Specific signatures of the gut microbiota and increased levels of butyrate in children treated with fermented cow's milk containing heat-killed Lactobacillus paracasei CBA L74

We recently demonstrated that cow's milk fermented with the probiotic L.paracasei CBA L74 (FM-CBAL74) reduces the incidence of respiratory and gastrointestinal tract infections in young children attending school. This effect apparently derives from a complex regulation of non-immune and immune protective mechanisms. We investigated wheter FM-CBAL74 could regulate gut microbiota composition and butyrate production.We randomly selected 20 healthy children (12-48 months) from the previous randomized controlled trial, before (t0) and after 3 months (t3) of dietary treatment with FM-CBAL74 (FM), or placebo (PL). Fecal microbiota was profiled using 16S rRNA gene amplicon sequencing and fecal butyrate concentration was also measured. Microbial alpha and beta-diversity were not significantly different between groups prior to treatment. FM-CBAL74 but not PL treatment increased the relative abundance of Lactobacillus. Individual Blautia, Roseburia and Faecalibacterium oligotypes were associated to FM-CBAL74 treatment and demonstrated correlative associations with immune biomarkers. Accordingly, PICRUSt analysis predicted an increase in the proportion of genes involved in butyrate production pathways, consistent with an increase in fecal butyrate observed only in the FM group. Dietary supplementation with FM-CBAL74 induces specific signatures in gut microbiota composition and stimulates butyrate production. These effects are associated with changes in innate and acquired immunity.Importance: The use of a fermented milk product containing the heat-killed probiotic strain L.paracasei CBAL74 induces changes in the gut microbiota, promoting the development of butyrate-producers. These changes in the gut microbiota composition correlate with increased levels of innate and acquired immunity biomarkers

Gut microbiota composition and butyrate production in children affected by non-IgE-mediated cow’s milk allergy

Cow’s milk allergy (CMA) is one of the earliest and most common food allergy and can be elicited by both IgE- or non-IgE-mediated mechanism. We previously described dysbiosis in children with IgE-mediated CMA and the effect of dietary treatment with extensively hydrolyzed casein formula (EHCF) alone or in combination with the probiotic Lactobacillus rhamnosus GG (LGG). On the contrary, the gut microbiota in non-IgE-mediated CMA remains uncharacterized. In this study we evaluated gut microbiota composition and fecal butyrate levels in children affected by non-IgE-mediated CMA. We found a gut microbiota dysbiosis in non-IgE-mediated CMA, driven by an enrichment of Bacteroides and Alistipes. Comparing these results with those previously obtained in children with IgE-mediated CMA, we demonstrated overlapping signatures in the gut microbiota dysbiosis of non-IgE-mediated and IgE-mediated CMA children, characterized by a progressive increase in Bacteroides from healthy to IgE-mediated CMA patients. EHCF containg LGG was more strongly associated with an effect on dysbiosis and on butyrate production if compared to what observed in children treated with EHCF alone. If longitudinal cohort studies in children with CMA will confirm these results, gut microbiota dysbiosis could be a relevant target for innovative therapeutic strategies in children with non-IgE-mediated CMA

Mediterranean diet intervention in overweight and obese subjects lowers plasma cholesterol and causes changes in the gut microbiome and metabolome independently of energy intake

ObjectivesThis study aimed to explore the effects of an isocaloric Mediterranean diet (MD) intervention on metabolic health, gut microbiome and systemic metabolome in subjects with lifestyle risk factors for metabolic disease.DesignEighty-two healthy overweight and obese subjects with a habitually low intake of fruit and vegetables and a sedentary lifestyle participated in a parallel 8-week randomised controlled trial. Forty-three participants consumed an MD tailored to their habitual energy intakes (MedD), and 39 maintained their regular diets (ConD). Dietary adherence, metabolic parameters, gut microbiome and systemic metabolome were monitored over the study period.ResultsIncreased MD adherence in the MedD group successfully reprogrammed subjects' intake of fibre and animal proteins. Compliance was confirmed by lowered levels of carnitine in plasma and urine. Significant reductions in plasma cholesterol (primary outcome) and faecal bile acids occurred in the MedD compared with the ConD group. Shotgun metagenomics showed gut microbiome changes that reflected individual MD adherence and increase in gene richness in participants who reduced systemic inflammation over the intervention. The MD intervention led to increased levels of the fibre-degrading Faecalibacterium prausnitzii and of genes for microbial carbohydrate degradation linked to butyrate metabolism. The dietary changes in the MedD group led to increased urinary urolithins, faecal bile acid degradation and insulin sensitivity that co-varied with specific microbial taxa.ConclusionSwitching subjects to an MD while maintaining their energy intake reduced their blood cholesterol and caused multiple changes in their microbiome and metabolome that are relevant in future strategies for the improvement of metabolic health

Dietary interventions and host-microbiome interaction

The term microbiome refers to the whole community of living microorganisms in a sample along with their potential activities that might influence the metabolic capabilities and functioning of such micro-environment. In particular, the human gut microbiome includes symbiotic bacteria, viruses, fungi and archaea located in the last part of our gastrointestinal tract. Each individual has a unique gut microbial composition as a peculiar fingerprint and it has been agreed that the development of several types of diseases in humans might be linked to gut microbiome perturbation in its microbial equilibrium, such condition known as dysbiosis. To this regard, in the last decades a worrisome increase in food allergy prevalence linked to a defect in immune tolerance mechanisms has been observed. The onset of such multifactorial disease is in turn associated to a gut microbiota alteration and mediated by both genetic and environmental risk factors, especially in pediatric age. Moreover, it's known that the immune system may control microbial composition and diversity. It should be considered that most of the knowledge on the associations between gut microbiota and immunity system derives from animal model studies. For this reason, the understanding of the relationship between food allergies and intestinal dysbiosis could be translated into advances regarding i) knowledge in prevention of onset of such diseases and ii) clinical practice with diet-directed therapeutic interventions using pro- or prebiotics, aimed at modulating the compromised immune system indirectly through gut microbiota activities. Indeed, the role of diet in influencing the gut microbiota composition and functions is widely recognized and the existence of the axis diet-microbiota-health is nowadays well established. The food-human interplay is of interest because the most recent trends are oriented towards a profitable use of diet to provide benefits to human health. In this respect, the Mediterranean diet received great attention as an appropriate strategy for the prevention and improvement of human health status. In fact, it has been demonstrated such dietary pattern being beneficial for the treatment of obesity, type II diabetes, inflammatory diseases, colorectal cancer and cardiovascular diseases. The significant and tangible evidence of the link between the Mediterranean diet and gut microbiota opens encouraging paths towards the establishments of diet-based health care and disease prevention. Despite such evidences, many challenges still exist since most of the studies assessed the impact of diet over human microbiome through correlation as well as association researches. Further efforts are necessary to understand the complicate host-microbiome interaction and to contribute to shed light on novel and different dietary strategies in order to beneficially impact human microbiome as well as human health. The overall aim of this thesis is to contribute to the evidences regarding the effects of dietary interventions in modulating the human microbiome which might in turn significantly impact human health, as to support the improvement of knowledge in such context

Different amplicon targets for sequencing-based studies of fungal diversity

Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes with the 16S rRNA being the gold standard for bacteria. As for Fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (ITS 1-2, 18S ribosomal small subunit RNA and D1/D2 domain of the 26S ribosomal large subunit RNA) both using biological samples and a mock community of common fungal species. All the three targets allowed an accurate identification of the species present. Nevertheless, high heterogeneity in ITS 1-2 length was found and this caused an over-estimation of the abundance of species with shorter ITS, while both 18S and 26S amplicons allowed a more reliable quantification. We demonstrated that ITS 1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities and efforts should be done for promoting the use of different targets in sequencing-based microbial ecology studies.Importance Amplicon-sequencing approaches for fungi may rely on different targets affecting diversity and abundance of the fungal species. An increasing number of studies will address the fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analysing a mock community and several biological samples we demonstrate that using different amplicon targets may change the results of fungal microbiota composition and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities

MODULAZIONE DEL MICROBIOTA INTESTINALE ATTRAVERSO UN INTERVENTO CON DIETA MEDITERRANEA IN SOGGETTI OBESI

Introduzione. Le diete tipiche dei paesi occidentali sono state associate all’alta incidenza di malattie metaboliche e cardiovascolari. In un recente studio osservazionale [1] è stato suggerito che gli effetti benefici spesso riportati per la dieta Mediterranea (DM) possano essere mediati dal microbioma intestinale. Pertanto è stato disegnato un intervento nutrizionale basato sui principi della DM per valutare l’effetto sul microbioma intestinale e sul relativo metaboloma. Metodi. Sono stati reclutati 80 soggetti sani, obesi e sovrappeso (età:18-60; BMI:25–35 kg/m2), con bassa aderenza alla DM. Quaranta soggetti hanno seguito per 2 mesi una dieta personalizzata ed isocalorica basata sulla DM, per aumentare il loro livello di aderenza senza avere perdite di peso, mentre 40 soggetti sono stati inseriti nel gruppo di controllo. È stato analizzato il microbiota fecale ed il metaboloma urinario e plasmatico. Risultati. Nonostante l’aderenza al protocollo è stata elevata in tutti i soggetti, è stata osservata una risposta diversa all’intervento nel gruppo DM, probabilmente legata alle caratteristiche individuali del microbiota. Nei soggetti DM che mostravano una migliore risposta metabolica all’intervento (riduzione dell’insulino-resistenza) è stato osservato un aumento di Faecalibacterium, Roseburia e altri Clostridia, riconosciuti come produttori di acidi grassi a corta catena. Inoltre, il gruppo di soggetti responders mostrava anche più bassi livelli di Prevotella prima del trattamento, rispetto ai non-responders, in accordo con recenti studi che correlano P. copri all’insulino-resistenza [2]. Conclusioni. Questo studio dimostra la possibilità di modulare il microbiota intestinale e le sue attività attraverso interventi nutrizionali mirati e che le caratteristiche individuali del microbiota possono influenzare la risposta al trattamento e pertanto devono essere prese in considerazione. Bibliografia [1] De Filippis F, et al. Gut 2016;65:1812-21 [2] Pedersen HK, et al. Nature 2016;535:376-8