Prevalence and distribution of Aeromonas hydrophila in the United States.

The abundance of Aeromonas hydrophila was measured in 147 natural aquatic habitats in 30 states and Puerto Rico. Viable cell counts were used to estimate density at all sites by using Rimler-Shotts medium, a differential presumptive medium for A. hydrophila. Temperature, pH, conductivity, salinity, and turbidity were measured simultaneously with water sample collection. The density of A. hydrophila was higher in lotic than in lentic systems. Saline systems had higher densities of A. hydrophila than did freshwater systems. A. hydrophila could not be isolated from extremely saline, thermal, or polluted waters, even though it was found over wide ranges of salinity, conductivity, temperature, pH, and turbidity. Of the water quality parameters measured, only conductivity was significantly regressed with density of A. hydrophila.     

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.     

Prevalence and distribution of Aeromonas hydrophila in the United States.

The abundance of Aeromonas hydrophila was measured in 147 natural aquatic habitats in 30 states and Puerto Rico. Viable cell counts were used to estimate density at all sites by using Rimler-Shotts medium, a differential presumptive medium for A. hydrophila. Temperature, pH, conductivity, salinity, and turbidity were measured simultaneously with water sample collection. The density of A. hydrophila was higher in lotic than in lentic systems. Saline systems had higher densities of A. hydrophila than did freshwater systems. A. hydrophila could not be isolated from extremely saline, thermal, or polluted waters, even though it was found over wide ranges of salinity, conductivity, temperature, pH, and turbidity. Of the water quality parameters measured, only conductivity was significantly regressed with density of A. hydrophila.     

Detection of metallo-β-lactamases-encoding genes in environmental isolates of Aeromonas hydrophila and Aeromonas jandaei

Aims:  To determine the prevalence and expression of metallo-β-lactamases (MBL)-encoding genes in Aeromonas species recovered from natural water reservoirs in southeastern Brazil.
Methods and Results:  Eighty - seven Aeromonas isolates belonging to Aeromonas hydrophila ( n  =   41) and Aer. jandaei ( n  =   46) species were tested for MBL production by the combined disk test using imipenem and meropenem disks as substrates and EDTA or thioglycolic acid as inhibitors. The presence of MBL genes was investigated by PCR and sequencing using new consensus primer pairs designed in this study. The cphA gene was found in 97·6% and 100% of Aer. hydrophila and Aer. jandaei isolates, respectively, whereas the acquired MBL genes bla _IMP , bla _VIM and bla _SPM-1 were not detected. On the other hand, production of MBL activity was detectable in 87·8% and 10·9% of the cphA -positive Aer. hydrophila and Aer. jandaei isolates respectively.
Conclusions:  Our results indicate that cphA seems to be intrinsic in the environmental isolates of Aer. hydrophila and Aer. jandaei in southeastern Brazil, although, based on the combined disk test, not all of them are apparently able to express the enzymatic activity.
Significance and Impact of the Study:  These data confirm the presence of MBL-producing Aeromonas species in natural water reservoirs. Risk of waterborne diseases owing to domestic and industrial uses of freshwater should be re-examined from the increase of bacterial resistance point of view.     

Starch-Ampicillin Agar for the Quantitative Detection of Aeromonas hydrophila

Interest in Aeromonas hydrophila as a food-borne and human pathogen is increasing. Isolation media from the clinical laboratory were evaluated for food use and either did not give quantitative recovery of A. hydrophila or did not permit ready differentiation of A. hydrophila from the background microflora. A new medium was developed which permitted quantitative recovery of A. hydrophila from foods. The medium consisted of phenol red agar base (Difco Laboratories), soluble starch (10 g/liter), and ampicillin (10 mg/liter). All foods surveyed contained A. hydrophila. Foods sampled included red meats, chicken, raw milk, and seafood (fish, shrimp, scallops, crab, and oysters). The count of A. hydrophila at the time of purchase ranged from 1 × 10²/g (lower limit of detection) to 5 × 10⁵/g. In most instances, the count of A. hydrophila increased during 1 week of storage at 5°C. The starch-ampicillin agar developed permitted rapid quantitative recovery of A. hydrophila from foods in the presence of very large numbers of competing microflora.     

Complete type III secretion system of a mesophilic Aeromonas hydrophila strain

We have investigated the existence and genetic organization of a functional type III secretion system (TTSS) in a mesophilic Aeromonas strain by initially using the Aeromonas hydrophila strain AH-3. We report for the first time the complete TTSS DNA sequence of an Aeromonas strain that comprises 35 genes organized in a similar disposition as that in Pseudomonas aeruginosa. Using several gene probes, we also determined the presence of a TTSS in clinical or environmental strains of different Aeromonas species: A. hydrophila, A. veronii, and A. caviae. By using one of the TTSS genes (ascV), we were able to obtain a defined insertion mutant in strain AH-3 (AH-3AscV), which showed reduced toxicity and virulence in comparison with the wild-type strain. Complementation of the mutant strain with a plasmid vector carrying ascV was fully able to restore the wild-type toxicity and virulence.     

RAPD analysis of Aeromonas salmonicida and Aeromonas hydrophila

The randomly amplified polymorphic DNA (RAPD) technique was used to analyse the genetic differentiation of 13 strains of Aeromonas salmonicida subsp. salmonicida , and seven strains of Aer. hydrophila. Reproducible profiles of genomic DNA fingerprints were generated by polymerase chain reaction (PCR) using a single randomly designed primer. The RAPD profiles of all the non-motile aeromonads, Aer. salmonicida subsp. salmonicida were identical. However, profiles of the motile aeromonads, Aer. hydrophila differed between isolates. These findings reveal genomic homogeneity in Aer. salmonicida subsp. salmonicida and genetic variety in Aer. hydrophila strains.     

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.     

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.     

Seasonal distribution of Aeromonas hydrophila in river water and isolation from river fish

The seasonal distribution of Aeromonas hydrophila in water and recovery rate from live river fish was investigated. The highest isolation rates of A. hydrophila occurred in water during the late winter followed by a progressive decline in density during the summer and monsoon seasons. The organism was recovered from fish throughout the period from which it was concluded that they form a reservoir which is unrelated to their density in water. The enterotoxigenicity of some environmental strains was tested in suckling mice and rabbit ileal loop.     

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.     

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.     

Seasonal distribution of Aeromonas hydrophila in river water and isolation from river fish

The seasonal distribution of Aeromonas hydrophila in water and recovery rate from live river fish was investigated. The highest isolation rates of A. hydrophila occurred in water during the late winter followed by a progressive decline in density during the summer and monsoon seasons. The organism was recovered from fish throughout the period from which it was concluded that they form a reservoir which is unrelated to their density in water. The enterotoxigenicity of some environmental strains was tested in suckling mice and rabbit ileal loop.     

Virulence potential and genetic diversity of Aeromonas caviae, Aeromonas veronii, and Aeromonas hydrophila clinical isolates from Mexico and Spain: a comparative study

A comparative study of 109 Aeromonas clinical isolates belonging to the 3 species most frequently isolated from patients with diarrhea in Mexico and Spain was performed to investigate the distribution of 3 prominent toxin genes and the gene encoding flagellin of lateral flagella; 4 well-established virulence factors in the genus Aeromonas. The aerolysin-hemolysin toxin genes were the most prevalent, being present in 89% of the total isolates. The ast toxin gene was conspicuously absent from the Aeromonas caviae and Aeromonas veronii groups but was present in 91% of the Aeromonas hydrophila isolates. Both the alt toxin gene and the lafA flagellin gene also had a low incidence in A. caviae and A. veronii. Differences in the prevalence of alt and lafA were observed between isolates from Mexico and Spain, confirming genus heterogeneity according to geographic location. Carriage of multiple toxin genes was primarily restricted to A. hydrophila isolates, suggesting that A. caviae and A. veronii isolates circulating in Mexico and Spain possess a limited array of virulence genes. Enterobacterial repetitive intergenetic consensus - polymerase chain reaction showed that the Aeromonas populations sampled lack dominant clones and were genetically heterogeneous, with A. caviae being the most diverse species. Further surveys of virulence determinants in genetically heterogeneous populations of Aeromonas isolates circulating worldwide are required to enhance the understanding of their capacity to cause disease.     

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:
     

Site-directed mutagenesis at histidines of aerolysin from Aeromonas hydrophila: a lipid planar bilayer study

The role of histidine residues in the formation of channels by the cytolytic toxin aerolysin has been studied in planar lipid bilayers by substituting each of the six histidines in the native protein with asparagine. His341 or His186 mutants had the same channel-forming ability as native toxin, whereas the His332 and His121 mutants were less active. Mutations at His132 and His107, which interfere with the oligomerization of the toxin, drastically reduce pore formation. These findings support the conclusion that oligomerization of the toxin must precede channel formation, and that at least two of the six histidine residues are essential for this to occur. The aerolysin channel is a water-filled pore with an approximate diameter of 9.3 +/- 0.4 A.     

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.     

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.     

Genomic study of polyhydroxyalkanoates producing Aeromonas hydrophila 4AK4

The complete genome of Gram-negative Aeromonas hydrophila 4AK4 that has been used for industrial production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) was sequenced and annotated. Its chromosome is 4,527,993 bp in size encoding 4,272 genes, including 28 rRNA genes and 104 tRNA genes. Comparative analysis indicated that genome of A. hydrophila 4AK4 was similar to that of the A. hydrophila ATCC 7966^T, an intensively studied aeromonad for its pathogenicity related to its genomic information. Genes possibly coming from other species or even other genus were identified in A. hydrophila 4AK4. A large number of putative virulent genes were predicted. However, a cytotonic enterotoxin (Ast) is absent in A. hydrophila 4AK4, allowing the industrial strain to be different from other A. hydrophila strains, indicating possible reduced virulence of strain 4AK4, which is very important for industrial fermentation. Genes involved in polyhydroxyalkanoate (PHA) metabolism were predicted and analyzed. The resulting genomic information is useful for improved production of PHA via metabolic engineering of A. hydrophila 4AK4.