STREPTOCOCCUS THERMOPHILUS UREASE ACTIVITY: PHYSIOLOGICAL ROLE AND TECHNOLOGICAL RELEVANCE IN DAIRY AND NON-DAIRY APPLICATIONS

The aim of this Ph.D. work was to investigate the physiological role and the technological relevance of the urease activity of the dairy bacterium Streptococcus thermophilus. It has been achieved a deeper comprehension of this peculiar enzymatic activity following different approaches. A milk-based medium that allows the discrimination between urease-positive S. thermophilus strains and urease-negative ones based on the colonies morphology had been developed; it was used as screening method looking for urease-defective mutants after UV mutagenesis of urease-positive strains of industrial interest. Moreover, a cytofluorimetric protocol for the evaluation of urease activity of various samples containing S. thermophilus was developed: the proposed applications are related to the evaluation of the urease activity of starter culture biomasses of S. thermophilus and to the enumeration of the S. thermophilus population in probiotic products containing this species, among others. We propose that the cytofluorimetric method should be seen as an innovative tool to put besides the standard ones to evaluate the quality of the products previously mentioned. The investigation of the physiological role of urease of S. thermophilus cells growing in milk highlighted that in presence of this enzymatic activity the overall metabolism of the species is boosted. Moreover, the cooperative role of urease, previously described for the yogurt consortium, has been extended also to the cooperation between a urease-positive S. thermophilus strain and a urease-negative one, supporting the proposal of the urease activity as an altruistic cooperative trait, which is costly for urease-positive species but provides a local benefit to the urease-negative species sharing the same environment, which can take advantage of the release of ammonia. At industrial level, urease activity is still considered more for its detrimental effects than for the positive effects exerted on S. thermophilus: we proposed different strategies to overcome this industrial problem. Firstly, we produced mutants of urease-positive strains of industrial interest, but they showed lower acidification rate compared to their wild types. So, we proposed to modify the production process of the biomasses with the aim of obtaining cells which carry a lower urease activity compared to those currently produced. In conclusion, the present work gives new insight in the comprehension of the urease activity of the dairy bacterium S. thermophilus, on how it can be exploited to improve the acidification performances of the strains and how it cannot, so far, be controlled during the milk acidification processes

Characterization of airborne viromes in cheese production plants

Aims To characterize airborne virus like particles isolated from two cheese production plants in order to reveal their complexity in terms of viral communities and microbial genes potentially mobilized by viruses. Methods and results Airborne virus like particles have been isolated from Grana Padano and Gorgonzola PDO cheese production plants and ripening cellars. A shotgun metagenomics analysis of the isolated viromes highlighted a high complexity of the viral communities both in terms of viral taxonomy and phage\u2010host associations. Bacterial reads in each of the viromes were confirmed to be abundant and their taxonomy appeared to be associated to the environmental parameters and the technological processes that characterize the sampling area. Antibiotic resistance genes have been identified in each virome thus confirming that phages could be involved in the mobilization of antimicrobial resistances among bacterial populations. Interestingly human viruses were also identified even if the contamination source was not revealed. Conclusions The environmental conditions, which are imposed by the technology of the dairy process, seams to shape the viral populations as a consequence of the adaptation of microbial taxa to those environments. The identification of sequences belonging to Legionella pneumophila and to the human papillomavirus, raised some considerations about the safety of cheese ripening cellars. Significance and impact of the study In conclusion, the analysis of the dairy airborne viromes, has revealed a high complexity of the viral communities even if the environments where the samples were collected were confined environments. Metagenomics of airborne viral population could be a promising monitoring tool for the biological characterization of dairy environments

Lean six sigma approach for reducing length of hospital stay for patients with femur fracture in a university hospital

Surgical intervention within 48 h of hospital admission is the gold standard procedure for the management of elderly patients with femur fractures, since the increase in preoperative waiting time is correlated with the onset of complications and longer overall length of stay (LOS) in the hospital. However, national evidence demonstrates that there is still the need to provide timely intervention for this type of patient, especially in some regions of central southern Italy. Here we discuss the introduction of a diagnostic–therapeutic assistance pathway (DTAP) to reduce the preoperative LOS for patients undergoing femur fracture surgery in a university hospital. A Lean Six Sigma methodology, based on the DMAIC cycle (Define, Measure, Analyze, Improve, Control), is implemented to evaluate the effectiveness of the DTAP. Data were retrospectively collected and analyzed from two groups of patients before and after the implementation of DTAP over a period of 10 years. The statistics of the process measured before the DTAP showed an average preoperative LOS of 5.6 days (standard deviation of 3.2), thus confirming the need for corrective actions to reduce the LOS in compliance with the national guidelines. The influence of demographic and anamnestic variables on the LOS was evaluated, and the impact of the DTAP was measured and discussed, demonstrating the effectiveness of the improvement actions implemented over the years and leading to a significant reduction in the preoperative LOS, which decreased to an average of 3.5 days (standard deviation of 3.60). The obtained reduction of 39% in the average LOS proved to be in good agreement with previously developed DTAPs for femur fracture available in the literature

Viromes as genetic reservoir for the microbial communities in aquatic environments : a focus on antimicrobial-resistance genes

Despite studies of viromes isolated from aquatic environments are becoming increasingly frequent, most of them are limited to the characterization of viral taxonomy. Bacterial reads in viromes are abundant but the extent to which this genetic material is playing a role in the ecology of aquatic microbiology remains unclear. To this aim, we developed of a useful approach for the characterization of viral and microbial communities of aquatic environments with a particular focus on the identification of microbial genes harbored in the viromes. Virus-like particles were isolated from water samples collected across the Lambro River, from the spring to the high urbanized Milan area. The derived viromes were analyzed by shotgun metagenomic sequencing looking for the presence, relative abundance of bacterial genes with particular focus on those genes involved in antimicrobial resistance mechanisms. Antibiotic and heavy metal resistance genes have been identified in all virome samples together with a high abundance of reads assigned to cellular processes and signaling. Virome data compared to those identified in the microbiome isolated from the same sample revealed differences in terms of functional categories and their relative abundance. To verify the role of aquatic viral population in bacterial gene transfer, water-based mesocosms were perturbed or not perturbed with a low dose of tetracycline. The results obtained by qPCR assays revealed variation in abundance of tet genes in the virome and microbiome highlighting a relevant role of viral populations in microbial gene mobilization

Targeting Food Allergy with Probiotics.

The dramatic increase in food allergy prevalence and severity globally is demanding effective strategies. Food allergy derives from a defect in immune tolerance mechanisms. Immune tolerance is modulated by gut microbiota composition and function, and gut microbiota dysbiosis has been associated with the development of food allergy. Selected probiotic strains could act on immune tolerance mechanisms. The mechanisms are multiple and still not completely defined. Increasing evidence is providing useful information on the choice of optimal bacterial species/strains, dosage, and timing for intervention. The increased knowledge on the crucial role played by gut microbiota-derived metabolites, such as butyrate, is also opening the way to a postbiotic approach in the stimulation of immune tolerance

Gut microbiota as target for innovative strategies against food allergy.

The dramatic increase in food allergy prevalence and severity globally requires effective strategies. Food allergy derives from a defect in immune tolerance mechanisms. Immune tolerance is modulated by gut microbiota function and structure, and microbiome alterations (dysbiosis) have a pivotal role in the development of food allergy. Environmental factors, including a low-fiber/high-fat diet, cesarean delivery, antiseptic agents, lack of breastfeeding, and drugs can induce gut microbiome dysbiosis, and have been associated with food allergy. New experimental tools and technologies have provided information regarding the role of metabolites generated from dietary nutrients and selected probiotic strains that could act on immune tolerance mechanisms. The mechanisms are multiple and still not completely defined. Increasing evidence has provided useful information on optimal bacterial species/strains, dosage, and timing for intervention. The increased knowledge of the crucial role played by nutrients and gut microbiota-derived metabolites is opening the way to a post-biotic approach in the stimulation of immune tolerance through epigenetic regulation. This review focused on the potential role of gut microbiome as the target for innovative strategies against food allergy

Myogenic commitment of human stem cells by myoblasts Co-culture: a static vs. a dynamic approach

An in-vitro model of human bone marrow mesenchymal stem cells (hBM-MSCs) myogenic commitment by synergic effect of a differentiation media coupled with human primary skeletal myoblasts (hSkMs) co-culture was developed adopting both conventional static co-seeding and perfused culture systems. Static co-seeding provided a notable outcome in terms of gene expression with a significant increase of Desmin (141-fold) and Myosin heavy chain II (MYH2, 32-fold) at day 21, clearly detected also by semi-quantitative immunofluorescence. Under perfusion conditions, myogenic induction ability of hSkMs on hBM-MSCs was exerted by paracrine effect with an excellent gene overexpression and immunofluorescence detection of MYH2 protein; furthermore, due to the dynamic cell culture in separate wells, western blot data were acquired confirming a successful cell commitment at day 14. A significant increase of anti-inflammatory cytokine gene expression, including IL-10 and IL-4 (15-fold and 11-fold, respectively) at day 14, with respect to the pro-inflammatory cytokines IL-12A (7-fold at day 21) and IL-1 beta (1.4-fold at day 7) was also detected during dynamic culture, confirming the immunomodulatory activity of hBM-MSCs along with commitment events. The present study opens interesting perspectives on the use of dynamic culture based on perfusion as a versatile tool to study myogenic events and paracrine cross-talk compared to the simple co-seeding static culture

The Other Side of Plastics: Bioplastic-Based Nanoparticles for Drug Delivery Systems in the Brain

: Plastics have changed human lives, finding a broad range of applications from packaging to medical devices. However, plastics can degrade into microscopic forms known as micro- and nanoplastics, which have raised concerns about their accumulation in the environment but mainly about the potential risk to human health. Recently, biodegradable plastic materials have been introduced on the market. These polymers are biodegradable but also bioresorbable and, indeed, are fundamental tools for drug formulations, thanks to their transient ability to pass through biological barriers and concentrate in specific tissues. However, this "other side" of bioplastics raises concerns about their toxic potential, in the form of micro- and nanoparticles, due to easier and faster tissue accumulation, with unknown long-term biological effects. This review aims to provide an update on bioplastic-based particles by analyzing the advantages and drawbacks of their potential use as components of innovative formulations for brain diseases. However, a critical analysis of the literature indicates the need for further studies to assess the safety of bioplastic micro- and nanoparticles despite they appear as promising tools for several nanomedicine applications

Streptococcus thermophilus urease activity boosts Lactobacillus delbrueckii subsp. bulgaricus homolactic fermentation

The proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in the yogurt consortium enhances the growth rate and size of each population. In contrast, the independent growth of the two species in milk leads to a slower growth rate and a smaller population size. In this study, we report the first evidence that the urease activity of S. thermophilus increases the intracellular pH of L. delbrueckii in the absence of carbon source. However, in milk, in the presence of lactose the alkalizing effect of urea-derived ammonia was not detectable. Nevertheless, based on glucose consumption and lactic acid production at different pHin, L. delbrueckii showed an optimum of glycolysis and homolactic fermentation at alkaline pH values. In milk, we observed that ammonia provided by urea hydrolysis boosted lactic acid production in S. thermophilus and in L. delbrueckii when the species were grown alone or in combination. Therefore, we propose that urease activity acts as an altruistic cooperative trait, which is costly for urease-positive individuals but provides a local benefit because other individuals can take advantage of urease-dependent ammonia release

3D Biomimetic Scaffold for Growth Factor Controlled Delivery: An In-Vitro Study of Tenogenic Events on Wharton's Jelly Mesenchymal Stem Cells.

The present work described a bio-functionalized 3D fibrous construct, as an interactive teno-inductive graft model to study tenogenic potential events of human mesenchymal stem cells collected from Wharton's Jelly (hWJ-MSCs). The 3D-biomimetic and bioresorbable scaffold was functionalized with nanocarriers for the local controlled delivery of a teno-inductive factor, i.e., the human Growth Differentiation factor 5 (hGDF-5). Significant results in terms of gene expression were obtained. Namely, the up-regulation of Scleraxis (350-fold, p ≤ 0.05), type I Collagen (8-fold), Decorin (2.5-fold), and Tenascin-C (1.3-fold) was detected at day 14; on the other hand, when hGDF-5 was supplemented in the external medium only (in absence of nanocarriers), a limited effect on gene expression was evident. Teno-inductive environment also induced pro-inflammatory, (IL-6 (1.6-fold), TNF (45-fold, p ≤ 0.001), and IL-12A (1.4-fold)), and anti-inflammatory (IL-10 (120-fold) and TGF-β1 (1.8-fold)) cytokine expression upregulation at day 14. The presented 3D construct opens perspectives for the study of drug controlled delivery devices to promote teno-regenerative events