Vibrio vulnificus and related species: a study of the evolution and emergence of new pathogenic variants
- Autores: Hector Carmona Salido
- Directores de la Tesis: Carmen Amaro González (dir. tes.), Fernando González Candelas (codir. tes.), Francisco Jose Roig Molina (codir. tes.)
- Lectura: En la Universitat de València ( España ) en 2024
- Idioma: inglés
- Número de páginas: 182
- Tribunal Calificador de la Tesis: Rosa Aznar Novella (presid.), Laura Gómez Valero (secret.), Jens Andre Hammerl (voc.)
- Programa de doctorado: Programa de Doctorado en Biodiversidad y Biología Evolutiva por la Universitat de València (Estudi General)
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
The advent of sequencing techniques and the creation of public databases has revolutionized the field of bacterial taxonomy. With more than 20,500 Vibrio genomes currently in the NCBI, we are able to shed light on several aspects of the biology of these fascinate organisms. The genus Vibrio comprises 149 valid species of Gram-negative, facultative anaerobic, oxidase positive bacteria that inhabit diverse aquatic environments worldwide, encompassing freshwater, estuarine, and marine ecosystems mainly located in temperate or warm zones. For this reason, climate change is favouring the spread of Vibrio species to traditionally colder areas such as the Baltic Sea or the North Sea. Many species are known to cause disease a series of diseases commonly known as vibriosis in fishes, bivalves, corals and even humans.
Among them, V. vulnificus stands out for being the deadliest pathogen. This is due to the presence of several virulence factors such as the RtxA1 toxin, the haemolysin VvhA or the capsule among many others. Additionally, there have been reported several outbreaks ligated with fish farms that are zoonotic by the presence of a plasmid. This plasmid encodes two key proteins that confers resistance to the innate immune system of the fish, Ftbp and Fpcrp. This species was classically divided into three biotypes by some phenotypical test. However, with access to genomic data, a phylogenomic study concluded that the species is divided into 5 evolutionary lineages. To demonstrate that V. vulnificus is therefore a pathogen whose zoonotic capacity was being underestimated and that HGT has a key role in the evolution of the species, we conducted this PhD thesis.
To achieve this we collected a dataset comprised of 364 genomes from NCBI, our own database and databases of collaborating laboratories. First, we reconstructed for the first time the core genome of the species to create a supermatrix. Then, we obtained a ML tree from this matrix and observed that, in addition to the previous 5 evolutionary lineages, there is one more with two clinical strains isolated from USA. We also observed that, among the core gene families, we could find key virulence factors involved in early tissue colonisation and inflammation. Next, we studied for the first time the relevance of recombination events inside the species. The PIM method revealed that almost every gene of the species had undergone at least one recombination event. This was further confirmed by the study of the genetic population structure and by analysing the rtxA gene in several groups.
To test the zoonotic capability of V. vulnificus, we compared a new group of strains isolated from diseased fish in eastern Mediterranean tilapia farms with control strains belonging to already described lineages and clades that we suspected could be zoonotic, L1-clade A, L3 and L5. This analysis showed that all these clades contained a family of virulence plasmid carrying the genes of the fish blood survival kit. Moreover, all groups were virulent to fish and mouse and resisted the bactericidal effect of human and tilapia serum, confirming their zoonotic character. However, complementary virulence studies in eel showed that tilapia strains were adapted to infect tilapia and eel strains were adapted to infect eel. In fact, we found that there was evidence of episodic diversifying selection in 7 codons of the gene ftbp. In addition to plasmids, we studied the presence of another MGE in our dataset, phages. Approximately half of the tested strains carried a potential prophage region. Hence, we decided to develop a new protocol to induce the lytic cycle based on mitomycin C. We successfully applied this protocol to a strain in our lab and confirmed the presence of a phage.
Two of the geographically restricted evolutionary lineages found in the ML tree were L3 and L4. Remarkably, these lineages emerged after recorded episodes of seawater warming. To facilitate epidemiological studies, we developed a multiplex PCR for the identification of the species and these groups. The new method was validated with more than 200 strains from different sources and we obtained 100% specificity. The new PCR can be used for monitoring purposes since it can be easily incorporated to other routine test of the species. We tested the presence of the bacterium in the Albufera lake and confirmed that it is present and can be isolated all year except during winter months.
Another objective of the present thesis was to find out whether there were clinical cases of V. vulnificus isolated from clinical samples in the Comunitat Valenciana. Although there were records of cases of human vibriosis and deaths due to human vibriosis, we were only able to recover one isolate that we received as putative V. vulnificus and which turned out to be another interesting species, V. metoecus. This species was very recently described and had been previously isolated in an ecosystem near the Albufera lake associated to eel surface mucus in 2016. We performed a genotypic and phenotypic characterisation as well as a study of its genome in comparison with related species. We found that it shares with V. vulnificus its ability to resist and proliferate in human serum if there was high iron concentration. In addition, we gathered evidence that this resistance depends on a sialylation system on the surface of the bacterium. However, the described virulence factors were common to genetically closer species such as V. cholerae and V. mimicus. To our knowledge, this is the first clinical case described for this species.
In conclusion, the evidence provided in this thesis points out that recombination is a major evolutionary force in Vibrio species. Specifically, in V. vulnificus recombination led to the emergence of new variants, some of which suppose a serious risk in public animal and human health. This emergence is further fuelled by fish farms, where the exchange of genetic information is frequent and natural selection favours the most adapted variants of the genes. Luckily, we have developed new powerful yet easy to apply detection methods to monitor and prevent the emergence of future outbreaks.
