Identification of Aeromonas Species Isolated from Freshwater Fish with the Microplate Hybridization Method

Aeromonas isolates were obtained from the intestinal tracts of six species of cultured freshwater fish and identified on the basis of their genotypic and phenotypic characters. The microplate hybridization method could differentiate type strains of Aeromonas species and related bacteria. DNA-DNA hybridization analysis showed that 65 aeromonad isolates were 72 to 100% related with either Aeromonas caviae, Aeromonas hydrophila, Aeromonas jandaei, Aeromonas sobria, or Aeromonas veronii. As many as 48% of the genotypically identified A. caviae, A. hydrophila, and A. sobria isolates differed from the type strains of corresponding species in one to three phenotypic characters. These results strongly suggest that not all aeromonad isolates from freshwater fish could be identified correctly on the basis of only the phenotypic characters, indicating the usefulness of the microplate hybridization method for the identification of aeromonads.     

Analysis of the interaction of Aeromonas caviae, A. hydrophila and A. sobria with mucins

Aeromonas species are known to be involved in human gastrointestinal diseases. These organisms colonize the gastrointestinal tract. Aeromonas hydrophila, A. caviae, and A. sobria have been demonstrated microscopically to adhere to animal cell lines that express mucous receptors, but quantitative studies of adherence to mucosal components such as mucin have not been published to date. Purified bovine submaxillary gland, hog gastric mucin, and fish skin mucin were used as a model to study mucin-binding activity among A. caviae, A. hydrophila, and A. sobria strains. Our findings revealed that binding of radiolabeled and enzyme-conjugated mucins to Aeromonas cells varied depending on the labeling procedure. The highest binding was observed when the three mucin preparations were labeled with horseradish peroxidase. Binding of the various horseradish peroxidase-labeled mucins by A. caviae, A. hydrophila, and A. sobria cells is a common property among Aeromonas species isolated from human infections, diseased fish, and from environmental sources. The proportion of Aeromonas strains which bind the various horseradish peroxidase-labeled mucins was significantly higher for A. hydrophila than for A. caviae and A. sobria. Bacterial cell-surface extracts containing active mucin-binding components recognized the horseradish peroxidase-labeled mucins. The molecular masses of the mucin-binding proteins were estimated by SDS-PAGE and Western blot as follows: A. caviae strain A4812 (95 and 44 kDa); A. hydrophila strain 48748 (97, 45, 33 and 22 kDa); and A. sobria strain 48739 (95 and 43 kDa). Mucin interaction with Aeromonas cells was also studied in terms of growth in mucin-rich media. The culture conditions greatly influence the expression of A. hydrophila mucin-binding activity.     

Isolation, Enumeration, and Characterization of Aeromonas from Polluted Waters Encountered in Diving Operations

Counts of total viable, aerobic, heterotrophic bacteria, indicator organisms, and Aeromonas spp. were made at a diver training site on the Anacostia River in Washington, D.C. The numbers of Aeromonas cells in Anacostia River sediment and water increased during periods of elevated water temperature, to maxima of 4 × 10⁵ cells per g of sediment and 300 cells per ml of water. Correspondingly, Aeromonas counts dropped 2 to 4 logs as the water temperature decreased to 0 to 0.5°C. Cultures taken by sterile swabs from the ears and face masks of divers after a 30-min swim in the Anacostia River yielded bacterial types and numbers similar to those found in the river. The nasal passages of the divers apparently did not become contaminated by swimming, possibly because of the protective effect of the face masks used by the divers. Properties associated with virulence in Aeromonas hydrophila and Aeromonas sobria strains isolated from the river, sediment, and divers were investigated. Nearly 40% of the strains of both species collected during the study produced cytotoxic activity for mouse Y-1 adrenal cells, as well as elastase. Enterotoxin activity, as detected by the Y-1 assay, was observed in 3% (1 of 35) of the strains of A. sobria and in 6% (19 of 330) of the A. hydrophila strains. Fluid accumulation in rabbit ileal loops induced by both species of Aeromonas varied greatly among the 17 strains examined. Fluid accumulation of at least 0.4 ml/cm was correlated with positive cytotoxin- or enterotoxin-like response in the Y-1 tissue culture assay.     

Effect of wastewater stabilization ponds on antimicrobial susceptibility and haemolysin occurrence among motile Aeromonas strains

Aeromonas strains (total=953) isolated from raw wastewater, stabilization pond effluent and sediments were evaluated for their susceptibilities to 17 antibiotics and for their ability to produce haemolysins. Stabilization ponds did not seem to select highly resistant strains of aeromonads. There were no differences in the resistance patterns of isolates from raw sewage, stabilization pond effluent and sediments. All strains were found to possess multiple resistance, most commonly to ampicillin, amoxicillin and novobiocin. Almost 90% of the strains of A. hydrophila and A. caviae were resistant to cephalothin, whereas more than 80% of A. sobria isolates were found to be susceptible to this antibiotic. Resistance to trimethoprim, oxytetracycline, nalidixic acid, chloramphenicol, tetracycline, trimethoprim-sulphamethoxazol, polymyxin B, kanamycin or erythromycin among all isolates did not exceed 10%. Moreover, no strain was found to be resistant to gentamycin and only 9 of the 953 isolates exhibited resistance to cefotaxim. The percentage of haemolytic strains was significantly higher in the stabilization pond effluent than in raw sewage. This high incidence of haemolytic activity was connected with a high proportion of A. sobria whereas, in samples from the raw sewage or stabilization pond sediments a high proportion of A. caviae decreased the total amount of haemolytic aeromonads. The high incidence of haemolytic activity (+) was associated particularly with A. sobria (93.3%) and A. hydrophila (88.7%) whereas A. caviae was found to be the lowest haemolytic species (16.3%).B. Imziln and Y.M.O. Lafdal are with Cadi Ayyad University, Faculty of Sciences Semialia, Department of Biology, Laboratory of Microbiology, BP S/15 Marrakech, Morocco. M. Jana is with the Hôpital Millitaire Avicenne Marrakech, Morocco.     

<Emphasis Type="Italic">Aeromonas</Emphasis> Spp. Human Isolates Induce Apoptosis of Murine Macrophages

Interactions of Aeromonas caviae, Aeromonas veronii biotype sobria, and Aeromonas hydrophila strains isolated from fecal specimens of humans with gastroenteritis on murine macrophages, J774 cells, were investigated. Analyses of cellular morphology and DNA fragmentation in phagocytes infected with these strains exhibited typical characteristic features of cells undergoing apoptosis. We observed the morphological changes, including condensation of nuclear chromatin, formation of apoptotic bodies and blebbing of cell membrane, and fragmentation of nuclear DNA into oligonucleosomal fragments. The lowest apoptotic index did not exceed 25%, whereas the highest reached 78% at 24 h and 96% at 48 h after infection. After incubation of J774 cells with cytotoxic enterotoxin isolated from A. veronii biotype sobria strain, we noted that the toxin was able to trigger cytotoxicity and apoptosis of macrophages. The results indicate that apoptosis could be one of the mechanisms contributing to the development of Aeromonas-associated diarrheal disease.     

Sequence Analysis of Amplified DNA Fragments Containing the Region Encoding the Putative Lipase Substrate-Binding Domain and Genotyping of Aeromonas hydrophila

DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.     

Molecular detection of the <Emphasis Type="Italic">Aeromonas</Emphasis> virulence aerolysin gene in retail meats from different animal sources in Egypt

Meat commonly contain the same Aeromonas spp. which occur in human diarrhoeal and non-diarrhoeal faecal samples. Motile Aeromonas were isolated from 5.6% of total 302 samples. The distribution of the isolates were 5.9 and 5.2% in fresh and frozen samples, respectively. Of the 302 samples taken of the four animal meat species investigated, the genus Aeromonas were isolated in 12.3% of the fresh samples collected from buffalo meat, in 6.5% of the samples collected from sheep meat and 14.0% from the samples collected from the cattle frozen meat samples. The camel meat did not reveal any Aeromonas isolates. Aeromonas hydrophila was isolated as the most prevalent species with 6.8%, followed by Aeromonas caviae with 2.7% and Aeromonas sobria with 2.1% from the total meat samples. Aerolysin toxin gene (aerA) was detected in 3/17 isolates of A. hydrophila isolated from contaminated meat. Infection due to bacterial pathogen with such virulent factor through contact with contaminated meat while handling them, poses health hazards to humans.     

Distribution and antibacterial drug resistance ofAeromonas spp. from fresh and brackish waters in Southern Turkey

The frequency of antibiotic resistance was compared inAeromonas spp. isolated from fresh and brackish water in Southern Turkey. A total of 97Aeromonas spp. strains were isolated from four zones (three from fresh and one from brackish water). Most of the strains isolated from all samples wereAeromonas hydrophila (79.4%), while the amount ofAeromonas sobria andAeromons caviae, were rather lower in the samples examined (17.5% and 3.1% respectively). A high proportion of isolates from all water sources showed resistance to cephalotin (86.6%) and trimethoprim-sulphamethoxazole (69%). On the other hand, a low proportion of bacteria showed resistance to tetracycline (14.4%), chloramphenicol (11.3%), gentamicin (7.2%) and nitrofurantoin (6.8%). Only one strain showing resistance to amikacin was found. Multiple Antibiotic Resistance Index (MARI) to at least two antibiotics was highest in brackish water (zone 4), followed by fresh water (zone 3). MARI values ranging from 0.2 to 0.8 for the bacteria isolated from brackish water. This study suggest that, multiple antibiotic resistantAeromonas spp., especiallyA. hydrophila, can be easily recovered from fresh and brackish water sources in Turkey and these sources may play as a reservoirs responsible for disease pathogen aeromonads.     

Pyrolysis mass spectrometry characterisation and numerical taxonomy ofAeromonas spp.

Reference strains (2) and 29 isolates ofAeromonas spp. from clinical material and environmental specimens were characterised in traditional biochemical tests, and in pyrolysis mass spectrometry, which gives data reflecting whole-cell composition. Numerical taxonomic analyses of the data sets were compared with conventional identification at species level, and pathogenic potential, as inferred from the origin of the isolates. Clustering with conventional test reaction patterns showed, for each of the species represented, a clearly defined core group of typical isolates, surrounded by a halo of aberrant strains. One further cluster comprised strains intermediate betweenA. caviae andA. hydrophila, and one strain was grossly atypical in both analyses. Clustering from pyrolysis data corresponded less well with species identification. Broadly, the biochemical division between core and halo strains was supported in pyrolysis forA. caviae andA. sobria, but the main group ofA. hydrophila in pyrolysis comprised strains clustering in the core and halo groups of this species, and three strains intermediate betweenA. hydrophila andA. caviae in biochemical tests. Two further pyrolysis clusters comprised core and halo strains ofA. hydrophila. However, pyrolysis clustering correlated well with inferred pathogenicity, showing four clusters of probable pathogens, six clusters of probable nonpathogens, and one two member cluster of doubtful status. Most strains that clustered in the species haloes, or in species-intermediate groups in biochemical tests, were non-human isolates, or were isolated in the absence of symptomatic infection. The correlation of inferred pathogenicity with biochemical clustering was poorer than that with pyrolysis clustering.Abbreviations CTRP conventional test reaction pattern - PyMS pyrolysis mass spectrometry     

Seasonal incidence of and antibiotic resistance among Aeromonas species isolated from domestic wastewater before and after treatment in stabilization ponds

The efficiency of stabilization pond treatment of domestic wastewater in removing culturable cells of motile Aeromonas and its influence on the incidence of resistance to seven antibiotics were investigated in this study. Removal efficiency was higher (P < 0.001) in the warm months (98.8%) than in the cold months (97%). Among the 264 isolates, 163 were Aeromonas caviae, 24 were A. hydrophila, and 54 were A. sobria. Twenty-three isolates could not be identified to the species level. In the influent, A. caviae dominated in both cold and warm months. In the water samples originating from the influent, A. sobria was present at higher percentages in the warm period. All the isolates were resistant to amoxicillin and most of them (73%) exhibited resistance to cephalothin. Of the three species tested, A. sobria was more susceptible to antibiotics than either A. caviae or A. hydrophila. The most striking difference among the species was seen in resistance to cephalothin. There were 91 % of A. caviae strains and 96% of A. hydrophila isolates that were resistant to cephalothin. However, only 9% of A. sobria strains exhibited resistance to this drug. The high incidence of resistance in raw sewage was connected with a high proportion of A. caviae, whereas in the water samples collected from the effluent during the warm months, a high proportion of A. sobria decreased the total amount of multiple-resistant bacteria. Results demonstrated the need for identification to the species level.Offprint requests to: L. Hassani.     

Aeromonas-Acanthamoeba interaction and early shift to a viable but nonculturable state of Aeromonas by Acanthamoeba

Aims: To investigate the hypothesis that amoeba may comprise a significant environmental reservoir for Aeromonas, Acanthamoeba–Aeromonas interaction experiments were performed. Methods and Results: Acanthamoeba were grown in monoculture and co-cultures with three different species of Aeromonas. Survival, invasion and viable but nonculturable state experiments were performed. We showed that at a low initial bacterial cell density, growth of Aeromonas spp. was inhibited by Acanthamoeba castellanii, while A. castellanii growth was unaffected. In contrast, a high initial bacterial cell density, Aeromonas hydrophila AEW44 and Aeromonas veronii biovar sobria AEW104 suppressed the growth of A. castellanii. Fluorescent and phase-contrast microscopic observations of GFP tagged Aer. hydrophila AEW44 demonstrated that the bacterial cells aggregated on A. castellanii cells after 15 min of incubation and internalized. Aeromonas hydrophila AEW44 cells were found to be actively moving. Interestingly, Aer. hydrophila AEW44 cells shifted more rapidly to a viable but nonculturable form when co-cultured with A. castellanii than in monoculture. Conclusions: We demonstrated that Aeromonas spp. are able to interact with and to infect the protozoan A. castellanii under laboratory conditions. Significance and Impact of the Study: Free-living amoeba might play a role as reservoir for Aeromonas, and thus may increase the transmission of Aeromonas by acting as a vehicle.     

广东中山地区气单胞菌环境株的分离、鉴定及系统发生关系

从广东省中山市的池塘水样、底泥、健康鱼、肠道及稻田土样中用Aeromonas的选择培养基分离到10株气单胞菌。通过生理生化测试、16S rDNA序列测定、与气单胞菌典型菌株的16S rDNA序列进行比对和聚类分析,对它们进行了鉴定,并研究了它们之间的系统发生关系。结果显示该地区环境中气单胞菌的优势种除A. hydrophila（HG1组）外, 还有A. caviae（HG4组）、A. jandaei(HG9组)和A. veronii(HG10组),其中后两种是国内新记录。这是国内首次对环境中气单胞菌多样性进行研究。     

Gastrointestinal Colonization Rates for Human Clinical Isolates of <Emphasis Type="Italic">Aeromonas Veronii</Emphasis> Using a Mouse Model

A variety of environment-associated gastrointestinal infections have been associated with the Aeromonas group of bacteria which contain both non-virulent strains as well as virulent strains within a particular species. This study monitors the colonization rates of colon tissue in a mouse-streptomycin dose/response model involving isolates of Aeromonas veronii biovar sobria obtained from human clinical specimens. The ability to successfully colonize mouse colon tissues by the human clinical isolates was then compared with the rates achieved in a previous study of Aeromonas isolates obtained from environmental drinking water samples. Results suggest that strains of Aeromonas isolated from drinking water environmental samples contain pathogenic and virulence capabilities similar to those seen in Aeromonas veronii clinical isolates from human infections.     

Salmonella,Arizona, Shigella and Aeromonas isolated from the snail Achatina achatina in Nigeria

The Salmonella, Arizona, Shigella and Aeromonas contents of 39 snails (Achatina achatina) were investigated. Serotyping of the Salmonella isolates revealed the presence of S. manhattan, S. ndolo, S. reading, S. uppsala, and S. typhimurium. Six of 18 Shigella isolates were identified as Sh. sonnei while all the Aeromonas proved to be A. hydrophila. Fifty eight percent of these A. hydrophila isolates were enterotoxigenic, and their toxin was shown to be heat labile. All the A. hydrophila strains were both protease positive and haemolytic: 66.7% being both and haemolytic while 33.3% showed only haemolysis.     

Evaluation of Different Assay Systems for Identification of Environmental Aeromonas Strains

Important biochemical reactions in conventional tests were compared with counterpart reactions in two multiple test systems, API-20E (Analytab Products, Plainview, N.Y.) and Aeromonas hydrophila medium, to evaluate their accuracy for the identification of motile Aeromonas spp. isolated from fish. In a total of 49 Aeromonas spp. isolates and 10 A. hydrophila reference strains, false-negative or -positive reactions were detected in the Voges-Proskauer test, indole production, gelatinase activity, production of gas, fermentation of arabinose, and lysine decarboxylase reaction. A good correlation was found, among the three identification systems, for the fermentation of mannitol and inositol as well as for the arginine dihydrolase and ornithine decarboxylase tests. The failure of A. hydrophila medium in the detection of gas indicates that this medium is not entirely suitable for defining aerogenic or anaerogenic strains. From the results of the present study, we consider that of the identification method and taxonomic scheme to be adopted for environmental Aeromonas spp. must be standardized.     

Self-pelleting autoagglutination on mesophilic aeromonads belonging to serotype O: 11

Self-pelleting autoagglutination of Aeromonas hydrophila and A. sobria strains from serogroup O:11 depends on the presence of the extracellular layer peripheral to the cell membrane (S layer) and motility. To show this phenotype, strains should not fimbriate during growth.     

Enterotoxigenicity,Hemolytic Activity and Antibiotic Resistance of Aeromonas Spp. Isolated from Freshwater Prawn Marketed in Dhaka,Bangladesh

Aeromonas spp. were isolated from gills, swimmerets, eggs, stomachs and ventral muscles of freshwater prawns (Macrobrachium malcolmsonii) available in the local fish market of Dhaka, Bangladesh. The density of Aeromonas spp. on these different body parts of the prawn samples ranged from 1.1±0.2 × 10⁴ to 1.5 ± 0.16 × 10⁷ cfu per gram. The viable counts of aeromonads, fecal coliforms (FC) and Escherichia coli gradually increased in prawn samples when stored at 4 C. At ?20 C, the viable counts gradually decreased and became zero on the 12th day of storage. The isolation of A. sobria (56%) was more frequent than that of A. hydrophila (31%) and A. caviae (13%). In the rabbit ileal loop (RIL) model, fluid accumulation induced by live cultures and cell-free culture filtrates of 11 strains ranged from 0.5 to 1.5 and 0.5 to 1.7 ml/cm of gut, respectively. Of 11 enterotoxigenic strains, 7 were A. sobria and 4 were A. hydrophila. Enterotoxigenicity correlated with hemolytic activity on blood agar. All enterotoxigenic strains were uniformly sensitive to chloramphenicol and gentamicin and resistant to novobiocin and vancomycin. Isolation of enterotoxigenic and antibiotic-resistant Aeromonas from these prawn samples indicates possible public health problems for their handlers as well for raw prawn consumers.     

High frequency of hemolytic and cytotoxic activity in Aeromonas spp. isolated from clinical, food and environmental in Rio de Janeiro, Brazil

Molecular study of aerolysin and cytotonic enterotoxin genes by PCR and colony blot hybridization was performed in 117 strains of Aeromonas spp. isolated from different sources. Homogeneous distribution of these genes in A. hydrophila complex strains was observed. For A. caviae and A. sobria complex strains, aerolysin genes were more frequent than cytotonic enterotoxins genes. Of 64 A. caviae complex strains, only one (1.5%) amplified the 451 bp product for the aer gene, however, the same primers detected a 400 bp product in 50 (78%) strains. This product was sequenced and had two short regions with homology to several hemolysin genes. The genotype aer ⁺/aerA⁺/hly ⁺/ast ⁺/alt ⁺ was detected in six A. hydrophila strains from food and environmental source. The most common genotype found in A. hydrophila strains was hly ⁺ (85%) and aerA⁺ (78.7%), while in A. caviae complex strains was aerA⁺ (32.8%). All A. veronii complex sobria strains were aer ⁺/aerA⁺. All A. caviae and A. hydrophila were positive when tested with aer probe using the colony blot test. Thirty-seven percent of A. hydrophila and 53% of A. caviae tested were positive for ast probe. Eighty-nine percent of samples were cytotoxic in Vero cells. Our data demonstrated that Aeromonas spp. can harbor and express virulence genes and reinforce the potential of Aeromonas as a human pathogen.     

Probiotic Potential of Autochthonous Bacteria Isolated from the Gastrointestinal Tract of Four Freshwater Teleosts

In this study, a total of 121 bacterial strains were isolated from the gastrointestinal tract of four teleostean species, namely striped snakehead (Channa striatus), striped dwarf catfish (Mystus vittatus), orangefin labeo (Labeo calbasu) and mrigal carp (Cirrhinus mrigala), among which 8 isolates showed promising antibacterial activity against four potential fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas sobria and Pseudomonas fluorescens and were non-hemolytic. The isolates were further screened in response to fish bile tolerance and extracellular digestive enzyme activity. Two bacterial strains MVF1 and MVH7 showed highest tolerance and extracellular enzymes activities, and selected for further studies. Antagonistic activity of these two isolates was further confirmed by in vitro growth inhibition assay against four selected fish pathogens in liquid medium. Finally, these two bacterial strains MVF1 and MVH7 were selected as potential probiotic candidates and thus identification by partial 16S rRNA gene sequence analysis. The bacterial isolates MVF1 and MVH7 were identified as two strains of Bacillus sp.     

Virulence ofAeromonas species as assessed through mouse lethality studies

The relative virulence of 32Aeromonas isolates, primarily of clinical origin, were evaluated for mouse lethality by intraperitoneal inoculation of 10⁷ CFU into albino mice. Three categories could be distinguished on the basis of this assay, including a highly virulent group (80%–100% mortality), a low to moderate virulence category (20%–60% mortality), and strains that were completely avirulent. Of theA. sobria isolates tested, 82% fell into the highly virulent category (P<0.005), whereasA. hydrophila strains were intermediate in virulence potential, andA. caviae strains studied were avirulent. There was no apparent correlation between highly virulentAeromonas isolates and phenotypes associated with enterotoxigenicity, hemolytic activity, cytotoxin production, or serum resistance; this suggests that a cell surface property may be important in mouse pathogenicity. The results of these studies indicate that mouse lethality assays may be an appropriate model for the study of invasive disease clinically produced byA. sobria andA. hydrophila.