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.     

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.     

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.     

Differential susceptibility to human serum byAeromonas spp.

A total of 91 strains ofAeromonas (A. hydrophila, A. sobria, andA. caviae) of clinical origin were challenged in vitro against pooled human serum. A majority of the isolates ofA. hydrophila andA. sobria were resistant to the bactericidal activity of human serum, as opposed to the more serum-sensitiveA. caviae species. This difference in serum sensitivity may potentially explain the greater association of the former species with bacteremia and invasive disease.     

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     

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.     

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.     

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.     

Application of a colorimetric DNA-DNA hybridization method for identification ofAeromonas genomic species,isolated from diarrheal stools

The colorimetric DNA-DNA hybridization method for the identification of 18 strains ofAeromonas spp. isolated from human stools was used. Bacterial isolates were also examined by phenotypic characteristics. On the basis of biochemical tests 13 strains were included in phenogroupA. caviœ and 5 strains inA. sobria. Identification to the species level was obtained by colorimetric hybridization method. DNA-DNA similarity values showed that isolates ofA. caviœ group belong to hybridization group (HG) 4 whereas isolates ofA. sobria belong to HG 8/10. DNA relatedness results obtained by the colorimetric method showed good agreement with values detected by the spectrophotometric method. The background in the colorimetric method is lower than in the spectrophotometric one. Results of this study indicate the usefulness of the colorimetric DNA-DNA hybridization in microplates method for the identification ofAeromonas genomic species, isolated from human diarrheal stools.     

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.     

Aeromonas species in stabilization ponds in the arid region of Marrakesh,Morocco, and relation to fecal-pollution and climatic factors

During the period 12 July 1985 to 23 December 1987, water samples were collected in two-week intervals for estimates ofAeromonas species in a waste treatment system located in the arid region of Marrakech, Morocco. Fecal coliforms, temperature, and chemical oxygen demand were measured simultaneously withAeromonas species densities. Statistical methods were utilized to analyze the significance of average differences and temporal patterns ofAeromonas species numbers. Removal ofAeromonas in the whole system did not exceed 1.14 log.Aeromonas densities showed significantly higher resistance to the treatment process when compared with fecal coliforms; however, abundance of the two groups presented a similar seasonal change. The highest numbers occurred during the cold months, while the lowest appeared in the warm months. Statistical time-series analyses of the densities data showed the seasonal and cyclic distribution ofAeromonas in this treatment plant. These temporal changes were simultaneously observed in all the stations investigated and were negatively correlated with water temperature values. Aeromonas populations were dominated byA. caviae andA. hydrophila in the inlet samples. These two species were rapidly eliminated in the treatment plant. The temporal distribution ofA. caviae was similar to the change in densities ofAeromonas and fecal coliforms. The seasonal fluctuations of abundance ofAeromonas were probably related to this species, which dominated in the winter samples but dropped during the summer. Meanwhile,A. sobria dominated all the final effluent samples. This greater survival ofA. sobria and its known pathogenicity may limit the re-use of treated water for irrigation of fodder plants.     

Characteristics of 0/129-sensitive motile Aeromonas strains isolated from freshwater on starch-ampicillin agar

Motile Aeromonas hydrophila strains were recovered from several freshwater sources by spread-plating water samples on starch-ampicillin agar, originally described as a medium for recovering Aeromonas hydrophila quantitatively from foods. Starch-ampicillin agar was compared with membrane Aeromonas medium and Rimler-Shotts medium for selectivity for, and recovery of, Aeromonas strains from freshwater. Thirty-four Aeromonas strains thus isolated were identified to species level by their phenotypic characteristics, and the Mol% G+C of representative strains was determined. Although resistant to 10 g of the vibriostatic agent 0/129, all these strains showed sensitivity to 150 g 0/129, which brings into question the use of this agent for distinguishing aeromonads from vibrios. The ability of these strains to produce extracellular virulence factors was generally similar to that reported for environmental strains isolated by other methods from various geographical locations within and beyond Australia. Ten of the 20 A. sobria strains, but none of the A. hydrophila or A. caviae strains, produced enterotoxin as shown by the suckling mouse test. Haemolysin was produced by 9/10 of the enterotoxigenic A. sobria strains and 2/9 A. hydrophila strains. Hemagglutinating activity was detected in 5/20 A. sobria and 7/9 A. hydrophila strains, and was inhibited by fucose and mannose, but not by galactose. The characteristics of these strains were comparable with those of Aeromonas strains isolated from other freshwater environments apart from their sensitivity to 0/129. Send offprint requests to: M. Cahill.     

Attachment of mesophilic aeromonads to cultured mammalian cells

The interaction between mesophilic aeromonads and cultured mouse adrenal cells was examined. Preliminary experiments indicated that aeromonad attachment was dependent upon inoculum size, incubation time, and incubation temperature. Optimal attachment was observed after 30 min of incubation at 37°C with an inoculum size of 1×10⁷ CFU. Heat-killed and formalin-treated organisms did not attach to the cultured cell system. The attachment of aeromonads to the mammalian cell surface was confirmed by light and scanning electron microscopy. Aeromonad attachment correlated both with the presence of pili and the specific aeromonad species, but not with hydrophobicity or the ability to autoagglutinate. Piliated strains were more likely to show high or moderate attachment.Aeromonas sobria, A. hydrophila, andA. veronii showed a greater ability to bind adrenal cells than didA. caviae. Removal of the pili from twoA. sobria isolates markedly reduced their attachment. In contrast, oneA. hydrophila isolate was strongly adherent after the removal of pili. The hemagglutination patterns produced byA. sobria and the other aeromonad species were distinctly different, but potentially predictive of the ability of aeromonads to attach to cultured mouse adrenal cells. These studies indicate that multiple mechanisms are important for the attachment of mesophilic aeromonads to mammalian cells. This model may prove useful for studying the pathogenesis of aeromonad infections.     

Characterization of <Emphasis Type="Italic">Aeromonas</Emphasis> Virulence Using an Immunocompromised Mouse Model

An immunocompromised mouse model was used to characterize Aeromonas strains for their ability to cause opportunistic, extraintestinal infections. A total of 34 isolates of Aeromonas (A. hydrophila [n = 12]), A. veronii biotype sobria [n = 7], A. caviae [n = 4], A. enchelia [n = 4], A. allosaccharophila [n = 2], A. salmonicida (n = 4), and A. bestiarum [n = 1]) were introduced by intraperitoneal injection into immunocompetent or chemically compromised (using cyclophosphamide) mice. The ability of each isolate to persist in the liver and spleen tissue was monitored at 24 hours after exposure. A majority of A. hydrophila and A veronii v. sobria strains, but none of the isolates of other Aeromonas species, were capable of persistent colonization (<300 cells/mg spleen and liver tissue at 24 hours). The presence or absence of several putative virulence factors (cytotoxicity to HEp-2, lipase activity, elastase activity, and hemolysis) were determined for each isolate using in vitro tests. There were no correlations between the presence or absence of biochemical test results for putative virulence factors and persistence of the isolate in spleen and liver tissue at 24 hours.     

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.     

Distribution and variation of bacitracin synthetase gene sequences in laboratory stock strains of Bacillus licheniformis

Distribution and variation of bacitracin synthetase gene (bac) sequences in 22 laboratory stock strains of Bacillus licheniformis were studied by Southern hybridization of bac gene probes from B. licheniformis ATCC 10716 to genomic PstI or HindIII restriction fragments. Eleven strains gave hybridization signals. These hybridization patterns were classed into two types. Eight strains showed similar patterns to that of ATCC 10716 and all, except one, produced bacitracin. The three strains showed fairly different hybridization patterns from that of ATCC 10716, and one (ATCC 33632) of them produced bacitracin. None of the remaining 11 strains, including ATCC 14580 (type strain), gave any hybridization signals. All strains carrying bac gene sequences were erythromycin resistant. With one exception, all strains without bac gene sequences were erythromycin sensitive. These results show that B. licheniformis strains are divided into two groups with respect to presence of bac gene sequences and erythromycin resistance. Received: 5 September 2001 / Accepted: 19 October 2001     

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.