The microbiota of the gastro-intestinal tract plays an important role in human health. In addition to their metabolic interactions with dietary constituents, gut bacteria may also be involved in more complex host interactions, such as modulation of the immune system. Changes in the microbiota during an individual’s lifespan are accompanied by modifications in multiple health parameters, and such observations have prompted intense scientific efforts aiming to understand the complex interactions between the microbiota and its human host, as well as how this may be influenced by diet (Milani et al., 2016). To investigate the role of diet of the microbiota we performed three studies. In the first study we investigated the human gut microbiota composition in obese and normal-weight children; in the second project we studied the role of polysaccharide and fiber privation in wild type mice to gain more insight into the role of diet on gut microbiota. In the third study we analyzed the role of another important microbiota communities: the oral microbiota, in association with diet and release of the inflammatory marker IL-8 from gingival epithelial cells (GEC). An altered gut microbiota has been linked to obesity in adulthood, though little is known about childhood obesity. In this study we characterized the composition of the gut microbiota in a large cohort of obese (n=42) and normal-weight school-aged children (n=36). The obese group was characterized by a more elevated intake of kcal/die and macronutrients (g/die) respect to normal-weight. Body mass index (BMI) z-score was significantly associated with gut microbiota composition, as determined with 16S rRNA gene amplicon sequencing, as well as levels of bacterial fermentation products (short chain fatty acids [SCFAs]). Obesity was associated with an altered gut microbiota characterized by elevated levels of Firmicutes and depleted levels of Bacteroidetes. Members of the Bacteroidetes were generally better predictors of BMI z-score and obesity than Firmicutes, which was likely due to discordant responses of Firmicutes OTUs. Correlation network analysis revealed that the gut microbiota of obese children also had increased correlation density and clustering of operational taxonomic units (OTUs). Moreover, we identified several bacterial taxa significantly associated with BMI z-score as well as SCFA levels. This suggests that alterations in the microbiota are linked to increased fermentation, which may play a role in promoting childhood obesity. To gain more insight into the role of diet on gut microbiota composition we studied the intestinal microbiota by using immunofluorescence images of fixed colon cross-sections for quantification and spatial organization of bacteria and laser capture microdissections (LCM) of specific areas from proximal to distal colon in wild-type mice. Subsequently, we sequenced LCM and stool samples by using 16S rRNA gene amplicon sequencing. We applied this protocol to three groups of mice with control diet (CON), fiber free (FF) and polysaccharide free (PF) demonstrating that elimination of microbiota-accessible carbohydrates from the diet, resulted in a decrease of diversity, changes in microbial composition along colon compartments and in stool samples, in a thinner mucus layer and in a reduced biovolume fraction of goblet cells. This high-resolution system to capture and examine spatial organization of intestinal microbes demonstrated the strong impact of diet on gut microbiota community but even on mucus layer structure and goblet cells. In the third study we examined the oral microbiota community in healthy humans and its association with nutrition, oral hygiene habits, and the release of the inflammatory marker IL-8 from GEC with and without stimulation with bacterial endotoxins to identify possible indicator operational taxonomic units (OTUs) for the initiation of oral diseases. GECs from twenty-one healthy participants were incubated with/without addition of bacterial lipopolysaccharides (LPS), and the oral microbiota was profiled using 16S rRNA gene-targeted sequencing. Members of the oral microbiota were associated with basal IL-8 levels, the intake of meat, tea, white wine, sweets and the use of chewing gum, as well as flossing habits, allergies, gender and BMI. Interestingly, the stimulation with bacterial endotoxin revealed eleven indicator bacterial OTUs, eight of which were associated with high basal levels of IL-8 and GEC response to LPS, two with high basal levels of IL-8, and one with low basal levels of IL8. The identification of indicator bacteria in healthy subjects with high levels of IL-8 release may be promising early-warning indicators for the possible onset of oral inflammations. In conclusion, in these three studies we demonstrated the importance of diet in the modulation of gut and oral microbiota in humans and wild-type mice and in health and disease, improving our understanding of host-microbes interactions.

THE MICROBIOTA: A COMPLEX INTERPLAY BETWEEN HOST,BACTERIAL COMMUNITIES AND DIET IN HEALTH AND DISEASE / A. Riva ; tutor: G.Morace ; coordinatore: G.Zuccotti. - : . DIPARTIMENTO DI SCIENZE DELLA SALUTE, 2017 Apr 11. ((29. ciclo, Anno Accademico 2016. [10.13130/riva-alessandra_phd2017-04-11].

THE MICROBIOTA: A COMPLEX INTERPLAY BETWEEN HOST,BACTERIAL COMMUNITIES AND DIET IN HEALTH AND DISEASE

A. Riva
2017-04-11

Abstract

The microbiota of the gastro-intestinal tract plays an important role in human health. In addition to their metabolic interactions with dietary constituents, gut bacteria may also be involved in more complex host interactions, such as modulation of the immune system. Changes in the microbiota during an individual’s lifespan are accompanied by modifications in multiple health parameters, and such observations have prompted intense scientific efforts aiming to understand the complex interactions between the microbiota and its human host, as well as how this may be influenced by diet (Milani et al., 2016). To investigate the role of diet of the microbiota we performed three studies. In the first study we investigated the human gut microbiota composition in obese and normal-weight children; in the second project we studied the role of polysaccharide and fiber privation in wild type mice to gain more insight into the role of diet on gut microbiota. In the third study we analyzed the role of another important microbiota communities: the oral microbiota, in association with diet and release of the inflammatory marker IL-8 from gingival epithelial cells (GEC). An altered gut microbiota has been linked to obesity in adulthood, though little is known about childhood obesity. In this study we characterized the composition of the gut microbiota in a large cohort of obese (n=42) and normal-weight school-aged children (n=36). The obese group was characterized by a more elevated intake of kcal/die and macronutrients (g/die) respect to normal-weight. Body mass index (BMI) z-score was significantly associated with gut microbiota composition, as determined with 16S rRNA gene amplicon sequencing, as well as levels of bacterial fermentation products (short chain fatty acids [SCFAs]). Obesity was associated with an altered gut microbiota characterized by elevated levels of Firmicutes and depleted levels of Bacteroidetes. Members of the Bacteroidetes were generally better predictors of BMI z-score and obesity than Firmicutes, which was likely due to discordant responses of Firmicutes OTUs. Correlation network analysis revealed that the gut microbiota of obese children also had increased correlation density and clustering of operational taxonomic units (OTUs). Moreover, we identified several bacterial taxa significantly associated with BMI z-score as well as SCFA levels. This suggests that alterations in the microbiota are linked to increased fermentation, which may play a role in promoting childhood obesity. To gain more insight into the role of diet on gut microbiota composition we studied the intestinal microbiota by using immunofluorescence images of fixed colon cross-sections for quantification and spatial organization of bacteria and laser capture microdissections (LCM) of specific areas from proximal to distal colon in wild-type mice. Subsequently, we sequenced LCM and stool samples by using 16S rRNA gene amplicon sequencing. We applied this protocol to three groups of mice with control diet (CON), fiber free (FF) and polysaccharide free (PF) demonstrating that elimination of microbiota-accessible carbohydrates from the diet, resulted in a decrease of diversity, changes in microbial composition along colon compartments and in stool samples, in a thinner mucus layer and in a reduced biovolume fraction of goblet cells. This high-resolution system to capture and examine spatial organization of intestinal microbes demonstrated the strong impact of diet on gut microbiota community but even on mucus layer structure and goblet cells. In the third study we examined the oral microbiota community in healthy humans and its association with nutrition, oral hygiene habits, and the release of the inflammatory marker IL-8 from GEC with and without stimulation with bacterial endotoxins to identify possible indicator operational taxonomic units (OTUs) for the initiation of oral diseases. GECs from twenty-one healthy participants were incubated with/without addition of bacterial lipopolysaccharides (LPS), and the oral microbiota was profiled using 16S rRNA gene-targeted sequencing. Members of the oral microbiota were associated with basal IL-8 levels, the intake of meat, tea, white wine, sweets and the use of chewing gum, as well as flossing habits, allergies, gender and BMI. Interestingly, the stimulation with bacterial endotoxin revealed eleven indicator bacterial OTUs, eight of which were associated with high basal levels of IL-8 and GEC response to LPS, two with high basal levels of IL-8, and one with low basal levels of IL8. The identification of indicator bacteria in healthy subjects with high levels of IL-8 release may be promising early-warning indicators for the possible onset of oral inflammations. In conclusion, in these three studies we demonstrated the importance of diet in the modulation of gut and oral microbiota in humans and wild-type mice and in health and disease, improving our understanding of host-microbes interactions.
MORACE, GIULIA
ZUCCOTTI, GIAN VINCENZO
Settore MED/07 - Microbiologia e Microbiologia Clinica
THE MICROBIOTA: A COMPLEX INTERPLAY BETWEEN HOST,BACTERIAL COMMUNITIES AND DIET IN HEALTH AND DISEASE / A. Riva ; tutor: G.Morace ; coordinatore: G.Zuccotti. - : . DIPARTIMENTO DI SCIENZE DELLA SALUTE, 2017 Apr 11. ((29. ciclo, Anno Accademico 2016. [10.13130/riva-alessandra_phd2017-04-11].
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/489400
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