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PubMed1998

Mesophilic Aeromonas strains from different serogroups: the influence of growth temperature and osmolarity on lipopolysaccharide and virulence.

Growth of mesophilic Aeromonas sp. strains from serogroups O:13, O:33 and O:44 at different temperatures and osmolarity resulted in changes in the lipopolysaccharide (LPS) and virulence of the strains tested, as we had previously reported for strains from serogroup O:34. The effect of osmolarity could be observed when the cells grew at 37 degrees C but not at 20 degrees C. Purified LPS from cells cultivated at 20 degrees C (high or low osmolarity) or at 37 degrees C at high osmolarity was smooth, whereas the LPS extracted from the cells cultivated on low osmolarity was rough. The smooth strains were resistant to the bactericidal activity of non-immune serum, while the rough strains were sensitive and showed better adhesion to Hep-2 cells than the rough strains. Furthermore, the smooth strains were more virulent for fish and mice than the rough strains. For mesophilic Aeromonas sp. strains from serogroups O:1 to O:44, these changes were not observed, except for serogroups O:13, O:33, O:34 and O:44.

Aeromonas↗
PubMed1991

Multilocus enzyme analysis of the genus Aeromonas and its use for species identification.

A total of 153 Aeromonas strains were analyzed by multilocus enzyme electrophoresis. All 11 genetic loci were polymorphic with three to 14 alleles per locus (average 6.55). The genetic diversity of each locus varied between 0.095 for indophenol oxidase and 0.881 for the fast variety of malic enzyme. Cluster analysis of the 122 enzyme types revealed a good correlation with taxonomic groupings as determined by DNA-DNA hybridization. In conjunction with biochemical analysis, as few as two enzymes may be sufficient for the identification of all species in the genus Aeromonas.

Aeromonas↗
PubMed1984

Methods for the isolation of Aeromonas hydrophila and Plesiomonas shigelloides from faeces.

Two solid selective media, xylose deoxycholate citrate agar (XDCA) and bile salts brilliant green agar (BBG) and an enrichment broth-alkaline peptone water, were evaluated for the isolation of Aeromonas hydrophila and Plesiomonas shigelloides. Alkaline peptone water and XDCA are useful for recovery of Aeromonas but not Plesiomonas, whereas BBG is satisfactory for both organisms.

Aeromonas↗
PubMed1987

A modification of bile salts brilliant green agar for isolation of motile Aeromonas from foods and environmental specimens.

Aeromonas has recently been recognized as an important enteropathogen. In faecal specimens the organism is readily identified and differentiated from coliforms by the positive oxidase reaction. However, few media have proved useful for foods and environmental specimens. Bile salts brilliant green starch agar (BBGS) was studied for this purpose by testing artificially contaminated foods as well as natural potential sources. The results indicate the suitability of the medium for the isolation of Aeromonas from foods and from environmental sources.

Aeromonas↗
PubMed1993

Adherence to HEp-2 cells and enteropathogenic potential of Aeromonas spp.

Aeromonas strains (total = 60) of clinical, water and food origin were tested for adherence to HEp-2 cells. Environmental strains were selected (except for A. caviae) to include primarily those expressing other virulence-associated properties. Adhesion was markedly species-dependent (A. veronii biotype sobria, 15 of 26 [58%]. A caviae, 4 of 12 [33%] and A. hydrophila, 2 of 8 [11%]). A. veronii biotype sobria were adhesive, irrespective of source (62 and 54% for clinical and environmental strains, respectively). Adherent strains of this species were enterotoxin-positive and most (13 of 15) grew at 43 degrees C. A. caviae isolated from clinical specimens contained a higher proportion (75%) of adherent strains than environmental strains (13%). Virulent subsets of A. veronii biotype sobria and A. caviae are adherent to HEp-2 cells. The HEp-2 assay is a useful model for investigating mechanisms of adherence and enteropathogenicity of virulent Aeromonas species.

Aeromonas↗
PubMed1986

Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates.

Aeromonas aminopeptidase contains two nonidentical metal binding sites that have been shown by both spectroscopy and kinetics to be capable of interacting with one another [Prescott, J.M., Wagner, F.W., Holmquist, B., & Vallee, B.L. (1985) Biochemistry 24, 5350-5356]. The effects of metal ion substitutions on the susceptibility of the p-nitroanilides of L-alanine, L-valine, and L-leucine--substrates that are hydrolyzed at widely differing rates by native Aeromonas aminopeptidase--were studied by determining values of kcat and Km for the 16 metalloenzymes that result from all possible combinations of Zn2+, Co2+, Ni2+, and Cu2+ in each of the two sites. The different combinations of metal ions and substrates yield a broad range in kinetic values; kcat varies by more than 1800-fold, Km by 3000-fold, and kcat/Km ratios by more than 10,000. L-Leucine-p-nitroanilide is by far the most susceptible of the three substrates, and the hyperactivation previously observed with aminopeptidase containing either Ni2+ or Cu2+ in the first binding site and Zn2+ in the second site occurs only with the two poorer substrates, L-alanine-p-nitroanilide and L-valine-p-nitroanilide. Although the enzyme with Zn2+ in both sites hydrolyzes the substrates with N-terminal alanine and valine poorly, it is extremely effective toward L-leucine-p-nitroanilide. Neither metal binding site can be identified as controlling either Km or kcat; both parameters are influenced by the identity of the metal ions, by the site each occupies, and, most strongly, by the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Aeromonas↗
PubMed1998

Clonal identification of Aeromonas hydrophila strains using randomly amplified polymorphic DNA analysis.

The suitability of arbitrary primer polymerase chain reaction (RAPD) as a typing technique was evaluated by comparing it with pulsed-field gel electrophoresis (PFGE) to characterize Aeromonas hydrophila strains isolated from a cluster of hospital-acquired infections. Five isolates from patients and 10 isolates from the water supply were compared to 10 epidemiologically unrelated strains isolated from patients and rivers. Two methods were used to prepare DNA and two primers (AP3 and AP5) were selected. The discriminatory power was better with the extractive DNA preparation than the boiling method. The discrimination of closely related from less related strains by PCR using AP3 was consistent with that by PFGE: water supply of Cholet hospital contaminated with Aeromonas species was not the source of the cluster of hospital infections and only two patients were infected with clonally-related strains. RAPD using primer AP3 was simpler, cheaper, and quicker to perform than pulsed-field gel electrophoresis and is well suited for the epidemiological study of A. hydrophila isolates.

Aeromonas hydrophila↗
PubMed1981

Exotoxins of Aeromonas hydrophila.

Eighty of 103 strains of Aeromonas hydrophila cultured at 100 rev./min produced heat-labile enterotoxins detected using the suckling mouse assay. Results in intestinal perfusion agreed with the suckling mouse test in all strains tested by both methods. Enterotoxic activity correlated with haemolysin and cytotoxin production, but 4% of strains would have been wrongly classified using haemolysin assay in place of the suckling mouse test and 11% misclassified on the basis of cytotoxin assay. There was a significant association between haemolytic and cytotoxic activity, but 15% of strains produced only one of these toxins. Haemolysin, cytotoxin and enterotoxin were not always associated in a given isolate. The time of appearance of exotoxins during bacterial growth and the effects of dialysis, heating and proteolytic enzymes also suggest that haemolysins, cytotoxins and enterotoxins of Aeromonas hydrophila are separate toxins and not different manifestations of the same toxin.

Aeromonas↗
PubMed1986

Haemagglutination patterns of Aeromonas spp. related to species and source of strains.

The study included 138 A. sobria and 182 A. hydrophila isolated in Perth from samples of diarrhoeal or non-diarrhoeal faeces or from domestic water. Strains were grouped in relation to agglutination of human, horse, rat and guinea pig erythrocytes and the effect of sugars on haemagglutination. Agglutination of red cells of all four species (primary group 1) was most commonly associated with A. sobria, particularly those strains isolated from faeces of patients with diarrhoea. Most A. hydrophila associated with diarrhoea also belonged to group 1 but A. hydrophila from non-diarrhoeal stools or from water most commonly agglutinated human and guinea pig cells but not horse erythrocytes (primary groups 2 and 3). Fucose-resistant haemagglutination (FRHA) of strains in primary group 1 occurred with about 68% of the strains of A. sobria associated with diarrhoea. Mannose-resistant haemagglutination (MRHA) was limited to 29% of strains of A. sobria associated with diarrhoea. The predominance of primary group 1 among strains of Aeromonas spp. associated with diarrhoea and the proportion of these strains showing FRHA suggest that haemagglutination of cells from human, horse, rat and guinea pig, particularly if fucose-resistant, should be considered in a search for characteristics, which possibly contribute to virulence of Aeromonas spp.

Aeromonas↗
PubMed1998

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae.

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot (Scophthalmus maximus) larvae exposed to V. pelagius and/or Aer. caviae. The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 10(3) ml-1 and 10(3) larva-1. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 x 10(2) bacteria larva-1 on day 3 post-hatching to approximately 10(5) bacteria fish-1 16 days post-hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 x 10(4) cfu larva-1 exposed to V. pelagius on days 2, 5 and 8 post-hatching. However, only 10(3) of the Vibrio spp. belonged to V. pelagius. When larvae were exposed to Aer. caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp. (10(4) larva-1), of which 9 x 10(3) belonged to Aer. caviae. Later in the experiment, at the time when high mortality occurred, 9 x 10(5) Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria (V. pelagius) to the rearing water.

Aeromonas↗
PubMed1998

Identification of Aeromonas strains of different origin to the genomic species level.

A total of 71 Aeromonas strains was identified with established genomic species by DNA-DNA hybridization. The strains were isolated from diarrhoeal stools, dead and live fish, drinking, lake, river and sea water, municipal sewage and aluminium rolling emulsion. The strains were allocated to seven hybridization groups (HGs) but the majority belonged to HG 4 (42%), HG 8/10 (30%) and HG 3 (18%). All strains were examined by 136 phenotypic tests. Useful phenotypic characters for separation of Aeromonas HG 1-3 genospecies were: utilization of DL-lactate, urocanic acid and growth at 40.5 degrees C. Few phenotypic differences were detected between strains of HG 4, HG 5 and HG 6. Most isolates of the Aer. veronii biotype sobria (HG 8) showed a characteristic biochemical profile: positive V.P. (Voges-Proskauer) reaction, oxidation of gluconate, production of gas from glucose, susceptibility to cephalotin, no hydrolysis of elastin, arbutin and aesculin, and no acid production from L-arabinose, arbutin and salicin.

Aeromonas↗
PubMed1999

DNA probes specific for Aeromonas hydrophila (HG1).

Aeromonas hydrophila (HG1)-specific RAPD-PCR fragments were investigated for their potential as DNA probes. From 20 RAPD-PCR fragment bands, it was found that two were specific to all isolates of Aeromonas hydrophila (HG1) tested. Cloning and nucleotide sequence determination of one of these bands showed that co-migration of similar sized amplicons had occurred and that this band (designated '7e') contained at least four fragments of different sequences. Three of these individual amplicons had a sequence specific to Aer. hydrophila (HG1) isolates. The sequence of one of these amplicons ('7e5') was used to design primers for a specific polymerase chain reaction (PCR). The specificity of the PCR was achieved using a modified hot-start procedure. The identity of the PCR amplicons was confirmed by high stringency hybridization with a digoxygenin-labelled 7e5 probe.

Aeromonas hydrophila↗
PubMed2000

Interaction between Aeromonas veronii and epithelial cells of spotted sand bass (Paralabrax maculatofasciatus) in culture.

An in vitro fish model to study the interaction between Aeromonas veronii and skin, gill and intestinal epithelial cells was developed using primary cultures of mucosal cells (isolated from healthy organisms). Primary cultures were exposed to Aeromonas veronii strain A186 isolated from a patient with severe gastrointestinal disease. Microbial adherence was assessed by a spectrophotometric evaluation of an enzyme-linked, biotin-streptavidin Aer. veronii cell-adhesion assay to confluent monolayers of epithelial cells on 96-well tissue culture plates. The three primary-culture cells are susceptible to Aer. veronii attachment, with the greatest binding affinity found in gills, and to a lesser extent, in skin and intestine epithelial cells. Aer. veronii adherence was dependent on bacterial load and incubation time. The effect of glycoconjugates on Aer. veronii adhesion was investigated by pre-incubating Aer. veronii cells with monosaccharides, sialic acid-rich glycoproteins and sulphated polysaccharides. In addition, the participation of a 48-kDa Aer. veronii lectin (MCBP - mucosal constituents binding protein), with affinity for mucosal constituents, was evaluated as a putative adhesion factor of Aer. veronii to the mucosal epithelial cells of spotted sand bass by pre-incubating bacterial cells with rabbit polyclonal antibodies to Aer. veronii MCBP. Our study shows that primary-culture fish mucosal cells provide a suitable model for the study of the interactions between Aer. veronii and epithelial cells of the fish mucosa, and to study putative virulence factors of fish pathogens.

Aeromonas↗
PubMed2001

Miniaturized tests for computer-assisted identification of motile Aeromonas species with an improved probability matrix.

AIMS: To develop miniaturized tests for the phenotypic identification of motile Aeromonas species using an improved probability matrix. METHODS AND RESULTS: Conventional tests were miniaturized for use in 96-well plates, and their performance assessed using 60 aeromonads comprising type and reference strains as well as clinical, fish and water isolates. A revised probability matrix for Aeromonas hybridization groups 1-14, including A. allosaccharophila, A. bestiarum, A. encheleia and A. popoffii, was developed. Using 26 tests, all the reference strains were correctly identified with the revised probability matrix, and 80% of the isolates were correctly identified at a Willcox probability level of 95%. CONCLUSION: The compact test format, coupled with a robust identification matrix, provides a convenient basis for identifying motile aeromonads. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification system for identifying aeromonads will be of use to medical and veterinary laboratories undertaking disease diagnosis.

Aeromonas↗
PubMed2002

The identification of the transcriptional regulator CRP in Aeromonas hydrophila JMP636 and its involvement in amylase production and the 'acidic toxicity' effect.

AIMS: The physiological examination of amylase production by Aeromonas hydrophila JMP636 and identification of the mechanism of regulation. METHODS AND RESULTS: Aeromonas hydrophila JMP636 was grown with single, then dual carbon sources; the growth cycle was followed and amylase activity throughout was monitored. The levels of cAMP, a known secondary messenger for the regulatory gene crp, were also examined. Amylase activity was regulated by catabolite repression. Physiological studies revealed that JMP636 exhibited both diauxic growth, with two carbon sources, and the 'acid toxicity' effect on glucose. The crp gene was cloned, expressed and inactivated from the JMP636 chromosome. Catabolite repression of amylase production and the 'acid toxicity' effect both require crp and were linked to cAMP levels. CONCLUSIONS: Regulation of amylase production was predicted to follow the model CRP-mediated cAMP-dependent Escherichia coli catabolite regulation system. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides an understanding of the physiology of the opportunistic pathogen Aer. hydrophila through identification of the mechanism of catabolite repression of amylase production and the existence of crp within this cell. It also provides a broader knowledge of global gene regulation and suggests regulatory mechanisms of other Aer. hydrophila gene/s.

Aeromonas hydrophila↗
PubMed2003

A novel type of family 19 chitinase from Aeromonas sp. No.10S-24. Cloning, sequence, expression, and the enzymatic properties.

A family 19 chitinase gene from Aeromonas sp. No.10S-24 was cloned, sequenced, and expressed in Escherichia coli. From the deduced amino acid sequence, the enzyme was found to possess two repeated N-terminal chitin-binding domains, which are separated by two proline-threonine rich linkers. The calculated molecular mass was 70 391 Da. The catalytic domain is homologous to those of plant family 19 chitinases by about 47%. The enzyme produced alpha-anomer by hydrolyzing beta-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the chitinase, the second glycosidic linkage from the nonreducing end was predominantly split producing (GlcNAc)2 and (GlcNAc)4. The evidence from this work suggested that the subsite structure of the enzyme was (-2)(-1)(+1)(+2)(+3)(+4), whereas most of plant family 19 chitinases have a subsite structure (-3)(-2)(-1)(+1)(+2)(+3). Thus, the Aeromonas enzyme was found to be a novel type of family 19 chitinase in its structural and functional properties.

Aeromonas↗
PubMed1998

Inhibition of Streptomyces griseus aminopeptidase and effects of calcium ions on catalysis and binding--comparisons with the homologous enzyme Aeromonas proteolytica aminopeptidase.

Streptomyces griseus aminopeptidase is a zinc metalloenzyme containing 2 mol zinc/mol protein, similar to the homologous enzyme Aeromonas proteolytica aminopeptidase. In addition, a unique Ca2+-binding site has been identified in the Streptomyces enzyme, which is absent in the Aeromonas enzyme. Binding of Ca2+ enhances stability of the Streptomyces enzyme and modulates its activity and affinity towards substrates and inhibitors in a structure-dependent manner. Among the three hydrophobic 4-nitroanilides of alanine, valine and leucine, the latter displays the largest overall activation (increase in k(cat)/Km). Large enhancements in affinity (1/Ki) upon Ca2+ binding have been observed for inhibitors with flexible (leucine-like) residues at their N-termini and smaller enhancements for inhibitors with rigid (phenylalanine-like) residues.

Aeromonas↗
PubMed1999

Aeromonas sobria infection with severe soft tissue damage and segmental necrotizing gastroenteritis in a patient with alcoholic liver cirrhosis.

A 49-year-old man, who had a 3-year history of liver dysfunction but had not been treated, was admitted to the hospital with a sudden onset of fever and generalized muscle pain. He subsequently developed generalized purpura with scattered hemorrhagic bullae of the skin and massive bloody stools. Aeromonas sobria was proven by culture of both blood and bullous fluid. In spite of the extensive treatment with antibiotics and other medications in the intensive care unit (ICU), the patient went into septic shock and died 2 days after admission. Pathological examination on autopsy revealed segmental necrotizing gastroenteritis with bacterial colonies and alcoholic liver cirrhosis, in addition to extensive severe soft tissue damage involving cellulitis and rhabdomyolysis and epidermolysis. Although the prognosis for Vibrio vulnificus infection with severe soft tissue damage in patients with liver cirrhosis, malignancy, diabetes mellitus or other pre-existing diseases is poor, the unfavorable progression of Aeromonas species, especially A. sobria infection is rare. This is thought to be the first report of an autopsied case.

Aeromonas↗