Aeromonas australiensis sp. nov., isolated from irrigation water.

A Gram-negative, facultatively anaerobic bacillus, designated strain 266(T), was isolated from an irrigation water system in the south-west of Western Australia. Analysis of the 16S rRNA gene sequence confirmed that strain 266(T) belonged to the genus Aeromonas, with the nearest species being Aeromonas fluvialis (99.6% similarity to the type strain, with 6 nucleotide differences) followed by Aeromonas veronii and Aeromonas allosaccharophila (both 99.5%). Analysis of gyrB and rpoD sequences suggested that strain 266(T) formed a phylogenetic line independent of other species in the genus. This was confirmed using the concatenated sequences of six housekeeping genes (gyrB, rpoD, recA, dnaJ, gyrA and dnaX) that also indicated that A. veronii and A. allosaccharophila were the nearest relatives. DNA-DNA reassociation experiments and phenotypic analysis further supported the conclusion that strain 266(T) represents a novel species, for which the name Aeromonas australiensis sp. nov. is proposed, with type strain 266(T) (=CECT 8023(T) =LMG 26707(T)). [corrected].

Multilocus phylogenetic analysis of the genus Aeromonas.

A broad multilocus phylogenetic analysis (MLPA) of the representative diversity of a genus offers the opportunity to incorporate concatenated inter-species phylogenies into bacterial systematics. Recent analyses based on single housekeeping genes have provided coherent phylogenies of Aeromonas. However, to date, a multi-gene phylogenetic analysis has never been tackled. In the present study, the intra- and inter-species phylogenetic relationships of 115 strains representing all Aeromonas species described to date were investigated by MLPA. The study included the independent analysis of seven single gene fragments (gyrB, rpoD, recA, dnaJ, gyrA, dnaX, and atpD), and the tree resulting from the concatenated 4705 bp sequence. The phylogenies obtained were consistent with each other, and clustering agreed with the Aeromonas taxonomy recognized to date. The highest clustering robustness was found for the concatenated tree (i.e. all Aeromonas species split into 100% bootstrap clusters). Both possible chronometric distortions and poor resolution encountered when using single-gene analysis were buffered in the concatenated MLPA tree. However, reliable phylogenetic species delineation required an MLPA including several "bona fide" strains representing all described species.

Aeromonas fluvialis sp. nov., isolated from a Spanish river.

A Gram-stain-negative, facultatively anaerobic bacterial strain, designated 717(T), was isolated from a water sample collected from the Muga river, Girona, north-east Spain. Preliminary analysis of the 16S rRNA gene sequence showed that this strain belonged to the genus Aeromonas, the nearest species being Aeromonas veronii (99.5 % similarity, with seven different nucleotides). A polyphasic study based on a multilocus phylogenetic analysis of five housekeeping genes (gyrB, rpoD, recA, dnaJ and gyrA; 3684 bp) showed isolate 717(T) to be an independent phylogenetic line, with Aeromonas sobria, Aeromonas veronii and Aeromonas allosaccharophila as the closest neighbour species. DNA-DNA reassociation experiments and phenotypic analysis identified that strain 717(T) represents a novel species, for which the name Aeromonas fluvialis sp. nov. is proposed, with type strain 717(T) (=CECT 7401(T) =LMG 24681(T)).

Aeromonas taiwanensis sp. nov. and Aeromonas sanarellii sp. nov., clinical species from Taiwan.

Two clinical Aeromonas strains (A2-50(T) and A2-67(T)) recovered from the wounds of two patients in Taiwan could not be assigned to any known species of this genus based on their 16S rRNA gene sequences, which showed similarities of 99.6-99.8 % to those of the type strains of Aeromonas caviae, A. trota and A. aquariorum. The rpoD phylogenetic tree allocated these strains to two novel and independent phylogenetic lines, the neighbouring species being A. caviae, the type strain of which showed 93.2 % similarity (56 bp differences) to strain A2-50(T) and 92.2 % (63 bp differences) to strain A2-67(T). A multilocus phylogenetic analysis of five housekeeping genes (gyrB, rpoD, recA, dnaJ and gyrA; 3684 bp) confirmed that the two strains formed independent phylogenetic lineages within the genus. These data, together with phenotypic characterization and DNA-DNA reassociation results, revealed that these strains represent novel Aeromonas species, for which the names Aeromonas taiwanensis sp. nov. (type strain A2-50(T) =CECT 7403(T) =LMG 24683(T)) and Aeromonas sanarellii sp. nov. (type strain A2-67(T) =CECT 7402(T) =LMG 24682(T)) are proposed.

Clinical relevance of the recently described species Aeromonas aquariorum.

Twenty-two human extraintestinal isolates (11 from blood) and three isolates recovered from patients with diarrhea were genetically characterized as Aeromonas aquariorum, a novel species known only from ornamental fish. The isolates proved to bear a considerable number of virulence genes, and all were resistant to amoxicillin (amoxicilline), cephalothin (cefalotin), and cefoxitin. Biochemical differentiation from the most relevant clinical species is provided.

Analysis of 16S rRNA gene mutations in a subset of Aeromonas strains and their impact in species delineation.

Characterization of 999 Aeromonas strains using a published 16S rDNA RFLP identification method showed that 8.1% of the strains produced unexpected (hereafter called "atypical") restriction patterns, making their identification uncertain. Atypical patterns were due to the presence of nucleotide polymorphisms among the rrn operons of the 16S rRNA gene (so-called microheterogeneities). Double sequencing signals at certain positions revealed the nucleotide composition was responsible for the microheterogeneities. Although the number of microheterogeneities was relatively low (0.06-0.66%), trees inferred from the 16S rRNA gene led either to a misidentification or to an inconclusive result for the majority of these strains. Strains with atypical patterns were, however, correctly identified using the rpoD gene sequences, as belonging to Aeromonas caviae, A. veronii, and A. media. All of them, but particularly the two former species, are associated with human disease. Microheterogeneities in 16S rRNA gene sequence were significantly (P 0.01) more prevalent in clinical than in environmental strains. This work also analyzed the effects of these microheterogeneities on the taxonomic position of the investigated strains. The results suggest the need for recording microheterogeneities in the 16S rRNA gene.

Analysis of 16S rRNA gene mutations in a subset of Aeromonas strains and their impact in species delineation

Characterization of 999 Aeromonas strains using a published 16S rDNA RFLP identification method showed that 8.1% of the strains produced unexpected (hereafter called «atypical») restriction patterns, making their identification uncertain. Atypical patterns were due to the presence of nucleotide polymorphisms among the rrn operons of the 16S rRNA gene (so-called microheterogeneities). Double sequencing signals at certain positions revealed the nucleotide composition was responsible for the microheterogeneities. Although the number of microheterogeneities was relatively low (0.06-0.66%), trees inferred from the 16S rRNA gene led either to a misidentification or to an inconclusive result for the majority of these strains. Strains with atypical patterns were, however, correctly identified using the rpoD gene sequences, as belonging to Aeromonas caviae, A. veronii, and A. media. All of them, but particularly the two former species, are associated with human disease. Microheterogeneities in 16S rRNA gene sequence were significantly (P 0.01) more prevalent in clinical than in environmental strains. This work also analyzed the effects of these microheterogeneities on the taxonomic position of the investigated strains. The results suggest the need for recording microheterogeneities in the 16S rRNA gene (AU)

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The recently proposed species Aeromonas sharmana sp. nov., isolate GPTSA-6T, is not a member of the genus Aeromonas.

A new species of the genus Aeromonas, Aeromonas sharmana sp. nov., was recently described on the basis of a single isolate, strain GPTSA-6T, obtained from a warm spring in India. The description of this new species included biochemical characterization, antibiotic susceptibility, cellular fatty-acid profiles, and 16S rRNA gene sequencing, but not DNADNA hybridization data. In the present article, phylogenetic analysis (branch distances in the tree and nucleotide signatures) of the 16S rRNA of isolate GPTSA-6T, together with certain phenotypic characteristics of the isolate reported in the earlier description, clearly indicate that this microorganism does not belong to the genus Aeromonas as known to date, although it falls within the radiation of the family Aeromonadaceae. Emendation from the List of Prokaryotic Names with Standing in Nomenclature is consequently proposed.

The recently proposed species Aeromonas sharmana sp. nov., isolate GPTSA-6T, is not a member of the genus Aeromonas

A new species of the genus Aeromonas, Aeromonas sharmana sp. nov., was recently described on the basis of a single isolate, strain GPTSA-6T, obtained from a warm spring in India. The description of this new species included biochemical characterization, antibiotic susceptibility, cellular fatty-acid profiles, and 16S rRNA gene sequencing, but not DNADNA hybridization data. In the present article, phylogenetic analysis (branch distances in the tree and nucleotide signatures) of the 16S rRNA of isolate GPTSA-6T, together with certain phenotypic characteristics of the isolate reported in the earlier description, clearly indicate that this microorganism does not belong to the genus Aeromonas as known to date, although it falls within the radiation of the family Aeromonadaceae. Emendation from the List of Prokaryotic Names with Standing in Nomenclature is consequently proposed (AU)

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Phenotypic, genotypic, and phylogenetic discrepancies to differentiate Aeromonas salmonicidafrom Aeromonas bestiarum / Discrepancias fenotípicas, genotípicas y filogenéticas para diferenciar Aeromonas salmonicida de Aeromonas bestiarum

The taxonomy of the «Aeromonas hydrophila» complex (comprising the species A. hydrophila, A. bestiarum, A. salmonicida, and A. popoffii) has been controversial, particularly the relationship between the two relevant fish pathogens A. salmonicida and A. bestiarum. In fact, none of the biochemical tests evaluated in the present study were able to separate these two species. One hundred and sixteen strains belonging to the four species of this complex were identified by 16S rDNA restriction fragment length polymorphism (RFLP). Sequencing of the 16S rDNA and cluster analysis of the 16S-23S intergenic spacer region (ISR)-RFLP in selected strains of A. salmonicida and A. bestiarum indicated that the two species may share extremely conserved ribosomal operons and demonstrated that, due to an extremely high degree of sequence conservation, 16S rDNA cannot be used to differentiate these two closely related species. Moreover, DNA-DNA hybridization similarity between the type strains of A. salmonicida subsp. salmonicida and A. bestiarum was 75.6 %, suggesting that they may represent a single taxon. However, a clear phylogenetic divergence between A. salmonicida and A. bestiarum was ascertained from an analysis based on gyrB and rpoD gene sequences, which provided evidence of a lack of congruence of the results obtained from 16S rDNA, 16S-23S ISR-RFLP, DNA-DNA pairing, and biochemical profiles (AU)

La taxonomía del complejo «Aeromonas hydrophila» (que comprende las especies A. hydrophila, A. bestiarum, A. salmonicida, y A. popoffii) ha sido controvertida, en particular las relaciones entre los dos patógenos de peces, A. salmonicida y A. bestiarum. De hecho, de las pruebas bioquímicas evaluadas en el presente estudio, ninguna fue capaz de separar estas dos especies. Ciento dieciséis cepas pertenecientes a las cuatro especies de este complejo se identificaron mediante el análisis del polimorfismo de la longitud de los fragmentos de restricción (RFLP) del 16S rDNA. La secuenciación del 16S rDNA y el análisis de grupos de RFLP de la región espaciadora intergénica (ISR) 16S-23S en cepas seleccionadas de A. salmonicida y A. bestiarum indicaron que las dos especies podrían compartir operones ribosómicos extremadamente conservados y demostraron que, debido a su elevado grado de conservación de secuencia, el 16S rDNA no puede utilizarse para diferenciar estas dos especies de relación tan estrecha. Además, la similitud de hibridación DNA-DNA entre las cepas tipo de A. salmonicida subsp. salmonicida y de A. bestiarum era del 75,6 %, lo que sugiere que pueden ser un único taxón. Sin embargo, el análisis simultáneo de las secuencias de los genes gyrB y rpoD puso de manifiesto una marcada divergencia filogenética entre A. salmonicida y A. bestiarum, lo cual aporta pruebas de la falta de congruencia de los resultados de 16S rDNA, ISR-RFLP, 16S-23S, apareamiento DNA-DNA y perfiles bioquímicos (AU)

Phenotypic, genotypic, and phylogenetic discrepancies to differentiate Aeromonas salmonicida from Aeromonas bestiarum.

The taxonomy of the "Aeromonas hydrophila" complex (comprising the species A. hydrophila, A. bestiarum, A. salmonicida, and A. popoffii) has been controversial, particularly the relationship between the two relevant fish pathogens A. salmonicida and A. bestiarum. In fact, none of the biochemical tests evaluated in the present study were able to separate these two species. One hundred and sixteen strains belonging to the four species of this complex were identified by 16S rDNA restriction fragment length polymorphism (RFLP). Sequencing of the 16S rDNA and cluster analysis of the 16S-23S intergenic spacer region (ISR)-RFLP in selected strains of A. salmonicida and A. bestiarum indicated that the two species may share extremely conserved ribosomal operons and demonstrated that, due to an extremely high degree of sequence conservation, 16S rDNA cannot be used to differentiate these two closely related species. Moreover, DNA-DNA hybridization similarity between the type strains of A. salmonicida subsp. salmonicida and A. bestiarum was 75.6 %, suggesting that they may represent a single taxon. However, a clear phylogenetic divergence between A. salmonicida and A. bestiarum was ascertained from an analysis based on gyrB and rpoD gene sequences, which provided evidence of a lack of congruence of the results obtained from 16S rDNA, 16S-23S ISR-RFLP, DNA-DNA pairing, and biochemical profiles.

Comparison of three molecular methods for typing Aeromonas popoffii isolates.

Three typing methods, restriction fragment length polymorphism (RFLP) of the 16S-23S intergenic spacer region (ISR), PCR amplification of the enterobacterial repetitive intergenic consensus (ERIC) and of the repetitive extragenic palindromic units (REP), were evaluated for typing 26 isolates of Aeromonas popoffii from different geographical origins. When the methods were independently studied, ERIC showed the highest discriminatory power. When the methods were combined, the best combination of two methods was ERIC with REP since strains showed a tendency to cluster according to their geographical origin. However, this tendency was reinforced with the addition of ISR-RFLP.

Potential virulence and antimicrobial susceptibility of Aeromonas popoffii recovered from freshwater and seawater.

Aeromonas popoffii is the most recent species within the genus Aeromonas described from freshwater. In our study this species was also recovered from this habitat and for the first time from seawater. Most of the virulence factors known in Aeromonas spp. (aerolysin/hemolysin, serine protease, lipases and DNases) were highly prevalent in this species. Third-generation cephalosporins and quinolones were the most active antimicrobial agents against A. popoffii.