Demonstration of a proteolytic activity on intracellular origin in Aeromonas hydrophila LP 50

Aeromonas hydrophila LP 50, isolated from packaged pasteurized milk, was grown in glucose-polypeptone medium at 30 degrees C. The proteolytic activity of A. hydrophila LP 50, optimum at the stationary phase of growth, is attributed to extracellular or membrane protease; no intracellular proteolytic activity was shown.     

Effect of a lactic starter culture on the growth and protease activity of Aeromonas hydrophila

Lactococcus lactis subsp. lactis strain Lac 388 (isolated from goats' cheese made without starter) had inhibitory activity against three strains of Aeromonas hydrophila either by the plating method or by the associative culture approach (broth, skim milk and ewes' milk). Low pH was only partially responsible for the antagonism. Inhibition due to hydrogen peroxide and nutrient depletion was excluded. This inhibitory compound(s) was not destroyed by proteolytic enzymes. In mixed cultures the strains of Aer. hydrophila did not produce detectable amounts of protease.     

Effects of cultural conditions on protease production by Aeromonas hydrophila

Production of extracellular proteolytic activity by Aeromonas hydrophila was influenced by temperature, pH, and aeration. Conditions which produced maximal growth also resulted in maximal protease production. Enzyme production appeared to be modulated by an inducer catabolite repression system whereby NH4+ and glucose repressed enzyme production and complex nitrogen and nonglucose, carbon energy sources promoted it. Under nutritional stress, protease production was high, despite poor growth.     

嗜水气单胞菌胞外蛋白酶的化学修饰

 蛋白酶是嗜水气单胞菌 (Aeromonashydrophila)的重要致病因子 .为研究其结构与功能之间的关系 ,用DEPC、EDC、PMSF、N AI等 9种化学修饰剂处理嗜水气单胞菌J 1株胞外蛋白酶ECPase54,然后检测残余酶活力 ,借以研究酶分子中氨基酸侧链基团与酶活性中心的关系 .结果表明 ,羧基、丝氨酸、ε 氨基、胍基等残基与酶活性无关 ;半胱氨酸残基与酶活性也无直接关系 ;而色氨酸、组氨酸、酪氨酸残基侧链以及二硫键的化学修饰引起酶活性的大幅度的下降 ,说明色氨酸、组氨酸、酪氨酸残基以及二硫键是酶活力所必需的基团     

Effects of cultural conditions on protease production by Aeromonas hydrophila.

Production of extracellular proteolytic activity by Aeromonas hydrophila was influenced by temperature, pH, and aeration. Conditions which produced maximal growth also resulted in maximal protease production. Enzyme production appeared to be modulated by an inducer catabolite repression system whereby NH4+ and glucose repressed enzyme production and complex nitrogen and nonglucose, carbon energy sources promoted it. Under nutritional stress, protease production was high, despite poor growth.     

Immunochemical analysis and possible biological role of an Aeromonas hydrophila surface array protein in septicaemia.

The biochemical, immunological, and biological properties of an S layer purified from an Aeromonas hydrophila strain (AH-342) involved in a case of bacteraemia were investigated. The S layer selectively removed from the cell surface was composed of a single acidic (pI 4.56) protein subunit (surface array protein, SAP) with a molecular mass of approximately 52 kDa. Amino acid analysis of this 52 kDa protein indicated a molecule composed of 498 amino acids with 46% hydrophobic residues. No cysteine residues were detected. The first 35 residues of the N-terminus were sequenced by Edman degradation; only 4-24% homology was noted between this sequence and those previously published for SAPs of Aeromonas salmonicida (A450) and a strain of A. hydrophila (TF7) originally isolated from a moribund fish. Polyclonal antibodies raised against AH-342 SAP were genospecific, reacting only against S layers produced by A. hydrophila strains and not those from Aeromonas veronii. Acute serum from the bacteraemic patient from whom AH-342 was isolated reacted strongly with the SAP of AH-342 in immunoblot studies. Purified SAP, when intraperitoneally co-inoculated with SAP- strains of A. hydrophila into Swiss-Webster mice, could reduce the 50% lethal dose by approximately 30-70 fold. The results suggest that the SAP of A. hydrophila strains may play an important role in systemic dissemination after invasion through the gastrointestinal mucosa.     

Solubilization of fish proteins using immobilized microbial cells

Cells of Bacillus megaterium, Aeromonas hydrophila, and Pseudomonas marinoglutinosa were immobilized in calcium alginate. The immobilized cells secreted protease when held in fish meat suspension in water. The enzyme synthesis by the entrapped cells was supported by small amounts of soluble nutrients present in the meat. The secreted protease solubilized the fish meat, solubilization being optimum at pH range of 7.5 to 9.5 and at 50 degrees C. Under these conditions immobilized B. megaterium was most efficient giving 30% solubilization of the meat, followed by A. hydrophila (18%), while immobilized P. marinoglutinosa was less effective. The optimum ratio of fish meat to beads was about 4:3 for B. megaterium and A. hydrophila. The beads had a storage life of 30 days at 4 degrees C. The results suggested potential for use of immobilized microbial cells having extracellular protease activity to enhance solubility of waste proteins. A prototype reactor with beads holding assembly was fabricated which could recover the beads from the meat slurry after the treatment.     

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.     

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.     

Experimental evidence for enteropathogenicity in Aeromonas veronii

Eleven ornithine-positive strains of Aeromonas (9 A. veronii and 2 provisionally classified as Aeromonas species ornithine positive) were tested for ability to cause fluid accumulation in the rabbit ileal loop. All eight beta-hemolytic strains caused fluid accumulation. Gel diffusion analysis revealed that the A. veronii beta-hemolysin was serologically related to the A. hydrophila beta-hemolysin, a known enterotoxic molecule. The biological activity of the A. veronii hemolysin was neutralized by antiserum to A. hydrophila hemolysin. One of three strains that were not beta-hemolytic caused fluid accumulation but only when the ileal loops were inoculated with live cultures. These results suggest that A. veronii is a potential enteropathogen that can cause diarrhea by means of a cell-freed enterotoxin (beta-hemolysin) or by a second mechanism that requires the presence of whole cells.     

Isolation of enterotoxigenic Aeromonas from fish.

This paper reports two different enterotoxigenic classes of the genus Aeromonas isolated from fishes. Strains of A. sobria were isolated from healthy fishes only wheras strains of A. hydrophila were isolated from both healthy and moribund fishes. Aeromonas sobria and hydrophilia strains produced a cytotoxic factor and were highly proteolytic. Seventy-five percent (75%) of the A. sobria strains produced only one type of hemolysis on 5% blood agar, but 92% of the A. hydrophilia produced two types of hemolysis. Enterotoxigenicity of all strains was tested by the rabbit gut loop technique and the suckling mouse test. Most of those Aeromonads were enterotoxigenic. Enterotoxigenic strains appeared to produce two different enterotoxins which were antigenically related, but they seemed to have different mechanisms of action. Enterotoxins of Aeromonas were shown to be antigenically different from those of E. coli.     

Detection of Aeromonas hydrophila in food with an enzyme-linked immunosorbent assay

A microtitration plate, antibody capture, enzyme-linked immunosorbent assay was developed for the detection of Aeromonas hydrophila serotype O : 11 (highly virulent strains). The assay utilizes a detector antibody which shows no cross-reactions with Aeromonas strains other than serotype O : 11 or non- Aeromonas competing organisms. The detector antibody is mixed with the sample and incubated for 1 h, microcentrifuged and the supernatant fluid (unadsorbed antibody) titred in a microtitre plate coated with A. hydrophila cells from serotype O : 11. All the A. hydrophila strains from serotype O : 11 tested reacted strongly with the detector antibody. Also by culturing and performing the immunoassay with the detector antibody we established and quantified the presence of A. hydrophila O : 11 in different foods.     

Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp.

Aims:  The main aims of this study were to clone and express an outer membrane protein (OMP), OmpW, of Aeromonas hydrophila and to study its distribution in Aeromonas spp.
Methods and Results:  The gene encoding OmpW in A. hydrophila has been cloned and expressed in Escherichia coli . Primers were designed for amplification of full-length ompW gene and used for identification of this gene in different Aeromonas spp. Of the 42 Aeromonas strains tested, all the isolates were positive by polymerase chain reaction (PCR) except one strain of Aeromonas veronii biovar veronii (VTE338). None of the other gram-negative bacteria were positive by PCR with primers specific to ompW gene of A. hydrophila . Polyclonal antibodies were raised in rabbit against the purified recombinant protein and the reaction of these antibodies was confirmed by western blotting using the purified recombinant protein and 42 Aeromonas cultures grown at various salt concentrations.
Conclusions:  The ompW -based PCR method developed in this study was found to be 100% specific and 97% sensitive. Expression of OmpW protein of Aeromonas was found to be salt-dependant. Recombinant OmpW protein was found to be highly immunogenic in fish.
Significance and Impact of the Study:  To our knowledge, this is the first report on cloning and expression of OmpW protein of A. hydrophila . Full-length ompW gene amplification by PCR can be used for the detection of Aeromonas . Recombinant OmpW protein can be useful for vaccination of fish against Aeromonas spp.     

Purification and characterization of a novel metalloprotease isolated from Aeromonas caviae

A novel protease produced by Aeromonas caviae was purified and characterized. The molecular weight of the protease (AP19) was estimated as 19 kDa on SDS-polyacrylamide gel electrophoresis. The protease activity was not inhibited completely by heating at 100 degrees C for 15 min. The proteolytic activities were inhibited by metalloprotease inhibitor. The N-terminal amino acid sequence of AP19 was VTASVSFSGRCTN. AP19 did not activate Aeromonas proaerolysin, and did not show fluid accumulation in the rabbit intestinal loop test. A high concentration of the protease showed cytotoxic activity against Vero cells.     

Bacteriocin-like substance (BLS) production in Aeromonas hydrophila water isolates

30 Aeromonas hydrophila water isolates were tested for bacteriocin-like substance (BLS) production using a target panel of closely related microorganisms and other Gram-positive and Gram-negative bacteria, including food-borne pathogens. A. hydrophila showed antibacterial activity against one or more indicator microorganisms, but the activity emerged only with non-phylogenetically related genera or species. In particular all A. hydrophila showed antibacterial activity against one or more of the tested Staphylococcus strains, five against Listeria spp. (Listeria seeligeri, Listeria welshimeri and Listeria ivanovii), and eight presented a weak antagonistic activity towards Streptococcus agalactiae and Lactobacillus spp. Inhibitory activity was not observed against the other Gram-positive (Listeria monocytogenes, Listeria innocua and Enterococcus spp.) and Gram-negative tested strains, including Aeromonas sobria, Aeromonas caviae and the same A. hydrophila, when used as indicator. Anti-staphylococcal activity was observed with a gradual increase of the inhibition zone during incubation and seemed to be influenced by A. hydrophila hemolytic expression. Extrachromosomal analysis showed the presence, in 70% of the strains, of one to five plasmids with molecular masses ranging from 2.1 to 41.5 MDa, but it was not possible to relate this result with BLS production.     

Distribution of virulence genes in Aeromonas spp. isolated from Sardinian waters and from patients with diarrhoea

AIMS: To characterize 46 isolates of different Aeromonas spp. strains (26 Aeromonas hydrophila, 13 Aeromonas sobria and 7 Aeromonas salmonicida) isolated from coastal water and clinical sources in Sardinia, Italy. METHODS AND RESULTS: The isolates were analysed for the production of the following virulence properties: slime, haemolysin, gelatinase and protease production, and adhesion to eucaryotic epithelial cells. The presence of known virulence genes: A. hydrophila cytolytic enterotoxin gene AHCYTOEN; type IV pilus gene Tap; Bundle forming pilus genes BfpA and BfpG were investigated using polymerase chain reaction (PCR). In addition the enterobacterial repetitive intergenic consensus sequences (ERIC)-PCR fingerprinting was used to further differentiate the strains. CONCLUSIONS: This study confirms the presence of virulent Aeromonas strains in the Mediterranean sea. The study also found a greater prevalence of haemolysin, protease and gelatinase production, as well as a higher adhesion capacity, among strains isolated from patients with diarrhoea. SIGNIFICANCE AND IMAPCT OF THE STUDY: This is the first time that Aeromonads have been isolated and characterized from Sardinian waters and from patients with diarrhoea in Sardinia. This study adds to our knowledge of the ecology of this micro-organism and may in the future help prevent infections both in fish and in humans.     

Interactions of clinical and environmental Aeromonas isolates with Caco-2 and HT29 intestinal epithelial cells

AIM: Evaluation of adherence and invasion of Aeromonas spp. to human colon carcinoma cell lines Caco-2 and HT29 and assessment of cytotoxic activity. METHODS AND RESULTS: A number of 27 strains of Aeromonas caviae and 23 strains of Aeromonas hydrophila was analysed. All strains were capable to adhere to sub-confluent monolayers of Caco-2 and HT29 cell types, presenting aggregative and diffuse adherence patterns cells, respectively. In the cytotoxic assays all strains showed cytopathic and/or cytotoxic activities to Vero cells. The evaluation of the tetrazolium salt (MTT test) reduction capability was carried out in Vero, Caco-2, and HT29 cells. MTT test showed that Vero cell line was the most sensitive cell type. In the invasion test, 13 strains were analysed on Caco-2 and HT29 monolayers. Only two (15%) of the 13 strains, A. hydrophila and A. caviae species, both isolated from vegetables were invasive to Caco-2 cells. No strains were able to invade the HT29 cells. CONCLUSIONS: A. hydrophila and A. caviae isolated from human diarrhoeic faeces, vegetables, and water, were able to adhere to and produce cytotoxic/cytopathic effects in intestinal epithelial cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Aeromonas spp. in food and water samples expressing virulence factors suggest that these sources may act as dissemination vehicles of human pathogen with implication in the public health.     

Immunoglobulin binding by the regular surface array of Aeromonas salmonicida

The cell surface of Aeromonas salmonicida is covered by a regular surface array composed of a single species of protein, the A-protein (Phipps, B. M., Trust, T. J., Ishiguro, E. E., and Kay, W. W. (1983) Biochemistry 22, 2934-2939). The array, known as the A-layer, is the key virulence factor for this organism. Cells containing the A-layer specifically bound rabbit IgG and human IgM with high affinity (KD = 1.0 X 10(-6) M and 3.3 X 10(-6) M, respectively), but neither isogenic A-protein-deficient strains nor an Aeromonas hydrophila strain also possessing a regular surface array had binding activity. Selective removal of A-protein at pH 2.2 inactivated IgG binding. Structurally intact IgG was requisite for binding since both Fab and Fc fragments were inactive. Aeromonas A-protein did not share the same IgG binding sites as Staphylococcus aureus protein A. Purified A-protein bound IgG only weakly, but reassembled A-layer regained binding activity. Protein modification and perturbation of the A-layer indicated that no single amino acid residue was critical for binding, and that the binding site consisted of a native arrangement of at least four A-protein monomers in the layer.     

Survival of Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria in soil

The survival of mesophilic Aeromonas spp. in soil in the presence or absence of indigenous microflora was evaluated in a laboratory study. Two cytotoxic ( Aer. hydrophila and Aer. caviae ) and one invasive ( Aer. sobria ) clinical isolate strains were selected for this study. After contamination of sterile or unsterilized soil with the three strains of Aeromonas , the number of living cells was determined over at least 5 months. For all strains the survival curves were characterized by an initial re-growth followed by a slow inactivation of bacteria, with significant differences due to the presence of indigenous microflora. The times necessary to achieve a 95% reduction of the initial population were > 140, 113 and 62 d in sterilized soil respectively for Aer. caviae, Aer. hydrophila and Aer. sobria , while the corresponding times in unsterilized soil were 42, 38 and 11 d. All strains preserved the virulence factors for the entire period of the study. These results suggest that the soil may be an important reservoir for Aeromonas spp. and, thus, may play an important role in the epidemiology of Aeromonas -associated human infections.     

Identification of Aeromonas hydrophila hybridization group 1 by PCR assays.

Synthetic oligonucleotide primers of 24 and 23 bases were used in a PCR assay to amplify a sequence of the lip gene, which encodes a thermostable extracellular lipase of Aeromonas hydrophila. A DNA fragment of approximately 760 bp was amplified from both sources, i.e., lysed A. hydrophila cells and isolated DNA. The amplified sequence was detected in ethidium bromide-stained agarose gels or by Southern blot analysis with an internal HindIII-BamHI 356-bp fragment as a hybridization probe. With A. hydrophila cells, the sensitivity of the PCR assay was < 10 CFU, and with the isolated target, the lower detection limit was 0.89 pg of DNA. Primer specificity for A. hydrophila was determined by the PCR assay with cells of 50 strains of bacteria, including most of the 14 currently recognized DNA hybridization groups of Aeromonas spp. as well as other human and environmental Aeromonas isolates. Detection of A. hydrophila by PCR amplification of DNA has great potential for rapid identification of this bacterium because it has proved to be highly specific.