Search

Vibrio vulnificus (Vv) is a multi-host pathogenic species currently subdivided into three biotypes (Bts). The three Bts are human-pathogens, but only Bt2 is also a fish-pathogen, an ability that is conferred by a transferable virulence-plasmid (pVvbt2). Here we present a phylogenomic analysis from the core genome of 80 Vv strains belonging to the three Bts recovered from a wide range of geographical and ecological sources. We have identified five well-supported phylogenetic groups or lineages (L). LI comprises a mixture of clinical and environmental Bt1 strains, most of them involved in human clinical cases related to raw seafood ingestion. LII is linked to the aquaculture industry and includes Bt3 strains exclusively, mostly related to wound infections or secondary septicemia after farmed-fish handling. LIII is formed by a mixture of Bt1 and Bt2 strains from various sources, including diseased fish, and is also related to the aquaculture industry. Lastly, LIV and LV include a few strains of Bt1 associated with specific geographical areas. The phylogenetic trees for ChrI and II are not congruent to one another, which suggests that inter- and/or intra-chromosomal rearrangements have been produced along Vv evolution. Further, the phylogenetic trees for each chromosome and the virulence plasmid were also not congruent, which also suggests that pVvbt2 has been acquired independently by different clones, probably in fish farms. From all these clones, the one with zoonotic capabilities (Bt2-Serovar E) has successfully spread worldwide. From these results, we propose a new updated classification of the species based on phylogenetic lineages rather than on Bts, as well as the inclusion of all Bt2 strains in a pathovar with the particular ability to cause fish vibriosis, for which we suggest the name 'piscis'.

Vibrio vulnificus biotype 2-serovar E is a zoonotic clonal complex that can cause death by sepsis in humans and fish. Unlike other biotypes, Bt2 produces a unique type of MARTXVv (Multifunctional-Autoprocessive-Repeats-in-Toxin; RtxA13), which is encoded by a gene duplicated in the pVvBt2 plasmid and chromosome II. In this work, we analyzed the activity of this toxin and its role in human sepsis by performing in vitro, ex vivo, and in vivo assays. First, we demonstrated that the ACD domain, present exclusively in this toxin variant, effectively has an actin-cross-linking activity. Second, we determined that the whole toxin caused death of human endotheliocytes and monocytes by lysis and apoptosis, respectively. Finally, we tested the hypothesis that RtxA13 contributes to human death caused by this zoonotic serovar by triggering an early cytokine storm in blood. To this end, we used a Bt2-SerE strain (R99) together with its rtxA13 deficient mutant, and a Bt1 strain (YJ016) producing RtxA11 (the most studied MARTXVv) together with its rtxA11 deficient mutant, as controls. Our results showed that RtxA13 was essential for virulence, as R99ΔΔrtxA13 was completely avirulent in our murine model of infection, and that R99, but not strain YJ016, induced an early, strong and dysregulated immune response involving the up-regulation of a high number of genes. This dysregulated immune response was directly linked to RtxA13. Based on these results and those obtained ex vivo (human blood), we propose a model of infection for the zoonotic serovar of V. vulnificus, in which RtxA13 would act as a sepsis-inducing toxin.

11 páginas, 6 figuras, 2 tablas.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms, The selection of an experimental animal model is of great importance in the study of bacterial virulence factors. Here, a bath infection of zebrafish larvae is proposed as an alternative model to study the virulence factors of Aeromonas hydrophila. Intraperitoneal infections in mice and trout were compared with bath infections in zebrafish larvae using specific mutants. The great advantage of this model is that bath immersion mimics the natural route of infection, and injury to the tail also provides a natural portal of entry for the bacteria. The implication of T3SS in the virulence of A. hydrophila was analyzed using the AH-1::aopB mutant. This mutant was less virulent than the wild-type strain when inoculated into zebrafish larvae, as described in other vertebrates. However, the zebrafish model exhibited slight differences in mortality kinetics only observed using invertebrate models. Infections using the mutant AH-11vapA lacking the gene coding for the surface S-layer suggested that this protein was not totally necessary to the bacteria once it was inside the host, but it contributed to the inflammatory response. Only when healthy zebrafish larvae were infected did the mutant produce less mortality than the wild-type. Variations between models were evidenced using the AH-11rmlB, which lacks the O-antigen lipopolysaccharide (LPS), and the AH-11wahD, which lacks the O-antigen LPS and part of the LPS outer-core. Both mutants showed decreased mortality in all of the animal models, but the differences between them were only observed in injured zebrafish larvae, suggesting that residues from the LPS outer core must be important for virulence. The greatest differences were observed using the AH- 11FlaB-J (lacking polar flagella and unable to swim) and the AH-1::motX (non-motile but producing flagella). They were as pathogenic as the wild-type strain when injected into mice and trout, but no mortalities were registered in zebrafish larvae. This study demonstrates that zebrafish larvae can be used as a host model to assess the virulence factors of A. hydrophila. This model revealed more differences in pathogenicity than the in vitro models and enabled the detection of slight variations in pathogenesis not observed using intraperitoneal injections of mice or fish, This work was partially funded by the projects CSD2007-00002 “Aquagenomics”, AGL2014-51773-C3, 201230E057 (CSIC) and BIO2013-47198-P, from the Spanish Ministerio de Economía y Competitividad, from Generalitat de Catalunya (Centre de Referència en Biotecnologia), and from ITN 289209 (FISHFORPHARMA) (EU). PR Saraceni received a Marie Curie Fellowship from the EU