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, 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.

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...

No abstract

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.

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 AlwNI and PstI for the identification of these species. A molecular frame for the identification of all known Aeromonas spp. is presented.

The ribosomal RNA multigene family in Escherichia coli comprises seven rrn operons of similar, but not identical, sequence. Four operons (rrnC, B, G, and E) contain genes in the 16S-23S intergenic spacer region (ISR) for tRNA(Glu-2) and three (rrnA, D, and H) contain genes for tRNA(Ile-1) and tRNA(Ala-1B). To increase our understanding of their molecular evolution, we have determined the ISR sequence of the seven operons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers designed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containing one or two tRNA genes were found at the same respective loci as those of strain K12. Intercistronic heterogeneity already found in K12 was representative of most variation among the strains studied and the location of polymorphic sites was the same. Dispersed nucleotide substitutions were very few but 21 variable sites were found grouped in a stem-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the pattern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest intercistronic heterogeneity. Most strains showed a strong tendency to homogenization. Concerted evolution could explain the notorious conservation of this region that is supposed to have low functional restrictions.

The phylogenetic interrelationships of members of the genus Carnobacterium and some atypical lactobacilli isolated from diseased salmonid fish were investigated by using reverse transcriptase sequencing of 16s rRNA. The four species Carnobacterium piscicola, Carnobacterium divergens, Carnobacterium gallinarum, and Carnobacterium mobile exhibited a high degree of sequence similarity with each other (ca. 96 to 98%) and formed a phylogenetically coherent group that was quite distinct from all other lactic acid bacteria. The sequence data clearly demonstrated that carnobacteria are phylogenetically closer to the genera Enterococcus and Vagococcus than to members of the genus Lactobacillus. The strains from fish were found to be phylogenetically related to the genus Vagococcus and represent a new species, Vagococcus salmoninarum. The type strain of Vagococcus salmoninarum is strain NCFB 2777.The genus Carnobacterium was proposed by Collins et al. (4) to accommodate the species Lactobacillus piscicola (17) and Lactobacillus divergens (18) and some so-called atypical lactobacilli isolated from poultry meat (24). Currently, four species, Carnobacterium piscicola (type species), Carnobacterium divergens, Carnobacterium gallinarum, and Carnobacterium mobile are recognized in this genus (4). Although these four species form a phenotypically coherent group (4, 9), the separateness of the genus Carnobacterium (in particular its relationship to the genus Lactobacillus) remains unclear. Members of the genus Carnobacterium differ from Lactobacillus species by their inability to grow on acetate medium (4, 9) and by their synthesis of oleic acid (c18:1A9,10) instead of cis-vaccenic acid (c18:1A11,12), which is produced by lactobacilli (4). At present these are the only criteria which distinguish the two genera.Sequencing of 16s rRNA by reverse transcriptase (20, 21) is currently the most rapid and powerful technique for elucidating the natural relationships of microorganisms (26). This method produces long stretches of sequence (ca. 95% of the total sequence), which enables precise phylogenetic relationships to be determined (26). In this study we determined 16s rRNA primary structures for members of the genus Carnobacterium by using reverse transcription in an attempt to clarify the relationship of these organisms to the genus Lactobacillus and other lactic acid bacteria. In addition, the phylogenetic position of two representative strains belonging to a group of atypical lactobacilli isolated from salmonid fish was also determined. MATERIALS AND METHODSCultures and cultivation. The test strains which we used are shown in Table 1. Strains of carnobacteria, enterococci, lactococci, leuconostocs, pediococci, and streptococci and fish isolates OS1-68T (T = type strain) and Rangen 128-81 were grown in YGPB broth (12) at 30°C. Strains OS1-68T and Rangen 128-81 were isolated by R.A. Holt, Oregon Department of Fish and Wildlife; strain OS1-68T was isolated in 1968 from an adult rainbow trout at the Oak Spring Hatchery * Corresponding...