Cloning, mutagenesis, and nucleotide sequence of a siderophore biosynthetic gene (amoA) from Aeromonas hydrophila.

Many isolates of the Aeromonas species produce amonabactin, a phenolate siderophore containing 2,3-dihydroxybenzoic acid (2,3-DHB). An amonabactin biosynthetic gene (amoA) was identified (in a Sau3A1 gene library of Aeromonas hydrophila 495A2 chromosomal DNA) by its complementation of the requirement of Escherichia coli SAB11 for exogenous 2,3-DHB to support siderophore (enterobactin) synthesis. The gene amoA was subcloned as a SalI-HindIII 3.4-kb DNA fragment into pSUP202, and the complete nucleotide sequence of amoA was determined. A putative iron-regulatory sequence resembling the Fur repressor protein-binding site overlapped a possible promoter region. A translational reading frame, beginning with valine and encoding 396 amino acids, was open for 1,188 bp. The C-terminal portion of the deduced amino acid sequence showed 58% identity and 79% similarity with the E. coli EntC protein (isochorismate synthetase), the first enzyme in the E. coli 2,3-DHB biosynthetic pathway, suggesting that amoA probably encodes a step in 2,3-DHB biosynthesis and is the A. hydrophila equivalent of the E. coli entC gene. An isogenic amonabactin-negative mutant, A. hydrophila SB22, was isolated after marker exchange mutagenesis with Tn5-inactivated amoA (amoA::Tn5). The mutant excreted neither 2,3-DHB nor amonabactin, was more sensitive than the wild-type to growth inhibition by iron restriction, and used amonabactin to overcome iron starvation.     

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.     

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.     

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.     

Clinical involvement of Aeromonas hydrophila.

Aeromonas hydrophila has for some time been regarded as an opportunistic pathogen in hosts with impaired local or general defence mechanisms. Infections in such individuals are generally severe. The organism is also being isolated with increasing frequency throughout the world from a variety of focal and systemic infections of varying severity in persons that are apparently immunologically normal. Most commonly it causes acute diarrheal disease by producing an enterotoxin. Thus the organism appears to have greater clinical significance that was hitherto suspected. The organism has been infrequently reported from humans in Canada, but its correct laboratory identification, together with increased awareness that it can contribute to illness, will undoubtedly lead to more reports of its isolation in Canada.     

Phospholipids and lipopolysaccharide of Aeromonas hydrophila.

The phospholipids and lipopolysaccharide of Aeromonas hydrophila were characterized. Phosphatidylethanolamine and phosphatidylglycerol were the major phospholipid components. The outer membrane contained more phosphatidylethanolamine and less phosphatidylglycerol than the inner membrane, and the phospholipids of the outer membrane contained a higher proportion of saturated fatty acids. Only four fatty acids (C14:0, C16:0, C16:1, and C18:1) were found in the phospholipids. The lipopolysaccharide of A. hydrophila did not contain the eight-carbon sugar 3-deoxyoctulosonic acid nor did it contain C16:0, both of which are typical constituents of the lipopolysaccharide of many other species.     

Amonabactin: a family of novel siderophores from a pathogenic bacterium

 Four peptide-based bis-catecholate siderophores, collectively known as the amonabactins, have been isolated from Aeromonas hydrophila. They have been fully characterized: tandem mass spectroscopy established the sequence of the amino acid components, chiral gas chromatographic mass spectra established the amino acid chirality, and two-dimensional NMR techniques determined the full connectivity and structure. Each of the amonabactins was synthesized and the synthetic material was compared to the natural product as a final proof of structure. These siderophores are bis-catecholates with the backbone composed of either tri- or tetrapeptides in the sequence (gly)-(L)-lys-(L)-lys-(D)-aro, where glycine is the optional amino acid attached to the N^ε of the N terminus lysine and aro is either tryptophan or phenylalanine. The ligand units, 2,3-dihydroxybenzamide groups, are attached to the N^ε amine of the C terminus lysine and either to glycine, if present, or to N^ε amines of the N terminus lysine. Each of the amonabactins supports growth of the organism under low-iron conditions in vitro and in serum. The architecture of these siderophores includes an inverted aromatic amino acid and unusual linkages which should prevent enzymatic hydrolysis of the peptide backbones. This, along with their ability to successfully compete for iron in serum, suggests a role in the pathogenicity of the organism. This paper is number 62 in the series Coordination Chemistry of Microbial Ion Transport. Details of the previous paper are given in [1]. Received: 6 June 1997 / Accepted: 9 September 1997     

Aeromonas hydrophila: analysis of 11 cases.

A retrospective analysis of 11 cases in which Aeromonas hydrophila was isolated indicated that the organsim caused local infection in 7 cases and asymptomatic colonization in 4. There were no cases of septicemia and none of the patients were known to have a malignant disease or immunosuppression. There were no deaths, although three of the patients required amputation of limbs because of myonecrosis. Chloramphenicol and aminoglycosides appeared to be appropriate therapeutic agents.     

Characterization of lactoferrin binding by Aeromonas hydrophila.

Various lactoferrin preparations (iron-saturated and iron-depleted human milk lactoferrins and bovine milk and colostrum lactoferrins) were bound by Aeromonas hydrophila. Binding was (i) reversible (65% of bound lactoferrin was displaced by unlabeled lactoferrin), (ii) specific (lactoferrin but not other iron-containing glycoproteins such as ferritin, transferrin, hemoglobin, and myoglobin inhibited binding), and (iii) significantly reduced by pepsin and neuraminidase treatment of the bacteria. The glycosidic domains of the lactoferrin molecule seem to be involved in binding since precursor monosaccharides of the lactoferrin oligosaccharides (mannose, fucose, and galactose) and glycoproteins which have homologous glycosidic moieties similar to those of the lactoferrin oligosaccharides (asialofetuin or fetuin) strongly inhibited lactoferrin binding. A. hydrophila also binds transferrin, ferritin, cytochrome c, hemin, and Congo red. However, binding of these iron-containing compounds seems to involve bacterial surface components different from those required for lactoferrin binding. Expression of lactoferrin binding by A. hydrophila was influenced by culture conditions. In addition, there was an inverse relationship between lactoferrin binding and siderophore production by the bacterium.     

Characterization of lactoferrin binding by Aeromonas hydrophila.

Various lactoferrin preparations (iron-saturated and iron-depleted human milk lactoferrins and bovine milk and colostrum lactoferrins) were bound by Aeromonas hydrophila. Binding was (i) reversible (65% of bound lactoferrin was displaced by unlabeled lactoferrin), (ii) specific (lactoferrin but not other iron-containing glycoproteins such as ferritin, transferrin, hemoglobin, and myoglobin inhibited binding), and (iii) significantly reduced by pepsin and neuraminidase treatment of the bacteria. The glycosidic domains of the lactoferrin molecule seem to be involved in binding since precursor monosaccharides of the lactoferrin oligosaccharides (mannose, fucose, and galactose) and glycoproteins which have homologous glycosidic moieties similar to those of the lactoferrin oligosaccharides (asialofetuin or fetuin) strongly inhibited lactoferrin binding. A. hydrophila also binds transferrin, ferritin, cytochrome c, hemin, and Congo red. However, binding of these iron-containing compounds seems to involve bacterial surface components different from those required for lactoferrin binding. Expression of lactoferrin binding by A. hydrophila was influenced by culture conditions. In addition, there was an inverse relationship between lactoferrin binding and siderophore production by the bacterium.     

O-antigen structure in a virulent strain of Aeromonas hydrophila

Abstract Lipopolysaccharide (LPS) was isolated from a strain of Aeromonas hydrophila which had displayed serological, bacteriophage attachment and virulence properties similar to those found in strains of Aeromonas salmonicida . The structure of the O-antigen was determined and had many points of similarity with that previously elucidated for the O-antigen of A. salmonicida . Methylation analysis, chromium trioxide oxidation and ¹H-n.m.r. were used to confirm that the repeating unit of the O-chain had the following structure:
     

Specific binding of lactoferrin to Aeromonas hydrophila.

The interaction of lactoferrin (Lf) with Aeromonas hydrophila (n = 28) was tested in a 125I-labeled protein-binding assay. The mean per cent binding values for human Lf (HLf) and bovine Lf (BLf) were 13.4 +/- 2.0 (SEM), and 17.5 +/- 2.7 (SEM), respectively. The Lf binding was characterized in type strain A. hydrophila subsp. hydrophila CCUG 14551. The HLf and BLf binding reached a complete saturation within 2 h. Unlabeled HLf and BLf displaced 125I-HLf binding in a dose-dependent manner, and more effectively by the heterologous (1 microgram for 50% inhibition) than the homologous (10 micrograms for 50% inhibition) ligand. Apo- and holo-forms of HLf and BLf both inhibited more than 80%, while mucin caused approx. 50% inhibition of the HLf binding. Various other proteins (including transferrin) or carbohydrates did not block the binding. Two HLf-binding proteins with an estimated molecular masses of 40 kDa and 30 kDa were identified in a boiled-cell-envelope preparation, while the unboiled cell envelope demonstrated a short-ladder pattern at the top of the separating gel and a second band at approx. 60 kDa position. These data establish a specific interaction of Lf and the Lf-binding proteins seem to be porins in A. hydrophila.     

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.     

Polar flagellum biogenesis in Aeromonas hydrophila

Mesophilic Aeromonas spp. constitutively express a single polar flagellum that helps the bacteria move to more favorable environments and is an important virulence and colonization factor. Certain strains can also produce multiple lateral flagella in semisolid media or over surfaces. We have previously reported 16 genes (flgN to flgL) that constitute region 1 of the Aeromonas hydrophila AH-3 polar flagellum biogenesis gene clusters. We identified 39 new polar flagellum genes distributed in four noncontiguous chromosome regions (regions 2 to 5). Region 2 contained six genes (flaA to maf-1), including a modification accessory factor gene (maf-1) that has not been previously reported and is thought to be involved in glycosylation of polar flagellum filament. Region 3 contained 29 genes (fliE to orf29), most of which are involved in flagellum basal body formation and chemotaxis. Region 4 contained a single gene involved in the motor stator formation (motX), and region 5 contained the three master regulatory genes for the A. hydrophila polar flagella (flrA to flrC). Mutations in the flaH, maf-1, fliM, flhA, fliA, and flrC genes, as well as the double mutant flaA flaB, all caused loss of polar flagella and reduction in adherence and biofilm formation. A defined mutation in the pomB stator gene did not affect polar flagellum motility, in contrast to the motX mutant, which was unable to swim even though it expressed a polar flagellum. Mutations in all of these genes did not affect lateral flagellum synthesis or swarming motility, showing that both A. hydrophila flagellum systems are entirely distinct.     

Isolation of Aeromonas hydrophila from the American alligator, Alligator mississippiensis.

Aeromonas hydrophila was isolated from the internal organs of nine adult alligators, Alligator mississippiensis, which died without apparent cause, suggesting the bacterium may have been a factor. One hundred and twenty-three alligators ranging in age from six months to over 10 years were captured from five locations in the southeastern United States and sampled for A. hydrophila. The bacterium was isolated from the oral cavity of 85% of the animals, on the external jaw area from over 50% and from 70% of the internal tissue samples. A. hydrophila is ubiquitous with alligators in their natural habitats, but apparently does not cause clinical disease. However, stress factors such as trapping, handling, and warm water temperatures may be conducive to the rapid proliferation of the bacteria, thereby facilitating disease.     

Characterization of an O-antigen bacteriophage from Aeromonas hydrophila.

A unique bacteriophage of Aeromonas hydrophila serotype O:34 was isolated, purified, and characterized. The bacterial surface receptor was shown to be the O-antigen polysaccharide component of lipopolysaccharide specific to serotype O:34, which was chemically characterized. The high molecular weight lipopolysaccharide fraction (a fraction enriched in O antigen) was fully able to inactivate bacteriophage PM1. Phage-resistant mutants of A. hydrophila O:34 were isolated and found to be specifically devoid of lipopolysaccharide O antigen. No other cell-surface molecules were involved in phage binding. The host range of bacteriophage PM1 was found to be very narrow, producing plaques only on A. hydrophila strains from serotype O:34.     

Survival of genetically-marked Aeromonas hydrophila in water

Survival of a genetically-marked Aeromonas hydrophila was monitored in water microcosms. There was no apparent loss of a marker plasmid which encoded the xylE reporter gene during prolonged incubation in lake water. Survival was best in sterile lake water but in sea water, cells died rapidly during the first 9 d, recovered up to day 12 and thereafter numbers fell up to 28 d accompanied by loss of the plasmid in a proportion of the cells.