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Nucleotide sequence and transcriptional analysis of the aerCaerA region of Aeromonas sobria encoding aerolysin and its regulatory region.

The nucleotide sequence of a 2510 base pair chromosomal fragment containing the aerolysin gene aerA, and its regulatory region aerC, from a clinical isolate of Aeromonas sobria was determined. The aerolysin gene coded for a 54.5 kD polypeptide and had a G + C content of 59%, indicating that it is endogenous to the genus Aeromonas. In contrast, the aerC region was characterized by its high A + T content (61%) and the presence of a core motif, aATAAAa, repeated eight times within 300 base pairs. A 12 base pair repeat, 5'AATAAAACCGGG3', present within this region occurred as a direct repeat 544 base pairs away, within the coding region of aerolysin. RNA polymerase binding studies and S1 mapping allowed the detection of two divergent non-overlapping promoters within aerC. Despite having identical transcriptional start sites in both A. sobria and Escherchia coli, the amount of aerolysin transcript produced in E. coli is 30-40 times less than that found in A. sobria. The signal peptide of preproaerolysin was shown by deletion to be essential for export of the toxin to the external medium. The mature toxin is a hydrophilic protein with no hydrophobic stretches long enough to cross a membrane. A search for similarities to the primary sequence of aerolysin revealed that the toxin may share a functional similarity to haemolysin (hlyA) of E. coli.

Aeromonas↗

Cloning and characterization of fxp, the flexible pilin gene of Aeromonas hydrophila.

The flexible pilus of Aeromonas hydrophila is a morphologically and biochemically unique organelle which binds eukaryotic cell surfaces and whose expression is induced by specific physiochemical conditions. fxp, the structural gene coding for the flexible pilus subunit, was localized on a 7.6kb plasmid of A. hydrophila strain AH26. A putative Shine-Dalgarno sequence and -10 and -35 regions were identified, a signal peptide sequence delineated, and the coding sequence compared with other bacterial sequences and found to be unique. Plasmid and chromosomal DNA was prepared from 66 other Aeromonas strains and 12 strains from other bacterial genera and examined by Southern blot hybridization using a labelled fxp oligonucleotide and the 7.6kb plasmid as probes. No hybridizing sequences were identified except in the original strain, AH26. It is proposed that fxp codes for a highly evolved organelle, possibly widely distributed in nature, but that it is carried on a genetic element that is rapidly lost from most strains upon in vitro cultivation and storage.

Aeromonas hydrophila↗

High-affinity binding of the basement membrane protein collagen type IV to the crystalline virulence surface protein array of Aeromonas salmonicida.

The surface of the fish pathogen Aeromonas salmonicida is covered by a paracrystalline array (the A-layer) which is a virulence factor for the organism. Quantification of the ability of A. salmonicida cells to bind collagen types I and IV in a 125I-radiolabelled liquid-phase assay showed that A-layer-positive cells bound high levels of collagen type IV, but significantly lower levels of collagen type I. Collagen type IV binding was confirmed using non-radiolabelled enzyme-linked immunosorbent assays. 125I-Collagen type IV binding was rapid, specific, saturable, high affinity, and essentially irreversible by unlabelled collagen type IV. The A-layer was responsible for collagen type IV binding because binding was inactivated by selective removal of the A-layer at pH 2.2, and neither isogenic A-layer-deficient A. salmonicida mutants nor strains of Aeromonas hydrophila possessing a morphologically similar paracrystalline array bound this basement membrane protein.

Aeromonas↗

[The occurrence of Escherichia coli, Aeromonas hydrophila, Plesiomonas shigelloides and Clostridium perfringens in the intestinal flora of gray herons (Ardea cinerea)].

The flora of the large intestine of 92 grey herons was examined for the frequency of aerobic and microaerobic growing bacteria. Clostridium perfringens, Aeromonas hydrophila, Plesiomonas shigelloides and E. coli were isolated from 55%, 48%, 14% and 35% of the birds, respectively. It could be demonstrated that the findings of these bacteria in the intestinal flora are depending on the age of the birds. The percentage of carriers of Clostridium perfringens, Aeromonas hydrophila and Plesiomonas shigelloides was highest in nestlings younger than 18 days, less high in older nestlings and lowest in adult grey herons. Contrary to those bacteria, E. coli was found more often in the intestinal flora at increasing age of the birds. Salmonella spp. were isolated from 6 birds. Two birds yielded positive for Yersinia enterocolitica and Campylobacter spp., respectively. Other aerobic and microaerobic bacteria play a less significant role as part of the intestinal flora.

Aeromonas↗

Typing of clinical and environmental Aeromonas veronii strains based on the 16S-23S rDNA spacers.

Genetic relationships of Aeromonas veronii strains isolated from human and environmental sources were investigated by restriction fragment length polymorphism (RFLP) of the polymerase chain reaction-amplified intergenic spacer region (ISR) flanked by the 16S and 23S rRNA genes. When using endonucleases AluI, HinfI and CfoI the 16S-23S rDNA-RFLP patterns showed considerable overall similarity, although most strains yielded specific profiles. Several intra-specific lines of descent comprised clinical strains linked to isolates from environmental sources. Strains having identical patterns may be individuals derived from highly similar, if not the same, microorganism. Results suggest that the ISR sequence-based method can be used to demonstrate colonization of a public water supply with a particular microorganism. In addition it could be very useful for tracing recurrent episodes of diarrhea and Aeromonas infection outbreaks.

Aeromonas↗

Identification of genes associated with copper tolerance in an adhesion-defective mutant of Aeromonas veronii biovar sobria.

TnphoA mutagenesis was used to identify adhesins of Aeromonas veronii biovar sobria 3767, a strain isolated from a diarrhoeal stool specimen. Six mutants, from a library of 154, exhibited significantly reduced levels of adhesion to HEp-2 cells. Primers to the terminal regions of TnphoA were used for inverse PCR and the product from one mutant was cloned into pBluescript and partial sequence data obtained. Scanning GenBank and EMBL data bases revealed DNA sequence similarity to the copA gene of Pseudomonas syringae pv. tomato which confers resistance to copper and other heavy metals. The transposon was located within the copA gene and the mutant exhibited a reduced tolerance to copper. Primer walking, using the inverse PCR product as a template, revealed three open reading frames (ORFs) copA, B and C in A. veronii biovar sobria 3767. The predicted amino acid sequences of ORFs A and B had significant homology (55 and 34% respectively) to the copA and B proteins of P. syringae. No amino acid or DNA sequence homology existed between ORF C of strain 3767 and any other gene in the data bases scanned. Further analysis of the nucleotide sequence failed to reveal the presence of typical copper regulatory genes within the vicinity of the Aeromonas sequence. The association between copper tolerance and adhesion in A. veronii biovar sobria requires further study.

Adhesins, Bacterial↗

Production and characterization of monoclonal antibodies to Aeromonas sobria surface antigens.

A panel of eight murine monoclonal antibodies (Mabs) was raised against the surface antigens of Aeromonas sobria isolated from a patient with diarrhoea. Antibodies to the LPS core and O-antigen and to protein antigens were generated. Three Mabs of the IgM isotype against either protein or LPS agglutinated 34/75 Aeromonas isolates from clinical and environmental sources. Similar numbers of A. hydrophila, A. sobria and A. caviae isolates were agglutinated. The Mabs were screened for their ability to inhibit A. sobria adhesion to HEp-2 cells, and haemagglutination (HA). Two Mabs directed against conformationally dependent epitopes on a 43-kDa protein blocked both functions. Of the anti-LPS Mabs one blocked adhesion only, and another blocked HA but not adhesion. Immunoprecipitation studies suggested that LPS-protein complexes may be involved in these potential virulence functions of A. sobria.

Aeromonas↗

Secreted enzymes of Aeromonas.

A hallmark characteristic of species of Aeromonas is their ability to secrete a wide variety of enzymes associated with pathogenicity and environmental adaptability. Among the most intensively studied are beta-lactamases, lipases, hemolytic enterotoxins, proteases, chitinases, nucleases and amylases. Multiple copies of genes encoding each type of enzyme provide additional biological diversity. Except for the chitinases, these multiple copies show little evolutionary relatedness at the DNA level and only limited similarity at the protein level. Indeed a number of the genes, such as nuclease H of A. hydrophila, have no similarity to known prokaryotic or eukaryotic sequences. The challenge is to determine how these genes evolved, where they originated and why Aeromonas possesses them in such abundance and variety.

Aeromonas↗

RFLP-PCR analysis of the aroA gene as a taxonomic tool for the genus Aeromonas.

The aroA gene has been identified as a target in screening for the presence of most Aeromonas species so far described by PCR. Synthetic oligonucleotide primers of 24 and 25 nucleotides were used by PCR assay to amplify a sequence of the aroA gene, which encodes 3-phosphoshikimate-1-carboxyvinyltransferase, a key enzyme of aromatic amino acids and folate biosynthetic pathway. A 1236-bp DNA fragment, representing most of the aroA gene, according to the nucleotide sequence of A. salmonicida, was amplified from all Aeromonas species tested, which represented most of the 14 hybridization groups. HaeII digestion of the 1236-bp fragment generated a restriction fragment length polymorphisms which could be used as a powerful tool for identification of aeromonads to the genus level.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

Enzyme-linked immunosorbent assay for detection of Aeromonas hydrophila serogroup O:19.

An enzyme-linked immunosorbent assay has been developed for the detection of Aeromonas hydrophila serogroup O:19 isolated from epizootics in eels. The enzyme-linked immunosorbent assay specificity was confirmed after testing A. hydrophila O:19 and non-O:19 strains from different origins, as well as other Aeromonas species and other fish pathogens such as Vibrio vulnificus biotype 2, V. furnisii, V. damsela, Yersinia ruckerii and Edwardsiella tarda. The detection limits for A. hydrophila O:19 cells were around 10(4)-10(5) cells/well. Artificially infected eels were analyzed and the immunodetection was confirmed by cultural methods. With this methodology A. hydrophila O:19 was successfully detected in infected eels and water samples. We described two subgroups within the serogroup O:19 (Guinée and Jansen system), one of them presents a 50 kDa outer membrane protein as a strong thermostable antigen which is not present in the other group.

Aeromonas hydrophila↗

Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.: correlation with virulence in a suckling mouse model.

Previous studies have shown that two hemolytic toxins, HlyA and AerA, contribute to the virulence of Aeromonas hydrophila. A survey was performed to gauge the distribution of hlyA and aerA genes in clinical and environmental Aeromonas isolates. For A. hydrophila, A. veronii biotype sobria and A caviae, 96%, 12% and 35% of strains, respectively, were hlyA positive, whereas, 78%, 97%, 41%, respectively, were aerA positive. All virulent A. hydrophila isolates were hlyA+ aerA+. This genotype was most common in A. hydrophila (75.4%) followed by A. caviae (29.4%) and A. veronii biotype sobria (9.6%). For A. hydrophila, a two-hemolytic toxin model of virulence provides the best prediction of virulence in an animal model.

Aeromonas↗

Aeromonas hydrophila vacuolating activity in the Caco-2 human enterocyte cell line as a putative virulence factor.

A non-enterotoxigenic strain of Aeromonas hydrophila isolated from diarrheic stools of an 8-month-old child was found to cause vacuolation in Caco-2 cells. The vacuoles became prominent 60 min after addition of the bacterial culture to the cell monolayers and, after 120 min, a complete disruption of the monolayers was observed. Cell vacuolation was not detected when Caco-2 monolayers were tested with sterile filtrates of overnight cultures of the A. hydrophila vacuolating strain AH14846. This strain produced a diffuse adherence pattern in Caco-2 cell monolayers, but did not produce detectable cytotonic enterotoxin in the suckling mouse test and only produced small quantities of aerolysin. By demonstrating the ability to induce vacuolation in mammalian cells of enterocytic lineage, the current study raises the possibility that such activity might contribute to gastrointestinal symptoms in infections involving Aeromonas strains which do not express well-established enterotoxins.

Aeromonas hydrophila↗

Lack of agreement between biochemical and genetic identification of Aeromonas spp.

Biochemical and genetic identification by RFLP (restriction fragment length polymorphism) of the PCR-amplified 16S r-RNA sequence were compared for a selection of 171 clinical and environmental isolates of Aeromonas spp. The investigation revealed large differences between the two methods. The species phenotypic identification scheme and the genetic technique applied to the environmental strains gave divergent results for 96% of the strains tested. There was 46% discrepancy between the two methods for the clinical isolates. The distribution of species differed between clinical and environmental isolates. A. hydrophila, A. caviae, A. jandaei and A. veronii dominated the clinical material (81% of isolates by RFLP), whilst only 21% of the environmental isolates belonged to those four species. From the environmental group A. salmonicida, A. bestiarum, A. sobria, A. media, and A. encheleia contributed 72% of the strains tested. The poor parity between the biochemical and the genetic identification of the environmental isolates, and to a lesser extent for the clinical isolates, underlines the fact that our current biochemical methods cannot adequately differentiate Aeromonas spp. This work also shows that the biochemical schemes derived from clinical isolates are incomplete for the identification of environmental strains.

Aeromonas↗

[Resistance to antimicrobial agents, hemolytic activity and plasmids in Aeromonas species].

A total of 174 Aeromonas isolates consisting of 100 strains from patients with diarrhea being mainly overseas travellers nd healthy subjects, and 74 strains from environmental sources including foods, fish, fresh water, sea water and river soil collected in the area of Tokyo Metropolis and Kanagawa Prefecture was examined for the antimicrobial resistance, presence of plasmids and hemolytic activity. Almost all the isolates (99.4%) were resistant to aminobenzyl penicillin. The isolation frequency of chloramphenicol- or tetracycline-resistant strain was low. Most environmental isolates of A. hydrophila were resistant to multiple antimicrobial agents. Thirty-seven percent of environmental isolates and 39% of human fecal ones carried plasmids. In environmental isolates, seven A. hydrophila and three A. sobria strains carried 63- to 150-kilobase pair (kb) conjugative R plasmids. Two A. hydrophila strains from both the healthy subject and domestic case with diarrhea carried 58- to 90-kb conjugative R plasmids, respectively. None of the isolates from the feces of overseas traveller's diarrhea carried the plasmid. Irrespective of the sources. A. hydrophila showed the highest hemolytic activity among three Aeromonas species. Eighty percent or more of A. hydrophila isolates were of hemolysin positive. The hemolytic titer of A. hydrophila strains from human feces was higher than that of the strains from environmental sources.

Aeromonas↗

[Rapid detection of the hemolysin genes in Aeromonas sobria by the polymerase chain reaction].

The hemolysin of Aeromonas sobria is one of the important virulence factors in this organism. Rapid detection and identification test for A. sobria is important for early and specific diagnosis of this infectious disease. We evaluated the polymerase chain reaction (PCR) for the rapid detection of A. sobria. Two pairs of synthetic oligonucleotide primers (ASA1-s and a; AerAAS-s and a) were used in PCR technique to detect the different hemolysin genes (ASA1 and aerAAS) in A. sobria. The PCR identified 91% of ASA1-positive and 23.4% of aerAAS-positive strains in beta-hemolytic A. sobria. Other species of Aeromonas, Plesiomonas shigelloides, Vibrio cholerae O1 and V. parahaemolyticus tested were negative in the PCR with two pairs of primers. The PCR technique for detection of two hemolysin genes suggested the possibility of application of this method for detection of A. sobria in A. sobria-associated infections.

Aeromonas↗

[Aeromonas hydrophila septicemia with necrotizing myofascitis in a patient with hypoplastic leukemia].

A 54-year-old male was admitted to Kawasaki Medical School Hospital with the complaint of fever. His diagnosis of hypoplastic leukemia had been made one year ago. After the admission, cecal mass with pain and high fever were noted. Four days later, he suddenly lost consciousness and died. Aeromonas hydrophila was isolated from blood cultures and also from the myofascitis specimen. Autopsy specimen of the iliopsoas muscle showed necrotizing myofascitis. The specimen obtained from the cecum showed submucosal hemorrhage with edema and these findings were compatible to ischemic colitis. This pathogen is widely distributed in nature, especially in water fields. Therefore, it would be advised to consider the Aeromonas hydrophila as one of the pathological organisms pathognomonic for the septicemia, when one may see febrile and gastrointestinal symptoms in a patient with hematological malignancies.

Aeromonas hydrophila↗

In vitro activity of cefoperazone against nonfermenters and Aeromonas hydrophila.

The in vitro activity of cefoperazone against 380 strains (33 species and unnamed groups) of nonferenters and 20 strains of Aeromonas hydrophila was studied by a microdilution method of determining minimal inhibitory concentrations. For comparison, the activities of ampicillin, ticarcillin, and cefamandole were simultaneously studied. Cefoperazone was the most active drug against Pseudomonas sp., Achromobacter xylosoxidans, Flavobacterium meningosepticum. Flavobacterium sp. (group IIb), Group IVc biotype 2, Group Ve, and Aeromonas hydrophila. Against other organisms it was equivalent in activity to or less active than comparative antibiotics. The only organisms which were usually resistant to 128 microgram of cefoperazone per ml were strains of Flavobacterium odoratum and Group IIk biotype 1.

Aeromonas↗

Antimicrobial susceptibility of Aeromonas species isolated from patients with diarrhea.

In vitro susceptibility to 17 antimicrobial agents was determined for Aeromonas caviae, A. hydrophila, and A. sobria isolated from feces of patients with diarrhea. The three Aeromonas species shared a similar susceptibility pattern, except to cephalothin. Of the oral antimicrobial agents, the quinolones, followed by chloramphenicol, were most active; trimethoprim-sulfamethoxazole and tetracycline had good but variable activity.

Adult↗