LACTOCOCCUS GARVIEAE AND MORGANELLA MORGANII: TWO BACTERIAL MODELS TO STUDY QUALITY AND SAFETY OF FISH PRODUCTS

This PhD thesis research project aims to improve the current systems of management and control of food safety, focusing the attention on the study of two contaminating emerging pathogens in fish products: Lactococcus garvieae, the etiological agent of a hemorrhagic septicemia in aquaculture sector, and Morganella morganii, the most active histamine-producing species. In the first part of the research the molecular characterization and genetic polymorphism of L. garvieae strains coming from different food niches were carried out revealing an early separation of L. garvieae population into two independent genetic lineages. Subsequent phylogenetic analyses through a MLST approach confirms the significant genetic heterogeneity in this species, the separation into two principal evolution lineages, and the presence of a third lineage totally separated from the others, that could be the ancestor branch of the main lineages. Genome sequencing of representative selected strains and the comparison with other L. garvieae sequences already present in databases, confirmed the subdivision in the three different subgroups. Within each subgroup the nucleotide similarity was 99%, while comparing subgroups the similarity decreased (94-95%). Moreover, this preliminary comparison reveals that the core genome of L. garvieae contains, in additions to genes codifying for the main metabolic pathways, genes encoding putative virulence factors, such as hemolysins and adhesin. Genes related to carbohydrate fermentation (such as lactose and sucrose) seemed part of the dispensable genome, as well as the gene cluster related to the capsule formation. Experiments carried out to evaluate the expression of virulence-related genes in representative L garvieae strains, indicated an adaptive response to different environmental conditions, specific at the strain-level. Regarding the second part of the research, we started exploring the bacterial population of fresh tuna samples and evaluated the histamine content. Despite low levels of histamine were detected (<10 ppm), many samples showed high total viable counts after temperature abuse. The 10% of the 141 enteric bacteria isolated from samples were identified by 16S rDNA sequence analysis, as M. morganii, the most active histamine-producing species. Subsequently a new quantitative PCR assay for the detection of M. morganii in potentially contaminated fish products before histamine accumulates was developed, with a detection limit of 0.563 pg of pure DNA, corresponding to DNA extracted from approximately nine cells. To improve the knowledge at genome level of the histidine decarboxylase of M. morganii (hdc gene), a region of 7681 bp was sequenced. Results showed the presence of genes encoding two putative histidine/histamine antiporters upstream and downstream the hdc gene, followed by an histidyl-tRNA synthetase. Gene expression analysis indicates that in M. morganii hdc was inducible, showing the highest expression under acidic conditions, mainly in presence of histidine supplementation. Finally, with the aim to prepare a significant collection of different M. morganii strains from different ecological niches, we compared several clinical biotypes, in addition to strains isolated from fish through REP and RAPD PCR analysis. The cluster analysis confirmed the existence of two main branches related to the habitat of isolation, which were separated at a level of similarity of 19%.