POLYPHASIC APPROACH TO STUDY THE EMERGING PATHOGEN LACTOCOCCUS GARVIEAE

This Ph.D. thesis research project aims to increase knowledge on emerging and opportunistic pathogens in food, to improve the current systems of management and control of food safety. During the project, the attention was focused on the study of Lactococcus garvieae, the aetiological agent of a hemorrhagic septicemia in aquaculture, but recently recognized as an opportunistic human pathogen. In the first part of the research, strains of L. garvieae coming from different niches were studied through a Multi Locus Sequence Typing approach and genome analysis and comparison. Data highlighted a high level of genetic heterogeneity within the species and the separation in two main genomic lineages, with the presence of a third lineage that might be considered the ancestor branch, and the evolutionary intermediate between Lactococcus lactis and L. garvieae main clusters. Moreover, the analysis of 11 sequenced genomes of L. garvieae and their comparison with L. lactis genomic sequences provided a first insight in the core and pan-genome of L. garvieae. The core genome, consisting of 1341 genes, contains genes related to virulence factors; about 70% of the total core genes were also conserved in the analysed Lactococcus genomes, suggesting a common ancestor. A dispensable genome constitute by many genes, could explain the cosmopolitan lifestyle of L. garvieae species. Subsequently to better clarify the polymorphic character of these strains, insertion sequences distribution and temperate bacteriophages were studied as mechanisms involved in genome plasticity. Insertion sequences (ISs) were found in many analysed genomes and a substantial homology to the Lactococcus lactis elements was found, suggesting the movement of ISs between these two phylogenetically closely related species. Moreover, five new elements occurring in Lactococcus garvieae where for the first time described. All the ISs are inserted in non-coding regions and their possible involvement in chromosomal rearrangement was described. The 50% of the 45 strains of L. garvieae, coming from different ecological niches, showed the presence of inducible temperate phages, belonging to Siphoviridae family; their differences in tail length and capside width group them in two morphotypes. Sequencing of a prophage from a dairy L. garvieae strain and in silico analysis of fourteen sequenced L. garvieae strains, revealed 7 complete phages disseminated along six genomes. The phage genomes have a length on average with other lactococcal phages, ranging from 30 to 40 kb; genome comparison revealed in some case homology with other L. lactis phages. Interestingly only one phage genome has a %GC similar to L. garvieae species: the other genomes showed a GC value similar to that of L. lactis. These findings led us to hypothesize that the phages infecting L. garvieae originated from L. lactis, highlighting that the latter species might have appeared long time ago in the environment. Being Lactococcus garvieae one of the most important pathogen in aquaculture sector, in the last part of this research, we isolated virulent phages to use in future strategies of phage therapy. Eleven lytic phages were isolated, and they showed a narrow lytic spectrum, in some case specific to few hosts. These results highlighted the importance of design a specific phage cocktail to improve the efficiency of phage therapy. Moreover, the genome sequencing of one of these phages showed homology with other L. lactis phages (c2 and Q54), suggesting that phages infecting different lactococcal species may have a common ancestor.