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Aeromonas hydrophila subsp. dhakensis subsp. nov., isolated from children with diarrhoea in Bangladesh, and extended description of Aeromonas hydrophila subsp. hydrophila (Chester 1901) Stanier 1943 (approved lists 1980).

A group of 10 Aeromonas hydrophila-like strains referred to as group BD-2, which mainly comprised haemolytic and cytotoxic isolates from diarrhoeal children in Bangladesh, was subjected to a polyphasic taxonomic study including fluorescent amplified fragment length polymorphism (FAFLP) and ERIC-PCR fingerprinting, microplate DNA-DNA hybridization and extensive phenotypic characterization. Comparison of FAFLP and ERIC-PCR genotypic profiles indicated that the group BD-2 isolates constituted a separate taxon that was most closely related to A. hydrophila DNA hybridization group (HG) 1. Determination of DNA-DNA hybridization values with type and reference strains of all known Aeromonas taxa revealed that the BD-2 group represented a homogeneous taxon (internal DNA homology, 77-94%) that exhibited 78-92% DNA relatedness with the type strain of A. hydrophila HG1. These results indicated that the BD-2 group belongs genotypically to A. hydrophila HG1, but examination of 152 phenotypic characteristics revealed that the group BD-2 isolates differed from representatives of HG1 in eight biochemical properties. The diagnostic value of at least three of these tests, namely utilization of urocanic acid and L-arabinose and acid production from L-arabinose, has been confirmed outside this study by other workers. Collectively, the genotypic and phenotypic data reported suggest that the BD-2 group represents a subspecies of A. hydrophila, for which the name Aeromonas hydrophila subsp. dhakensis subsp. nov. is proposed. Its type strain is P21T (= LMG 19562T= CCUG 45377T). An extended description of A. hydrophila subsp. hydrophila (Chester 1901) Stanier 1943 (Approved Lists 1980) is given.

Aeromonas hydrophila↗

Aeromonas jandaei and Aeromonas veronii dual infection of a human wound following aquatic exposure.

Exudate removed from an infection that developed below the left eye of a 10-year-old male following a previously inflicted wound after aquatic exposure was cultured and revealed two different Aeromonas spp. Further characterization showed that one strain was phenotypically identical to Aeromonas veronii, while the other strain was confirmed by DNA hybridization analysis to be Aeromonas jandaei sp. nov. This is the first report of these more recently described aeromonads, thus far rarely reported from clinical disease, occurring simultaneously in a human infection.

Aeromonas↗

Detection and characterization of the hemolysin genes in Aeromonas hydrophila and Aeromonas sobria by multiplex PCR.

A multiplex PCR assay was designed to amplify the Aeromonas hydrophila and A. veronii bv. sobria hemolysin and aerolysin genes. The assay was evaluated by using 121 clinical isolates and 7 reference strains of Aeromonas spp., and these were divided into five genotypes on the basis of the results of the multiplex PCR. The five genotypes were characterized as type 1 for those carrying the ahh1 gene only (36% of isolates), type 2 for those carrying the asa1 gene only (8.5% of isolates), type 3 for those carrying both the ahh1 and the asa1 genes (4% of isolates), type 4 for those carrying the ahh1 gene and the A. hydrophila aerA (aerolysin) gene (37.5% of isolates), and type 5 for those in which no hemolysin genes were detected (14% of isolates). The most common single hemolysin gene carried among all the Aeromonas isolates examined was ahh1, with 99 of 128 (77%) of isolates testing positive for this gene either alone or in combination with other hemolysin genes. Phenotypic expression of toxins was evaluated in a Vero cell culture cytotoxicity assay. These results indicated that there is a statistically significant correlation between the cytotoxin titers and the hemolysin genotype. Isolates belonging to genotype 4 (carrying both the ahh1 gene and the aerolysin and hemolysin aerA genes) expressed higher cytotoxin titers than isolates of the other genotypes (P < 0.001). These isolates were more cytotoxic in cell culture and may have greater clinical significance.

Aeromonas hydrophila↗

Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case.

AIMS: To evaluate the public health significance of representative strains of two Aeromonas spp., mainly from freshwater fish, on the basis of production of virulence-associated factors and presence of the haemolytic genes aerA and hlyA. METHODS AND RESULTS: Eleven strains of Aer. hydrophila, three strains of Aer. veronii biovar sobria (all from freshwater fish) and one strain of Aer. hydrophila from human diarrhoea were tested for potential virulence traits and for the presence of the haemolytic genes aerA and hlyA. Ten Aer. hydrophila isolates were aerA(+)hlyA(+) and two aerA(+)hlyA(-). Aeromonas veronii biovar sobria isolates were aerA(-)hlyA(-). Strains from the three genotypes showed enterotoxic activity in the suckling mouse assay. At 28 degrees C, four Aer. hydrophila fish strains could be considered as potentially virulent (possessing at least two of these characteristics: haemolytic, cytotoxic and enterotoxic). One Aer. veronii biovar sobria strain and the clinical isolate were cytotoxic on Vero cells. When grown at 4 degrees C, these six isolates fulfilled virulence criterion, but at 37 degrees C, only one fish strain, an Aer. hydrophila, did. CONCLUSIONS: The potential health risk derived from the presence of Aer. hydrophila and Aer. veronii biovar sobria in ice-stored freshwater fish should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of virulence factors is affected by temperature incubation and not always related to the presence of haemolytic genes.

Aeromonas↗

[Isolation of Aeromonas species from patients with sporadic diarrhea and characterization of Aeromonas hydrophila isolates].

A total of 16 strains of Aeromonas species were isolated from feces of 348 patients with sporadic diarrhea in western Kanagawa, Japan from 1996 to 1998. Of the 16 isolates, 7 were Aeromonas hydrophila, 1 was A. sobria and 8 were A. caviae. The strains of A. hydrophila were examined for hemolytic activities, hemolysin gene types and O-serogroups. Although all 7 strains of A. hydrophila showed hemolytic activities on sheep blood agar, in the test for hemolytic activities in culture supernatant, only 1 of the these strains showed no hemolytic activity against sheep erythrocytes. From the results of PCR assay, the tested strains of A. hydrophila were grouped into 2 hemolysin gene types of [ahh1 + ahh3 + aerA] (n = 6) and [ahh1 + aerA] (n = 1) both of which are recognized to be enteropathogenic. Five of the 7 strains of A. hydrophila belonged to serogroup O11. These results suggest that 7 strains of A. hydrophila isolates are recognized to be enteropathogenic strains and serogroup O11 is the major O-serogroup of enteropathogenic A. hydrophila in humans.

Aeromonas↗

Antigenic diversity of the S-layer proteins from pathogenic strains of Aeromonas hydrophila and Aeromonas veronii biotype sobria.

The antigenic relatedness of paracrystalline surface array proteins with subunit molecular weights of approximately 52,000 from isolates of Aeromonas hydrophila and Aeromonas veronii biotype sobria belonging to a single heat-stable serogroup was examined. Enzyme-linked immunosorbent assay and immunoblotting with two different polyclonal antisera against surface exposed and non-surface-exposed epitopes of the S-layer protein from A. hydrophila TF7 showed that the S-layer proteins of the mesophilic aeromonads were antigenically diverse. NH2-terminal amino acid sequence analysis of four antigenically different proteins showed that while the proteins were structurally related, they differed in primary sequence. Absorption experiments with heterologous live cells showed that cross-reactive epitopes were in non-surface-exposed regions of the S-layer proteins, while absorption with homologous live cells showed that the immunodominant epitopes of the S-layer protein of strain TF7 were strain specific and exposed on the surface of the native, tetragonal array produced by this strain. Proteolytic digestion of the TF7 S-layer protein with trypsin, chymotrypsin, or endoproteinase Glu-C produced an amino-terminal peptide of approximate Mr 38,000 which was refractile to further proteolytic cleavage under nondenaturing conditions. This peptide carried the immunodominant surface-exposed region of the protein, and chemical cleavage with cyanogen bromide further mapped the portion of these surface-exposed epitopes to a peptide of approximate Mr 26,000, part of which maps within the Mr 38,000 protease-resistant NH2-terminal peptide.

Aeromonas↗

Hemolytic and proteolytic activities of Aeromonas hydrophila and Aeromonas veronii biovar sobria in broth and salmon extract at different temperatures.

Expression of hemolytic and proteolytic activities throughout the growth cycle was investigated with two enterotoxic aeromonad strains assigned to the species Aeromonas hydrophila and Aeromonas veronii biovar sobria. Although growth kinetic data were dependent on strain, temperature, and substrate, maximum populations attained were higher than 9 log CFU/ml in aerated tryptone soya broth plus yeast extract (TSBYE) and salmon extract within the range 4 to 28 degrees C. For both strains in TSBYE, variable amounts of hemolytic activity were first detected at any temperature when aeromonad counts were over 9 log CFU/ml. Afterwards, this activity increased up to similar levels (109 to 112 hemolytic units per ml) without a significant increase in populations. Salmon extract supported hemolysin synthesis at 28 but not 4 degrees C. Proteolytic activity of the A. hydrophila strain was only expressed in salmon extract at 28 degrees C, whereas A. veronii biovar sobria did at 28 degrees C in both substrates and at 10 degrees C in TSBYE.

Aeromonas↗

Purification and characterization of cytolytic toxins produced by Aeromonas hydrophila and Aeromonas veronii biotype sobria strains.

Cytolytic toxins produced by Aeromonas hydrophila and Aeromonas veronii biotype sobria strains were partially purified from culture filtrates by two steps of purification: ammonium sulfate precipitation and hydrophobic chromatography using Phenyl-Sepharose CL-4B. Hemolytic activity was detected in one or two peaks in elution profile. Purified toxins were also cytotoxic to Vero and CHO cells. Moreover, these toxins revealed cytotonic activity to CHO cells.

Aeromonas↗

The type IV Aeromonas pilus (Tap) gene cluster is widely conserved in Aeromonas species.

Nothing is known regarding the expression or function of the type IV Aeromonas pilus (Tap), which was recently identified following the cloning of a pilus biogenesis gene cluster (tapABCD). As a first step to determine the possible significance of Tap for Aeromonas virulence, the distribution of the tapA and tapD genes in hybridization group reference strains and clinical (n=42) and environmental (n=29) isolates was determined. Homologues of tapA and tapD were present in all strains tested. Hybridization with the tapA probe enabled us to differentiate between clinical and environmental isolates of A. veronii biovar sobria.

Aeromonas↗

Seasonal prevalence of nosocomial Aeromonas hydrophila infection related to aeromonas in hospital water.

A seasonal variation in nosocomial Aeromonas hydrophila infection was correlated with the number of aeromonas in the hospital water supply. The high summer prevalence of A. hydrophila infection coincided with periods when water counts from storage tanks were highest. The waterborne origin of these infections highlights the importance of maintaining clean water supplies, especially where storage tanks are used. Monitoring A. hydrophila in hospital water, particularly during the summer months, may prove helpful.

Aeromonas↗

Chemical structure of aeromonas gum--extracellular polysaccharide from Aeromonas nichidenii 5797.

Aeromonas (A) gum, an extracellular heteropolysaccharide produced by the bacterium Aeromonas nichidenii strain 5797, was studied by 1H and 13C NMR spectroscopy including 2D COSY, TOCSY, 1H, 13C HMQC, HMBC and ROESY experiments after O-deacetylation and Smith degradation. These investigations revealed the presence of an O-acetylated pentasaccharide repeating unit composed of mannose, glucose, xylose and glucuronic acid, and it has the following structure: [Image: see text]

Aeromonas↗

Physical map of the chromosome of Aeromonas salmonicida and genomic comparisons between Aeromonas strains.

I-Ceul and Pmel physical maps of the Aeromonas salmonicida A449 chromosome were constructed using PFGE. The circular chromosome of A. salmonicida A449 was estimated to be 4658 +/- 30 kb. The approximate location of several genes, including those encoding proteins implicated in virulence, were identified. The map showed that the known virulence-factor-encoding genes were not clustered. The I-Ceul genomic digestion fingerprints of several typical and atypical strains of A. salmonicida were compared. The results confirmed the homogeneity of typical strains, which provided further support for the clonality of the population structure of this group. Extensive diversity was observed in the I-Ceul digestion fingerprint of atypical strains, although a clonality was observed in the strains isolated from diseased goldfish. The results suggest that comparison of I-Ceul digestion fingerprints could be used as a powerful taxonomic tool to subdivide the atypical strains and also help clarify some of the current confusion associated with the taxonomy of the genus Aeromonas.

Aeromonas↗

A taxonomic study of the Aeromonas hydrophila-Aeromonas punctata group.

A total of 203 characters has been determined for 68 strains of Aeromonas belonging to the Aeromonas hydrophila-A. punctata group. The results have been subjected to computer analysis using the coefficient of Jaccard-Sneath and the strains clustered by the method of aggregation according to the variance. The 68 strains can be divided into two well-segregated classes on the basis of 59 variable characters, of which seven are of diagnostic value. The two classes are considered as two separate species. The first one (42 strains) is assigned to the type species of the genus, A. hydrophila, and it appears that the species name, A. punctata, is an illegitimate synonym for A. hydrophila. The second (26 strains) constitutes a new species for which the name A. sobria sp. nov. is proposed. The type strain of this new species has been deposited under the reference CIP7433 (our strain 208).

Aeromonas↗

[Studies on motile-Aeromonas infection 3). Phage typing of motile Aeromonas isolated from patients with diarrhea].

Phage types were determined for 102 strains of motile Aeromonas isolated from patients with diarrhea at four metropolitan hospitals in Tokyo. The following results were obtained. 1) Of the 102 strains examined, 52 (51.0%) were divided into 28 phage types. This rate was considerably higher, compared to our previous results, namely, 21.7% for strains isolated from natural environments and 25% for those isolated from meats. By bacterial species, phage types could be determined for 33 (52.4%) of 63 strains of A. hydrophila, for 16 (45.7%) of 35 strains of A. sobria, 2 (50.0%) of 4 strains of A. caviae and 1 (100%) strain of Aeromonas spp. 2) Of the 52 strains for which the phage types could be determined, the greatest number (16 strains, 30.8%) were identified as belonging to Type I group. These are followed by 5 strains (9.6%) which were identified as Type I/III group and 2 strains (3.4%) each identified as Type I/II, I/II/V, IV, V and VI groups. The remaining 21 strains were identified as belonging to one of the other phage type groups. Thirty-five (67.3%) of the strains for which the phage types were identified, were found to belong either to Type I group or to combinations with Type I. This demonstrated that 34.0% of the isolates from patients with diarrhea were related to Type I.

Aeromonas↗

Value of blood agar for primary plating and clinical implication of simultaneous isolation of Aeromonas hydrophila and Aeromonas caviae from a patient with gastroenteritis.

The simultaneous recovery of Aeromonas hydrophila and Aeromonas caviae from the stool of a 49-year-old woman with watery diarrhea was facilitated through the use of a blood agar medium which detected the hemolytic capability of A. hydrophila. In vitro phenotypic tests support the conclusion that only the A. hydrophila isolate was clinically significant.

Aeromonas↗

Growth of Aeromonas spp. on Butzler Campylobacter selective agar and evaluation of the agar for the primary isolation of Aeromonas spp. from clinical specimens.

The fortuitous finding that Aeromonas spp. grew well on Butzler Campylobacter selective medium (BCSA) in a microaerobic atmosphere at 42 degrees C prompted us to evaluate the performance of BCSA for selective isolation of aeromonads in comparison with ampicillin (30 micrograms/ml) sheep blood agar (ASBA30). Overall recovery rates of aeromonads from 563 stool samples from patients with acute diarrhea were higher on ASBA30 (70.4%) than on BCSA (56.3%); however, 21 (29.5%) grew only on BCSA. The three human-associated Aeromonas spp. could be recovered on BCSA and ASBA30. We recommend the use of BCSA to laboratories reluctant to include a specific selective medium for aeromonads.

Aeromonas↗

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.

Aeromonas↗