| Abstract
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Vibrio vulnificus is a heterogeneous gram-negative bacterium from brackish and saline water. The species comprise avirulent and human/fish virulent strains from environment or clinical sources. Based on some phenotypic differences the species has been subdivided into three biotypes; biotype 1 includes environmental (water, selfish...) and human strains isolated worldwide, biotype 2 clinical strains from fish and a few human strains (serovar E isolates) all of them isolated worldwide, and biotype 3 clinical strains from human outbreaks geographically restricted to Israel. However, this classification does not reflect the real situation of the species as the characters used for the biotyping are not longer useful for the correct characterization of new isolates. The goal of this work was to analyze the real structure of the species and the relationships between the isolates of the different biotypes. To this end, we firstly increased our V. vulnificus collection with strains of the three biotypes isolated from different sources and geographical regions. We focused our work on the biotype 2 of the species because its high incidence in eel culture in Spain. This biotype was considered an obligate eel pathogen although previous works of our group demonstrated that this pathogen was able to survive for long periods of time in the water. Our first objective was developing a new isolation protocol that allows the specific isolation of the biotype 2 strains from the environment. Using eel serum in a saline buffer we were able to isolate biotype 2 strains from different water samples as well as from healthy eels. In parallel to this studies, we performed a genomic comparison (SSH technique) between biotype 1 and 2 strains in order to found some genetic loci related to host specificity. These analyses were performed in a collaborative project between our group and the laboratory of the Dr. Lien-I Hor (Tainan, Taiwan). Few differences were found, emphasizing the high similarity between biotypes 1 and 2 of V. vulnificus. Although this, we found three sequences specific for biotype 2 isolates and three more for serovar E isolates. The first ones are located in a plasmid that later studies have demonstrated that is essential for host specificity and fish virulence. Using some of these sequences we designed a Multiplex PCR assay that permits the rapid identification of the species, the biotype 2 and the zoonotic serovar. Once we increased the genetic diversity of our collection we carry out different analysis that included phenotypic, molecular (ribotyping) and genetic (selected genetic typing and MLST of housekeeping and virulence genes) tests for typing bacterial isolates. The results clearly demonstrate that the species is highly heterogeneous. Phenotipically we were not able to find a specific profile that could be associated with biotype, serovar, or origin of the isolate. The DNA polymorphisms studied prone to divide the V. vulnificus into two groups, one of them mainly associated with human septicaemic isolates and the other with environmental ones. Similar divisions were observed with the other techniques. Based on the results of these genetic studies we could conclude that the biotype 1 is the most heterogeneous, whereas the biotype 3 is quite homogeneous forming a separated clade that could have evolved recently. Regarding the biotype 2, the phylogenies obtained revealed that those isolates could have emerged from different environmental isolates and that the fish virulence would have been acquired with the virulence plasmid. In conclusion, our study does not support the actual subdivision of the species into biotypes. Our data show that there are two distinct V. vulnificus populations that correlated very well with all the genetic typing methods applied. These two divisions could be suitable for a new classification scheme of the species.
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