Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Nutritional strategies to control mycotoxin damages in swine

Mycotoxins are contaminants of agricultural products both in the field and during storage and can enter the food chain through contaminated cereals and foods (milk, meat, and eggs) obtained from animals fed mycotoxin contaminated feeds. Mycotoxins are genotoxic carcinogens that cause health and economic problems. Ochratoxin A and fumonisin B1 have been classified by the International Agency for Research on Cancer in 1993, as “possibly carcinogenic to humans” (class 2B). To control mycotoxins induced damages, different strategies have been developed to reduce the growth of mycotoxigenic fungi as well as to decontaminate and/or detoxify mycotoxin contaminated foods and animal feeds. Critical points, target for these strategies, are: prevention of mycotoxin contamination, detoxification of mycotoxins already present in food and feed, inhibition of mycotoxin absorption in the gastrointestinal tract, reduce mycotoxin induced damages when absorption occurs. Decontamination processes, as indicate by FAO, needs the following requisites to reduce toxic and economic impact of mycotoxins: it must destroy, inactivate, or remove mycotoxins; it must not produce or leave toxic and/or carcinogenic/mutagenic residues in the final products or in food products obtained from animals fed decontaminated feed; it must be capable of destroying fungal spores and mycelium in order to avoiding mycotoxin formation under favorable conditions; it should not adversely affect desirable physical and sensory properties of the feedstuff; it has to be technically and economically feasible. One important approach to the prevention of mycotoxicosis in livestock is the addition in the diets of the non-nutritionally adsorbents that bind mycotoxins preventing the absorption in the gastrointestinal tract. Activated carbons, hydrated sodium calcium aluminosilicate (HSCAS), zeolites, bentonites, and certain clays, are the most studied adsorbent and they possess a high affinity for mycotoxins. In recent years, there has been increasing interest on the hypothesis that the absorption in consumed food can be inhibited by microorganisms in the gastrointestinal tract. Numerous investigators showed that some dairy strains of LAB and bifidobacteria were able to bind aflatoxins effectively. There is a strong need for prevention of the mycotoxin-induced damages once the toxin is ingested. Nutritional approaches, such as supplementation of nutrients, food components, or additives with protective effects against mycotoxin toxicity are assuming increasing interest. Since mycotoxins have been known to produce damages by increasing oxidative stress, the protective properties of antioxidant substances have been extensively investigated. Purpose of the present study was to investigate in vitro and in vivo, strategies to counteract mycotoxin threat particularly in swine husbandry. The Ussing chambers technique was applied in the present study that for the first time to investigate in vitro the permeability of OTA and FB1 through rat intestinal mucosa. Results showed that OTA and FB1 were not absorbed from rat small intestine mucosa. Since in vivo absorption of both mycotoxins normally occurs, it is evident that in these experimental conditions Ussing diffusion chambers were not able to assess the intestinal permeability of OTA and FB1. A large number of LAB strains isolated from feces and different gastrointestinal tract regions of pigs and poultry were screened for their ability to remove OTA, FB1, and DON from bacterial medium. Results of this in vitro study showed low efficacy of isolated LAB strains to reduce OTA, FB1, and DON from bacterial medium. An in vivo trial in rats was performed to evaluate the effects of in-feed supplementation of a LAB strain, Pediococcus pentosaceus FBB61, to counteract the toxic effects induced by exposure to OTA contaminated diets. The study allows to conclude that feed supplementation with P. pentosaceus FBB61 ameliorates the oxidative status in liver, and lowers OTA induced oxidative damage in liver and kidney if diet was contaminated by OTA. This P. pentosaceus FBB61 feature joined to its bactericidal activity against Gram positive bacteria and its ability to modulate gut microflora balance in pigs, encourage additional in vivo experiments in order to better understand the potential role of P. pentosaceus FBB61 as probiotic for farm animals and humans. In the present study, in vivo trial on weaned piglets fed FB1 allow to conclude that feeding of 7.32 ppm of FB1 for 6 weeks did not impair growth performance. Deoxynivalenol contamination of feeds was evaluated in an in vivo trial on weaned piglets. The comparison between growth parameters of piglets fed DON contaminated diet and contaminated diet supplemented with the commercial product did not reach the significance level but piglet growth performances were numerically improved when the commercial product was added to DON contaminated diet. Further studies are needed to improve knowledge on mycotoxins intestinal absorption, mechanism for their detoxification in feeds and foods, and nutritional strategies to reduce mycotoxins induced damages in animals and humans. The multifactorial approach acting on each of the various steps could be a promising strategy to counteract mycotoxins damages.

Studies on biotic and abiotic elicitors inducing defense responses in tomato

Tomato (Lycopersicon esculentum Mill., Solanum lycopersicon L.) is one of the most popular vegetable throughout the world, and the importance of its cultivation is threatened by a wide array of pathogens. In the last twenty years this plant has been successfully used as a model plant to investigate the induction of defense pathways after exposure to fungal, bacterial and abiotic molecules, showing triggering of different mechanisms of resistance. Understanding these mechanisms in order to improve crop protection is a main goal for Plant Pathology. The aim of this study was to search for general or race-specific molecules able to determine in Solanum lycopersicon immune responses attributable to the main systems of plant defense: non-host, host-specific and induced resistance. Exopolysaccharides extracted by three fungal species (Aureobasidium pullulans, Cryphonectria parasitica and Epicoccum purpurascens), were able to induce transcription of pathogenesis-related (PR) proteins and accumulation of enzymes related to defense in tomato plants cv Money Maker,using the chemical inducer Bion® as a positive control. During the thesis, several Pseudomonas spp. strains were also isolated and tested for their antimicrobial activity and ability to produce antibiotics. Using as a positive control jasmonic acid, one of the selected strain was shown to induce a form of systemic resistance in tomato. Transcription of PRs and reduction of disease severity against the leaf pathogen Pseduomonas syringae pv. tomato was determined in tomato plants cv Money Maker and cv Perfect Peel, ensuring no direct contact between the selected rhizobacteria and the aerial part of the plant. To conclude this work, race-specific resistance of tomato against the leaf mold Cladosporium fulvum is also deepened, describing the project followed at the Phytopathology Laboratory of Wageningen (NL) in 2007, dealing with localization of a specific R-Avr interaction in transfected tomato protoplast cultures through fluorescence microscopy.

Characterisation of probiotic strains for the control and prevention of enteropathogens in the food chain

60 strains (belonging to the genera Lactobacillus, Bifidobacterium, Leuconostoc and Enterococcus) were tested for their capacity to inhibit the growth of 3 strains of Campylobacter jejuni: Lactobacilli and bifidobacteria were left to grow in MRS or TPY broth at 37°C overnight in anaerobic conditions; Campylobacter jejuni was inoculated in blood agar plates at 37°C for 24-48 hours in microaerophilic conditions. The inhibition experiments were carried out in vitro using ”Spot agar test” and “Well diffusion assay” techniques testing both cellular activity and that of the surnatant. 11 strains proved to inhibit the growth of Campylobacter jejuni. These strains were subsequently analised analised in order to evaluate the resistance to particular situations of stress which are found in the gastrointestinal tract and during the industrial transformation processes (Starvation stress, osmotic stress, heat stress, resistance to pH and to bile salts). Resistance to starvation stress: all strains seemed to resist the stress (except one strain). Resistance to osmotic stress: all strains were relatively resistant to the concentrations of 6% w/v of NaCl (except one strain). Resistance to heat stress: only one strain showed little resistance to the 55°C temperature. Resistance to pH: In the presence of a low pH (2.5), many strains rapidly lost their viability after approximately 1 hour. Resistance to bile salts: Except for one strain, all strains seemed to be relatively resistant to the 2% w/v concentration of bile salts. Afterward, strains were identified by using phenotipic and molecular techniques. Phenotipic identification was carried out by using API 50 CHL (bioMérieux) and API 20 STREP identification system (bioMérieux); molecular identification with species-specific PCR: the molecular techniques confirmed the results by phenotipic identification. For testing the antibiotic resistance profile, bacterial strains were subcultured in MRS or TPY broth and incubated for 18 h at 37°C under anaerobic conditions. Antibiotics tested (Tetracycline, Trimethoprim, Cefuroxime, Kanamycin, Chloramphenicol, Vancomycin, Ampycillin, Sterptomycin, Erythromycin) were diluted to the final concentrations of: 2,4,8,16,32,64,128,256 mg/ml. Then, 20 μl fresh bacterial culture (final concentration in the plates approximately 106 cfu/ml) were added to 160 μl MRS or TPY broth and 20 μl antibiotic solution. As positive control the bacterial culture (20 ul) was added to broth (160 ul) and water (20 ul). Test was performed on plates P96, that after the inoculum were incubated for 24 h at 37oC, then the antibiotic resistance was determined by measuring the Optical Density (OD) at 620 nm with Multiscan EX. All strains showed a similar behaviour: resistance to all antibiotic tested. Further studies are needed.

Use of sub-lethal high pressure homogenization (HPH) treatments to enhance functional properties of lactic acid bacteria probiotic strains

The aim of this PhD thesis was to evaluate the effect of a sub-lethal HPH treatment on some probiotic properties and on cell response mechanisms of already-known functional strains, isolated from Argentinean dairy products. The results achieved showed that HPH treatments, performed at a sub-lethal level of 50 MPa, increased some important functional and technological characteristics of the considered non intestinal probiotic strains. In particular, HPH could modify cell hydrophobicity, autoaggregation and resistance to acid gastric conditions (tested in in vitro model), cell viability and cell production of positive aroma compounds, during a refrigerate storage in a simulated dairy product. In addition, HPH process was able to increase also some probiotic properties exerted in vivo and tested for two of the considered strains. In fact, HPH-treated cells were able to enhance the number of IgA+ cells more than other not treated cells, although this capacity was time dependent. On the other hand, HPH treatment was able to modify some important characteristics that are linked to the cell wall and, consequently, could alter the adhesion capacity in vivo and the interaction with the intestinal cells. These modifications, involving cell outermost structures, were highlighted also by Trasmission Electron Microscopy (TEM) analysis. In fact, the micrographs obtained showed a significant effect of the pressure treatment on the cell morphology and particularly on the cell wall. Moreover, the results achieved showed that composition of plasma membranes and their level of unsaturation are involved in response mechanisms adopted by cells exposed to the sub-lethal HPH treatment. Although the response to the treatment varied according to the characteristics of individual strains, time of storage and suspension media employed, the results of present study, could be exploited to enhance the quality of functional products and to improve their organoleptic properties.

Study of Italian isolates of Alternaria spp.: molecular and morphological characterization and pathogenesis on apple tree

It was decided to carry out a morphological and molecular characterization of the Italian Alternaria isolatescollected from apple , and evaluate their pathogenicity and subsequently combining the data collected. The strain collection (174 isolates) was constructed by collecting material (received from extension service personnel) between June and August of 2007, 2008, and 2009. A Preliminary bioassays were performed on detached plant materials (fruit and leaf wounded and unwounded), belonging to the Golden cultivar, with two different kind of inoculation (conidial suspension and conidial filtrate). Symptoms were monitored daily and a value of pathogenicity score (P.S.) was assigned on the basis of the diameter of the necrotic area that developed. On the basis of the bioassays, the number of isolates to undergo further molecular analysis was restricted to a representative set of single spore strains (44 strains). Morphological characteristics of the colony and sporulation pattern were determined according to previous systematic work on small-spored Alternaria spp. (Pryor and Michaelides, 2002 and Hong et al., 2006). Reference strains (Alternaria alternata, Alternaria tenuissima, Alternaria arborescens and four Japanese strains of Alternaria alternata mali pathotype), used in the study were kindly provided by Prof. Barry Pryor, who allows a open access to his own fungal collection. Molecular characterization was performed combining and comparing different data sets obtained from distinct molecular approach: 1) investigation of specific loci and 2) fingerprinting based on diverse randomly selected polymorphic sites of the genome. As concern the single locus analysis, it was chosen to sequence the EndoPG partial gene and three anonymous region (OPA1-3, OPA2- and OPa10-2). These markers has revealed a powerful tool in the latter systematic works on small-spored Alternaria spp. In fact, as reported in literature small-spored Alternaria taxonomy is complicated due to the inability to resolve evolutionary relationships among the taxa because of the lack of variability in the markers commonly used in fungi systematic. The three data set together provided the necessary variation to establish the phylogenetic relationships among the Italian isolates of Alternaria spp. On Italian strains these markers showed a variable number of informative sites (ranging from 7 for EndoPg to 85 for OPA1-3) and the parsimony analysis produced different tree topologies all concordant to define A. arborescens as a mophyletic clade. Fingerprinting analysis (nine ISSR primers and eight AFLP primers combination) led to the same result: a monophyleic A. arborescens clade and one clade containing both A. tenuissima and the A. alternata strains. This first attempt to characterize Italian Alternaria species recovered from apple produced concordant results with what was already described in a similar phylogenetic study on pistachio (Pryor and Michaelides, 2002), on walnut and hazelnut (Hong et al., 2006), apple (Kang et al., 2002) and citurus (Peever et al., 2004). Together with these studies, this research demonstrates that the three morphological groups are widely distributed and occupy similar ecological niches. Furthermore, this research suggest that these Alternaria species exhibit a similar infection pattern despite the taxonomic and pathogenic differences. The molecular characterization of the pathogens is a fundamental step to understanding the disease that is spreading in the apple orchards of the north Italy. At the beginning the causal agent was considered as Alteraria alternata (Marshall and Bertagnoll, 2006). Their preliminary studies purposed a pathogenic system related to the synthesis of toxins. Experimental data of our bioassays suggest an analogous hypothesis, considering that symptoms could be induced after inoculating plant material with solely the filtrate from pathogenic strains. Moreover, positive PCR reactions using AM-toxin gene specific primers, designed for identification of apple infecting Alternaria pathovar, led to a hypothesis that a host specific toxin (toxins) were involved. It remains an intriguing challenge to discover or not if the agent of the “Italian disease” is the same of the one previously typified as Alternaria mali, casual agent of the apple blotch disease.

Intensificazione di processi biologici per la Bioremediation aerobica di suoli contaminati

Enzyveba, a partially characterized complex consortium of not-adapted microorganisms developed through prolonged stabilization of organic wastes, was found to markedly intensify the aerobic remediation of aged PAH- and PCB-contaminated soil by acting as a source of exogenous specialized microorganisms and nutrients. Thus, Enzyveba was tested in the bioremediation of Diesel (G1) and HiQ Diesel (G2) contaminated soils under aerobic slurry-phase conditions by means of a chemical, microbiological, ecotoxicological integrated analytical procedure. The addition of Enzyveba resulted in a higher availability of cultivable specialized bacteria and fungi but this resulted in a slight intensification of soil remediation, probably because of the high content of nutrients and specialized microorganisms of the soil. In many cases, the biotreatability of soils impacted by diesel fuel is limited by their poor content of autochthonous pollutant-degrading microorganisms. Thus, bioaugmentation with stable and reproducible cultures with the required broad substrate specificity might be the solution for a successful remediation. Two microbial consortia, ENZ-G1 and ENZ-G2, were enriched from Enzyveba on G1 and G2. Both consortia consist of a similar composition of bacterial and fungal species. They exhibited a comparable and significant biodegradation capability by removing about 90% of 1 g/l of diesel fuel under liquid culture conditions. Given their remarkable biodegradation potential, richness of quite diverse microbes, stability and resistance after cryopreservation at -20 °C for several months, both consortia appear very interesting candidates for bioaugmentation on site. The mycoflora of a soil historically contaminated by high concentration of PCBs was characterised before, at the beginning and at the end of the biotreatment mentioned above. Several mitosporic fungi isolated from soil grew in presence of a mixture of three PCBs congeners when also glucose was provided. This is the first study in which 5 strains of mitosporic species able to biodegrade PCB are reported in the literature.

Therapeutic microbiology: characterization of Bifidobacterium strains for the treatment of enteric disorders in newborns.

Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastro-intestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to non tumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify 3 B. breve strains and a B. longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. The formulation of a synbiotic product with an appropriate prebiotic fiber capable of supporting the growth of the selected Bifidobacterium strains was also considered in this study. In this respect the ability of the 4 selected Bifidobacterium strains to use as the sole carbon source and energy source different polisaccharide fibers was investigated The last phase of the work has been dedicated to the evaluation of the gut microbial diversity in newborns whose mothers has been subjected to antibiotic therapy a few hours before the delivery because of a Streptococcus type B infection. These newborns can represent a possible target for the probiotic strains selected in this work.

Characterization and exploiment of Microbial Strains to be used in Meat Processing and Fermentation

The principal aim of this research project has been the evaluation of the specific role of yeasts in ripening processes of dry-cured meat products, i.e. speck and in salami produced by adding Lactobacilli starter cultures, i.e. L. sakei, L. casei, L. fermentum, L. rhamnosus, L.sakei + S.xylosus. In particular the contribution of the predominant yeasts to the hydrolytic patterns of meat proteins has been studied both in model system and in real products. In fact, although several papers have been published on the microbial, enzymatic, aromatic and chemical characterization of dry-cured meat e.g. ham over ripening, the specific role of yeasts has been often underestimated. Therefore this research work has been focused on the following aspects: 1. Characterization of the yeasts and lactic acid bacteria in samples of speck produced by different farms and analyzed during the various production and ripening phases 2. Characterization of the superficial or internal yeasts population in salami produced with or without the use of lactobacilli as starter cultures 3. Molecular characterization of different strains of yeasts and detection of the dominant biotypes able to survive despite environmental stress factors (such as smoke, salt) 4. Study of the proteolytic profiles of speck and salami during the ripening process and comparison with the proteolytic profiles produced in meat model systems by a relevant number of yeasts isolated from speck and salami 5. Study of the proteolytic profiles of Lactobacilli starter cultures in meat model systems 6. Comparative statistical analysis of the proteolytic profiles to find possible relationships between specific bands and peptides and specific microorganisms 7. Evaluation of the aromatic characteristics of speck and salami to assess relationships among the metabolites released by the starter cultures or the dominant microflora

Fusarium species responsible for mycotoxin production in wheat crop: involvement in food safety

Fusarium Head Blight (FHB) is a worldwide cereal disease responsible of significant yield reduction, inferior grain quality, and mycotoxin accumulation. Fusarium graminearum and F. culmorum are the prevalent causal agents. FHB has been endemic in Italy since 1995, while there are no records about its presence in Syria. Forty-eight and forty-six wheat kernel samples were collected from different localities and analyzed for fungal presence and mycotoxin contamination. Fusarium strains were identified morphologically but the molecular confirmation was performed only for some species. Further differentiation of the chemotypes for trichothecene synthesis by F. graminearum and F. culmorum strains was conducted by PCR assays. Fusarium spp. were present in 62.5% of Syrian samples. 3Acetyl-Deoxynivalenol and nivalenol chemotypes were found in F. culmorum whilst all F. graminearum strains belonged to NIV chemotype. Italian samples were infected with Fusarium spp for 67.4%. 15Ac-DON was the prevalent chemotype in F. graminearum, while 3Ac-DON chemotype was detected in F. culmorum. The 60 Syrian Fusarium strains tested for mycotoxin production by HPLC-MS/MS have shown the prevalence of zearalenone while the emerging mycotoxins were almost absent. The analysis of the different Syrian and Italian samples of wheat kernels for their mycotoxin content showed that Syrian kernels were mainly contaminated with storage mycotoxins, aflatoxins and ochratoxin whilst Italian grains with mainly Fusarium mycotoxins. The aggressiveness of several Syrian F. culmorum isolates was estimated using three different assays: floret inoculation in growth chamber, ear inoculation in the field and a validated new Petri-dish test. The study of the behaviour of different Syrian wheat cultivars, grown under different conditions, has revealed that Jory is a FHB Syrian tolerant cultivar. This is the first study in Syria on Fusarium spp. associated to FHB, Fusarium mycotoxin producers and grain quality.

Phenotype and genotype characterization of Monilinia spp. isolates and preformed antifungal compounds in peach peel fruit at different developmental stages

The brown rot fungi belong to a group of fungal pathogens that causes considerable damage to cultivated fruits trees, particularly stone fruits and apples in the temperate regions of the World and during the postharvest with an important economic impact. In particular in Italy, it is important to monitor the Monilinia population to control economic losses associated to the peach and nectarine market. This motivates the research steps presented in this dissertation on Monilinia Italian isolates. The Monilinia species collected from stone fruits have been identified using molecular analysis based on specific primers. The relevant role of M. fructicola was confirmed and, for the first time, it was found also on apple fruits. To avoid the development of resistant strains and implement valid treatment strategies, the understanding of the fruit natural resistance during different developmental stages and the assessment of the Monilinia sensitivity/resistance to fungicides are required. The relationship between the inhibition spots and the phenolic compounds in peach fruit peel was highlighted in this research. Three methods were used to assess isolate resistance/sensitivity, the amended medium, the Spiral Gradient Endpoint Method (SGD) and the Alamar Blue method. The PCR was used to find possible mutation points in the b-tubulin gene that is responsible for fungicide resistance. Interestingly, no mutation points were observed in resistant M. laxa isolates, suggesting that the resistance could be stimulated by environmental factors. This lead to the study of the effect of the temperature on the resistance and the preliminary results of in vitro tests showed that maximum inhibition was observed at 30°C.

Molecular variability of Meningococcal antigens in carriage and disease strains and in other Neisseria species

This PhD thesis is focused on the study of the molecular variability of some specific proteins, part of the outer membrane of the pathogen Neisseria meningitidis, and described as protective antigens and important virulence factors. These antigens have been employed as components of the vaccine developed by Novartis Vaccines against N. meningitidis of serogroup B, and their variability in the meningococcal population is a key aspect when the effect of the vaccine is evaluated. The PhD project has led to complete three major studies described in three different manuscritps, of which two have been published and the third is in preparation. The thesis is structured in three main chapters, each of them dedicated to the three studies. The first, described in Chapter 1, is specifically dedicated to the analysis of the molecular conservation of meningococcal antigens in the genomes of all species classified in the genus Neisseria (Conservation of Meningococcal Antigens in the Genus Neisseria. A. Muzzi et al.. 2013. mBio 4 (3)). The second study, described in Chapter 2, focuses on the analysis of the presence and conservation of the antigens in a panel of bacterial isolates obtained from cases of the disease and from healthy individuals, and collected in the same year and in the same geographical area (Conservation of fHbp, NadA, and NHBA in carrier and pathogenic isolates of Neisseria meningitidis collected in the Czech Republic in 1993. A. Muzzi et al.. Manuscript in preparation). Finally, Chapter 3 describes the molecular features of the antigens in a panel of bacterial isolates collected over a period of 50 years, and representatives of the epidemiological history of meningococcal disease in the Netherlands (An Analysis of the Sequence Variability of Meningococcal fHbp, NadA and NHBA over a 50-Year Period in the Netherlands. S. Bambini et al.. 2013. PloS one e65043).

Characterization of West Nile virus strains isolated in Italy

West Nile virus (WNV) is a neurotropic flavivirus that is maintained in an enzootic cycle between mosquitoes and birds, but can also infect and cause disease in humans and other vertebrate species. Most of WNV infections in humans are asymptomatic, but approximately 20% of infected people develop clinical symptoms, although severe neurological diseases are observed in less than 1% of them. WNV is the most widely distributed arbovirus in the world and has been recently associated with outbreaks of meningo-encephalitis in Europe, including Italy, caused by different viral strains belonging to distinct lineages 1 and 2. The hypothesis is that genetic divergence among viral strains currently circulating in Italy might reflect on their pathogenic potential and that the rapid spread of WNV with increased pathogenicity within naïve population suggest that epidemic forms of the virus may encode mechanisms to evade host immunity. Infection with WNV triggers a delayed host response that includes a delay in the production of interferon-α (IFN-α). IFNs are a family of immuno-modulatory cytokines that are produced in response to virus infection and serve as integral signal initiators of host intracellular defenses. The increased number of human cases and the lack of data about virulence of European WNV isolates highlight the importance to achieve a better knowledge on this emerging viral infection. In the present study, we investigate the phenotypic and IFN-α-regulatory properties of different WNV lineage 1 and 2 strains that are circulating in Europe/Italy in two cell lines: Vero and 1321N1. We demonstrate that: Vero and 1321N1 cells are capable of supporting WNV replication where different WNV strains show similar growth kinetics; WNV lineage 2 strain replicated in Vero and 1321N1 cells as efficiently as WNV lineage 1 strains; and both lineages 1 and 2 were highly susceptible to the antiviral actions of IFN-α.

Aureobasidium pullulans as biological control agent: modes of action

The postharvest phase has been considered an environment very suitable for successful application of biological control agents (BCAs). However, the tri-interaction between fungal pathogen, host (fruit) and antagonist is influenced by several parameters such as temperature, oxidative stresses, oxygen composition, water activity, etc. that could be determining for the success of biocontrol. Knowledge of the modes of action of BCAs is essential in order to enhance their viability and increase their potentialities in disease control. The thesis focused on the possibility to explain the modes of action of a biological control agent (BCA): Aureobasidium pullulans, in particular the strains L1 and L8, control effective against fruit postharvest fungal pathogen. In particular in this work were studied the different modes of action of BCA, such as: i) the ability to produce volatile organic compounds (VOCs), identified by SPME- gas chromatography-mass spectrometry (GC-MS) and tested by in vitro and in vivo assays against Penicillium spp., Botrytis cinerea, Colletotrichum acutatum; ii) the ability to produce lytic enzymes (exo and endo chitinase and β-1,3-glucanase) tested against Monilinia laxa, causal agent of brown rot of stone fruits. L1 and L8 lytic enzymes were also evaluated through their relative genes by molecular tools; iii) the competition for space and nutrients, such as sugars (sucrose, glucose and fructose) and iron; the latter induced the production of siderophores, molecules with high affinity for iron chelation. A molecular investigation was carried out to better understand the gene regulation strictly correlated to the production of these chelating molucules. The competition for space against M. laxa was verified by electron microscopy techniques; iv) a depth bibliographical analysis on BCAs mechanisms of action and their possible combination with physical and chemical treatments was conducted.