Functional and Genetic characterization of new genomic islands from an E. coli strain associated with neonatal meningitis.

Enterobacteriaceae genomes evolve through mutations, rearrangements and horizontal gene transfer (HGT). The latter evolutionary pathway works through the acquisition DNA (GEI) modules of foreign origin that enhances fitness of the host to a given environment. The genome of E. coli IHE3034, a strain isolated from a case of neonatal meningitis, has recently been sequenced and its subsequent sequence analysis has predicted 18 possible GEIs, of which: 8 have not been previously described, 5 fully meet the pathogenic island definition and at least 10 that seem to be of prophagic origin. In order to study the GEI distribution of our reference strain, we screened for the presence 18 GEIs a panel of 132 strains, representative of E. coli diversity. Also, using an inverse nested PCR approach we identified 9 GEI that can form an extrachromosomal circular intermediate (CI) and their respective attachment sites (att). Further, we set up a qPCR approach that allowed us to determine the excision rates of 5 genomic islands in different growth conditions. Four islands, specific for strains appertaining to the sequence type complex 95 (STC95), have been deleted in order to assess their function in a Dictyostelium discoideum grazing assays. Overall, the distribution data presented here indicate that 16 IHE3034 GEIs are more associated to the STC95 strains. Also the functional and genetic characterization has uncovered that GEI 13, 17 and 19 are involved in the resistance to phagocitation by Dictyostelium d thus suggesting a possible role in the adaptation of the pathogen during certain stages of infection.

The role of bifidobacteria in newborn health and the intestinal microbial balance

Gut microbial acquisition during the early stage of life is an extremely important event since it affects the health status of the host. In this contest the healthy properties of the genus Bifidobacterium have a central function in newborns. The aim of this thesis was to explore the dynamics of the gut microbial colonization in newborns and to suggest possible strategies to maintain or restore a correct balance of gut bacterial population in infants. The first step of this work was to review the most recent studies on the use of probiotics and prebiotics in infants. Secondly, in order to prevent or treat intestinal disorders that may affect newborns, the capability of selected Bifidobacterium strains to reduce the amount of Enterobacteriaceae and against the infant pathogen Streptococcus agalactiae was evaluated in vitro. Furthermore, the ability of several commercial fibers to stimulate selectively the growth of bifidobacterial strains was checked. Finally, the gut microbial composition in the early stage of life in response to the intrapartum antibiotic prophylaxis (IAP) against group B Streptococcus was studied using q-PCR, DGGE and next generation sequencing. The results globally showed that Bifidobacterium breve B632 strain is the best candidate for the use in a synbiotic product coupled to a mixture of two selected prebiotic fibers (galactooligosaccharides and fructooligosaccharides) for gastrointestinal disorders in infants. Moreover, the early gut microbial composition was affected by IAP treatment with infants showing lower counts of Bifidobacterium spp. and Bacteroides spp. coupled to a decrement of biodiversity of bacteria, compared to control infants. These studies have shown that IAP could affect the early intestinal balance in infants and they have paved the way to the definition of new strategies alternative to antibiotic treatment to control GBS infection in pregnant women.

Definition of Food Safety Criteria for Bacteria Food-Borne Pathogens in Ready to Eat products

The aims of this research study is to explore the opportunity to set up Performance Objectives (POs) parameters for specific risks in RTE products to propose for food industries and food authorities. In fact, even if microbiological criteria for Salmonella and Listeria monocytogenes Ready-to-Eat (RTE) products are included in the European Regulation, these parameters are not risk based and no microbiological criteria for Bacillus cereus in RTE products is present. For these reasons the behaviour of Salmonella enterica in RTE mixed salad, the microbiological characteristics in RTE spelt salad, and the definition of POs for Bacillus cereus and Listeria monocytogenes in RTE spelt salad has been assessed. Based on the data produced can be drawn the following conclusions: 1. A rapid growth of Salmonella enterica may occurr in mixed ingredient salads, and strict temperature control during the production chain of the product is critical. 2. Spelt salad is characterized by the presence of high number of Lactic Acid Bacteria. Listeria spp. and Enterobacteriaceae, on the contrary, did not grow during the shlef life, probably due to the relevant metabolic activity of LAB. 3. The use of spelt and cheese compliant with the suggested POs might significantly reduce the incidence of foodborne intoxications due to Bacillus cereus and Listeria monocytogenes and the proportions of recalls, causing huge economic losses for food companies commercializing RTE products. 4. The approach to calculate the POs values and reported in my work can be easily adapted to different food/risk combination as well as to any changes in the formulation of the same food products. 5. The optimized sampling plans in term of number of samples to collect can be derive in order to verify the compliance to POs values selected.