Whole-Genome Sequencing and Comparative Genomics of Three Helicobacter pylori Strains Isolated from the Stomach of a Patient with Adenocarcinoma.

Helicobacter pylori is a common pathogen associated with several severe digestive diseases. Although multiple virulence factors have been described, it is still unclear the role of virulence factors on H. pylori pathogenesis and disease progression. Whole genome sequencing could help to find genetic markers of virulence strains. In this work, we analyzed three complete genomes from isolates obtained at the same point in time from a stomach of a patient with adenocarcinoma, using multiple available bioinformatics tools. The genome analysis of the strains B508A-S1, B508A-T2A and B508A-T4 revealed that they were cagA, babA and sabB/hopO negative. The differences among the three genomes were mainly related to outer membrane proteins, methylases, restriction modification systems and flagellar biosynthesis proteins. The strain B508A-T2A was the only one presenting the genotype vacA s1, and had the most distinct genome as it exhibited fewer shared genes, higher number of unique genes, and more polymorphisms were found in this genome. With all the accumulated information, no significant differences were found among the isolates regarding virulence and origin of the isolates. Nevertheless, some B508A-T2A genome characteristics could be linked to the pathogenicity of H. pylori.

Detection of Helicobacter pylori Microevolution and Multiple Infection from Gastric Biopsies by Housekeeping Gene Amplicon Sequencing.

Despite the great efforts devoted to research on Helicobacter pylori, the prevalence of single-strain infection or H. pylori mixed infection and its implications in the mode of transmission of this bacterium are still controversial. In this study, we explored the usefulness of housekeeping gene amplicon sequencing in the detection of H. pylori microevolution and multiple infections. DNA was extracted from five gastric biopsies from four patients infected with distinct histopathological diagnoses. PCR amplification of six H. pylori-specific housekeeping genes was then assessed on each sample. Optimal results were obtained for the cgt and luxS genes, which were selected for amplicon sequencing. A total of 11,833 cgt and 403 luxS amplicon sequences were obtained, 2042 and 112 of which were unique sequences, respectively. All cgt and luxS sequences were clustered at 97% to 9 and 13 operational taxonomic units (OTUs), respectively. For each sample from a different patient, a single OTU comprised the majority of sequences in both genes, but more than one OTU was detected in all samples. These results suggest that multiple infections with a predominant strain together with other minority strains are the main way by which H. pylori colonizes the human stomach.

Health Benefits of Heat-Killed (Tyndallized) Probiotics: An Overview.

Nowadays, the oral use of probiotics is widespread. However, the safety profile with the use of live probiotics is still a matter of debate. Main risks include: Cases of systemic infections due to translocation, particularly in vulnerable patients and pediatric populations; acquisition of antibiotic resistance genes; or interference with gut colonization in neonates. To avoid these risks, there is an increasing interest in non-viable microorganisms or microbial cell extracts to be used as probiotics, mainly heat-killed (including tyndallized) probiotic bacteria (lactic acid bacteria and bifidobacteria). Heat-treated probiotic cells, cell-free supernatants, and purified key components are able to confer beneficial effects, mainly immunomodulatory effects, protection against enteropathogens, and maintenance of intestinal barrier integrity. At the clinical level, products containing tyndallized probiotic strains have had a role in gastrointestinal diseases, including bloating and infantile coli-in combination with mucosal protectors-and diarrhea. Heat-inactivated probiotics could also have a role in the management of dermatological or respiratory allergic diseases. The reviewed data indicate that heat-killed bacteria or their fractions or purified components have key probiotic effects, with advantages versus live probiotics (mainly their safety profile), positioning them as interesting strategies for the management of common prevalent conditions in a wide variety of patients´ characteristics.

Marinobacter maroccanus sp. nov., a moderately halophilic bacterium isolated from a saline soil.

During the taxonomic investigation of exopolymer-producing halophilic bacteria, a rod-shaped, motile, Gram-stain-negative, halophilic bacterium, designated strain N4T, was isolated from a saline soil located in northern Morocco. Optimal growth of the isolate was at 30-37 ºC and at pH 7.0-8.0, in the presence of 5-7 % (w/v) NaCl. Useful characteristics for the phenotypic differentiation of strain N4T from other Marinobacter species included α-chymotrypsin and α-glucosidase activities and the carbohydrate assimilation profile. The major fatty acids detected in strain N4T were C16:0 and C18:1ω9c and the predominant respiratory quinone was ubiquinone-9. Sequence analysis of the 16S rRNA gene indicated that strain N4T belonged to the genus Marinobacter and was closely related to the type strains of Marinobacter adhaerens (99.04 % similarity), Marinobacter salsuginis (98.97 %) and Marinobacter flavimaris (98.36 %). Phylogenetic analysis of the rpoD gene sequence also showed that the nearest neighbours of strain N4T were M. salsuginis (91.49 % similarity), M. adhaerens and M. flavimaris (90.63 %). Strain N4T showed 87.98 % average nucleotide identity with M. flavimaris and M. salsuginis, and 87.47 % with M. adhaerens. Regarding in-silico genome-to-genome distance, strain N4T showed DNA-DNA hybridization values of 33.30 % with M. adhaerens, 34.60 % with M. flavimaris and 34.70 % with M. salsuginis. The DNA G+C content of strain N4T was 57.3 mol%. Based on the results of phenotypic characterization, phylogenetic analysis and genome comparison, strain N4T represents a novel species of the genus Marinobacter, for which the name Marinobacter maroccanus sp. nov. is proposed. The type strain is N4T (=CECT 9525T=LMG 30466T).

Complete Genome Sequence of Marinobacter flavimaris LMG 23834T, Which Is Potentially Useful in Bioremediation.

The complete genome sequence of the halophilic strain Marinobacter flavimaris LMG 23834T is presented here. The genomic information of this type strain will be useful for taxonomic purposes and for its potential use in bioremediation studies.

Usefulness of Housekeeping Genes for the Diagnosis of Helicobacter pylori Infection, Strain Discrimination and Detection of Multiple Infection.

BACKGROUND: Helicobacter pylori infects human stomachs of over half the world's population, evades the immune response and establishes a chronic infection. Although most people remains asymptomatic, duodenal and gastric ulcers, MALT lymphoma and progression to gastric cancer could be developed. Several virulence factors such as flagella, lipopolysaccharide, adhesins and especially the vacuolating cytotoxin VacA and the oncoprotein CagA have been described for H. pylori. Despite the extensive published data on H. pylori, more research is needed to determine new virulence markers, the exact mode of transmission or the role of multiple infection. MATERIALS AND METHODS: Amplification and sequencing of six housekeeping genes (amiA, cgt, cpn60, cpn70, dnaJ, and luxS) related to H. pylori pathogenesis have been performed in order to evaluate their usefulness for the specific detection of H. pylori, the genetic discrimination at strain level and the detection of multiple infection. A total of 52 H. pylori clones, isolated from 14 gastric biopsies from 11 patients, were analyzed for this purpose. RESULTS: All genes were specifically amplified for H. pylori and all clones isolated from different patients were discriminated, with gene distances ranged from 0.9 to 7.8%. Although most clones isolated from the same patient showed identical gene sequences, an event of multiple infection was detected in all the genes and microevolution events were showed for amiA and cpn60 genes. CONCLUSIONS: These results suggested that housekeeping genes could be useful for H. pylori detection and to elucidate the mode of transmission and the relevance of the multiple infection.

Potential pathogenicity of Aeromonas hydrophila complex strains isolated from clinical, food, and environmental sources.

Aeromonas are autochthonous inhabitants of aquatic environments, including chlorinated and polluted waters, although they can also be isolated from a wide variety of environmental and clinical sources. They cause infections in vertebrates and invertebrates and are considered to be an emerging pathogen in humans, producing intestinal and extra-intestinal diseases. Most of the clinical isolates correspond to A. hydrophila, A. caviae, and A. veronii bv. Sobria, which are described as the causative agents of wound infections, septicaemia, and meningitis in immunocompromised people, and diarrhoea and dysenteric infections in the elderly and children. The pathogenic factors associated with Aeromonas are multifactorial and involve structural components, siderophores, quorum-sensing mechanisms, secretion systems, extracellular enzymes, and exotoxins. In this study, we analysed a representative number of clinical and environmental strains belonging to the A. hydrophila species complex to evaluate their potential pathogenicity. We thereby detected their enzymatic activities and antibiotic susceptibility pattern and the presence of virulence genes (aer, alt, ast, and ascV). The notably high prevalence of these virulence factors, even in environmental strains, indicated a potential pathogenic capacity. Additionally, we determined the adhesion capacity and cytopathic effects of this group of strains in Caco-2 cells. Most of the strains exhibited adherence and caused complete lysis.

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.

Genome Sequence of Plesiomonas shigelloides Strain 302-73 (Serotype O1).

Plesiomonas shigelloides, the only species of the genus, is an emergent pathogenic bacterium associated with human diarrheal and extraintestinal disease. We present the whole-genome sequence analysis of the representative strain for the O1 serotype (strain 302-73), providing a tool for studying bacterial outbreaks, virulence factors, and accurate diagnostic methods.

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.

Enhanced polyhydroxyalkanoates accumulation by Halomonas spp. in artificial biofilms of alginate beads

Microbial mats are complex but stable, multi-layered and multi-functional biofilms, which are the most frequent bacterial formations in nature. The functional strategies and physiological versatility of the bacterial populations growing in microbial mats allow bacteria to resist changing conditions within their environment. One of these strategies is the accumulation of carbon- and energy-rich polymers that permit the recovery of metabolic activities when favorable conditions are restored. In the present study, we systematically screened microbial mats for bacteria able to accumulate large amounts of the ester carbon polymers polyhydroxyalkanoates (PHA). Several of these strains were isolated from Ebro Delta microbial mats and their ability to accumulate PHA up to 40-60% of their dry weight was confirmed. According to two identification approaches (16S rRNA and rpoD genes), these strains were identified as Halomonas alkaliphila (MAT-7, -13, -16), H. neptunia (MAT-17), and H. venusta (MAT-28). To determine the mode of growth yielding maximum PHA accumulation, these three different species were cultured in an artificial biofilm in which the cells were immobilized on alginate beads. PHA accumulation by cells that had detached from the biofilm was compared with that of their planktonic counterparts. Experiments in different culture media showed that PHA accumulation, measured as the relative fluorescence intensity after 48 h of incubation at 30 degrees C, was higher in immobilized than in planktonic cells, with the exception of cells growing in 5% NaCl, in which PHA accumulation was drastically lower in both. Therefore, for obtaining high PHA concentrations, the use of immobilized cells may be a good alternative to the PHA accumulation by bacteria growing in the classical, planktonic mode. From the ecological point of view, increased PHA accumulation in detached cells from biofilms would be a natural strategy to improve bacterial dispersion capacity and, consequently, to increase survival in stressed environments (AU)

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Enhanced polyhydroxyalkanoates accumulation by Halomonas spp. in artificial biofilms of alginate beads.

Microbial mats are complex but stable, multi-layered and multi-functional biofilms, which are the most frequent bacterial formations in nature. The functional strategies and physiological versatility of the bacterial populations growing in microbial mats allow bacteria to resist changing conditions within their environment. One of these strategies is the accumulation of carbon- and energy-rich polymers that permit the recovery of metabolic activities when favorable conditions are restored. In the present study, we systematically screened microbial mats for bacteria able to accumulate large amounts of the ester carbon polymers polyhydroxyalkanoates (PHA). Several of these strains were isolated from Ebro Delta microbial mats and their ability to accumulate PHA up to 40-60% of their dry weight was confirmed. According to two identification approaches (16S rRNA and rpoD genes), these strains were identified as Halomonas alkaliphila (MAT-7, -13, -16), H. neptunia (MAT-17), and H. venusta (MAT-28). To determine the mode of growth yielding maximum PHA accumulation, these three different species were cultured in an artificial biofilm in which the cells were immobilized on alginate beads. PHA accumulation by cells that had detached from the biofilm was compared with that of their planktonic counterparts. Experiments in different culture media showed that PHA accumulation, measured as the relative fluorescence intensity after 48 h of incubation at 30 degrees C, was higher in immobilized than in planktonic cells, with the exception of cells growing in 5% NaCl, in which PHA accumulation was drastically lower in both. Therefore, for obtaining high PHA concentrations, the use of immobilized cells may be a good alternative to the PHA accumulation by bacteria growing in the classical, planktonic mode. From the ecological point of view, increased PHA accumulation in detached cells from biofilms would be a natural strategy to improve bacterial dispersion capacity and, consequently, to increase survival in stressed environments.

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)).

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.

Prediction of whole-genome DNA G+C content within the genus Aeromonas based on housekeeping gene sequences.

Different methods are available to determine the G+C content (e.g. thermal denaturation temperature or high performance liquid chromatography, HPLC), but obtained values may differ significantly between strains, as well as between laboratories. Recently, several authors have demonstrated that the genomic DNA G+C content of prokaryotes can be reliably estimated from one or several protein coding gene nucleotide sequences. Few G+C content values have been published for the Aeromonas species described and the data, when available, are often incomplete or provide only a range of values. Our aim in this current work was twofold. First, the genomic G+C content of the type or reference strains of all species and subspecies of the genus Aeromonas was determined with a traditional experimental method in the same laboratory. Second, we wanted to see if the sequence-based method to estimate the G+C content described by Fournier et al. [7] could be applied to determine the G+C content of the different species of Aeromonas from the sequences of the genes used in taxonomy or phylogeny for this genus.

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.

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)).

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)).

Divergent evolution and purifying selection of the flaA gene sequences in Aeromonas.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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. REVIEWERS: This article was reviewed by Alexey Kondrashov, John Logsdon and Olivier Tenaillon (nominated by Laurence D Hurst).

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