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.

Recent phylogenetic studies of the genus Aeromonas based on gyrB and rpoD gene sequences have improved the phylogeny based on 16S rRNA gene sequences first published in 1992, particularly in the ability to split closely related species. These studies did not include the recently described species Aeromonas simiae and Aeromonas molluscorum and only a single strain of Aeromonas culicicola was available for analysis at that time. In the present work, these Aeromonas species and newly isolated strains of A. culicicola were examined. Sequence analysis indicates that A. simiae and A. molluscorum belong to non-described phylogenetic lines of descent within this genus, which supports the original description of both species. The most closely related species are Aeromonas schubertii and Aeromonas encheleia, respectively, which is consistent with 16S rRNA gene sequencing results. However, while the five strains of A. molluscorum showed nucleotide differences in their gyrB and rpoD gene sequences, the only two known A. simiae strains exhibited identical gene sequences, suggesting that they are isolates of the same strain. On the basis of the rpoD gene sequence phylogeny, A. culicicola strains from the original description and new isolates from drinking water and ornamental fish clustered within the species Aeromonas veronii, suggesting inconsistencies with previous results. Other strains with previously controversial taxonomy and new isolates from other studies were included in this study in order to clarify their phylogenetic affiliation at the species level.

Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.

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 sigma70 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 taxonomic diversity and antibiotic resistance phenotypes of aeromonads were examined in samples from drinking and waste water treatment plants (surface, ground and disinfected water in a drinking water treatment plant, and raw and treated waste water) and tap water. Bacteria identification and intra-species variation were determined based on the analysis of the 16S rRNA, gyrB and cpn60 gene sequences. Resistance phenotypes were determined using the disc diffusion method. Aeromonas veronii prevailed in raw surface water, Aeromonas hydrophyla in ozonated water, and Aeromonas media and Aeromonas puntacta in waste water. No aeromonads were detected in ground water, after the chlorination tank or in tap water. Resistance to ceftazidime or meropenem was detected in isolates from the drinking water treatment plant and waste water isolates were intrinsically resistant to nalidixic acid. Most of the times, quinolone resistance was associated with the gyrA mutation in serine 83. The gene qnrS, but not the genes qnrA, B, C, D or qepA, was detected in both surface and waste water isolates. The gene aac(6')-ib-cr was detected in different waste water strains isolated in the presence of ciprofloxacin. Both quinolone resistance genes were detected only in the species A. media. This is the first study tracking antimicrobial resistance in aeromonads in drinking, tap and waste water and the importance of these bacteria as vectors of resistance in aquatic environments is discussed.

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.

The reconstruction of correct genealogies among biological entities, the estimation of the divergence time between organisms or the study of the different events that occur along evolutionary lineages are not always based on suitable genes. For reliable results, it is necessary to look at full-length sequences of genes under stabilizing selection (neutral or purifying) and behaving as good molecular clocks. In bacteria it has been proved that the malate dehydrogenase gene (mdh) can be used to determine the inter- and intraspecies divergence, and hence this gene constitutes a potential marker for phylogeny and bacterial population genetics. We have sequenced the full-length mdh gene in 36 type and reference strains of Aeromonas. The species grouping obtained in the phylogenetic tree derived from mdh sequences was in agreement with that currently accepted for the genus Aeromonas. The maximum likelihood models applied to our sequences indicated that the mdh gene is highly conserved among the Aeromonas species and the main evolutionary force acting on it is purifying selection. Only two sites under potential diversifying selection were identified (T 108 and S 193). In order to determine if these two residues could have an influence on the MDH structure, we mapped them in a three-dimensional model constructed from the sequence of A. hydrophila using the human mitochondrial MDH as a template. The presence of purifying selection together with the linear relationship between substitutions and gene divergence makes the mdh an excellent candidate gene for a phylogeny of Aeromonas and probably for other bacterial groups.

The bacterial flagellum is the most important organelle of motility in bacteria and plays a key role in many bacterial lifestyles, including virulence. The flagellum also provides a paradigm of how hierarchical gene regulation, intricate protein-protein interactions and controlled protein secretion can result in the assembly of a complex multi-protein structure tightly orchestrated in time and space. As if to stress its importance, plants and animals produce receptors specifically dedicated to the recognition of flagella. Aside from motility, the flagellum also moonlights as an adhesion and has been adapted by humans as a tool for peptide display. Flagellar sequence variation constitutes a marker with widespread potential uses for studies of population genetics and phylogeny of bacterial species. We sequenced the complete flagellin gene (flaA) in 18 different species and subspecies of Aeromonas. Sequences ranged in size from 870 (A. allosaccharophila) to 921 nucleotides (A. popoffii). The multiple alignment displayed 924 sites, 66 of which presented alignment gaps. The phylogenetic tree revealed the existence of two groups of species exhibiting different FlaA flagellins (FlaA1 and FlaA2). Maximum likelihood models of codon substitution were used to analyze flaA sequences. Likelihood ratio tests suggested a low variation in selective pressure among lineages, with an omega ratio of less than 1 indicating the presence of purifying selection in almost all cases. Only one site under potential diversifying selection was identified (isoleucine in position 179). However, 17 amino acid positions were inferred as sites that are likely to be under positive selection using the branch-site model. Ancestral reconstruction revealed that these 17 amino acids were among the amino acid changes detected in the ancestral sequence. The models applied to our set of sequences allowed us to determine the possible evolutionary pathway followed by the flaA gene in Aeromonas, suggesting that this gene have probably been evolving independently in the two groups of Aeromonas species since the divergence of a distant common ancestor after one or several episodes of positive selection. This article was reviewed by Alexey Kondrashov, John Logsdon and Olivier Tenaillon (nominated by Laurence D Hurst).

An analysis of the universal target (UT) sequence from the cpn60 gene was performed in order to evaluate its usefulness in phylogenetic and taxonomic studies and as an identification marker for the genus Aeromonas. Sequences of 555 bp, corresponding to the UT region, were obtained from a collection of 35 strains representing all of the species and subspecies of Aeromonas. From the analysis of these sequences, a range of divergence of 0-23.3% was obtained, with a mean of 11.2+/-0.9%. Comparative analyses between cpn60 and gyrB, rpoD and 16S rRNA gene sequences were carried out from the same Aeromonas strain collection. Sequences of the cpn60 UT region showed similar discriminatory power to gyrB and rpoD sequences. The phylogenetic relationships inferred from cpn60 sequence distances indicated an excellent correlation with the present affiliation of Aeromonas species with the exception of Aeromonas hydrophila subsp. dhakensis, which appeared in a separate phylogenetic line, and Aeromonas sharmana, which exhibited a very loose phylogenetic relationship to the genus Aeromonas. Sequencing of cpn60 from 33 additional Aeromonas strains also allowed us to establish intra- and interspecific threshold values. Intraspecific divergence rates were

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To evaluate the usefulness of partial recA sequences for the identification of Aeromonas strains at the genotype level. A partial recA sequence was obtained from 21 type or reference strains and 33 Aeromonas isolates, collected in the South of Switzerland from human, animal and aquatic environments. The 272 bp long recA fragments showed a mean interspecies divergence of 7.8% and allowed the classification of strains at genotype level. However, some discrepancies could be observed with other gene sequence based analyses in the classification of some strains. The 272 bp long recA fragment is a good molecular marker to infer taxonomy of members of the genus Aeromonas, even if the primers we chose for the amplification did not allow its direct sequencing. In the genus Aeromonas, nucleotide sequences of some protein-encoding genes have already been evaluated as molecular markers to be used in taxonomical and epidemiological researches. This study suggests the usefulness of a recA fragment as a further sequence to investigate for these purposes.

Aeromonas spp. are versatile bacteria that exhibit a wide variety of lifestyles. In an attempt to improve the understanding of human aeromonosis, we investigated whether clinical isolates displayed specific characteristics in terms of genetic diversity, population structure and mode of evolution among Aeromonas spp. A collection of 195 Aeromonas isolates from human, animal and environmental sources was therefore genotyped using multilocus sequence analysis (MLSA) based on the dnaK, gltA, gyrB, radA, rpoB, tsf and zipA genes. The MLSA showed a high level of genetic diversity among the population, and multilocus-based phylogenetic analysis (MLPA) revealed 3 major clades: the A. veronii, A. hydrophila and A. caviae clades, among the eleven clades detected. Lower genetic diversity was observed within the A. caviae clade as well as among clinical isolates compared to environmental isolates. Clonal complexes, each of which included a limited number of strains, mainly corresponded to host-associated subsclusters of strains, i.e., a fish-associated subset within A. salmonicida and 11 human-associated subsets, 9 of which included only disease-associated strains. The population structure was shown to be clonal, with modes of evolution that involved mutations in general and recombination events locally. Recombination was detected in 5 genes in the MLSA scheme and concerned approximately 50% of the STs. Therefore, these recombination events could explain the observed phylogenetic incongruities and low robustness. However, the MLPA globally confirmed the current systematics of the genus Aeromonas. Evolution in the genus Aeromonas has resulted in exceptionally high genetic diversity. Emerging from this diversity, subsets of strains appeared to be host adapted and/or disease specialized" while the A. caviae clade displayed an atypical tempo of evolution among aeromonads. Considering that A. salmonicida has been described as a genetically uniform pathogen that has adapted to fish through evolution from a variable ancestral population, we hypothesize that the population structure of aeromonads described herein suggested an ongoing process of adaptation to specialized niches associated with different degrees of advancement according to clades and clusters."

The genus Aeromonas has been described as comprising several species associated with the aquatic environment, which represents their principal reservoir. Aeromonas spp. are commonly isolated from diseased and healthy fish, but the involvement of such bacteria in human infection and gastroenteritis has frequently been reported. The primary challenge in establishing an unequivocal link between the Aeromonas genus and pathogenesis in humans is the extremely complicated taxonomy. With the aim of clarifying taxonomic relationships among the strains and phenotypes, a multilocus sequencing approach was developed and applied to characterize 23 type and reference strains of Aeromonas spp. and a collection of 77 field strains isolated from fish, crustaceans, and mollusks. All strains were also screened for putative determinants of virulence by PCR (ast, ahh1, act, asa1, eno, ascV, and aexT) and the production of acylated homoserine lactones (AHLs). In addition, the phenotypic fingerprinting obtained from 29 biochemical tests was submitted to the nonparametric combination (NPC) test methodology to define the statistical differences among the identified genetic clusters. Multilocus sequence typing (MLST) achieved precise strain genotyping, and the phylogenetic analysis of concatenated sequences delineated the relationship among the taxa belonging to the genus Aeromonas, providing a powerful tool for outbreak traceability, host range diffusion, and ecological studies. The NPC test showed the feasibility of phenotypic differentiation among the majority of the MLST clusters by using a selection of tests or the entire biochemical fingerprinting. A Web-based MLST sequence database (http://pubmlst.org/aeromonas) specific for the Aeromonas genus was developed and implemented with all the results.

The exeF-exeG intergenic regions from different hybridization groups (HG) of Aeromonas were studied by PCR amplification using a single pair of primers. Six main classes of PCR products were identified according to size: 360 bp, 320 bp, 280 bp, 230-240 bp, 220 bp and 160 bp. Direct sequencing of the PCR products indicated that the shorter intergenic regions had probably originated from deletion of DNA segments between direct repeats. Correlation of certain PCR products with Aeromonas caviae (HG4), A. caviae (HG5), A. veronii (HG8) and A. salmonicida (HG3) was revealed. The PCR reaction was also shown to be generally specific for Aeromonas spp. Thus, the usefulness of this rapid, single colony-based PCR test for both identification and preliminary differentiation of Aeromonas spp. is demonstrated.

It is well known that water constitutes an important contamination route for microorganisms. This is especially true for Aeromonas which are widespread in untreated and treated waters. In this study, Portuguese untreated waters not regularly monitored were screened for the presence and diversity of aeromonads. A total of 206 isolates were discriminated by RAPD-PCR and 80 distinct strains were identified by gyrB based phylogenetic analysis. The most frequently detected species were Aeromonas hydrophila, Aeromonas bestiarum and Aeromonas media. The antibiotic susceptibility profile of these strains was determined and showed a typical profile of the genus. Nonetheless, the percentage of resistant strains to tetracycline, chloramphenicol and/or trimethoprim/sulfamethoxazole was lower than that reported for clinical isolates and isolates recovered from aquacultures and other environments historically subjected to antibiotic contamination. This suggests that the existence of such pressures in those environments selects for resistant Aeromonas. A similar trend for integron presence was found. Genes coding for CphA and TEM, and tet(A), (E), (C) or (D) genes were found in 28%, 1%, and 10% of the strains, respectively. 10% of the strains contained an integron. Variable regions of seven class 1 integrons and one class 2 integron were characterised. Furthermore, strains displayed virulence related phenotypes such as extracellular lipolytic and proteolytic activities as well as aerolysin related genes (43% of strains). The ascV and aexT genes were found in 16% and 3% of strains respectively and, in some cases, concomitantly in the same specimen. This study shows that diverse Aeromonas spp. presenting distinct antibiotic resistance features and putative virulence traits are frequently present in waters for human and animal consumption in Portugal. Genes associated to antibiotic resistance and microbial virulence previously identified in organisms with human health significance were detected in these aeromonads, suggesting that these waters may act as a pivotal route for infections.