Il polietilene (PE) rappresenta più del 60% di tutte le plastiche derivate dal petrolio a livello mondiale, e si sta accumulando ad un tasso di diversi milioni di tonnellate per anno a causa della sua riluttanza alla degradazione biotica e abiotica. La degradazione microbica è stata proposta come possibile strada alternativa nella riduzione dei rifiuti plastici. Lo scopo generale di questo lavoro è stata l'identificazione di ceppi batterici in grado di metabolizzare il PE e di identificare le vie metaboliche coinvolte in tale processo di biodegradazione. Abbiamo analizzato mediante approccio metagenomico diversi campioni di plastica raccolti in una discarica abbandonata, ed è stata scoperta una forte relazione tra le proprietà della plastica (inclusa la presenza di coloranti) e la comunità microbica Analizzando la comunità microbica esposta al PE nell’ ambiente, abbiamo isolato 10 ceppi batterici in grado di crescere utilizzando il PE come unica fonte di energia e di carbonio. Uno di questi ceppi, Pseudomonas aeruginosa UC4003, ha mostrato la più alta capacità di crescere in terreno minimo e polietilene. Quando cresce su PE, questo ceppo produce un enzima extracellulare, proteina-attivatore per l’ossidazione degli n-alcani (PA), coinvolto nelle prime fasi di degradazione di polietilene. / Plastics production, use and degradation are hot topics that have come to the forefront over recent years. Polyethylene (PE) represents more than 60% of all petroleum-derived plastics worldwide and is accumulating at rates of several millions of tons per year because of its strong recalcitrance to biotic and abiotic degradation. Microbial degradation has been proposed as a possible alternative way to reduce plastic wastes. The general aim of this work was the identification of bacterial strains able to metabolize PE and to identify the biochemical pathways of this biodegradation process. In an abandoned landfill we collected different plastic samples; using a metagenomic approach, we found a strong relationship between the plastic properties (including the presence of colorants) and the microbial community By screening the natural microbial community exposed to PE in environment, we isolated 10 bacteria which revealed the ability to grow on PE as only energy and carbon source. A bacterium, Pseudomonas aeruginosa UC4003, showed the highest growth rate in minimal salt medium and polyethylene. When grown on PE, this strain produced an extracellular enzyme, protein-like activator for n-alkane oxidation (PA), involved in the first step of polyethylene degradation.

La presente attività di ricerca ha avuto come oggetto di studio la formazione di biofilm in condizioni dinamiche: per raggiungere tale obiettivo, è stato costruito un idoneo apparato che permettesse di ottenere una valutazione quantitativa sia delle cellule presenti nella soluzione di crescita sia di quelle adese su tre materiali di prova(acciaio inossidabile, PET e rame). Oltre ad esprimere una valutazione dell’adesione batterica alle superfici, si è proceduto ad indagare sui possibili parametri sperimentali che potessero influenzare la formazione di biofilmdi Listeria su superfici abiotiche. Allo stesso tempo l’efficacia battericida di alcuni biocidi è stata valutata sulle cellule planctoniche così come su biofilm batterici sviluppati su superfici abiotiche. Il ruolo del sistema di quorum sensing basato su luxS nella formazione di biofilm batterici: un ceppo di L. innocua con buone capacità adesive è stato confrontato con il suo ceppo deficiente di luxS per la sua suscettibilità ad agenti biocidi così come per la sua capacità di adesione superficiale L’effetto della precendentemente menzionata inattivazione genetica sulla cinetica di crescita batterica e, quindi, sull’efficienza metabolica complessiva è stata esaminate mediante lo strumento BioscreenC. L’effetto inibitorio di due composti polifenolici è stato valutato sul ceppo deficiente di luxS così come sul ceppo originale. / The current research activity was focused on biofilm formation in dynamic conditions. A suitable device was thus constructed, in order to have a quantitative evaluation of the cells freely suspended in the flowing nutrient solution used as well as the sessile cells which adhered on the three tested surfaces (stainless steel, polyethylene therephthalate and copper). During this project, other than of a quantitative assessment of bacterial adhesion, we investigated also on the parameters which could affect significantly the biofilm formation of Listeria on abiotic surfaces. At the same time antimicrobial efficiency of selected biocides was investigated on planktonic cells as well as on bacterial biofilms grown on abiotic surfaces. The role of luxS-based quorum sensing system in the biofilm development process was evaluated: a luxS-null mutant of an adhesive strain of L. innocua was obtained through Campbell-like genetic inactivation and then evaluated for its sensitivity to biocidal agents and adhesion to surfaces, in comparison with its parental strains. Furthermore the effect of genetic inactivation on bacterial growth kinetic and thus on overall bacterial metabolic efficiency was assessed through the apparatus of BioscreenC. Finally two polyphenolic compounds were investigated for their inhibitory effect on Listeria.

Nowadays, exciting bioanalytical microsystems are currently receiving increasing attention in biology since they can comply with the considerable demand for reliable, sensitive and low-cost analysis tools. Small reagents volumes, low power consumption, portability, fast analysis, high throughput and systems integration are the key aspects that make these systems more and more appealing within both the academic and industrial communities. In the last years, many microdevices were developed for a wide range of biological applications, particularly dedicated to cellu-lar or molecular analysis. Many efforts were devoted to the realization of Cell-Based Biosensors (CBBs) to monitor the dynamic behaviour of cell cultures for pharmacological screening and basic research. Other researchers focused their interests in the development of so-called Lab-on-a-Chip (LOC) systems for DNA analysis mostly applied to clinical diagnosis. This thesis deals with the investigation of two miniaturized devices – a cell-based biosensor and a DNA amplification system – for the cellular and molecular analysis of wine yeasts, respectively. The first device consists of integrated electrochemical sensors – Ion-Sensitive Field-Effect Transistor (ISFET), impedimetric and temperature sensors – for the real time evaluation of pH and cell settling of yeasts under batch culture conditions. The assessment of yeast performance and robustness has been focused on ethanol tolerance, as it is one of the main stress factors acting in wine, and thus, one of the major causes of stuck fermentations. A good agreement between extracellular acidification and cell growth trends at different ethanol concentration has been demonstrated, significantly reducing the time of the traditional assays. Moreover, resistivity measurements have shown the possibility to follow progressive settling of the cell suspension. Concerning the second system, a Polymerase Chain Reaction (PCR) microdevice has been biologically validated by successfully amplifying yeast genomic DNA fragments. Additionally, the outcome of PCR has been positively assessed with diluted samples and boiled yeast cultures, demonstrating the possibility to skip the time-consuming purification process for potential LOC applications with very little or no pre-PCR sample manipulations. The encouraging results from both microsystems have demonstrated their suitability for wine yeast analysis, aimed at quality improvements of the winemaking process.

The human microbiome represents the community of archaea, bacteria, micro-eukaryotes, and viruses present in and on the human body. Metagenomics is the most recent and advanced tool that allows the study of the microbiome at high resolution by sequencing the whole genetic content of a biological sample. The computational side of the metagenomic pipeline is recognized as the most challenging one as it needs to process large amounts of data coming from next-generation sequencing technologies to obtain accurate profiles of the microbiomes. Among all the analyses that can be performed, phylogenetics allows researchers to study microbial evolution, resolve strain-level relationships between microbes, and also taxonomically place and characterize novel and unknown microbial genomes. This thesis presents a novel computational phylogenetic approach implemented during my doctoral studies. The aims of the work range from the high-quality visualization of large phylogenies to the reconstruction of phylogenetic trees at unprecedented scale and resolution. Large-scale and accurate phylogeny reconstruction is crucial in tracking species at strain-level resolution across samples and phylogenetically characterizing unknown microbes by placing their genomes reconstructed via metagenomic assembly into a large reference phylogeny. The proposed computational phylogenetic framework has been used in several different metagenomic analyses, improving our understanding of the complexity of microbial communities. It proved, for example, to be crucial in the detection of vertical transmission events from mothers to infants and for the placement of thousands of unknown metagenome-reconstructed genomes leading to the definition of many new candidate species. This poses the basis for large-scale and more accurate analysis of the microbiome.

Durante la mia tesi di dottorato ho selezionato nuovi potenziali ceppi probiotici, combinando l'approccio convenzionale con l’utilizzo di piattaforme di nuova concezione. Mi sono concentrata inizialmente sul cosiddetto "screening tradizionale" finalizzato all'isolamento di nuovi ceppi batterici di lattobacilli e bifidobatteri e alla valutazione della loro sicurezza per il consumo umano e della loro efficacia. I ceppi selezionati sono stati quindi sottoposti ad uno screening più specifico, a seconda dell'applicazione a cui sarebbero stati destinati. In questa fase sono stati utilizzati sia test in vitro che modelli animali, al fine di valutare l'applicabilità dei ceppi di recente selezionati come probiotici per la promozione della salute umana e il loro possibile impiego in campo alimentare. Ho valutato il potenziale utilizzo dei ceppi nell’integrazione alimentare di soggetti che seguono diete ad alto contenuto proteico per ridurre il rischio di accumulo intestinale di ammine biogene, e ho individuato un ceppo di Lactobacillus con presunta attività ammino-degradativa. L'obiettivo principale di un’altra attività di screening è stato lo studio degli effetti immunomodulanti di nuovi ceppi: combinazioni di probiotici sono risultati buoni candidati per il trattamento delle malattie infiammatorie e autoimmuni (miastenia grave e sclerosi multipla) nel modello di topo. Infine, i ceppi sono stati sottoposti a screening per una potenziale applicazione in campo alimentare, finalizzata ad indagare la possibilità di produrre formulazioni di probiotici “atomizzati” da utilizzare insieme alla base commerciale del gelato. / During my PhD thesis I screened new potential probiotic strains by combining conventional approach to newly designed platforms. I focused first on the so-called "conventional screening” aimed to the isolation of new bacterial strains of lactobacilli and bifidobacteria and to assess their safety for human consumption and their efficacy by in-vitro methods. Selected strains were then submitted to a targeted screening by specifically designed models, depending on the application to which they would be destined. These models combined in vitro tests and animal models in order to assess the applicability of newly selected strains as probiotics for the promotion of human health and their possible use in food field. I evaluated the potential use of strains in the food supplementation of subjects that follow high-protein diets to reduce the risk of intestinal accumulation of biogenic amines, and I identified a strain of Lactobacillus with alleged amino degradative activity. The main objective of another screening activity was the study of the immunoregulatory effects of new strains: combinations of probiotics resulted good promising candidates for the treatment of inflammatory and autoimmune diseases (myasthenia gravis and multiple sclerosis) in rat model. Finally, strains were screened for a potential application in food field, in order to investigate the possibility of producing spray-dried probiotic formulations to be used together with commercial ice-cream bases.

In recent years, the world has faced major health challenges, from the rise of antibiotic resistance to the emergence of new pathogens with pandemic potential. This highlights the importance of considering human health as inextricably intertwined with that of other animals and the environment in which they live. This paradigm is known as “One Health”, which is the integration of environmental sciences, veterinary science, and medicine. Within this perspective, viruses, the most abundant biological entities on Earth, play a central role in connecting different organisms, deeply influencing the health of their hosts. Despite their great importance, most viruses are still poorly understood, mainly because of the technical and economic limitations posed by isolation, cultivation, and single colony sequencing. However, recently developed genomic technologies offer a cheaper and more sensitive alternative to study viruses, allowing a better integration of data from various sources and making it possible to explore how they circulate among different hosts and environments. In this thesis, I hypotesise that, by combining different classes of genomic methods with One Health practices, it is possible to reveal much more of the entire picture of viral diversity and evolution that by simply using them in a separated way. I show that this is the case for each one of the scientific questions addressed in this work, which are organised in three main chapters: - In the second chapter I analysed 22 metatranscriptomes from tick samples from different parts of Italy, obtaining a set of 91 viral contigs for which I reconstructed the phylogeny, with the aim to identify the presence of possible pathogens and characterise the unexplored viral diversity in the country. This analysis not only clarified the molecular epidemiology of well-known pathogens such as Tick-borne encephalitis virus, but also allowed the discovery of at least 10 novel viral species. - In the third chapter, I investigated the origin and spread of West Nile virus, an emerging pathogen causing neurological disease worldwide. The goal was to expand the current knowledge of this virus by increasing the number of sequenced genomes and to reconstruct how the virus moved between Africa and Europe. Phylogeographic and phylodynamic methods showed that this pathogen originated in Africa and repeatedly invaded the European continent, revealing the dynamics of its evolution through space and time. - In the last chapter, I contributed to obtaining a set of DNA phages assembled from human microbiomes and viromes by manual curation and comparative genomics and developed a new approach to study their evolution in relation to their bacterial hosts. I found that the strength of co-phylogenetic patterns between viruses and their hosts is generally weak, suggesting that their ecological relationships emerge on short evolutionary timescales. Taken together, these results show how the integration of viral metagenomics and phylogenomics in One Health is essential to answer fundamental questions about the diversity of viruses and how they spread and evolve. Furthermore, the methods and protocols developed in these studies can be applied to similar cases, allowing a systematic exploration of many other datasets to expand our knowledge of the virosphere. This information can be used to implement containment strategies, public health policies, therapies, and biotechnologies.

In the last two decades several studies have been trying to explore a possible role for gut microbiota in Autism Spectrum Disorders (ASD), supported by the high incidence of gastrointestinal disorders among ASD children and by the now well recognized existence of the brain-gut-microbiota axis (the complex system of bidirectional interactions between central nervous system, gastrointestinal tract and microorganisms inhabiting the gut). Nevertheless, results about alterations in gut microbiota composition and/or activity in ASD are to date strongly contrasting. A possible explanation could be that these studies tend to treat ASD as a unique pathology, whereas it includes different cognitive-behavioural phenotypes. Moreover, they do not consider factors which are important for children’s gut flora development, such as type of delivery, nutritional history (e.g. formula milk during lactation) and medical history (e.g. antibiotics intake) as well as factors that may affect the present composition of microbiota, such as the current diet (e.g. the strong food selectivity that often occurs in ASD children) and the presence of gastrointestinal disorders. In this study, I developed an interview to parents to assess whether there are differences related to the above mentioned aspects between ASD children and typically developing children and among ASD themselves, considering differences in cognitive level and severity of autistic traits. I also explored the use of special diets such as gluten-, lactose and casein free diets, the reasons for their adoption and the possible benefits for the child. Moreover, I decided to include in this interview also a section dedicated to parental difficulties in managing mealtime in order to collect information useful to plan future interventions. I found differences between ASD- and typical children in the incidence of gastrointestinal disorders and food selectivity. Especially, some children initially eat everything and then switch to a more and more restricted diet. This could be considered an early symptom of the pathology. I also found an association between gastrointestinal disorders and severity of autistic traits. Furthermore, I collected faecal samples from ASD families (two parents, an ASD child and a typically developing sibling) and analysed them with metaproteomics and bioinformatics techniques in order to assess microbiota activity and evaluate it in light of ASD phenotype, nutritional habits, gastrointestinal disorders and genetic proximity. Demonstrating the existence of a different microbiota composition in ASD or at least in a subgroup could allow to identify a biomarker of a possible development of ASD and to design preventive interventions, even through probiotics intake. Moreover, it could help to better understand the molecular mechanism underlying this pathology.

Quando le condizioni sono sfavorevoli alla crescita, membri dei generi Bacillus e Clostridia (incluso Clostridium botulinum, l’agente eziologico del botulismo) formano endospore, forme cellulari estremamente resistenti, metabolicamente dormienti e difficili da distruggere. Tuttavia, le spore attraverso il processo di germinazione, riattivano il ciclo vegetativo non appena le condizioni tornano favorevoli. Questa capacità di “riattivazione” delle spore è causa di “food spoilage” e di intossicazioni alimentari. Considerando che le specie Clostridium botulinum e Clostridium sporogenes sono filogeneticamente correlate, in questo lavoro, il ceppo Clostridium sporogenes UC9000, isolato da latte crudo, è stato utilizzato come modello non-patogeno di Clostridium botulinum per studiare la germinazione. Studi fisiologici hanno rivelato che le spore del ceppo UC9000 germinano in presenza di L-alanina/ L-cisteina in combinazione con L-lattato, mentre un analisi in silico ha permesso di identificare omologhi dei recettori coinvolti nella risposta all’L-alanina in Bacillus. Attraverso l’analisi del genoma sono stati identificati gli enzimi SleB, CwlJ e SleL, responsabili della degradazione del cortex. CwlJ è stato localizzato nel coat della spora grazie ad uno studio di proteomica, è stato espresso in forma solubile in E. coli ed un test di attività in vitro ha evidenziato la sua capacità di indurre la germinazione di spore “decoated” / When environmental conditions are unfavorable to the growth, Bacillus and Clostridium bacteria (including Clostridium botulinum, the causative agent of foodborne botulism) form endospores, metabolically dormant cell types resistant to several adverse conditions and difficult to kill. However, under suitable conditions, spores resume the vegetative life by triggering the germination process. Thus, spores are dangerous agents of human foodborne disease and food spoilage. In this work, the strain Clostridium sporogenes UC9000, isolated from raw milk, was used like not-pathogenic model of Clostridium botulinum to better understand the mechanisms underpinning the Clostridium germination. Clostridium sporogenes is a species phylogenetically related to Clostridium botulinum and often used like its surrogate. Physiological studies revealed that UC9000 spores germinate in presence of L-alanine/L-cysteine in combination with L-lactate, while in silico analyses allowed the identification of homologues of the Bacillus germinant receptors responsive to L-alanine. The genome screening also detected genes coding for SleB, CwlJ and SleL, enzymes participating to the cortex degradation. CwlJ was found resident in the spore coat by performing a proteomic analysis, it was expressed in soluble form in E. coli and an in vitro assay of activity revealed its capability to induce germination when added exogenously to decoated spores

Enterococcus faecium è un componente fondamentale del microbiota di diversi alimenti fermentati quali formaggi e salumi e viene spesso isolato in alto numero in alimenti pronti al consumo. É inoltre largamente utilizzato come probiotico sia per l’uomo che per gli animali. Allo stesso tempo, però, questa specie batterica rappresenta una delle cause principali di infezioni nosocomiali quali endocarditi ed infezioni al tratto urinario. Studi recenti hanno dimostato che la specie E. faecium è costituita da due sub-popolazioni principali: la prima è denominate hospital associated (HA) clade “A” ed include la maggior parte dei ceppi responsabili di infezioni umane; la seconda è chiamata community associated (CA) clade “B”, e contiene principalmente ceppi commensali dell’uomo. Analisi più approfondite hanno rivelato un ulteriore suddivisione all’interno del clade A, nel sub-clade A1 (che raggruppa la maggioranza dei ceppi clinici) e nel sub-clade A2, associato agli animali e più sporadicamente ad infezioni umane. Nel 2012, EFSA ha redatto una linea guida per la valutazione della sicurezza di E. faecium usato come probiotico per gli animali, concludendo che i cepi appartenenti all’hospital-associated clade non devono essere utilizzati in nutrizione animale. Comunque, la distinzione tra le due sub-popolazioni è stata fatta utilizzando dati ottenuti prevalentemente da isolati umani e animali e solo un numero limitato di ceppi isolati dagli alimenti è stato considerato. Obiettivo di questa tesi di dottorato è stato quello di valutare la sicurezza di E. faecium negli alimenti fermentati, considerando ceppi isolati da formaggi artigianali e prodotti carnei e utilizzando sia tecniche di genomica che analisi fisiologiche. Nessuno dei ceppi alimentari studiati è risultato parte del clade A1, ma un ceppo isolato da un salame stagionato pronto al consumo ha rivelato diversi tratti tipici dei ceppi A1, tra cui particolari IS, transposase e geni di resistenza agli antibiotici. Questi risultati, così come altri dati, sottolineano la necessità di approfondire le conoscenze circa il ruolo dei ceppi di E. faecium isolati da alimenti come fattore di rischio per la salute umana. / Enterococcus faecium is commonly found in high numbers in ready to eat foods, being a member of the bacterial communities of a variety of fermented foods, including cheese and sausages, and is widely used as human and animal probiotic. However, this bacterial species is a leading cause of nosocomial infection, mainly endocarditis and urinary tract infections. Recent studies have demonstrated that E. faecium species consists of two very distinct clades: the hospital associated (HA) clade “A”, which includes most of the strains responsible for human infections, and the community associated (CA) clade “B”, that contains primarily human commensal isolates. Deeper analysis revealed a further split within clade A into sub-clade A1 (which groups the vast majority of clinical isolates), and sub-clade A2, associated with animals and sporadic human infections. In 2012, the European Food Safety Authority has issued a guideline for the safety assessment of E. faecium used as animal probiotics, concluding the strains belonging to the hospital-associated clade should not be used in animal nutrition. However, the differentiation of the two clades has been performed using data mainly deriving from human and animal isolates, and only a limited number of strains from the food chain were considered. Aim of this doctoral thesis was to assess the safety of E. faecium in fermented food, considering strains isolated from artisanal cheese and meat products, and using both whole genome-based techniques and physiological studies. None of the food isolates studied in this work belong to the epidemic clade A1, however a strain isolated from a ready to eat salami revealed several A1-specific traits, such as specific IS, transposases and antibiotic resistance genes. These results, as well as other data, underline the emergency of deeper understanding the role of E. faecium isolated from fermented foods as risk factor for human health.