The phylogenetic interrelationships of members of the genera Aeromonas and Phiomonas were investigated by using small-subunit ribosomal DNA (rDNA) sequencing. Members of the genus Aeromonas formed a distinct line within the gamma subclass of the Proteobucteriu. Plesiomonas shigelloides also clustered within the confines of the gamma subclass of the Proteobucteriu but exhibited a closer association with members of the family Enterobucteriuceue than with members of the family Aeromonuduceue. Species of the genus Aeromonas exhibited very high levels of overall sequence similarity (ca. 98 to 100%) with each other. Several of the relationships derived from an analysis of the rDNA sequence data were in marked disagreement with the results of chromosomal DNA-DNA pairing experiments. Diagnostic rDNA signatures that have possible value for differentiating most Aeromonas species were discerned.

The phylogenetic relationships of all known species of the genus Aeromonas were investigated by using the sequence of gyrB, a gene that encodes the B-subunit of DNA gyrase. Nucleotide sequences of gyrB were determined from 53 Aeromonas strains, including some new isolates, which were also characterized by analysis of the 16S rDNA variable regions. The results support the recognition of the family Aeromonadaceae, as distinct from Plesiomonas shigelloides and other enteric bacteria. This phylogenetic marker revealed strain groupings that are consistent with the taxonomic organization of all Aeromonas species described to date. In particular, gyrB results agreed with 16S rDNA analysis; moreover, the former showed a higher capacity to differentiate between species. The present analysis was useful for the elucidation of reported discrepancies between different DNA-DNA hybridization sets. Additionally, due to the sequence diversity found at the intraspecies level, gyrB is proposed as a useful target for simultaneous identification of species and strains. In conclusion, the gyrB gene has proved to be an excellent molecular chronometer for phylogenetic studies of the genus Aeromonas. INTRODUCTIONThe genus Aeromonas comprises a collection of oxidaseand catalase-positive, glucose-fermenting, facultatively anaerobic, Gram-negative, rod-shaped bacteria that are resistant to the vibriostatic agent O/129 and are generally motile by means of polar flagella (Popoff, 1984). Aeromonads are autochthonous to aquatic environments worldwide and have been implicated in the aetiology of a variety of fish and human diseases, frequently including diarrhoea and occasionally systemic infections (Janda, 1991). Over the past few years, interest in the genus Aeromonas as an emergent human pathogen has increased significantly Joseph & Carnahan, 2000).The classification of the genus Aeromonas has been dogged by confusion and controversy. In Bergey's Manual of Systematic Bacteriology (Popoff, 1984), the genus was divided into three mesophilic and motile species (Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria) and the psychrophilic, non-motile species Aeromonas salmonicida. Extensive DNA-DNA hybridization studies (Popoff et al., 1981;Hickman-Brenner et al., 1987Kuijper et al., 1989;Carnahan et al., 1991) have resulted in the recognition of 14 so-called DNA homology groups (HGs): A. hydrophila (HG1), Aeromonas sp. (unnamed; HG2), A. salmonicida (HG3), A. caviae (HG4), Aeromonas media (HG5), Aeromonas eucrenophila (HG6), A. sobria (HG7), Aeromonas veronii biogroup sobria (HG8), Aeromonas jandaei (HG9), A. veronii biogroup veronii (HG10), Aeromonas sp. (unnamed; HG11), A. schubertii (HG12), Aeromonas group 501 (HG13; formerly Enteric group 501) and Aeromonas trota (HG14). The name Aeromonas bestiarum has been proposed for strains included in HG2 (Ali et al., 1996). During the past decade, three novel species have been described: Aeromonas allosaccharophila (Martínez-Murcia et al., 1992b), Aeromonas encheleia (Esteve et al., 1995b) and A...

The phylogenetic relationships of all known species of the genus Aeromonas, and especially Aeromonas bestiarum and Aeromonas salmonicida, were investigated on 70 strains using the rpoD sequence, which encodes the s 70 factor. This analysis was complemented with the sequence of gyrB, which has already proven useful for determining the phylogenetic relationships in the genus. Nucleotide sequences of rpoD and gyrB showed that both genes had similar substitution rates (<2 %) and a similar number of variable positions (34 % for rpoD versus 32 % for gyrB). Strain groupings by analysis of rpoD, gyrB and a combination of both genes were consistent with the taxonomic organization of all Aeromonas species described to date. However, the simultaneous analysis of both clocks improved the reliability and the power to differentiate, in particular, closely related taxa. At the inter-species level, gyrB showed a better resolution for differentiating Aeromonas sp. HG11/Aeromonas encheleia and Aeromonas veronii/Aeromonas culicicola/Aeromonas allosaccharophila, while rpoD more clearly differentiated A. salmonicida from A. bestiarum. The analysis of rpoD provided initial evidence for clear phylogenetic divergence between the latter two species.

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Identification of Aeromonas species, emergent pathogens for humans, has long been controversial due to their phenotypic and genomic heterogeneities. Computer analysis of the published 16S rRNA gene sequences revealed that restriction fragment length polymorphism of the PCR-amplified 16S rRNA gene is a good and rapid way of assessing the identities of all known species of Aeromonas. The method was evaluated with the reference strains of all species (or DNA homology groups) and 76 clinical isolates of diverse origin. Most results from the two approaches were in agreement, but some discrepancies were discerned. Advantages over previous phenotypic and genetic methods are discussed.