Purification and characterization of an Aeromonas hydrophila hemolysin.

A hemolysin produced by Aeromonas hydrophila CA-11, isolated from an environmental source, was purified by sulfopropyl-Sephadex C-25 chromatography at pH 5.0. This hemolysin caused fluid accumulation in infant mouse intestines and rabbit intestinal loops and killed Vero cells, as did the hemolysin produced by strain AH-1, isolated from a diarrheal case. In polyacrylamide gel electrophoreses at pHs 4.0 and 9.4 and in thin-layer isoelectric focusing, CA-11 hemolysin migrated as a single band to a position different from that of AH-1 hemolysin. Immunodiffusion tests indicated that CA-11 hemolysin was immunologically related to AH-1 hemolysin but possessed unique antigenic determinants. Neutralization tests with antihemolysin sera also demonstrated immunological cross-reactivity between AH-1 and CA-11 hemolysins. These results apparently indicate that the hemolysins produced by the two strains of A. hydrophila are immunologically and physicochemically different from each other.     

Pathogenicity factors and virulence for rainbow trout (Salmo gairdneri) of motile Aeromonas spp. isolated from a river

Ninety-seven motile Aeromonas strains were isolated over a period of a year from samples of water and sediment collected at different sites along a river. Strains were regularly recovered from all samples, regardless of the source of isolation or seasonal conditions. Isolates were biochemically characterized by the API 20NE system (Analytab Products, Plainview, N.Y.) and classified as Aeromonas hydrophila (74 strains), Aeromonas sobria (11 strains), and Aeromonas caviae (12 strains). Despite the high level of homogeneity observed in their biochemical patterns, they displayed different degrees of virulence for fish; 72.02% of A. hydrophila isolates and 63% of A. sobria isolates were virulent for fish by intramuscular challenge, but lower frequencies of virulence were observed when intraperitoneal injections were used. All A. caviae strains proved to be avirulent. Caseinases, hemolysins, and Vero cytotoxins were produced by 100, 91, and 94.59%, respectively, of A. hydrophila strains and with lower frequencies and lower caseinase activities by A. sobria isolates. No correlation was found between these activities and the degree of virulence of the strains for fish. Most hydrophobic strains seem to be concentrated in A. caviae, A. sobria, and avirulent A. hydrophila groups. Known virulence markers commonly associated with virulent strains (acriflavine negative and self-pelleting negative and precipitation after boiling positive phenotypes) had a low representation in the total of strains studied and were not associated with virulence.     

Demonstration of cholera toxin-related factor in cultures of Aeromonas species by enzyme-linked immunosorbent assay.

The production of toxins by Aeromonas species was examined by the suckling mouse test, the hemolysin test, and the enzyme-linked immunosorbent assay with anticholera enterotoxin. A factor that was immunologically related to cholera enterotoxin was produced by 5 of 14 strains of Aeromonas hydrophila and 4 of 15 strains of Aeromonas sobria. Analysis by these assays and by a test for heat stability suggested that the factor differed from hemolysin and from toxin that was active in the suckling mouse test.     

Activities of Aeromonas hydrophila hemolysins and their interaction with erythrocyte membranes.

The activity of hemolysin produced by Aeromonas hydrophila CA-11, isolated from an environmental source, was more sensitive to temperature than that of hemolysin produced by strain AH-1, isolated from a diarrheal case. CA-11 hemolysin failed to elicit hemolysis below 10 degrees C. Immunoblotting analyses showed that both hemolysins formed into oligomers in rabbit erythrocyte membrane even when no hemolysis occurred. suggesting that the binding and the subsequent oligomerization are temperature independent. Sodium salicylate inhibited lysis of rabbit erythrocytes by both hemolysins, but selected monosaccharides and oligosaccharides did not. Thin-layer immunostaining indicated that both hemolysins bound to phosphoglycerides with net negative charge but weakly to the ones with no net negative charge. Neither sphingomyelin nor lysophosphoglyceride reacted with the hemolysins, whereas the hemolysins bound to free fatty acids. These results suggest that the binding of either hemolysin to the membrane component, probably phospholipid, requires both negative charge of the polar head group and suitable hydrophobicity of the nonpolar tails.     

Detection and characterization of the hemolysin genes in Aeromonas hydrophila and Aeromonas sobria by multiplex PCR

A multiplex PCR assay was designed to amplify the Aeromonas hydrophila and A. veronii bv. sobria hemolysin and aerolysin genes. The assay was evaluated by using 121 clinical isolates and 7 reference strains of Aeromonas spp., and these were divided into five genotypes on the basis of the results of the multiplex PCR. The five genotypes were characterized as type 1 for those carrying the ahh1 gene only (36% of isolates), type 2 for those carrying the asa1 gene only (8.5% of isolates), type 3 for those carrying both the ahh1 and the asa1 genes (4% of isolates), type 4 for those carrying the ahh1 gene and the A. hydrophila aerA (aerolysin) gene (37.5% of isolates), and type 5 for those in which no hemolysin genes were detected (14% of isolates). The most common single hemolysin gene carried among all the Aeromonas isolates examined was ahh1, with 99 of 128 (77%) of isolates testing positive for this gene either alone or in combination with other hemolysin genes. Phenotypic expression of toxins was evaluated in a Vero cell culture cytotoxicity assay. These results indicated that there is a statistically significant correlation between the cytotoxin titers and the hemolysin genotype. Isolates belonging to genotype 4 (carrying both the ahh1 gene and the aerolysin and hemolysin aerA genes) expressed higher cytotoxin titers than isolates of the other genotypes (P < 0.001). These isolates were more cytotoxic in cell culture and may have greater clinical significance.     

Production of "Asao toxin" by Aeromonas strains isolated from feces and drinking water

Cultures of Aeromonas species were tested for production of a toxin recently purified by Asao et al. (T. Asao, Y. Kinoshita, S. Kozaki, T. Uemura, and G. Sukaguchi, Infect. Immun. 46:122-127, 1984) and described as a hemolysin with enterotoxic and cytotoxic activity. The toxin was produced by only 63% of Aeromonas sobria strains and by 93% of Aeromonas hydrophila strains. Also, 54% of A. hydrophila strains produced another cytotoxic entity.     

Identification and characterization of the Aeromonas sobria hemolysin glycoprotein receptor on intestine 407 cells.

Aeromonas sobria hemolysin is important in the pathogenesis of diarrhoea caused by this enteropathogenic bacterium. By immunoprecipitation analysis using hemolysin and anti-hemolysin antibody, a 66 kDa protein (p66) was identified as a receptor for A. sobria hemolysin on Intestine 407 cells. Treatment of p66 with N-glycosidase F reduced the apparent sized of p66 to 60 kDa on SDS-polyacrylamide gels. p66, released from Intestine 407 cells following incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, bound A. sobria hemolysin. Thus treatment of Intestine 407 cells with PI-PLC resulted in the remarkable decrease of the sensitivity to A. sobria hemolysin. These results are consistent with the hypothesis that p66, the binding protein for A. sobria hemolysin, is a glycosylphosphatidylinositol-anchored glycoprotein expressed on the surface of Intestine 407 cells and probably plays a role as a receptor for A. sobria hemolysin on the intestinal cells.     

Phenotypic characterization and DNA relatedness in human fecal isolates of Aeromonas spp.

Phenotypic characteristics were used to identify 189 Aeromonas strains isolated from human feces. One hundred forty-two of these strains were placed in 11 DNA hybridization groups, and the genetic and phenotypic data were compared. According to the criteria of Popoff, 66% of the strains were identified as Aeromonas caviae, 18% were identified as A. sobria, and 16% were identified as A. hydrophila. Some biochemical characteristics differed from the criteria of Popoff; 19 of 40 (48%) of tested strains were encapsulated, 42 of 124 (34%) of A. caviae strains were nonmotile, and all A. sobria strains were resistant to KCN. Gas production from D-glucose was temperature dependent; 11 of 64 (17%) A. hydrophila and A. sobria strains produced gas only at 22 degrees C. Of 142 Aeromonas strains, 57% belonged to hybridization group 4, 25% belonged to group 8, 11% belonged to group 1, 4% belonged to group 5A, 2% belonged to group 3, and 1% belonged to group 2. Of 26 strains phenotypically identified as A. hydrophila, 8 (31%) were in hybridization group 8, which contains strains of the new species A. veronii. It therefore appears that our ability to identify Aeromonas strains phenotypically is not sufficiently specific. Either additional definitive biochemical markers must be found or phenotypic identification, at least for some Aeromonas groups, must be regarded as only presumptive.     

Haemolysin occurrence among Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria strains isolated from different aquatic ecosystems

A total of 909 Aeromonas spp. isolates from different aquatic ecosystems were tested for haemolysin production by both sheep and horse blood agar-plate assays and by rabbit erythrocytes in broth assay. A comparison of these different methods was undertaken in order to appreciate their capacity to evaluate the haemolytic activity of Aeromonas spp. isolated from aquatic ecosystems. The haemolytic activity was associated particularly with A. hydrophila and A. sobria (about 95% of strains), whereas A. caviae did not produce haemolysin (about 95% of strains). A method suitable for use in routine diagnostic microbiology laboratories is proposed for quantifying both groups of A. hydrophila/A. sobria and A. caviae in environmental water.     

Different clinical characteristics among Aeromonas hydrophila, Aeromonas veronii biovar sobria and Aeromonas caviae monomicrobial bacteremia

This study aimed to compare the clinical presentations of Aeromonas hydrophila, A. veronii biovar sobria and A. caviae monomicrobial bacteremia by a retrospective method at three hospitals in Taiwan during an 8-yr period. There were 87 patients with A. hydrophila bacteremia, 45 with A. veronii biovar sobria bacteremia and 22 with A. caviae bacteremia. Compared with A. hydrophila and A. veronii biovar sobria bacteremia, A. caviae bacteremia was more healthcare-associated (45 vs 30 and 16%; P = 0.031). The patients with A. caviae bacteremias were less likely to have liver cirrhosis (27 vs 62 and 64%; P = 0.007) and severe complications such as shock (9 vs 40 and 47%; P = 0.009) and thrombocytopenia (45 vs 67 and 87%; P = 0.002). The APACHE II score was the most important risk factor of Aeromonas bacteremia-associated mortalities. The APACHE II scores of A. caviae bacteremias were lower than A. hydrophila bacteremia and A. veronii biovar sobria bacteremia (7 vs 14 and 16 points; P = 0.002). In conclusion, the clinical presentation of A. caviae bacteremia was much different from A. hydrophila and A. veronii biovar sobria bacteremia. The severity and mortality of A. caviae bacteremia were lower than A. hydrophila or A. veronii biovar sobria bacteremia.     

Partial characterisation of a soluble haemagglutinin from human diarrhoeal isolates of Aeromonas

A soluble haemagglutinin has been identified in cell-free culture supernates of human diarrhoeal isolates of Aeromonas sobria, A. hydrophila and A. caviae. It was oligomeric; a major peak of haemagglutinating activity had an apparent mol. wt of 780,000 but there was haemagglutinating activity throughout the mol. wt range less than 40,000- greater than 10(6). Human group O, A and B, horse, rabbit, chicken and rat erythrocytes, but not those of sheep and cow, were agglutinated by the soluble haemagglutinin, in contrast to the cell-bound agglutinin. Agglutination was inhibited by fetuin, a complex glycoprotein, but not by simple sugars. The haemagglutinating activity was not affected by 0.5 M NaCl, dithiothreitol or the presence or absence of Ca++. It was unrelated to the haemolytic, enterotoxigenic and proteolytic activities present in cell-free extracts of A. sobria. All A. sobria, 73% of A. hydrophila and 68% of A. caviae strains tested produced this soluble haemagglutinin. A. caviae does not appear to be an enteric pathogen, therefore this soluble haemagglutinin alone is unlikely to be a virulence factor in Aeromonas spp.     

Aeromonas spp. and their association with human diarrheal disease.

Between January 1988 and December 1989 Aeromonas species were isolated from 45 (1.8%) of 2,480 patients with acute gastroenteritis. No other bacterial enteric pathogens were found in any of these 45 patients. Of the 45 Aeromonas isolates, 35 strains (77.8%) were Aeromonas hydrophila, 7 (15.5%) were Aeromonas sobria, and 3 (6.7%) were Aeromonas caviae. Most of the patients were under 5 years of age. No bacterial enteric pathogens, including Aeromonas species, were isolated from 512 age- and sex-matched control subjects. Examination of the Aeromonas isolates for exotoxin production (enterotoxin and hemolysin) indicated that all strains, irrespective of species, were enterotoxin positive (rabbit ileal loop model) and hemolysin positive (rabbit erythrocyte model). These results suggest that Aeromonas species are potential enteric pathogens in our geographical region.     

Correlation of the suicide phenomenon in Aeromonas species with virulence and enteropathogenicity.

Certain strains of mesophilic aeromonads (Aeromonas hydrophila, A. sorbria, and A. caviae), when grown in broth containing 0.5% glucose, undergo growth inhibition concomitant with acetate accumulation. Because these strains are nonviable after 24 h, this phenomenon is termed suicide. We investigated suicidal strains of Aeromonas species as a means of understanding animal virulence and enteropathogenicity. To assess virulence, batches of five white mice were inoculated intraperitoneally with 10(7) cells (washed) of suicidal and nonsuicidal strains of A. hydrophila and A. sobria and suicidal strains of A. caviae. The three nonsuicidal strains of A. sobria tested showed lethality as early as 12 h and were uniformly fatal within 36 h postinoculation. After 36 h, the three suicidal strains killed only 1 of 15 mice inoculated. Four A. hydrophila strains tested which showed the suicide phenomenon at 37 degrees C were variably lethal (40 to 100%). None of three suicidal strains of A. caviae were lethal. Enteropathogenicity was studied by orally inoculating three white mice each with the same Aeromonas strains (10(8) cells, in skim milk) and assessing diarrhea and intestinal fluid accumulation. Diarrhea and fluid accumulation were present in all mice inoculated with two nonsuicidal strains of A. sobria and in 4 of 12 mice given four suicidal strains of A. hydrophila. Two suicidal strains each of A. sorbria and A. caviae failed to elicit any gastrointestinal disturbances. These data suggest that the suicide phenomenon may explain strain-specific (A. sobria and A. hydrophila) and species-specific (A. caviae) virulence and enteropathogenicity.     

Association of Aeromonas sobria with human infection.

Fifteen Aeromonas isolates from various human infections and nine isolates from polluted water were identified as either Aeromonas hydrophila or Aeromonas sobria and examined for cytotoxigenicity, enterotoxigenicity, adherence to epithelial cells, and other virulence-associated factors, including proteases, lipases, elastases, and hemolysins. Two groups of organisms (I and II) were distinguishable based on differences in median lethal doses in mice and cytotoxicity for Y-1 adrenal cells. Group I clinical and environmental strains had median lethal doses of less than 10(7) colony-forming units, were cytotoxic, frequently possessed several virulence-associated factors, and had lysine decarboxylase-positive or Voges-Proskauer-positive phenotypes or both. Piliation of Aeromonas was associated strongly with ability to adhere to human buccal cells, and these characteristics were associated with group I strains. Group II clinical and environmental strains had median lethal doses of greater than or equal to 10(7) colony-forming units, were not cytotoxic, and usually were lysine decarboxylase negative or Voges-Proskauer negative or both. Clinical strains in group II exhibited enterotoxigenicity, which was not detected in group II environmental strains. A sobria was more frequently associated with human infections; 13 of the 15 clinical strains were A. sobria, and 2 were A. hydrophila. On the other hand, the majority of the environmental strains (seven of nine) were A. hydrophila.     

Growth of Aeromonas species on enteric agars.

The efficacy of eight routine enteric agars for supporting the growth of 32 strains of Aeromonas spp. (17 A. hydrophila strains, 8 A. sobria strains, and 7 A. caviae strains) was investigated. The plating efficiency of Aeromonas spp. on these media varied greatly (range, 0 to 100%), as did their colony size when compared with that on noninhibitory medium (5% sheep blood agar). Plating efficiency on seven of these eight media appeared to be strain- and not species dependent. Overall, eosin-methylene blue and Hektoen enteric agars showed low plating efficiencies for A. hydrophila, whereas both A. sobria and A. caviae were severely inhibited on brilliant green agar. When all these species are considered collectively, deoxycholate, MacConkey, and xylose lysine deoxycholate appeared to be the most satisfactory routine agars for Aeromonas spp. recovery when used in conjunction with blood agar.     

The Aeromonas genus]

Aeromonas hydrophila manifested itself since its discovery in 1891 as a pathogen of cold-blooded and warm-blooded animals and man. Aeromonads cause intestinal and non-intestinal disease. The genus Aeromonas comprises: A. hydrophila, A. sorbria, A. caviae, A. veronii, A. schubertii, i.e. mesophilie species and as to psychrophilie immobile species. A. salmonicida and A. media. For warm-blooded animals and man mesophilie motile species are important as pathogens. A. veronii is an ornithine-decarboxylase positive species, A. schubertii is mannitol-negative, A. caviae is non-haemolytic and VP-negative. It is difficult to differentiate. A. hydrophila with aerogenic and anaerogenic strains from A. sobria. Several practical differential diagnostic tests were suggested by Janda et al. and Joseph et al.: hydrolysis of esculine, KCN, arabinose and salicin are usually positive in A. hydrophila, in A. sobria usually negative. Existing species of mesophilie aeromonads, however, do not correspond to some strains which are found. Therefore Arduino et al. divided their aeromonads into DNA-hybridization groups: for A. hydrophila there were 5, for A. caviae 2 and for A. sobria 1 hybridization group. Biochemisal characteristics corresponded to the hybridization groups. For isolation of aeromonads from faeces selective media with ampicillin must be used and possibly enrichment in alkaline peptone water. Evidence of pathogenity factors is similar as in E. coli,: detection of adhesins and enterotoxin or cytotoxin by means of tests commonly used in cholera and E. coli. The types of adhesins are differentiated by means of fucose- galactose- and mannose resistant haemagglutination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship of toxin production to species in the genus Aeromonas

Ninety-five strains of Aeromonas were divided into three species--A. sobria, A. hydrophila and A. caviae--on the basis of results in 13 biochemical tests. The minimum number of tests necessary to distinguish these species was determined. Culture filtrate of the strains were tested for cytotoxin and cytotonin, haemolysin and protease. One filtrate with high-titre cytotoxin, haemolysin and protease activities was subjected to gel filtration on Sephadex G75 and isoelectric focussing. Of the five cell lines tested, Vero cells were most sensitive to the cytotoxin; no reproducible cytotonic effects were observed. The haemolysin effect appeared to be equivalent to cytotoxin. At least two distinct protease activities were found that might be responsible for the cytotonic effects described. Cytotoxin production was species related; it was present in A. sobria and A. hydrophila but not in A. caviae.     

Nucleotide sequences and characterization of haemolysin genes from Aeromonas hydrophila and Aeromonas sobria.

Extracellular haemolysin is thought to be one of the important virulence factors in Aeromonas infection. Two extracellular haemolysin genes (AHH3 and AHH4) from Aeromonas hydrophila strain 28SA, one (AHH5) from A. hydrophila strain AH-1 and one (ASA1) from Aeromonas sobria strain 33 were cloned into cosmid and plasmid vector DNA in Escherichia coli. The nucleotide sequences of the open reading frames of AHH3 and AHH4 are both 1476 basepairs (bp), whereas AHH5 and ASA1 are 1455 and 1467 bp in length, respectively. The deduced amino acid sequences of AHH3, AHH4, AHH5 and the previously reported aerolysin from A. hydrophila showed a significant degree of sequence homology of over 90% each. The amino acid identity of the ASA1 haemolysin and those from A. hydrophila and Aeromonas trota aerolysins ranged from 58-68%. From DNA hybridization analysis using our cloned haemolysin genes as probes, we found that the AHH5 and ASA1 DNA probes hybridized with about 31 and 75% strains of motile Aeromonas species, respectively. The activity of haemolysins of cloned genes were different in medium agar containing various erythrocytes.     

Purification and characterization of Aeromonas sobria Ae24 pili: a possible new colonization factor.

Pili of Aeromonas sobria Ae24 were purified and characterized. The molecular mass of the pilin was estimated to be about 19 kDa by SDS-PAGE. The Ae24 pili were electrophoretically distinguishable from previously reported Aeromonas hydrophila Ae6 W pili and A. sobria Ae1 pili, although all three had indistinguishable morphology and shared a high degree of homology in the N-terminal amino acid sequences. Strain Ae24 and its purified pili adhered to rabbit intestine and agglutinated human and rabbit erythrocytes. Hemagglutination was inhibited by D-galactose and D-mannose, but not by L-fucose. Organisms pretreated with Fab fraction of the antipilus antibody failed to adhere to the intestine. Organisms did not adhere to intestine pretreated with the purified pili. These findings suggest that the pili are a colonization factor of A. sobria Ae24 for the rabbit intestine, and that the receptor is galactose- and mannose-containing structure.     

Purification and some properties of Aeromonas hydrophila hemolysin

A hemolysin produced by a strain of Aeromonas hydrophila isolated from a patient with diarrhea was purified by acid precipitation and quarternary aminoethyl-Sephadex chromatography. The molecular weight of the hemolysin was estimated at 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at 48,000 by Sephadex G-100 gel filtration. In polyacrylamide gel electrophoresis at pH 4.0 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the hemolysin migrated as a single band, whereas electrophoresis at pH 9.4 and thin-layer isoelectric focusing demonstrated multiple bands. The results may indicate charge isomers of the hemolysin. The purified hemolysin had a hemolytic activity of 134 hemolytic units per microgram of protein on rabbit erythrocytes. It caused fluid accumulation in infant mouse intestines and rabbit ligated ileal loops. Purified hemolysin also elicited cytotoxicity to Vero cells and lethal toxicity to mice. All these biological activities were lost on heating for 5 min at 56 degrees C. These findings support the notion that A. hydrophila hemolysin is a cytotoxic enterotoxin.