Typing of Aeromonas strains by DNA restriction endonuclease analysis and polyacrylamide gel electrophoresis of cell envelopes.

The outer membrane protein (OMP) composition (OMP typing) of 46 fecal Aeromonas strains from hybridization groups (HGs) 1 (A. hydrophila; n = 10), 4 (A. caviae; n = 16), and 8 (A. veronii; n = 20) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a phenotypic typing method. Almost every isolate of HG-1 and HG-8 had a unique OMP profile, in contrast to isolates of HG-4, which were separated into five different OMP types. It was possible to recognize HGs 1, 4, and 8 by OMP profiles. Twenty-three Aeromonas strains from HGs 1 (n = 5), 4 (n = 10), and 8 (n = 8) were tested by whole-cell DNA restriction endonuclease analysis (REA) as a genetic typing method. All strains tested by REA (with SmaI) had different DNA digestion patterns. Although additional DNA-rRNA hybridization analyses with SmaI and 16S and 23S rRNAs from Escherichia coli showed a reduction in the number of restriction bands to 8 to 13 hybridized fragments, the discriminative value was less when compared with that obtained by REA. The individual differences found by REA were used to analyze whether patients remained colonized by the same Aeromonas strain. Of 11 patients with diarrhea, 2 had a different isolate on repeat culture. In addition, one of nine tested fecal samples contained two Aeromonas isolates with different REA patterns. These results indicate that during diarrheal disease the intestinal tract may be colonized simultaneously with different Aeromonas isolates.     

Microbiologic and clinical evidence supporting the role of Aeromonas caviae as a pediatric enteric pathogen.

Aeromonas caviae was recovered as the sole potential enteric pathogen from the stools of 14 of 17 symptomatic children (10 younger than 1 year of age) while Aeromonas hydrophila, Aeromonas sobria, and Plesiomonas shigelloides were isolated once each. The infants from whom A. caviae was isolated all presented with a watery diarrhea lasting 1 to 3 weeks. None of these infants was breast-fed, and all had a stool pH of greater than 7.5. All of the A. caviae isolates, including a reference strain (ATCC 15468), adhered to HEp-2 cells, and preliminary data showed that they produced a cytotoxin as well. Because A. caviae can survive at an elevated pH, as found in the gastrointestinal tract of formula-fed infants, and because of the adherence and cytotoxin production capabilities of the species, it should be regarded as an enteric pathogen in pediatric patients and most probably among adults as well.     

Susceptibilities of motile Aeromonas sp. to antimicrobial agents

Resistance to antimicrobial agents of 106 isolates of motile Aeromonas sp. was characterized. The results indicated that in vitro susceptibilities among the three species of the motile Aeromonas sp. were similar, and only the distribution of susceptibility to cephalothin was different. The percentage of resistance of A. sobria strains was lower than the percentage of the resistant strains of A. hydrophila and A. caviae. All of the isolates were susceptible to kanamycin, nalidixic acid, tobramycin, amikacin, netilmicin, cefuroxime, ceftriaxone, cefoperazone and cefotaxime. Three of the tested strains (two A. hydrophila and one A. caviae) transferred resistance plasmids to the Aeromonas hydrophila recipient.     

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.     

食品与环境中豚鼠气单胞菌系统发育分析以及毒力和耐药性研究

目的食品与环境中49株豚鼠气单胞菌菌种鉴定、毒力与耐药性检测。方法VITEK、API 20NE生化鉴定,gyrB、rpoD系统发育分析;PCR检测act、alt、ast、lip、ahp、aerA、hlyA、ompA1、fla基因;AST-GN16药敏试验。结果生化鉴定为豚鼠/嗜水气单胞菌株54株,经系统发育分析豚鼠气单胞菌49株,嗜水气单胞菌4株,中国台湾省气单胞菌1株。毒力基因alt、lip、ompA1、fla、act、aerA、hlyA检出率依次为100.00%、100.00%、79.59%、14.29%、2.04%、2.04%、2.04%,未检出ast、ahp;存在4种毒力基因组合,优势组合为alt/lip/ompA1(32/49)。豚鼠气单胞菌氨苄西林、头孢唑林、头孢西丁、阿莫西林/克拉维酸、头孢曲松、复方新诺明、安曲南耐药率依次为79.59%、14.29%、10.20%、6.12%、4.08%、4.08%、2.04%,哌拉西林/他唑巴坦、亚胺培南、阿米卡星、庆大霉素、左氧氟沙星、替加环素、呋喃妥英均敏感;2株多重耐药菌株。结论gyrB、rpoD可有效鉴定豚鼠/嗜水气单胞菌,rpoD可区分近亲缘关系豚鼠与中国台湾省气单胞菌;所有豚鼠气单胞菌均携带2种以上毒力基因,1株环境源菌携带7种毒力基因;青霉素类普遍耐药,产生β-内酰胺酶是最主要耐药机制;未发现碳青霉烯类、氨基糖苷类、氟喹诺酮类、四环素类、呋喃类耐药菌株;食品和生活饮用水存在多重耐药菌。     

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.     

Aeromonas species exhibit aggregative adherence to HEp-2 cells.

Clinical and environmental isolates of Aeromonas species (five A. hydrophila isolates, three A. caviae isolates, and two A. sobria isolates) were tested for their adherence to HEp-2 cells. Clinical isolates of A. hydrophila and A. sobria exhibited aggregative adherence similar to that presented by enteroadherent-aggregative Escherichia coli. Bacterial aggregates adhered to cells with a typical "stacked-brick" appearance. In contrast, A. caviae strains showed a diffuse adherence pattern.     

Differentiation of motile and mesophilic Aeromonas strains into species by testing for a CAMP-like factor.

Motile and mesophilic Aeromonas strains can presumptively be differentiated into species in 18 to 24 h by testing the isolates for the production of a CAMP-like factor. Aeromonas hydrophila strains were positive either aerobically or anaerobically, Aeromonas sobria strains were positive only aerobically, and Aeromonas caviae strains were always negative.     

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 haemolysis and its correlation with enterotoxicity in Aeromonas spp.

A total of 147 clinical and environmental isolates of Aeromonas that included 14 A. hydrophila, 60 A. sobria and 73 A. caviae strains was tested for haemolysin production and its correlation with enterotoxicity; 108 isolates produced beta-haemolysis. For A. hydrophila and A. sobria, titres of haemolysin were 16-128 HU/ml and for A. caviae, 16-64 HU/ml. In the ileal loop test, 82 (55.8%) strains of Aeromonas spp. produced enterotoxin. Of the beta-haemolytic strains, 72.7% of A. hydrophila, 58.6% of A. sobria and 68.6% of A. caviae isolates caused fluid accumulation in rabbit ileal loops. One strain each of alpha-haemolytic A. sobria and A. caviae, one of non-haemolytic A. sobria and nine of non-haemolytic A. caviae also caused a secretory response. The beta-haemolytic strains caused significantly more (p < 0.05) fluid accumulation than the alpha- and non-haemolytic isolates regardless of their species designation. The remaining 65 (44.2%) isolates belonging to the three species included alpha-, beta- and non-haemolytic strains: they failed to cause fluid accumulation in the initial experiments but did so after one to three consecutive passages through rabbit ileal loops. Two alpha- and 13 non-haemolytic strains switched to production of beta-haemolysis when they showed positive ileal loop reactions. However, on repeated subcultures or on storage in the laboratory, all of them reverted to their original haemolytic character and no longer produced enterotoxic activity.     

Biochemical identification of Aeromonas genospecies isolated from humans

One hundred phenotypic characteristics were determined for 138 clinical and environmental Aeromonas strains. Cluster analysis revealed three major phenons equivalent to the A. hydrophila, A. caviae, and A. sobria groups, each of which contained more than one genospecies and more than one named species. An excellent correlation was found between phenotypic identification and classification based on DNA relatedness. DNA hybridization groups within each of the phenotypic groups were also separable by using a few biochemical characteristics. Key tests were production of acid from or growth on D-sorbitol (which separated DNA hybridization group 3 from groups 1 and 2 within the A. hydrophila phenogroup), growth on citrate (which essentially separated DNA hybridization group 4 from groups 5A and 5B within the A. caviae phenogroup), and growth on DL-lactate (which separated DNA hybridization group 1 from groups 2 and 3 within the A. hydrophila phenogroup as well as group 5A from groups 4 and 5B within the A. caviae phenogroup). All except one strain in the A. sobria phenogroup belonged to DNA hybridization group 8. DNA hybridization groups were not equally distributed among clinical and environmental isolates, suggesting that strains of certain DNA hybridization groups might be less virulent than others.     

Phenotypic characteristics of Aeromonas species isolated from adult humans.

The phenotypic characteristics of 89 Aeromonas strains, most of which had been isolated from feces, were examined. Eighty-two percent of the isolates could be placed into one of four groups on the basis of five tests. The relationship of these groups to the three motile species of Aeromonas (Aeromonas caviae, A. hydrophila, and A. sobria) that have been isolated from humans is unclear. Because the means for identification of Aeromonas strains to the species level appear to be imprecise and because the role of each of these species in human diarrhea is unclear at this time, we recommend that identification of enteric Aeromonas isolates to the species level not be done routinely.     

Type III secretion system genes in clinical Aeromonas isolates

We have identified the genes ascF and ascG, which encode components of a putative type III secretion system (TTSS) in AEROMONAS: We investigated the distribution of these and other TTSS genes in 84 clinical isolates and found hybridizing sequences in 50% of the strains, with a higher prevalence in Aeromonas hydrophila and Aeromonas veronii than in Aeromonas caviae.     

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.     

Assessment of a chemiluminescent universal probe for taxonomical and epidemiological investigations of Aeromonas sp isolates.

AIMS--To assess a chemiluminescent universal probe for taxonomical and epidemiological investigations of Aeromonas sp isolates. METHODS--Total DNA was extracted from 69 well characterised Aeromonas sp strains and digested with the restriction endonucleases Sma I or Pst I. Following electrophoresis, the resulting fragments were transferred to a nylon membrane where they were hybridised to a commercially available universal probe of 16S + 23S rRNA. The banding patterns (ribotypes) were made visible by enhanced chemiluminescence. RESULTS--Both restriction endonucleases produced heterogeneous ribotypes so that no allocation could be made to any of the control genospecies tested. For A hydrophila and A caviae, however, groups of strains (mostly from the same patient) could be identified by indistinguishable banding patterns. A relatively high proportion (36%) of A sobria strains were non-typable. CONCLUSIONS--Although this universal chemiluminescent probe is user friendly, it is unsuitable for taxonomical investigations of Aeromonas sp. It is useful in epidemiological studies of A hydrophila and A caviae, but is of less value for A sobria.     

Methods for the detection of haemagglutinins in Aeromonas

When grown in specified conditions and tested by a rocked-tile method, 40 of 41 isolates of two species of Aeromonas formed simultaneously at least two haemagglutinins among which were: (i) a mannose-sensitive haemagglutinins with strongest activity for guinea-pig or fowl red cells, formed by all of 31 isolates of A. hydrophila and 9 of 10 isolates of A. punctata ss. caviae; (ii) a haemagglutinin, sensitive to L-fucose or D-mannose, that reacted with human red cells and which was formed by all 41 isolates; and (iii) a mannose-resistant 'tanned red cell' haemagglutinin formed by 29 isolates of A. hydrophila and one isolate of A. punctata ss. caviae. Results emphasise that for the fullest possible identification of haemagglutinins produced by Aeromonas spp., strains should be cultured in a variety of conditions and tested with a wide range of red-cell species.     

Toxin production by Aeromonas spp. from different sources

One hundred and eleven isolates of Aeromonas from water and from human sources were identified to species level and tested for the production of cytotoxin. These results were correlated with the source of each isolate and, for those from human faeces, with the clinical history of diarrhoea. A. caviae predominated in water, comprising 16 of 32 isolates; only one isolate from water was A. sobria. In human faecal samples 21 of 76 isolates were A. sobria; this was a significant difference. Cytotoxin producing strains were significantly more common in patients with no known cause for their gut symptoms. It is concluded that gastro-enteritis caused by Aeromonas is related to species and to production of cytotoxin.     

Biochemical characteristics, enterotoxigenicity and susceptibility to antimicrobial agents of clinical isolates of Aeromonas species encountered in the western region of Saudi Arabia

The biochemical characteristics, enterotoxigenicity and susceptibility to antibiotics are reported for 22 strains of Aeromonas species isolated from clinical specimens in the Western Region of Saudi Arabia. Aeromonas caviae was the species most frequently observed; a high proportion of these strains fermented lactose, whereas lactose fermentation was not observed in strains of A. hydrophila and A. sobria. Enterotoxigenicity, as judged by cytotoxicity in tissue culture was observed in three of four A. hydrophila strains and six of seven A. sobria strains, but in only one of 11 A. caviae strains. Two schemes for the biochemical assessment of enterotoxigenicity were found to be in 91% and 86% agreement respectively with cytotoxicity studies and in 95% agreement with each other. No single biochemical test correlated fully with enterotoxigenicity, but 86% of strains that oxidized gluconate produced a cytotoxin. Most strains were inhibited by concentrations of gentamicin, amikacin, chloramphenicol and tetracycline achievable in plasma. Most strains were resistant to broad-spectrum penicillins and many were also resistant to cefuroxime and cefoxitin.     

Species identification of Aeromonas strains based on carbon substrate oxidation profiles.

Twenty clinical strains each of Aeromonas hydrophila, Aeromonas caviae, and Aeromonas sobria were evaluated for their abilities to oxidize one or more of 95 carbon sources on a GN Microplate (BIOLOG, Hayward, Calif.). Nine substrates yielded good, discriminatory values for the three species tested. The panel appears to be useful for the species identification of Aeromonas isolates originating from human material.     

Invasiveness of Aeromonas spp. in relation to biotype, virulence factors, and clinical features.

Of 69 fecal isolates of Aeromonas spp., 18 had the ability to invade HEp-2 cells. Invasiveness correlated with biotype; of the 18 invasive strains, 16 were A. sobria and 2 were A. hydrophila. No invasive strains were found among the A. caviae. Of the 18 invasive strains, 13 were enterotoxigenic. Of the enterotoxigenic and invasive strains, 12 were A. sobria, but enterotoxicity was also more common among noninvasive strains of A. sobria. Fucose-resistant hemagglutination was also more common in A. sobria, but invasive strains were equally divided between fucose-resistant hemagglutination and other patterns. Detailed clinical information was available for 27 of the 69 strains. All 15 strains of A. sobria or A. hydrophila associated with diarrhea were enterotoxigenic; 6 of the 10 strains of A. sobria were also invasive. Blood was present in the stool samples of five of the six patients with invasive A. sobria and in none of the patients with noninvasive strains. Although limited, these observations suggest that dysenteric symptoms may be produced by invasive Aeromonas spp.