The 16S rRNA gene sequences of the type strains of Aeromonas enteropelogenes and Aeromonas ichthiosmia were determined by polymerase chain reaction direct sequencing in order to clarify their interrelationships with other aeromonad species. On the basis of 16S rRNA gene sequence analysis, A. enteropelogenes and A. ichthiosmia were found to be identical to Aeromonas trota and Aeromonas veronii, respectively.

In 1983, the vernacular name Enteric Group 77 was coined for a group of strains that had been referred to our laboratory as "possible Vibrio cholerae except for gas production." By DNA-DNA hybridization (hydroxyapatite, 32P), 8 of 10 strains of Enteric Group 77 were very highly related to the labeled strain 1169-83 (74 to 100% at 60 degrees C and 75 to 100% at 75 degrees C; percent divergence, 0.0 to 2.5). Type strains of six other Aeromonas species were 45 to 66% related (60 degrees C) to strain 1169-83, but type strains of 27 Vibrio species were only 2 to 6% related. The name Aeromonas veronii is proposed for the highly related group of nine strains formerly known as Enteric Group 77. The type strain is designated as ATCC 35604 (CDC 1169-83). Strains of A. veronii grew well at 36 degrees C and had positive reactions at this temperature for indole, methyl red, Voges-Proskauer, citrate, lysine and ornithine decarboxylases, DNase, lipase, and motility; the strains had negative reactions for arginine decarboxylase, H2S, urea, and malonate. The following sugars were fermented: D-glucose (acid and gas), cellobiose (seven of nine strains), D-galactose, maltose, D-mannitol, D-mannose, alpha-methyl-D-glucoside (eight of nine strains), salicin, sucrose, and trehalose. The following sugars were not fermented: adonitol, L-arabinose, D-arabitol, dulcitol, erythritol, myo-inositol, lactose, raffinose, L-rhamnose, D-sorbitol, and D-xylose. The positive ornithine decarboxylase reaction differentiates A. veronii from other Aeromonas species. The antibiogram of A. veronii is typical of other Aeromonas strains (resistance to ampicillin and carbenicillin and susceptibility to most other agents). A. veronii strains were isolated from three clinical sources: respiratory secretions of four victims of drowning or near drowning in fresh water (probably not clinically significant); infected wounds of two patients previously exposed to fresh water (unknown clinical significance); and stools from three patients with diarrhea (probably clinically significant).

We investigated the ability of a recently developed genomic fingerprinting technique, named AFLP, to differentiate the 14 currently defined DNA hybridization groups (HGs) in the genus Aeromonas. We also determined the taxonomic positions of the phenospecies Aeromonas allosaccharophila, Aeromonas encheleia, Aeromonas enteropelogenes, and Aeromonas ichthiosmia, which have not been assigned to HGs yet. A total of 98 Aeromonas type and reference strains were included in this study. For the AFLP analysis, the total genomic DNA of each strain was digested with restriction endonucleases ApaI and TaqI. Subsequently, restriction fragments were selectively amplified under high-stringency PCR conditions. The amplification products were electrophoretically separated on a polyacrylamide gel and visualized by autoradiography. Following high-resolution densitometric scanning of the resulting band patterns, AFLP data were further processed for a computer-assisted comparison. A numerical analysis of the digitized fingerprints revealed 13 AFLP clusters which, in general, clearly supported the current Aeromonas taxonomy derived from DNA homology data. In addition, our results indicated that there is significant genotypic heterogeneity in Aeromonas eucrenophila (HG6), which may lead to a further subdivision of this species. A. allosaccharophila and A. encheleia did not represent a separate AFLP cluster but were found to be genotypically related to HG8/10 and HG6, respectively. In addition, the results of the AFLP analysis also confirmed the phylogenetic findings that A. enteropelogenes and A. ichthiosmia are in fact identical to Aeromonas trota (HG13) and Aeromonas veronii (HG8/10), respectively. The results of this study clearly show that the AFLP technique is a valuable new high-resolution genotypic tool for classification of Aeromonas species and also emphasize that this powerful DNA fingerprinting method is important for bacterial taxonomy in general.

We re-investigated the taxonomic affiliation between Aeromonas culicicola Pidiyar et al. 2002 and Aeromonas veronii Hickman Brenner et al. 1987, two species that have previously been shown to be closely related on the basis of gyrB and rpoD gene sequencing and amplified fragment length polymorphism fingerprinting. From extended biochemical characterization and fatty acid analysis, it was concluded that A. culicicola did not constitute a unique phenotypic group in the genus Aeromonas but instead clearly fitted the phenotypic description of A. veronii biovar (bv.) sobria. Concordant with the phenotypic results, new DNA-DNA hybridizations revealed reassociation values in the range of 79-88% between the type strains of A. culicicola and A. veronii which indicates that both taxa belong to the same DNA hybridization group. Together with previously reported evidence, we conclude from the new taxonomic data that A. culicicola is a typical member of A. veronii bv. sobria and that its status as a new Aeromonas species is not justified. On the basis of nomenclatural priority, it is therefore proposed that A. culicicola Pidiyar et al. 2002 should be considered as later subjective synonym of A. veronii Hickman-Brenner et al. 1987.

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.

Previously, a DNA fingerprinting study based on Amplified Fragment Length Polymorphism (AFLP) analysis has revealed a possible genotypic resemblance of the species Aeromonas ichthiosmia and Aeromonas allosaccharophila to Aeromonas veronii (Huys et al., Int. J. Syst. Bacteriol. 46, 572-580 [19961). Currently, two genotypically indistinguishable biovars are known to exist in the latter species, namely A. veronii biovar sobria and A. veronii biovar veronii. In the current study, new DNA-DNA hybridization experiments showed that the type strain of A. ichthiosmia, LMG 12645T (= DSM 6393T), and that of A. allosaccharophila, LMG 14059T (= CECT 4199T), were 84-96% and 78-82% related to A. veronii strain LMG 9075T (= ATCC 35624T), respectively. Based upon phenotypic characterization including a total of 151 tests, the type strain of A. ichthiosmia could be clearly allocated to A. veronii biovar sobria. On the other hand, the three strains constituting the species A. allosaccharophila were found to be phenotypically heterogeneous. None of these strains clearly fitted the biochemical description of either of the two A. veronii biovars or tightly clustered with any of the A. veronii reference strains. On the basis of published taxonomic evidence (including AFLP and phylogenetic data) and the newly reported results, there is compiling evidence to conclude that A. ichthiosmia Schubert et al. 1990 is a later synonym of A. veronii Hickman-Brenner et al. 1987. However, due to the lack of agreement encountered between the new DNA reassociation results and previously reported DNA homology and phylogenetic data, a conclusive proposal on the genotypic position of A. allosaccharophila should await further studies.

Fourteen sucrose-positive Aeromonas jandaei-like isolates from fresh water and reared European eels were subjected to a polyphasic study to determine their taxonomic position. Numerical taxonomy was used to analyse phenotypic data obtained for these isolates and 43 type and reference strains representative of recognized Aeromonas species. The A. jandaei cluster (phenon 1) was defined at 81.6 % similarity (S(J)); this included the A. jandaei-like isolates, the sucrose-positive strain Aeromonas veronii biogroup sobria CECT 4910 and nearly all A. jandaei reference strains used in the study. Four other reference strains of A. veronii biogroup sobria and the type strain of Aeromonas ichthiosmia were peripheral to the A. jandaei cluster. The supra-group 'A. jandaei-A.veronii biogroup sobria-A. ichthiosmia' was linked at 80.7 % similarity (S(J)) and was clearly segregated from the phenotypic core of the A. veronii biogroup sobria species, which was related to the reference strain Popoff 224 (CECT 4835). DNA relatedness between strains grouped in the A. jandaei cluster (phenon 1) and A. jandaei CECT 4228(T) ranged from 70 to 100 %, but was below 50 % when DNAs from A. veronii biogroup sobria CECT 4835, A. veronii biogroup veronii CECT 4257(T) and A. ichthiosmia CECT 4486(T) were used. In addition, DNA relatedness between peripheral A. veronii biogroup sobria strains and the species A. jandaei (CECT 4228(T)), A. veronii (CECT 4257(T), CECT 4835) and A. ichthiosmia (CECT 4486(T)) was always below 54 %, as it was between the species A. ichthiosmia (CECT 4486(T)) and A. veronii (CECT 4257(T), CECT 4835). Emendation of A. jandaei is proposed; this taxon now includes sucrose-positive clinical and environmental strains as well as environmental isolates that are pathogenic for fish and humans. Other new traits for this species are the ability to grow at 4-42 degrees C, acid production from glycerol but not from lactose, D-melibiose or D-raffinose, the use of D-gluconate, L-glutamate or L-proline but not L-lactate, L-alanine, L-arabinose or L-arginine, hydrolytic activity against casein, elastin, starch and lecithin and the inability to lyse arbutin. The DNA G+C content of A. jandaei is also reported for the first time; it ranges from 58.1 to 61.1 mol%. On the other hand, the DNA relatedness data support the classification of peripheral reference strains of A. veronii biogroup sobria outside this taxon, indicating that biogroup sobria requires further revision.