Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR genes The central clade of the genus Vibrio, also called the Vibrio core group, comprises six species that are tightly related (DNA-DNA reassociation values are very close to 70 % for most species pairs). Identification of novel strains to the species level within this group is troublesome and results are quite often dependent on the methodology employed. Therefore, this group represents an excellent framework to test the robustness of multilocus sequence analysis (MLSA) not only for inferring phylogeny but also as an identification tool without the need for DNA-DNA hybridization assays. The genes selected, 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR, were amplified by direct PCR from 44 Vibrio core-group strains. Subsequent analysis allowed us to recognize toxR and rpoD as the most resolving individual genes and showed that concatenated sequences of rpoD, rctB and toxR were more useful than concatenated sequences of all seven genes. To validate our conclusions, MLSA similarities have been correlated with DNA-DNA relatedness values obtained in this study and values taken from the literature. Although the seven concatenated genes gave the best correlation, the concatenated sequences of rpoD, rctB and toxR have the practical advantage of showing a considerable gap between the maximal interspecies similarity and the minimal intraspecies similarity recorded, meaning that they can be used quite conveniently for species identification of vibrios. INTRODUCTIONMLSA (multilocus sequence analysis) is a recently developed technique derived from multilocus sequence typing that has been used successfully in epidemiology studies. The use of MLSA as an alternative method for species delineation in bacteriology is currently being evaluated, following a recommendation of the ad hoc committee for the re-evaluation of the species definition in bacteriology (Stackebrandt et al., 2002). In MLSA studies, several housekeeping genes (more than five) are analysed and relationships between taxa are established. One important advantage of this kind of approach is the availability of genomic sequences from any laboratory, avoiding the problems of lack of comparability that are associated with DNA-DNA reassociation data. However, the usefulness of MLSA for describing bacterial species has to be demonstrated for each of the taxa or group of species studied and for the selected housekeeping genes. Authors should prove that there is a sufficient degree of congruence between the alternative technique (i.e. MLSA) and DNA-DNA reassociation data. To date, the technique has been applied to discrete bacterial taxa, mainly genera of lactic acid bacteria (Naser et al. et al., 2007, 2008) and the Vibrionaceae (Goarant et al., 2006;Urbanczyk et al., 2007;Thompson et al., 2008;Rameshkumar et al., 2008).In the family Vibrionaceae, sequence similarities for the 16S rRNA gene are ¢97.6 % among members of the so-called Vib...

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The virulence mechanisms of Vibrio vulnificus biotype 2 have been studied and compared with those of biotype 1 in mice as the experimental animals. Biotype 2 isolates from European eels were as virulent for mice as biotype 1 strains (509% lethal dose, about 105 CFU per mouse); a septicemic infection developed in less than 24 h. These strains had several properties in common with biotype 1 organisms including capsule expression, uptake of various iron sources, and production of exoproteins, whose role in mouse virulence has been demonstrated. We also discuss the implication of biotype 2 strains in human infections.

Strains of Vibrio vulnificus biotype 2, isolated from internal organs of diseased European eels as pure cultures of opaque cells, together with some reference strains from Japanese eels, were used in this study. Spontaneous translucent-phase variants were obtained from the corresponding parent strains and compared for a variety of phenotypic traits related to virulence for eels. The rate of colony dissociation from opaque to translucent cells was higher (around 10-2) than that observed for translucent to opaque cells (10-3 to 10-4). Electron microscopy with ruthenium red revealed the presence of a capsule of variable thickness on opaque cells, whereas translucent-type colonies had no observable capsular materials. No differences in plasmid profiles were detected between the two cell types so that plasmids do not seem to be implicated in the mechanism of phase shift of biotype 2 strains. No apparent difference in outer membrane protein and lipopolysaccharide patterns could be observed between the cell types. Both isogenic morphotypes were able to grow in eel serum and minimal medium supplemented with ethylenediamine di(O-hydroxyphenyl-acetic acid) or transferrin. Therefore, the presence of capsule was not required for the acquisition of iron from iron chelators or for resistance to serum bactericidal action. Both morphotypes were highly virulent for elvers, although the 50% lethal dose for translucent cells was higher than that for the corresponding opaque cells. The latter observation, together with the overall data, suggests that the production of capsular materials by biotype 2 of V. vulnificus is not essential for the development of vibriosis in eels, at least when cells are injected intraperitoneally.

Phenotypic and genetic studies were performed on some atypical aeromonas strains of uncertain taxonomic position. 16S rRNA gene sequence analysis revealed that these strains represent a hitherto unknown genetic line within the genus Aeromonas, for which the name Aeromonas allosaccharophila sp. nov. is proposed. The type strain is CECT 4199.