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PubMed2000

Extended method for discrimination of Aeromonas spp. by 16S rDNA RFLP analysis.

A previously described molecular method, based on 16S rDNA RFLP analysis, for the identification of Aeromonas spp. was unable to separate the species Aeromonas salmonicida, Aeromonas bestiarum and the recently described Aeromonas popoffii. In this study, the method has been extended with endonucleases AIwNI and PstI for the identification of these species. A molecular frame for the identification of all known Aeromonas spp. is presented.

Aeromonas↗

PubMed2002

Aeromonas culicicola sp. nov., from the midgut of Culex quinquefasciatus.

The taxonomic position was examined of three isolates, MTCC 3249T, SH and SLH, from the midgut of female Culex quinquefasciatus and Aedes aegyptii mosquitoes. Numbers of cells of these isolates increased 2000-fold after a blood meal of the mosquitoes. 16S rRNA gene sequence analysis of the novel strains showed that they were highly homologous to strains of Aeromonas. DNA-DNA hybridization studies showed that DNA of strain MTCC 3249T was 96 and 88% similar to that of strains SH and SLH, respectively, and showed 54% relatedness to Aeromonas jandaei and 61% relatedness to Aeromonas sobria, which is below the cut-off value for species differentiation. The biochemical profiles of all three novel strains were identical. On the basis of a polyphasic approach using phenotypic analysis, 16S rRNA gene sequencing and DNA-DNA hybridization studies, a novel species is proposed for these isolates, Aeromonas culicicola sp. nov., with the type strain MTCC 3249T (= NCIM 5147T). Isolation of A. culicicola from the midgut of mosquitoes might help to explain the origin of Aeromonas infections caused without exposure to contaminated water, soil or food.

Aedes↗

PubMed1984

Media for isolation of Aeromonas spp. from faeces.

Five solid media were evaluated for isolation of Aeromonas spp. from faeces: desoxycholate citrate agar (DCA), MacConkey's agar (MAC), xylose-desoxycholate-citrate agar (XDCA), Rogol's medium (ROG), which contained ampicillin 20 mg/L and p-nitrophenyl-glycerine 25 mg/L as inhibitors, and blood agar (BA) with ampicillin 10 mg/L. False negative oxidase tests limited the usefulness of DCA and MAC and, although the use of XDCA avoided the problem of fermentation of lactose, some Aeromonas spp. failed to grow on XDCA or produced minute colonies unsuitable for oxidase tests. BA yielded the highest rate of isolation for Aeromonas spp. from 323 faecal samples--15.2% for all Aeromonas spp. and 9.3% for enterotoxigenic (ENT+) strains. This compares with 10.8% for all strains and 6.5% for ENT+ strains isolated on DCA, 7.1% for all strains and 4% for ENT+ strains on MAC and 4% for all strains and 1.5% for ENT+ strains on ROG. Blood agar with ampicillin is recommended for isolation of Aeromonas spp. from faeces.

Aeromonas↗

PubMed1990

Effects of incorporating ampicillin, bile salts and carbohydrates in media on the recognition and selection of Aeromonas spp. from faeces.

The effects of incorporating ampicillin, some bile salts and sugars into media on the primary recognition and selection of aeromonads from faeces were examined. Most (88%) of the 101 Aeromonas strains examined had an ampicillin MIC of greater than or equal to 40 mg/L, and would be isolated on blood agar containing ampicillin 30 mg/L. The strains with an ampicillin MIC of less than 40 mg/L were all of human origin and predominantly A. caviae. Although ampicillin at 10, 20 or 30 mg/L in blood agar inhibited faecal bacteria, the ability to detect Aeromonas strains with a high ampicillin MIC was less when the lower concentrations of ampicillin were used, without any improvement in the isolation of those strains with a low ampicillin MIC. Thirty-seven strains were tested for sensitivity to several different bile salts and found to be at least as resistant to them as Escherichia coli NCTC 10418. Bile salt sensitivity was not related to the species or source of a strain. There were minor differences in sensitivity to bile salts between some strains which related to whether strains had been isolated originally in the presence of bile salt or not. The effects of the presence of E. coli, Klebsiella spp. and Enterococcus faecalis on the growth of Aeromonas strains in mixed culture on media with and without carbohydrate were examined. The colony size of some Aeromonas strains was reduced in mixed culture but colony counts were not affected with any Aeromonas strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Aeromonas↗

PubMed1996

Aeromonas trota strains, which agglutinate with Vibrio cholerae O139 Bengal antiserum, possess a serologically distinct fimbrial colonization factor.

Pili of Aeromonas trota strain 1220, which agglutinates with Vibrio cholerae O139 Bengal antiserum, were purified and characterized. The molecular mass of the subunit protein was estimated to be 20 kDa and the pl was 5 center dot 4. The pili were immunologically unrelated to the other Aeromonas pili reported so far. However, the N-terminal amino acid sequence of the subunit pilin was similar to those of the pilins from other Aeromonas pili reported previously. Neither A. trota cells nor pili purified from strain 1220 agglutinated human and rabbit erythrocytes, but both adhered to the rabbit intestine. Bacterial cells pretreated with antipilus antibody (Fab portion) failed to adhere to the rabbit intestine. Moreover, bacteria did not adhere to the rabbit intestine pretreated with the purified pili. This pilus antigen was not detected in V. cholerae O139 Bengal and other Aeromonas spp. These findings suggest that the pilus of the A. trota strain is a novel colonization factor of Aeromonas spp.

Aeromonas↗

PubMed2003

Aeromonas hydrophila subsp. ranae subsp. nov., isolated from septicaemic farmed frogs in Thailand.

A group of seven sucrose-negative Aeromonas strains (referred to as group Au) isolated from the internal organs of septicaemic farmed frogs (Rana rugulosa) in Thailand was subjected to a polyphasic taxonomic study including fluorescent amplified fragment length polymorphism (FAFLP) and ERIC-PCR fingerprinting, 16S rDNA sequencing, microplate DNA-DNA hybridizations and extensive phenotypic characterization. Comparison of FAFLP and ERIC-PCR fingerprints indicated that the group Au isolates belonged to the species Aeromonas hydrophila DNA hybridization group (HG) 1 in which they represent a genotypic subgroup closely affiliated to A. hydrophila subsp. hydrophila and subsp. dhakensis. One representative of the Au group exhibited > or = 99.0% 16S rDNA sequence similarity with the type strains of the two A. hydrophila subspecies. DNA-DNA hybridization with type and reference strains of all known Aeromonas taxa revealed that the Au group represented a homogeneous taxon that exhibited the highest relatedness with members of the two A. hydrophila subspecies, ranging from 75 to 93%. Phenotypic characterization on the basis of 152 features further revealed that the Au group isolates differed from A. hydrophila subsp. hydrophila or subsp. dhakensis in a total of 13 biochemical properties. Of these, assimilation of L-glycine and isobutyrate as sole carbon source, acid production from salicin and D-sucrose, and aesculin hydrolysis were of diagnostic value. From the results of this study, it can be concluded that the Aeromonas frog isolates of the Au group represent a new subspecies of A. hydrophila, for which the name Aeromonas hydrophila subsp. ranae subsp. nov. is proposed. Its type strain is Au-1D12(T) (=LMG 19707(T) = CCUG 46211(T)).

Aeromonas hydrophila↗

PubMed1990

Distribution and characterization of hemolytic, and enteropathogenic motile Aeromonas in aquatic environment.

A year-long survey on the distribution of motile Aeromonas species in the surface waters of riverine and marine environments was conducted. The filtered membranes were directly placed onto the modified Pril-xylose-ampicillin agar for the enumeration of Aeromonas species. High counts of motile aromonads were found in riverine stations and this bacterial population was also observed in significant quantities in polluted marine samples. In the identification of 2,444 isolates, three species of motile Aeromonas were observed. A. caviae (43%) was prevalent followed by A. sobria (35%) and A. hydrophila (20%). A. hydrophila was high in clean riverine samples, A. sobria was predominantly isolated from a stagnant water sampling area, and A. caviae was distributed more in marine samples. Statistical analyses suggested that the densities of Aeromonas were related to the cumulative effect of various physicochemical parameters rather than to a single factor. Among the species of Aeromonas, A. hydrophila, and A. sobria were highly hemolytic whereas only 11% of A. caviae were observed to lyse sheep erythrocytes. Suckling-mouse assay was performed to elucidate the enterotoxicity of motile aeromonads and 21% of the tested strains (one A. caviae strain) were found to produce enterotoxin.

Aeromonas↗

PubMed1996

Immunological analysis of extracellular products and cell surface components of motile Aeromonas isolated from fish.

The present work describes the characterization of antigens present in the extracellular products (ECP) and cell wall of strains of motile Aeromonas isolated from rainbow trout culture systems. The relationships among virulence for fish, O-serogroup and profile of LPS were also examined. The slide agglutination test showed that most of the virulent strains of motile Aeromonas (72%) were included in the serotypes O3, O6, O11 and O19 (Guinée and Jansen System). However, there were also non-pathogenic strains within these groups. Electrophoretic analysis of lipopolysaccharides (LPS) and proteins from cell envelope and ECP showed heterogeneity not only among the different serogroups but also within the same serotype. Immunoblot assays of cell envelope components, and of LPS present in the ECP demonstrated a close relationship among Aeromonas strains from the same serotype, while strains from different serotypes were not immunologically related. Moreover, this assay showed that motile Aeromonas belonging to distinct serotypes produced extracellular proteins immunologically related. On the other hand, antigenic cross reactivity was observed between the LPS obtained from cell envelope and those obtained from the ECP. The present results point out the need to include strains representative of each of the serotypes which predominates in a particular area and their ECPs in the formation of vaccines against motile Aeromonas septicaemia.

Aeromonas↗

PubMed1995

Attachment of non-culturable toxigenic Vibrio cholerae O1 and non-O1 and Aeromonas spp. to the aquatic arthropod Gerris spinolae and plants in the River Ganga, Varanasi.

Non-cultivable, pathogenic O1 and non-O1 Vibrio cholerae and Aeromonas spp. were resuscitated from aquatic arthropods and plant homogenate respectively, by rabbit ileal loop (RIL) assay. These organisms adhered to the aquatic arthropod Gerris spinolae and various species of phytoplankton in the River Ganga, but failed to grow after direct inoculation on artificial media except for only 10 homogenates of the arthropod. The number of non-O1 V. cholerae and Aeromonas recovered on direct inoculation of G. spinolae homogenates were in the order of 10(5)-10(6) whereas those of the Ganga water were 10(2)-10(3) ml-1. A total of 119 strains of O1 and non-O1 V. cholerae and Aeromonas spp. (69 isolates from G. spinolae and 50 from aquatic plants) were recovered from the loop contents. The results indicate that production of the enzyme chitinase by O1 and non-O1 V. cholerae and Aeromonas spp. might facilitate their adsorption and multiplication on different species of zoo- and phyto-plankton. Most of the isolates were enterotoxic, haemolytic and resistant to different antibiotics. This study suggests that species of zoo- and phyto-planktons, until now not reported to be associated with O1 and non-O1 V. cholerae, may act as reservoirs of these organisms as well as different species of Aeromonas in a fresh-water riverine ecosystem.

Aeromonas↗

PubMed2002

Incidence of toxic Aeromonas isolated from food and human infection.

One hundred and ninety four Aeromonas isolates (99 from food and 95 from clinical sources) were analyzed as to the species involved and the toxins produced. Of the clinical isolates of Aeromonas, 29.4% were enterotoxigenic, 43.1% were hemolytic and 89% were cytotoxigenic. Among the food isolates, 18.2% were enterotoxigenic, 17.1% were hemolytic and 72.7% were cytotoxigenic. Aeromonas sobria and Aeromonas veronii produced more enterotoxin and cytotoxin than the other isolates, whereas A. veronii and Aeromonas salmonicida produced cell-free hemolysin. Most of the isolates produced cytotoxins (81%) active on Vero (green monkey kidney) and Chinese hamster ovary cells, but only the culture supernatant of A. sobria produced vacuolation in these cell lines.

Aeromonas↗

PubMed1981

Serogrouping of motile Aeromonas species isolated from healthy and moribund fish.

A total of 195 strains of motile Aeromonas isolated from fish were characterized as Aeromonas hydrophila and Aeromonas sobria. In view of the frequency of isolation and the importance of motile Aeromonas species as fish pathogens, a serological classificaton of these organisms was attempted. Antisera were prepared in rabbits against formalinized whole cell suspensions and against boiled cells of 12 different isolates. Seventy-six strains could be grouped by tube agglutination with whole cells as antigen and anti-whole cell antiserum. However, only 39 strains were typable with anti-O serum. Differentiation was made between heat-stable antigens and heat-labile antigens which did not block the O agglutination reaction. The same heat-labile antigen could be associated with different heat-stable particulate antigens, and a relationship was observed between the heat-stable particulate antigens and the virulence of A. hydrophila for fish. In addition to these two types of antigen, motile Aeromonas possessed heat-stable soluble antigens which could be detected by indirect hemagglutination. One strain seemed to possess various heat-stable soluble antigens; so far, however, it does not appear to be feasible to use these antigens for serology. Finally, we also observed cross-reactions between some A. hydrophila and A. sobria strains.

Aeromonas↗

PubMed1999

PCR detection, characterization, and distribution of virulence genes in Aeromonas spp.

We found 73.1 to 96.9% similarity by aligning the cytolytic enterotoxin gene of Aeromonas hydrophila SSU (AHCYTOEN; GenBank accession no. M84709) against aerolysin genes of Aeromonas spp., suggesting the possibility of selecting common primers. Identities of 90 to 100% were found among the eight selected primers from those genes. Amplicons obtained from Aeromonas sp. reference strains by using specific primers for each gene or a cocktail of primers were 232 bp long. Of hybridization group 4/5A/5B (HG4/5A/5B), HG9, and HG12 or non-Aeromonas reference strains, none were positive. PCR-restriction fragment length polymorphism (PCR-RFLP) with HpaII yielded three types of patterns. PCR-RFLP 1 contained two fragments (66 and 166 bp) found in HG6, HG7, HG8, HG10, and HG11. PCR-RFLP 2 contained three fragments (18, 66, and 148 bp) found in HG1, HG2, HG3, and HG11. PCR-RFLP 3, with four fragments (7, 20, 66, and 139 bp), was observed only in HG13. PCR-amplicon sequence analysis (PCR-ASA) revealed three main types. PCR-ASA 1 had 76 to 78% homology with AHCYTOEN and included strains in HG6, HG7, HG8, HG10, and HG11. PCR-ASA 2, with 82% homology, was found only in HG13. PCR-ASA 3, with 91 to 99% homology, contained the strains in HG1, HG2, HG3, and HG11. This method indicated that 37 (61%) of the 61 reference strains were positive with the primer cocktail master mixture, and 34 (58%) of 59 environmental isolates, 93 (66%) of 141 food isolates, and 100 (67%) of 150 clinical isolates from around the world carried a virulence factor when primers AHCF1 and AHCR1 were used. In conclusion, this PCR-based method is rapid, sensitive, and specific for the detection of virulence factors of Aeromonas spp. It overcomes the handicap of time-consuming biochemical and other DNA-based methods.

Aeromonas↗

PubMed2002

Identification and characterization of pathogenic Aeromonas veronii biovar sobria associated with epizootic ulcerative syndrome in fish in Bangladesh.

Sparse information is available on the virulence factors of Aeromonas strains isolated from diseased fish, from the environment, and from humans. In the present study, 52 Aeromonas isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate [PhP] typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors. The isolates from the fish exhibiting EUS symptoms were identified to be Aeromonas veronii biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting. Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi Aeromonas isolates. The 52 Aeromonas isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines. All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes. Our results suggest that a clonal group of A. veronii biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.

Aeromonas↗

PubMed2001

Role of flm locus in mesophilic Aeromonas species adherence.

The adherence mechanism of Aeromonas caviae Sch3N to HEp-2 cells was initially investigated through four mini-Tn5 mutants that showed a 10-fold decrease in adherence. These mutants lost motility, flagella, and their lipopolysaccharide (LPS) O antigen (O-Ag). Three genes, flmB-neuA-flmD, were found to be interrupted by the transposon insertions; additionally, two other genes, one lying upstream (flmA) and one downstream (neuB), were found to be clustered in the same operon. While the flmA and flmB genes were present in all mesophilic Aeromonas spp. (A. hydrophila, A. caviae, A. veronii bv. veronii, and A. veronii bv. sobria) tested, this was not the case for the neuA-flmD-neuB genes. Construction and characterization of flmB insertion mutants in five other mesophilic Aeromonas strains revealed the loss of motility, flagella, and adherence but did not alter the LPS composition of these strains. Taking the above findings into consideration, we conclude (i) that flagella and possibly the LPS O-Ag are involved in the adherence of the mesophilic Aeromonas to human epithelial cells; (ii) flmA and flmB are genes widely distributed in the mesophilic Aeromonas and are involved in flagella assembly, and thus adherence; and (iii) in A. caviae Sch3N the flmA and flmB genes are found in a putative operon together with neuA, flmD, and neuB and are involved in LPS O-Ag biosynthesis and probably have a role in flagellum assembly.

Aeromonas↗

PubMed2004

Aeromonas hydrophila beta-hemolysin induces active chloride secretion in colon epithelial cells (HT-29/B6).

The diarrheal mechanisms in Aeromonas enteritis are not completely understood. In this study we investigated the effect of aeromonads and of their secretory products on ion secretion and barrier function of monolayers of human intestinal cells (HT-29/B6). Ion secretion was determined as a short-circuit current (I(SC)) of HT-29/B6 monolayers mounted in Ussing-type chambers. Transepithelial resistance (R(t)) served as a measure of permeability. A diarrheal strain of Aeromonas hydrophila (strain Sb) added to the mucosal side of HT-29/B6 monolayers induced a significant I(SC) (39 +/- 3 microA/cm(2)) and decreased the R(t) to approximately 10% of the initial value. A qualitatively identical response was obtained with sterile supernatant of strain Sb, and Aeromonas supernatant also induced a significant I(SC) in totally stripped human colon. Tracer flux and ion replacement studies revealed the I(SC) to be mainly accounted for by electrogenic Cl(-) secretion. Supernatant applied serosally completely abolished basal I(SC). The supernatant-induced I(SC) was inhibited by the protein kinase C inhibitor chelerythrine, whereas a protein kinase A inhibitor (H8) and a Ca(2+) chelator (BAPTA-AM) had no effect. Physicochemical properties indicated that the supernatant's active compound was an aerolysin-related Aeromonas beta-hemolysin. Accordingly, identical I(SC) and R(t) responses were obtained with Escherichia coli lysates harboring the cloned beta-hemolysin gene from strain SB or the aerA gene encoding for aerolysin. Sequence comparison revealed a 64% homology between aerolysin and the beta-hemolysin cloned from Aeromonas sp. strain Sb. In conclusion, beta-hemolysin secreted by pathogenic aeromonads induces active Cl(-) secretion in the intestinal epithelium, possibly by channel insertion into the apical membrane and by activation of protein kinase C.

Aeromonas hydrophila↗

PubMed1986

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.

Acetoin↗

PubMed1989

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.

Aeromonas↗

PubMed1989

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.

Aeromonas↗