EVALUATION OF THE IMPACT OF DIETARY INTERVENTIONS ON THE HUMAN INTESTINAL MICROBIAL ECOSYSTEM THROUGH IMPROVED BIOINFORMATICS AND STATISTICS

The aim of this PhD work was the characterization of the intestinal microbial ecosystem through bioinformatic and statistical analyses of the microbiomics data originated from three studies carried out on different human populations: healthy (non-diseased) adults, hyperlipidemic and normo-lipidemic children and adolescents, and subjects with diagnosed irritable bowel syndrome (IBS). Specifically, the three studies presented in this PhD theses were as follows: \u2022 Probiotic crossover intervention study: A randomized, double-blind, crossover, placebo-controlled intervention study was carried out to determine the impact of a Bifidobacterium bifidum strain on the IME of adult healthy volunteers. \u2022 Children\u2019s dyslipidemia single arm intervention study: In this trial, the IME of children and adolescents with primary hyperlipidemia (a risk factor for cardiovascular diseases) was compared with the IME of control (normo-lipidemic) group. In addition, the modulatory effect of the regular intake of hazelnuts on hyperlipidemic subjects\u2019s IME was examines in order to evaluate if the modulation of IME by hazelnuts ameliorates the lipid profile. \u2022 Irritable bowel syndrome (IBS) observational study: The aim of this trial was the characterization of the IME in human subjects affected by IBS. The characterization was performed through the analysis of the bacterial communities present in the faecal samples collected during a multicentre intervention trial (Cremon et al., 2017). Irritable bowel syndrome is the most common gastrointestinal disorder in western countries. IBS is conventionally classified in four different subtypes based on bowel clinical symptoms: constipation, diarrhoea, alternating constipation and diarrhoea, and unsubtyped IBS. Clinical and immunological data collected during the trial were used to investigate the potential correlations between the IME characteristic for a specific IBS subtypes and the physiological and clinical parameters of the host, including bowel symptoms, faecal levels of IgA and cytokines, and depression/anxiety scores. The analysis of the data collected during the three studies presented in this PhD thesis revealed significant correlations between the intestinal microbial ecosystem and specific physiological parameters, characteristic for the physiological conditions under study. Therefore, these findings suggest that the differential abundance of specific OTUs could be used as a biomarker for a specific host condition. Conclusion of the probiotic B. bifidum Bb crossover intervention trial The intervention trial based on the administration of B. bifidum strain Bb has shown that the probiotic treatment modified the relative abundances of bacterial taxa that have often been associated with healthy conditions. The treatment modulated the faecal levels of butyrate, a microbial metabolite exerting multiple effects on gut health. Therefore, the daily consumption of B. bifidum Bb cells may positively affect human health; however, as for most dietary interventions, the current state of knowledge does not allow us to better define the significance of any taxonomic or metabolite changes of the intestinal microbial ecosystem on the host health. Conclusion of children\u2019s dyslipidemia single arm intervention trial The results of this study support the hypothesis that young individuals with primary hyperlipidemia possess an dysbiotic intestinal microbial ecosystem, which could plausibly contribute to the abnormal lipid profile of these subjects. A limitation of this study is the small sample size, which may reduce the potential robustness of the obtained results. Moreover, further studies focusing on the mechanisms involved in such hypothesized association are warranted. Our study is the first human trial investigating the potential role of hazelnuts as IME modulator and, in specific, suggests that a dietary intervention with hazelnut could be an effective and practical strategy to positively modulate the IME of hyperlipidemic subjects. Conclusion of the IBS-subtypes observational study This descriptive study demonstrates that the altered distribution of bacteria within the Gram-positive order Clostridiales distinguishes the intestinal microbial ecosystem of IBS subtypes, plausibly contributing to the observed altered faecal levels of the SCFAs acetate, butyrate and propionate. Our study proposes intestinal Clostridiales and colonic SCFAs as IBS subtypes biomarkers that can also potentially represent therapeutic targets. In addition, this study supports the notion that distinct therapeutic approaches should be developed for the different IBS subtype

Impact of a multistrain probiotic formulation with high bifidobacterial content on the fecal bacterial community and short-chain fatty acid levels of healthy adults

The consumption of probiotic products is continually increasing, supported by growing scientific evidence of their efficacy. Considering that probiotics may primarily affect health (either positively or negatively) through gut microbiota modulation, the first aspect that should be evaluated is their impact on the intestinal microbial ecosystem. In this study, we longitudinally analyzed the bacterial taxonomic composition and organic acid levels in four fecal samples collected over the course of four weeks from 19 healthy adults who ingested one capsule a day for two weeks of a formulation containing at least 70 billion colony-forming units, consisting of 25% lactobacilli and 75% Bifidobacterium animalis subsp. lactis. We found that 16S rRNA gene profiling showed that probiotic intake only induced an increase in a single operational taxonomic unit ascribed to B. animalis, plausibly corresponding to the ingested bifidobacterial strain. Furthermore, liquid chromatography/mass spectrometry revealed a significant increase in the lactate and acetate/butyrate ratio and a trend toward a decrease in succinate following probiotic administration. The presented results indicate that the investigated probiotic formulation did not alter the intestinal bacterial ecosystem of healthy adults and suggest its potential ability to promote colonization resistance in the gut through a transient increase in fecal bifidobacteria, lactic acid, and the acetate/butyrate ratio

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

Hemifacial microsomia: Case report and literature review

Hemifacial microsomia (HFM) is a sporadic congenital malformation of the craniofacial structures derived from the first and second branchial arches. The incidence of HFM has been reported to range from 1 in 3,0001 to 1 in 26,0002 live births, making HFM the second most common congenital malformation in the face after cleft lip and/or palate. An 11-year-old girl came at Galeazzi Institute (Milan) in January 2017. She presented left hemifacial microsomia with absence of the left ramus of mandible and the left temporomandibular joint (tmj), part of the zygomatic arch, hypoplasia of the lateral and inferior orbital bone and of the zygomatic bone. She also presented a medial canthal dystopia. She underwent to costochondral bone graft and calvaria bone graft for reconstruction of part of the mandible and the TMJ. An emi-Le Fort I, emi-Le Fort III, and sagittal segmental osteotomy of the right mandible were performed to improve the correct occlusion. Traditionally, the costochondral graft has been considered the gold standard for ramus-condyle reconstruction in the pediatric mandible when appropriate. Some studies cite growth unpredictability and ankylosis as concerns with rib. Further studies examining carefully the factors predicting graft growth, such as size of cartilage cap, surgical technique, and postoperative physiotherapy, are warranted

Enrichment of intestinal Lactobacillus by enhanced secretory IgA coating alters glucose homeostasis in P2rx7 −/− mice

The secretory immunoglobulin A (SIgA) in mammalian gut protects the organism from infections and contributes to host physiology by shaping microbiota composition. The mechanisms regulating the adaptive SIgA response towards gut microbes are poorly defined. Deletion of P2rx7, encoding for the ATP-gated ionotropic P2X7 receptor, leads to T follicular helper (Tfh) cells expansion in the Peyer\u2019s patches (PPs) of the small intestine, enhanced germinal centre (GC) reaction and IgA secretion; the resulting alterations of the gut microbiota in turn affects host metabolism. Here, we define gut microbiota modifications that correlate with deregulated SIgA secretion and metabolic alterations in P2rx7 12/ 12 mice. In particular, Lactobacillus shows enhanced SIgA coating in P2rx7 12/ 12 with respect to wild-type (WT) mice. The abundance of SIgA-coated lactobacilli positively correlates with Tfh cells number and body weight, suggesting Lactobacillus-specific SIgA response conditions host metabolism. Accordingly, oral administration of intestinal Lactobacillus isolates from P2rx7 12/ 12 mice to WT animals results in altered glucose homeostasis and fat deposition. Thus, enhanced SIgA production by P2X7 insufficiency promotes Lactobacillus colonization that interferes with systemic metabolic homeostasis. These data indicate that P2X7 receptor-mediated regulation of commensals coating by SIgA is important in tuning the selection of bacterial taxa, which condition host metabolism

Evidence of dysbiosis in the intestinal microbial ecosystem of children and adolescents with primary hyperlipidemia and the potential role of regular hazelnut intake

Hyperlipidemia starts at a pediatric age and represents an unquestionable risk factor for cardiovascular disease. Modulation of the intestinal microbial ecosystem (IME), in principle, can ameliorate lipid profiles. In this study, we characterized the IME of children and adolescents with primary hyperlipidemia by analyzing fecal samples through 16S rRNA gene profiling (n\ua0=\ua015) and short chain fatty acid (SCFA) quantification (n\ua0=\ua032). The same analyses were also carried out on age-matched normolipidemic controls (n\ua0=\ua015). Moreover, we evaluated the modulatory effect of regular hazelnut intake (approximately 0.43 g of hazelnuts with skin per kg of body weight) on the IME of 15 children and adolescents with hyperlipidemia for eight weeks. We found alterations of numerous operational taxonomic units potentially associated with SCFA-producing bacteria and reductions in the fecal levels of acetate, butyrate and propionate in hyperlipidemic subjects. Furthermore, we observed that an eight-week hazelnut intervention may induce limited changes in fecal microbiota composition but can significantly modulate the fecal levels of predominant intestinal SCFAs, such as acetate. Finally, correlation analyses indicated that changes in lipidemic parameters are linked to modifications of the abundance of specific bacterial taxa, such as the families Lachnospiraceae and Ruminococcaceae and the genera Akkermansia, Bacteroides, Roseburia, and Faecalibacterium. This study suggests that children and adolescents with primary hyperlipidemia possess an altered IME. The promising results presented here support the need for future dietary interventions aimed at positively modulating the IME of hyperlipidemic subjects

Proteomic peptide scan of porphyromonas gingivalis fima type II for searching potential B-cell epitopes

Purpose. To identify potential antigenic targets for Porphyromonas gingivalis vaccine development. Materials and methods. In the present study, we analyzed the Porphyromonas gingivalis, fimA type II primary amino acid sequence and characterized the similarity to the human proteome at the pentapeptide level. Results. We found that exact peptide-peptide profiling of the fimbrial antigen versus the human proteome shows that only 19 out of 344 fimA type II pentapeptides are uniquely owned by the bacterial protein. Conclusions. The concept that protein immunogenicity is allocated in rare peptide sequences and the search the Porphyromonas gingivalis fimA type II sequence for peptides unique to the bacterial protein and absent in the human host, might be used in new therapeutical approaches as a significant adjunct to current periodontal therapies

Mechanical influence of tissue culture plates and extracellular matrix on mesenchymal stem cell behavior: A topical review

Tissue engineering applications need a continuous development of new biomaterials able to generate an ideal cell-extracellular matrix interaction. The stem cell fate is regulated by several factors, such as growth factors or transcription factors. The most recent literature has reported several publications able to demonstrate that environmental factors also contribute to the regulation of stem cell behavior, leading to the opinion that the environment plays the major role in the cell differentiation. The interaction between mesenchymal stem cells (MSCs) and extracellular environment has been widely described, and it has a crucial role in regulating the cell phenotype. In our laboratory (Tecnologica Research Institute, Crotone, Italy), we have recently studied how several physical factors influence the distribution and the morphology of MSCs isolated from dental pulp, and how they are able to regulate stem cell differentiation. Mechanical and geometrical factors are only a small part of the environmental factors able to influence stem cell behavior, however, this influence should be properly known: in fact, this assumption must be clearly considered during those studies involving MSCs; furthermore, these interactions should be considered as an important bias that involves an high number of studies on the MSCs, since in worldwide laboratories the scientists mostly use tissue culture plates for their experiments

Effect of oral consumption of capsules containing Lactobacillus paracasei LPC-S01 on the vaginal microbiota of healthy adult women: a randomized, placebo-controlled, double-blind crossover study

Oral consumption of probiotics is practical and can be an effective solution to preserve vaginal eubiosis. Here, we studied the ability of orally administered Lactobacillus paracasei LPC-S01 (DSM 26760) to affect the composition of the vaginal microbiota and colonize the vaginal mucosa in nondiseased adult women. A total of 40 volunteers took oral probiotic (24 billion CFU) or placebo capsules daily for 4 weeks, and after a 4-week washout, they switched to placebo or probiotic capsules according to the crossover design. A total of 23 volunteers completed the study according to the protocol. Before and after capsule ingestion, vaginal swabs were collected for qPCR quantification to detect L. paracasei LPC-S01 and for 16S rRNA gene sequencing. Vaginal swabs were grouped according to their bacterial taxonomic structure into nine community state types (CSTs), four of which were dominated by lactobacilli. Lactobacillus paracasei LPC-S01 was detected in the vagina of two participants. Statistical modeling (including linear mixed-effects model analysis) demonstrated that daily intake of probiotic capsules reduced the relative abundance of Gardnerella spp. Quantitative PCR with Gardnerella vaginalis primers confirmed this result. Considering the pathogenic nature of G. vaginalis, these results suggest a potential positive effect of this probiotic capsule on the vaginal microbial ecosystem

Surface Layer of Lactobacillus helveticus MIMLh5 Promotes Endocytosis by Dendritic Cells

Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-\u3b2), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-\u3b1) or IL-1\u3b2 were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production.IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs