spp. and fecal coliforms in the sewage treatment ponds of an urban wastewater center were studied after 20 months of sampling from five stations in these ponds. Isolation and identification of 247 Aeromonas strains were undertaken over four seasons at the inflow and outflow of this pond system. The hemolytic activity of these strains was determined. The Aeromonas spp. and the fecal coliform distributions showed seasonal cycles, the amplitude of which increased at distances further from the wastewater source, so that in the last pond there was an inversion of the Aeromonas spp. cycle in comparison with that of fecal coliforms. The main patterns in these cycles occurred simultaneously at all stations, indicating control of these bacterial populations by seasonal factors (temperature, solar radiation, phytoplankton), the effects of which were different on each bacterial group. The analysis of the Aeromonas spp. population structure showed that, regardless of the season, Aeromonas caviae was the dominant species at the pond system inflow. However at the outflow the Aeromonas spp. population was dominated by A. caviae in winter, whereas Aeromonas sobria was the dominant species in the treated effluent from spring to fall. Among the Aeromonas hydrophila and A. sobria strains, 100% produced hemolysin; whereas among the A. caviae strains, 96% were nonhemolytic.

Validation of flow cytometry for rapid enumeration of bacterial concentrations in pure cultures.

Flow cytometry was investigated as a rapid detection and counting method for bacteria in pure cultures. A simple two-parameter detection scheme was employed: particle size was measured by forward angle light scatter and nucleic acid content by fluorescence of the DNA/RNA-binding dye ethidium bromide. The technique gave results that correlated exceptionally well with conventional plate counting for four species of bacteria, and concentrations in the range 10(2) to 10(7) cfu/ml. Cytometric counts were obtained in a few minutes, as compared with 2 d required for the plate counts. Under ideal conditions, each bacterial species examined exhibited a characteristic 'signature' on the cytometer, which could be explained by its known properties and morphology.

Particle agglutination assays to identify fibronectin and collagen cell surface receptors and lectins in Aeromonas and Vibrio species.

A rapid particle agglutination assay (PAA) utilizing latex beads coated with connective tissue and serum proteins was evaluated for its ability to identify fibronectin, collagen (types I and IV), fibrinogen, and transferrin cell surface receptors on Vibrio and Aeromonas strains isolated from diseased fish, human infections, and the environment. Similar tests were performed to screen for cell surface lectins. Vibrio as well as Aeromonas strains were found to bind connective tissue proteins (collagen types I, II, and IV and fibronectin), serum proteins (i.e., fibrinogen), and glycoproteins (bovine submaxillary mucin, hog gastric mucin, orosomucoid, and fetuin) immobilized on the latex particles. The specificity of the agglutination reaction was studied by particle agglutination inhibition assays performed by preincubating bacterial suspensions in solutions containing either gelatin (for the various connective tissue protein PAA reagents) or sialic acid-rich glycoproteins (for the various glycoprotein PAA reagents). Expression of cell surface receptors for connective tissue proteins was found to depend on culture methods.

Identification of the cell surface receptor for bacteriophage 18 from Aeromonas hydrophila.

Bacteriophage 18 was previously isolated by multiplication in Aeromonas hydrophila. The bacteriophage receptor was shown to be the lipopolysaccharide (LPS), specifically the low MW polysaccharide fraction (LPS core oligosaccharide) A. hydrophila mutants, resistant to this phage were isolated and found to be devoid of LPS O antigen by several criteria and had alterations in the lipopolysaccharide core.

The quantitative relationship of lethality between extracellular protease and extracellular haemolysin of Aeromonas salmonicida in Atlantic salmon (Salmo salar L.).

An additive relationship of lethality between purified protease and haemolysin of the extracellular products (ECP) of Aeromonas salmonicida was demonstrated by i.p. injection in Atlantic salmon (Salmo salar L.). The lethal toxicity of the combinations of protease and haemolysin follow a linear regression line y = -54.54x + 2400. The LD50 of protease and haemolysin when injected separately was 2400 ng/g fish and 44 ng protein/g fish, respectively.

Characterization of Aeromonas sobria hemolysin by use of monoclonal antibodies against Aeromonas hydrophila hemolysins.

Aeromonas sobria produces hemolysin in a form activable with trypsin under defined cultural conditions. In immunoblotting analyses with the culture supernatant of A. sobria, the monoclonal antibody reacting specifically to Aeromonas hydrophila CA-11 hemolysin bound to the 53,000- and 49,000-dalton bands before and after trypsinization, respectively. The monoclonal antibody reacting to A. hydrophila AH-1 hemolysin did not bind either band. A. sobria hemolysin is, therefore, related antigenically to CA-11 hemolysin, while the molecular weights before and after activation differ from those of A. hydrophila hemolysins, being 54,000 and 51,000, respectively. The hemolytic and enterotoxigenic activities of A. sobria hemolysin were both neutralized by the monoclonal antibody against CA-11 hemolysin. It seems, therefore, that the same site on A. sobria hemolysin is responsible for both biological activities.

Typing of Aeromonas strains by DNA restriction endonuclease analysis and polyacrylamide gel electrophoresis of cell envelopes.

The outer membrane protein (OMP) composition (OMP typing) of 46 fecal Aeromonas strains from hybridization groups (HGs) 1 (A. hydrophila; n = 10), 4 (A. caviae; n = 16), and 8 (A. veronii; n = 20) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a phenotypic typing method. Almost every isolate of HG-1 and HG-8 had a unique OMP profile, in contrast to isolates of HG-4, which were separated into five different OMP types. It was possible to recognize HGs 1, 4, and 8 by OMP profiles. Twenty-three Aeromonas strains from HGs 1 (n = 5), 4 (n = 10), and 8 (n = 8) were tested by whole-cell DNA restriction endonuclease analysis (REA) as a genetic typing method. All strains tested by REA (with SmaI) had different DNA digestion patterns. Although additional DNA-rRNA hybridization analyses with SmaI and 16S and 23S rRNAs from Escherichia coli showed a reduction in the number of restriction bands to 8 to 13 hybridized fragments, the discriminative value was less when compared with that obtained by REA. The individual differences found by REA were used to analyze whether patients remained colonized by the same Aeromonas strain. Of 11 patients with diarrhea, 2 had a different isolate on repeat culture. In addition, one of nine tested fecal samples contained two Aeromonas isolates with different REA patterns. These results indicate that during diarrheal disease the intestinal tract may be colonized simultaneously with different Aeromonas isolates.

Characterization of a pilus produced by Aeromonas hydrophila.

A pilus produced by Aeromonas hydrophila was purified and partially characterized. The pilin monomers had an apparent molecular weight of 17,000. Agglutination studies indicated serological cross-reactivity in the pili of A. hydrophila strains. Presence of pili did not correlate with hydrophobicity or haemagglutinating ability of the bacteria.

Typing of Aeromonas species by protein fingerprinting: comparison of radiolabelling and silver staining for visualising proteins.

A method for typing Aeromonas species by silver staining of total soluble proteins separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis is described. There was good agreement with the results obtained by autoradiography of whole-cell proteins for isolates examined by both methods.

Amonabactin, a novel tryptophan- or phenylalanine-containing phenolate siderophore in Aeromonas hydrophila.

Aeromonas hydrophila 495A2 excreted two forms of amonabactin, a new phenolate siderophore composed of 2,3-dihydroxybenzoic acid, lysine, glycine, and either tryptophan (amonabactin T) or phenylalanine (amonabactin P). Supplementing cultures with L-tryptophan (0.3 mM) caused exclusive synthesis of amonabactin T, whereas supplements of L-phenylalanine (0.3 to 30 mM) gave predominant production of amonabactin P. The two forms of amonabactin were separately purified by a combination of production and polyamide column chromatographic methods. Both forms were biologically active, stimulating growth in iron-deficient medium of an amonabactin-negative mutant. Of 43 additional siderophore-producing isolates of the Aeromonas species that were tested, 76% (19 of 25) of the A. hydrophila isolates were amonabactin positive, whereas only 19% (3 of 16) of the A. sobria isolates and all (3 of 3) of the A. caviae isolates produced amonabactin, suggesting a predominant synthesis of amonabactin in certain Aeromonas species.

Bioactivity and immunological characterization of a cholera toxin-cross-reactive cytolytic enterotoxin from Aeromonas hydrophila.

A cytolytic enterotoxin of molecular weight 52,000 was isolated and purified from culture supernatants of a human diarrheal isolate (SSU) of Aeromonas hydrophila. The toxin reacted with cholera antitoxin when tested in an enzyme-linked immunosorbent assay and by Western blot (immunoblot) analysis. The appearance of cytotoxic and hemolytic activities in culture supernatant occurred simultaneously 8 h after the initial inoculation of the culture. Loss of hemolytic activity and cholera toxin cross-reactivity was correlated with heat and pH inactivation. Homologous antibodies neutralized the cytotoxic and hemolytic activities associated with the toxin, but cholera antitoxin did not neutralize these activities. The toxin also possessed enterotoxic activity as demonstrated by fluid accumulation in rabbit ligated intestinal loops. When purified cytolytic enterotoxin was injected intravenously into mice, death occurred within 2 min, whereas mice injected with whole cells or sonicated cell fragments died after several hours or days. Results from 51Cr release experiments demonstrated that the cytolytic enterotoxin had significant membrane-damaging capability. These results indicated that the cytolytic and enterotoxic activities expressed by the described A. hydrophila toxin may contribute significantly to the pathogenesis of disease associated with A. hydrophila.

Purification and characterization of Aeromonas hydrophila beta-hemolysin.

Aeromonas hydrophila hemolysin was excreted in our culture conditions during the stationary growth phase. The toxin was purified to homogeneity by a three-step method: ultrafiltration, acid precipitation in the presence of RNA and anion exchange chromatography with FPLC apparatus. Beta-hemolysin is a protein not associated with lipids, carbohydrates or nucleic acids whose subunit mol. wt is 51,000. The mol. wt determined by polyacrylamide gel electrophoresis suggests that the molecule is in a trimeric form. The toxin is thermolabile and inactivated by proteolytic enzymes such as trypsin, chymotrypsin, pronase, subtilisin and proteinase K. Antibodies raised against the beta-hemolysin neutralize both hemolytic and cytotoxic activities. When injected at high dose, this purified hemolytic protein causes a positive rabbit ileal loop test, thus indicating that beta-hemolysin could be the main virulence factor involved in intestinal symptoms.

Aeromonas: identificación de especies y correlación de marcadores bioquímicos y pruebas de enteropatogenidad / Aeromonas: identification of species and relationship of markers and enterotoxicity

La patogenicidad intestinal de Aeromonas para el humano ha sido bien establecida y se conoce que producen enteroxinas, citotoxinas y tienen capacidad de adherencia a las células del epitelio intestinal. En nuestro medio constituyen un importante patógeno intestinal, aislándoseles en 185 (15%) de 1264 coprocultivos positivos, constituyéndose en el quinto patógeno intestinal en frecuencia. Se estudian 185 cepas de Aeromonas, aisladas en el C.R.B. del H.U.M., durante los años 1983-1986. Para la especiación se usa el esquema Popoff y Veron, se sigue a Dean, mediante la utilización de las pruebas de ratones lactantes, para detectar enterotoxina y a Burke y cols. para la hemolisina. De las 185 cepas estudiadas, 84 (45.41%) son A. hydrophila, 21 (11.35%) A. sobria y 80 (43.24%) A. caviae. Este estudio ratifica que en A. hydrophila el patrón VP + L + A- y está presente en cepas que producen enterotoxina capaces, con este mecanismo conocido, de producir infección diarréica. En A. Sobria el patrón VP + L + A- presente en 66.67% no concuerda con la producción de hemolisina y enterotoxina. Esta especie que se reporta también frecuente como enteropatógeno, pudiera no siempre serlo a poseer otros mecanismos distintos de patogenicidad a los estudiados. El estudio es coincidente con otros en que ese patrón no está presente con frecuencia en A. caviae, no obstante, es interesante que 2 son hemolisina+ y 3 enterotoxina+, ello pudiera explicar la enteropatogenicidad que esporádicamente se le reporta

Characterization of an Aeromonas hydrophila strain isolated on a septicemic out-break in a fish-farm of Spain.

An Aeromonas hydrophila strain (AH-3) was isolated from a septicemic out-break on a gold-fish farm near Barcelona (Spain). On the bases of their virulence and surface characteristics was classified as moderate to weakly virulent.