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.

Highlights d Cities possess a consistent ''core'' set of non-human microbes d Urban microbiomes echo important features of cities and city-life d Antimicrobial resistance genes are widespread in cities d Cities contain many novel bacterial and viral species

Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 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 primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies. Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address 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 analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

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.

We recently demonstrated that cow's milk fermented with the probiotic 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 whether FM-CBAL74 could regulate gut microbiota composition and butyrate production. We randomly selected 20 healthy children (12 to 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 the fecal butyrate concentration was also measured. Microbial alpha and beta diversities were not significantly different between groups prior to treatment. FM-CBAL74 but not PL treatment increased the relative abundance of Individual ,, and oligotypes were associated with 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. The use of a fermented milk product containing the heat-killed probiotic strain 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.

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Purpose To investigate whether a Mediterranean diet (MD) affected the plasma concentrations of endocannabinoids (ECs), N-acylethanolamines (NAEs) and their specific ratios in subjects with lifestyle risk factors for metabolic diseases. To identify the relationship between circulating levels of these compounds and gut microbiome, insulin resistance and systemic inflammation. Methods A parallel 8-week randomised controlled trial was performed involving 82 overweight and obese subjects aged (mean ± SEM) 43 ± 1.4 years with a BMI of 31.1 ± 0.5 kg/m2, habitual Western diet (CT) and sedentary lifestyle. Subjects were randomised to consume an MD tailored to their habitual energy and macronutrient intake (n = 43) or to maintain their habitual diet (n = 39). Endocannabinoids and endocannabinoid-like molecules, metabolic and inflammatory markers and gut microbiome were monitored over the study period. Results The MD intervention lowered plasma arachidonoylethanolamide (AEA, p = 0.02), increased plasma oleoylethanolamide/palmitoylethanolamide (OEA/PEA, p = 0.009) and OEA/AEA (p = 0.006) and increased faecal Akkermansia muciniphila (p = 0.026) independent of body weight changes. OEA/PEA positively correlated with abundance of key microbial players in diet–gut–health interplay and MD adherence. Following an MD, individuals with low-plasma OEA/PEA at baseline decreased homeostatic model assessment of insulin resistance index (p = 0.01), while individuals with high-plasma OEA/PEA decreased serum high-sensitive C-reactive protein (p = 0.02). Conclusions We demonstrated that a switch from a CT to an isocaloric MD affects the endocannabinoid system and increases A. muciniphila abundance in the gut independently of body weight changes. Endocannabinoid tone and microbiome functionality at baseline drives an individualised response to an MD in ameliorating insulin sensitivity and inflammation. Clinical Trial Registry number and website NCT03071718; www.clinicaltrials.gov

The human oral cavity is a complex ecosystem, and the alterations in salivary microbial communities are associated with both oral and nonoral diseases. The Mediterranean diet (MD) is a healthy dietary pattern useful for both prevention and treatment of several diseases. To further explore the effects of the MD on human health, in this study, we investigated the changes in the salivary microbial communities in overweight/obese subjects after an individually tailored MD-based nutritional intervention. Healthy overweight and obese subjects were randomized between two intervention groups. The MD group (Med-D group) increased their MD adherence during 8 weeks of intervention while the control diet (control-D) group did not change their dietary habits. The salivary microbiota was assessed at baseline and after 4 and 8 weeks of intervention. Despite no observed changes in the overall salivary microbiota composition, we found a significant decrease in the relative abundances of species-level operational taxonomic units annotated as Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola in the Med-D group compared to that in the control-D group after 8 weeks of intervention, which are known to be associated with periodontal disease. Such variations were significantly linked to dietary variables such as MD adherence rates and intakes of animal versus vegetable proteins. In addition, increased levels of Streptococcus cristatus were observed in the Med-D group, which has been reported as an antagonistic taxon inhibiting P. gingivalis gene expression. Our findings suggest that an MD-based nutritional intervention may be implicated in reducing periodontal bacteria, and an MD may be a dietary strategy supportive of oral homeostasis. IMPORTANCE Changes in dietary behavior with increased adherence to a Mediterranean diet can determine a reduction of periodontopathogenic bacterial abundances in the saliva of overweight subjects with cardiometabolic risk due to an unhealthy lifestyle, without any change in individual energy intake, nutrient intake, and physical activity.