David Miñana-Galbis

Reclassification of Geobacillus pallidus (Scholz et al. 1988) Banat et al. 2004 as Aeribacillus pallidus gen. nov., comb. nov.

Although Anoxybacillus and Geobacillus, two genera of thermophilic bacteria close to the genus Bacillus, have only been described recently, the number of species in these genera has increased rapidly. Four thermophilic, lipolytic strains (DR01, DR02, DR03 and DR04) isolated from a hot spring in Veracruz (Mexico), which could not be identified phenotypically, were subjected to 16S rRNA gene sequence analysis. Three strains were identified as belonging to the genus Anoxybacillus, but strain DR03 was identified as Geobacillus pallidus. This result led us to perform a phylogenetic analysis of the genera Anoxybacillus and Geobacillus based on 16S rRNA gene sequences from all the type strains of these genera. Phylogenetic trees showed three major clusters, Anoxybacillus-Geobacillus tepidamans, Geobacillus sensu stricto and Geobacillus pallidus, while the 16S rRNA gene sequences of G. pallidus (DR03 and the type strain) showed low similarity to sequences of Anoxybacillus (92.5-95.1 %) and Geobacillus (92.8-94.5 %) species, as well as to Bacillus subtilis (92.2-92.4 %). In addition, G. pallidus could be differentiated from Anoxybacillus and Geobacillus on the basis of DNA G+C content and fatty acid and polar lipid profiles. From these results, it is proposed that Geobacillus pallidus should be classified in a novel genus, for which we propose the name Aeribacillus, as Aeribacillus pallidus gen. nov., comb. nov. The type strain of Aeribacillus pallidus is H12(T) (=ATCC 51176(T) =DSM 3670(T) =LMG 19006(T)).

Biochemical identification and numerical taxonomy of Aeromonas spp. isolated from environmental and clinical samples in Spain

Aims: To study the phenotypic characteristics of Aeromonas spp. from environmental and clinical samples in Spain and to cluster these strains by numerical taxonomy. 
Methods and Results: A collection of 202 Aeromonas strains isolated from bivalve molluscs, water and clinical samples was tested for 64 phenotypic properties; 91% of these isolates were identified at species level. Aeromonas caviae was predominant in bivalve molluscs and Aerom. bestiarum in freshwater samples. Cluster analyses revealed eight different phena: three containing more than one DNA‐DNA hybridization group but including strains that belong to the same phenospecies complex (Aerom. hydrophila, Aerom. sobria and Aerom. caviae), Aerom. encheleia, Aerom. trota and three containing unidentified Aeromonas strains isolated from bivalve molluscs. 
Conclusions:Aeromonas spp. are widely distributed in environmental and clinical sources. A selection of 16 of the phenotypical tests chosen allowed the identification of most isolates (91%), although some strains remain unidentified, mainly isolates from bivalve molluscs, suggesting the presence of new Aeromonas species. Numerical taxonomy was not in total concordance with the identification of the studied strains. 
Significance and Impact of the Study: Numerical taxonomy of Aeromonas strains isolated from different sources revealed the presence of potentially pathogenic Aeromonas spp., especially in bivalve molluscs, and phena with unidentified strains that suggest new Aeromonas species.

Phylogenetic analysis and identification of Aeromonas species based on sequencing of the cpn60 universal target

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 <or=3.5%, while interspecific divergence rates fell between 3.7 and 16.9%, excluding A. sharmana. In this study, cpn60 UT sequencing was shown to be a universal, useful, simple and rapid method for the identification and phylogenetic affiliation of Aeromonas strains.

Proposal to assign Aeromonas diversa sp. nov. as a novel species designation for Aeromonas group 501

The Aeromonas group 501, also named Aeromonas sp. HG13, is taxonomically close to A. schubertii. Results obtained in previous studies, including DNA-DNA hybridization and DNA fingerprinting, suggest that Aeromonas group 501 could constitute a different Aeromonas species. In this work we have performed a polyphasic study with the two strains comprising the Aeromonas sp. HG13 in order to propose a formal species name. They could be differentiated from A. schubertii by the indole and lysine decarboxylase tests and the utilization of l-lactate. Phenotypically, both strains were also easily separated from the other Aeromonas species. Sequence analysis of the 16S rRNA gene showed high sequence similarities (>97%) between Aeromonas group 501 and all Aeromonas species. Nevertheless, sequence divergences of cpn60, dnaJ, gyrB and rpoD genes were higher than the intraspecific threshold values established for each gene (3.5%, 3.3%, 2.3% and 2.6%, respectively), while sequence divergences between strains CDC 2478-85(T) and CDC 2555-87 were low (0.6-1.1%). The DNA G+C content of the type strain was 62.2mol%. Phenotypic and genotypic evidence strongly suggests that the Aeromonas group 501 is a novel species of the genus Aeromonas, for which the name Aeromonas diversa sp. nov. is proposed. The type strain is CDC 2478-85(T) (=CECT 4254(T)=ATCC 43946(T)=LMG 17321(T)).

Pseudomonas deceptionensis sp. nov., a psychrotolerant bacterium from the Antarctic.

During the taxonomic investigation of cold-adapted bacteria from samples collected in the Antarctic area of the South Shetland Islands, one Gram-reaction-negative, psychrotolerant, aerobic bacterium, designated strain M1(T), was isolated from marine sediment collected on Deception Island. The organism was rod-shaped, catalase- and oxidase-positive and motile by means of a polar flagellum. This psychrotolerant strain grew at temperatures ranging from -4 °C to 34 °C. Phylogenetic studies based on 16S rRNA gene sequences confirmed that Antarctic isolate M1(T) was a member of the genus Pseudomonas and was located in the Pseudomonas fragi cluster. 16S rRNA gene sequence similarity values were >98 % between 13 type strains belonging to the Pseudomonas fluorescens lineage. However, phylogenetic analysis of rpoD gene sequences showed that strain M1(T) exhibited high sequence similarity only with respect to Pseudomonas psycrophila (97.42 %) and P. fragi (96.40 %) and DNA-DNA hybridization experiments between the Antarctic isolate M1(T) and the type strains of these two closely related species revealed relatedness values of 58 and 57 %, respectively. Several phenotypic characteristics, together with the results of polar lipid and cellular fatty acid analyses, were used to differentiate strain M1(T) from related pseudomonads. Based on the evidence of this polyphasic taxonomic study, strain M1(T) represents a novel species, for which the name Pseudomonas deceptionensis sp. nov. is proposed. The type strain is M1(T) ( = LMG 25555(T)  = CECT 7677(T)).

Virulence Factors of Erwinia amylovora: A Review

Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS), the exopolysaccharide (EPS) amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3′-5′)-cyclic di-GMP (c-di-GMP) and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus), have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them.

Shewanella vesiculosa sp. nov., a psychrotolerant bacterium isolated from an Antarctic coastal area.

Two strains of psychrotolerant bacteria, designated M7(T) and M5, isolated from Antarctic coastal marine environments were studied to determine their taxonomic position. The organisms comprised Gram-negative, rod-shaped, facultatively anaerobic cells that were motile by means of single polar flagella. Neither of the bacterial isolates had a requirement for Na(+). These two psychrotolerant strains grew at temperatures ranging from -4 to 30 degrees C. Both strains were capable of producing H(2)S from thiosulfate and were able to use sodium nitrate and trimethylamine N-oxide as terminal electron acceptors during anaerobic growth. 16S rRNA gene sequence analysis placed M7(T) and M5 within the genus Shewanella; the strains showed the highest similarity (99.9 and 99.2 % respectively) with respect to the type strains of Shewanella livingstonensis and Shewanella frigidimarina. However the levels of gyrB sequence similarity between strain M7(T) and the type strains of S. livingstonensis and S. frigidimarina were 87.6 and 87.4 %, respectively. DNA-DNA hybridization experiments performed between the Antarctic isolate M7(T) and S. livingstonensis LMG 19866(T) and S. frigidimarina LMG 19475(T) revealed levels of relatedness of 32 and 35 %, respectively. Strain M5 showed 100 % DNA relatedness with respect to strain M7(T). The DNA G+C content of these bacteria was 42 mol%. Several phenotypic characteristics, the cellular fatty acid compositions and the quinone content of strains M7(T) and M5 served to differentiate them from related shewanellae. On the basis of the data from this polyphasic taxonomic study, M7(T) and M5 constitute a single genospecies. They represent a novel species of the genus Shewanella, for which the name Shewanella vesiculosa sp. nov. is proposed. The type strain is M7(T) (=LMG 24424(T) =CECT 7339(T)).

Malate dehydrogenase: a useful phylogenetic marker for the genus Aeromonas.

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.

Reclassification of Aeromonas hydrophila subspecies anaerogenes

Technological advances together with the continuous description of new taxa have led to frequent reclassifications in bacterial taxonomy. In this study, an extensive bibliographic revision, as well as a sequence analysis of nine housekeeping genes (cpn60, dnaJ, dnaX, gyrA, gyrB, mdh, recA, rpoB and rpoD) and a phenotypic identification of Aeromonas hydrophila subspecies anaerogenes were performed. All data obtained from previous physiological, phylogenetic, and DNA-DNA hybridization studies together with those presented in this study suggested that A. hydrophila subspecies anaerogenes belonged to the species Aeromonas caviae rather than A. hydrophila. Therefore, the inclusion of A. hydrophila subsp. anaerogenes in the species A. caviae is proposed.

The reference strain Aeromonas hydrophicla CIP 57.50 should be reclassified as Aeromonas salmonicida CIP 57.50.

The use of reference strains is a critical element for the quality control of different assays, from the development of molecular methods to the evaluation of antimicrobial activities. Most of the strains used in these assays are not type strains and some of them are cited erroneously because of subsequent reclassifications and descriptions of novel species. In this study, we propose that the reference strain Aeromonas hydrophila CIP 57.50 be reclassified as Aeromonas salmonicida CIP 57.50 based on phenotypic characterization and sequence analyses of the cpn60, dnaJ, gyrB and rpoD genes.