The disease vibriosis is caused by the bacterial pathogen Vibrio anguillarum and results in large losses in aquaculture both in Denmark and around the world. Antibiotics have been widely used in antimicrobial prophylaxis and treatment of vibriosis. Recently, numerous multidrug-resistant strains of V. anguillarum have been isolated, indicating that antibiotic use has to be restricted and alternatives have to be developed. Lytic phages have been demonstrated to play an essential role in preventing bacterial infection. However, phages are also known to play a critical role in the evolution of bacterial pathogenicity development. Therefore, successful application of phage therapy in the treatment of vibriosis requires a detailed understanding of phage-host interactions, especially with regards to anti-phage defense mechanisms in the host.
Part I. As a first approach, 24 V. anguillarum and 13 Vibrio sp. strains, representing considerable temporal (20 years) and geographic (9 countries) variation in regards to their origins of isolation, and 11 vibriophages representing three different families (Myoviridae, Siphoviridae, and Podoviridae), were characterized with respect host range, morphology, genome size and lytic properties. Together the host range of the 11 vibriophages covered all the 37 Vibrio strains in the collection. In addition, the occurrence of unique susceptibility patterns of the individual host isolates, as well as key phenotypic properties related to phage susceptibility that were common to the isolated strains were studied.
Part II. In vitro phage-host interactions in two V. anguillarum strains (BA35 and PF430-3) and their corresponding phages (ΦH20 (Siphoviridae) and KVP40 (Myoviridae)), during growth in the settings of micro-colonies, biofilms, and the free-living phase were investigated, in order to explore the resistance/tolerance mechanisms responsible for the different outcomes of these two phage-host interactions. The study demonstrated large intraspecific differences in phage-protection mechanisms, as strain BA35 obtained genetic resistance through mutational changes, whereas strain PF430-3 was protected from phage infection by formation of a biofilm. These different protection mechanisms have important implications for the phage-host dynamics and efficiency of phage infection.
Part III. To gain an insight into whether bacteria of the Vibrio genus use quorum sensing (QS) to control their phage-susceptibility, a study of the QS-mediated interaction between phage KVP40 and V. anguillarum PF430-3 was carried out. The data show that QS does indeed regulate phage-host interactions in V. anguillarum PF430-3, potentially by using QS transcription factor VanT to repress ompK expression. It was demonstrated that QS controls the choice of anti-phage defense strategies in the V. anguillarum strain PF430-3, suggesting the presence of dynamic, temporary adaptations to phage infection pressure, while still securing the ability to produce a functional OmpK receptor.
In conclusion, this thesis provides a first insight into the dynamic vibriophage-host interactions, indicating the complexity of phage therapy in the treatment of vibriosis, regarding the evolution of anti-phage defense mechanisms, gene regulation, quorum sensing, biofilm formation, as well as pathogenesis. Together, these discoveries will enable us to evaluate these potential factors which regulate phage-host interactions, and on that background, to optimize phage therapeutic strategies against vibriosis with regards to the composition of applied phage cocktails, the timing of treatment, and the likely outcome of phage therapy and QS inhibiting molecules.