Draft Genome Sequence of Aeromonas lusitana sp. nov. Strain DSM 24905T, Isolated from a Hot Spring in Vila-Real, Portugal.

Aeromonas lusitana sp. nov. is an isolate derived from a study aimed at characterizing Aeromonas spp. from water sources used for recreation and agricultural purposes and assessing the implications these organisms have for human and animal health. We present here the 4.52-Mbp draft genome sequence of this novel species.

Draft Genome Sequence of Aeromonas cavernicola sp. nov. DSM 24474T, Isolated from a Cavern Brook in the Moravia Region of the Czech Republic.

Species of the Aeromonas genus can be found in numerous environmental milieus, including various water sources, and some species cause disease in animals. We present here the draft genome sequence for Aeromonas cavernicola DSM 24474T, a novel species isolated from a freshwater brook within a cavern in the Czech Republic.

Phylogenetic diversity of Aeromonas from "alheira," a traditional Portuguese meat product.

"Alheira" is a traditional smoked meat sausage produced in the north of Portugal, representing an important economic resource for the region. This meat product has been subjected to research studies with the aim of detecting the presence of common foodborne pathogens, but, to our knowledge, isolation of emerging foodborne Aeromonas from alheira has never been previously described. Present work attempts to evaluate the Aeromonas species diversity of 84 isolates of Aeromonas spp. collected from 32 alheira samples. All presumptive Aeromonas isolates were subjected to genotyping by enterobacterial repetitive intergenic consensus-polymerase chain reaction analysis. The isolates presenting a different pattern were subjected to gyrB gene sequencing for species classification, and the species A. hydrophila, A. salmonicida, A. caviae, A. media, and A. allosaccharophila were identified. The Aeromonas species diversity found has not been previously described in any other meat product evaluated in previous studies. It is also important to highlight the presence of A. hydrophila and A. caviae because they were previously associated with illness in humans, including gastroenteritis.

Phylogenetic identification of Aeromonas from pigs slaughtered for consumption in slaughterhouses at the North of Portugal.

In the present study, 710 isolates of Aeromonas spp. have been collected from pig carcasses, diaphragm muscle, faeces, dehairing equipment and water in slaughterhouses at the North of Portugal. The isolates were obtained from a total of 154 samples. All presumptive Aeromonas isolates were subjected to ERIC-PCR analysis and those which presented a different pattern were taken and the species classified by gyrB gene sequencing. We have found the species A. hydrophila, A. salmonicida, A. bestiarum, A. caviae, A. media, A. veronii, A. allosaccharophila, A. simiae and A. aquariorum. To our knowledge, this extent of Aeromonas species diversity has not been previously described from meat or from the slaughter environment, perhaps due to the unreliability of available identification methods. A noticeable level of isolate redundancy (strains with identical gyrB sequence) from different samples collected in different dates was also obtained, indicating that only a few predominant strains of these species persist at the slaughter system. It is also important to emphasise the presence of Aeromonas species previously associated with illness in man.

Aeromonas rivuli sp. nov., isolated from the upstream region of a karst water rivulet.

Two freshwater isolates (WB4.1-19(T) and WB4.4-101), sharing 99.9 % 16S rRNA gene sequence similarity, were highly related to Aeromonas sobria (99.7 % similarity; 6 bp differences). A phylogenetic tree derived from a multi-locus phylogenetic analysis (MLPA) of the concatenated sequences of five housekeeping genes (gyrB, rpoD, recA, dnaJ and gyrA; 3684 bp) revealed that both strains clustered as an independent phylogenetic line next to members of Aeromonas molluscorum and Aeromonas bivalvium. The DNA-DNA reassociation value between the two new isolates was 89.3 %. Strain WB4.1-19(T) had a DNA-DNA relatedness value of <70 % with the type strains of the other species tested. Phenotypic characterization differentiated the two novel strains from all other type strains of species of the genus Aeromonas. It is concluded that the two new strains represent a novel species of the genus Aeromonas, for which the name Aeromonas rivuli sp. nov. is proposed, with the type strain WB4.1-19(T) (=CECT 7518(T)=DSM 22539(T)=MDC 2511(T)).

Aeromonas aquariorum sp. nov., isolated from aquaria of ornamental fish.

During a survey to determine the prevalence of Aeromonas strains in water and skin of imported ornamental fish, 48 strains presumptively identified as Aeromonas were isolated but they could not be identified as members of any previously described Aeromonas species. These strains were subjected to a polyphasic approach including phylogenetic analysis derived from gyrB, rpoD and 16S rRNA gene sequencing, DNA-DNA hybridization, MALDI-TOF MS analysis, genotyping by RAPD and extensive biochemical and antibiotic susceptibility tests in order to determine their taxonomic position. Based on the results of the phylogenetic analyses and DNA-DNA hybridization data, we describe a novel species of the genus Aeromonas, for which the name Aeromonas aquariorum sp. nov. is proposed, with strain MDC47T (=DSM 18362T =CECT 7289T) as the type strain. This is the first Aeromonas species description based on isolations from ornamental fish.

Updated phylogeny 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.

First record of the rare species Aeromonas culicicola from a drinking water supply.

We describe the recovery of the rare species Aeromonas culicicola, so far known only in mosquitoes in India, from a drinking water supply in Spain. Typing, using enterobacterial repetitive intergenic consensus-PCR, revealed that the 27 new isolates belonged to 3 very closely related strains. These strains were genetically identified by 16S rRNA gene sequencing. Spanish strains differed from the mosquito strains in three nucleotide positions. The AHCYTOEN gene was present in these water strains, which may have a public health significance.

Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes.

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.

Phylogenetic analysis of members of the genus Aeromonas based on gyrB gene sequences.

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.

Sequence microdiversity at the ribosomal RNA operons of Escherichia coli pyelonephritogenic strains.

OBJECTIVE: To determine whether Escherichia coli strains isolated from patients with uncomplicated acute pyelonephritis can be distinguished from those isolated from patients with complicated acute pyelonephritis on the basis of the genetic background. METHODS: In total, 103 E. coli strains isolated from patients with acute pyelonephritis (59 uncomplicated pyelonephritis (UAP) and 44 complicated pyelonephritis (CAP)) were characterized by RFLP of the intergenic spacer region 16S-23S rRNA, the presence of three alternative sequences found in the polymorphic V6 loop of the 16S rRNA gene, the presence of the pap gene, and antibiotic susceptibility. RESULTS: At similarity levels of 70%, four RFLP groups (alpha1, alpha2, beta1 and beta2) were discerned. Strains from UAP were statistically significant for alpha RFLP, with a strong association with the presence of the pap gene, V6-I sequence and antibiotic multisensitivity. Strains from CAP randomly belonged to the alpha or beta RFLP groups, with a very low presence of the pap gene, and random presence of V6 sequences, and were multiresistant to antibiotics. When the CAP strains were distributed according to underlying pathology, non-obstructive cases had RFLP and V6 polymorphisms similar to those of UAP cases, while obstructive cases were clearly distinct. CONCLUSIONS: UAP and non-obstructive CAP E. coli strains are sensitive to antimicrobials, show a high level of the pap gene and belong to the selective, homogeneous and highly protected molecular alpha2 group, where no recombinations, deletions or insertions are present. On the contrary, obstructive and vesicorenal reflux E. coli strains show significant antimicrobial resistance, high intercistronic heterogenicity (wide presence of block nucleotidic substitutions, deletions or insertions) and significantly lower virulence.

Características moleculares de cepas de escherichia coli causantes de pielonefritis agudas / Molecular characteristics of Escherichia coli that cause acute pielonefritis

A un total de 103 cepas de E. Boli aisladas de enfermos con pielonefritis aguda (59 cepas de pielonefritis no complicadas (PNAnoC) y 44 de pielonefritis complicadas (PNAC)) y 72 cepas de la colección ECOR se les ha determinado el tipo de secuencia V6 (posiciones 1000-1040) del gen 16S-rRNA, la posesión del gen pap y la secuencia espacio intergénico 16S-23S-rRNA mediante amplificación por PCR usando 7 cebadores específicos, digestión con endonucleasas, electroforesis y análisis de los polimorfismos de longitud de fragmentos de restricción (RFLP). Con niveles de similaridad del 70 por ciento se han formado 4 grupos denominados a-1, a-2, beta-1 y beta-2. El subgrupo a-2 se ha correlacionado con el B-2 de MLEE considerado de maxima virulencia.Las cepas aisladas de PNAnoC mostraron una asociación causal significativa (p <0,01) con el subgrupo molecular a-2 (74 por ciento) , secuencia V6-1(91,2 por ciento) y presencia del gen pap (85,3 por ciento). Por contra, en las cepas aisladas de PNAC no se observó relación de causalidad (p = n. s.) entre los grupos moleculares, la secuencia V6-I y la presencia del gen pap. La agrupación de las cepas de PNAC según el diagnóstico de la patología de base (no obstructiva: 13 casos, obstructiva: 20 casos y reflujo vesico-renal: 11 casos) demostró que para los casos no obstructivos se conservaba una estructura molecular estrechamente relacionada a los casos de PNAnoC, mientras que para el resto la pertenencia al grupo alfa/beta y tipo de secuencia era aleatorio con una presencia del gen pap muy baja (12,9 por ciento). La incidencia de polirresistencia a los antibióticos en el grupo a fue bastante menor que en el grupo beta (5,6 por ciento versus 25 por ciento respectivamente).En el grupo beta la frecuencia del gen pap en cepas polirresistentes disminuyó más de 4 veces respecto a las cepas sensibles o con monorresistencia de su propio grupo o más de 6 veces respecto a las del grupo a Se concluye que las cepas de PNAnoC poseen una elevada virulencia y homogeneidad y están altamente protegidas de recombinaciones o reordenaciones con patrón molecular de: subgrupo a-2 + presencia gen pap + secuencia V6-I + multisensibilidad a los antibióticos. Las cepas de PNAC (excepto los casos no obstructivos) tienen gran variabilidad intercistrónica (grupo alfa/beta, tipo de secuencia V6), poseen una baja virulencia y son mas polirresistentes a los antibióticos. (AU)

Typing of clinical and environmental Aeromonas veronii strains based on the 16S-23S rDNA spacers.

Genetic relationships of Aeromonas veronii strains isolated from human and environmental sources were investigated by restriction fragment length polymorphism (RFLP) of the polymerase chain reaction-amplified intergenic spacer region (ISR) flanked by the 16S and 23S rRNA genes. When using endonucleases AluI, HinfI and CfoI the 16S-23S rDNA-RFLP patterns showed considerable overall similarity, although most strains yielded specific profiles. Several intra-specific lines of descent comprised clinical strains linked to isolates from environmental sources. Strains having identical patterns may be individuals derived from highly similar, if not the same, microorganism. Results suggest that the ISR sequence-based method can be used to demonstrate colonization of a public water supply with a particular microorganism. In addition it could be very useful for tracing recurrent episodes of diarrhea and Aeromonas infection outbreaks.

Extended method for discrimination of Aeromonas spp. by 16S rDNA RFLP analysis.

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

Phylogenetic positions of Aeromonas encheleia, Aeromonas popoffii, Aeromonas DNA hybridization group 11 and Aeromonas group 501.

The 16S rDNA sequences of the recently described Aeromonas encheleia and Aeromonas popoffii, were determined and compared with data from all known Aeromonas sp. Diagnostic 16S rDNA regions were also sequenced for some strains previously considered as an extension of A. encheleia and a strain of Aeromonas Group 501 (formerly Enteric Group 501). Results indicated that A. encheleia and A. popoffii are phylogenetically separated species as originally described. A conclusion about HG11 taxonomic status is not recommended until previous discrepancies are clarified by further DNA-DNA hybridization and sequencing studies.

Patterns of sequence variation in two regions of the 16S rRNA multigene family of Escherichia coli.

Sequence heterogeneities of variable positions located at regions V1 and V6 of 56 cloned 16S rRNA genes were determined from six Escherichia coli strains. These nucleotides were involved in secondary structure base-pairing of stem-loops. Compensatory and single mutations have occurred but secondary structure was conserved. Eight different sequences were found in the stem at region V1 indicating that in these sites mutation rates are higher than those of homogenizatin processes. Region V6 showed two different structures (V6-I and V6-II) although heterogeneities were determined in nine sites. Strains ECOR52 and ECOR56 only showed the V6-I sequence, ECOR35 showed V6-II, whereas clones from ECOR42 and ECOR49 showed both types of V6 structures. Results were confirmed by PCR using V6 sequence-specific probes. Stem V6-II was also found in 16S rRNA sequences deposited in the RDP (Ribosomal Database Project) belonging to distantly related taxa; ancestral sequence V6-II seems to be homogenized in all rrn operons of the multigene family of strain ECOR35 producing effects of distortion in the molecular clock, similar to those that homoplasies could produce. V6 sequence-specific probes were applied to the 72 ECOR strains: half showed both V6-I and V6-II, and the rest had one or another. Only strain ECOR24 did not yield products in the PCR test and sequencing of 12 cloned 16S rRNA genes revealed a third form, V6-III, also found in the RDP. Concerted evolution by homogenization of the rRNA family may induce chronometric distortions responsible for a loss of ultrametricity in phylogenetic trees, particularly, of very closely related micro-organisms.

Intraspecific diversity of the 23S rRNA gene and the spacer region downstream in Escherichia coli.

The molecular microevolution of the 23S rRNA gene (rrl) plus the spacer downstream has been studied by sequencing of different operons from some representative strains of the Escherichia coli ECOR collection. The rrl gene was fully sequenced in six strains showing a total of 67 polymorphic sites, a level of variation per nucleotide similar to that found for the 16S rRNA gene (rrs) in a previous study. The size of the gene was highly conserved (2902 to 2905 nucleotides). Most polymorphic sites were clustered in five secondary-structure helices. Those regions in a large number of operons were sequenced, and several variations were found. Sequences of the same helix from two different strains were often widely divergent, and no intermediate forms existed. Intercistronic variability was detected, although it seemed to be lower than for the rrs gene. The presence of two characteristic sequences was determined by PCR analysis throughout all of the strains of the ECOR collection, and some correlations with the multilocus enzyme electrophoresis clusters were detected. The mode of variation of the rrl gene seems to be quite similar to that of the rrs gene. Homogenization of the gene families and transfer of sequences from different clonal lines could explain this pattern of variation detected; perhaps these factors are more relevant to evolution than single mutation. The spacer region between the 23S and 5S rRNA genes exhibited a highly polymorphic region, particularly at the 3' end.

Sequence diversity in the 16S-23S intergenic spacer region (ISR) of the rRNA operons in representatives of the Escherichia coli ECOR collection.

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.

Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes.

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.

Intraspecific genetic diversity of Oenococcus oeni as derived from DNA fingerprinting and sequence analyses.

The intraspecific genetic diversity of Oenococcus oeni, the key organism in the malolactic fermentation of wine, has been evaluated by random amplified polymorphic DNA (RAPD), ribotyping, small-plasmid content, and sequencing of RAPD markers with widespread distribution among the strains. Collection strains representing the diversity of this species have been studied together with some new isolates, many of which were obtained from wines produced by spontaneous malolactic fermentation. The RAPD profiles were strain specific and discerned two main groups of strains coincident with clusters obtained by macrorestriction typing in a previous work. Ribotyping and the conservation of RAPD markers indicates that O. oeni is a relatively homogeneous species. Furthermore, identical DNA sequences of some RAPD markers among strains representative of the most divergent RAPD clusters indicates that O. oeni is indeed a phylogenetically tight group, probably corresponding to a single clone, or clonal line of descent, specialized to grow in the wine environment and universally spread.