Characterization of the gut bacterial and viral microbiota in latent autoimmune diabetes in adults.

Latent autoimmune diabetes in adults (LADA) is a heterogeneous disease characterized by autoantibodies against insulin producing pancreatic beta cells and initial lack of need for insulin treatment. The aim of the present study was to investigate if individuals with LADA have an altered gut microbiota relative to non-diabetic control subjects, individuals with type 1 diabetes (T1D), and individuals with type 2 diabetes (T2D). Bacterial community profiling was performed with primers targeting the variable region 4 of the 16S rRNA gene and sequenced. Amplicon sequence variants (ASVs) were generated with DADA2 and annotated to the SILVA database. The gut virome was sequenced, using a viral particle enrichment and metagenomics approach, assembled, and quantified to describe the composition of the viral community. Comparison of the bacterial alpha- and beta-diversity measures revealed that the gut bacteriome of individuals with LADA resembled that of individuals with T2D. Yet, specific genera were found to differ in abundance in individuals with LADA compared with T1D and T2D, indicating that LADA has unique taxonomical features. The virome composition reflected the stability of the most dominant order Caudovirales and the families Siphoviridae, Podoviridae, and Inoviridae, and the dominant family Microviridae. Further studies are needed to confirm these findings.

Association of general health and lifestyle factors with the salivary microbiota - Lessons learned from the ADDITION-PRO cohort.

Introduction: Previous research indicates that the salivary microbiota may be a biomarker of oral as well as systemic disease. However, clarifying the potential bias from general health status and lifestyle-associated factors is a prerequisite of using the salivary microbiota for screening. Materials & Methods: ADDDITION-PRO is a nationwide Danish cohort, nested within the Danish arm of the Anglo-Danish-Dutch Study of Intensive treatment in People with Screen-Detected Diabetes in Primary Care. Saliva samples from n=746 individuals from the ADDITION-PRO cohort were characterized using 16s rRNA sequencing. Alpha- and beta diversity as well as relative abundance of genera was examined in relation to general health and lifestyle-associated variables. Permutational multivariate analysis of variance (PERMANOVA) was performed on individual variables and all variables together. Classification models were created using sparse partial-least squares discriminant analysis (sPLSDA) for variables that showed statistically significant differences based on PERMANOVA analysis (p < 0.05). Results: Glycemic status, hemoglobin-A1c (HbA1c) level, sex, smoking and weekly alcohol intake were found to be significantly associated with salivary microbial composition (individual variables PERMANOVA, p < 0.05). Collectively, these variables were associated with approximately 5.8% of the observed differences in the composition of the salivary microbiota. Smoking status was associated with 3.3% of observed difference, and smoking could be detected with good accuracy based on salivary microbial composition (AUC 0.95, correct classification rate 79.6%). Conclusions: Glycemic status, HbA1c level, sex, smoking and weekly alcohol intake were significantly associated with the composition of the salivary microbiota. Despite smoking only being associated with 3.3% of the difference in overall salivary microbial composition, it was possible to create a model for detection of smoking status with a high correct classification rate. However, the lack of information on the oral health status of participants serves as a limitation in the present study. Further studies in other cohorts are needed to validate the external validity of these findings.

Metformin-induced changes of the gut microbiota in healthy young men: results of a non-blinded, one-armed intervention study.

AIMS/HYPOTHESIS: Individuals with type 2 diabetes have an altered bacterial composition of their gut microbiota compared with non-diabetic individuals. However, these alterations may be confounded by medication, notably the blood-glucose-lowering biguanide, metformin. We undertook a clinical trial in healthy and previously drug-free men with the primary aim of investigating metformin-induced compositional changes in the non-diabetic state. A secondary aim was to examine whether the pre-treatment gut microbiota was related to gastrointestinal adverse effects during metformin treatment. METHODS: Twenty-seven healthy young Danish men were included in an 18-week one-armed crossover trial consisting of a pre-intervention period, an intervention period and a post-intervention period, each period lasting 6 weeks. Inclusion criteria were men of age 18-35 years, BMI between 18.5 kg/m2 and 27.5 kg/m2, HbA1c < 39 mmol/mol (5.7%) and plasma creatinine within the normal range. No prescribed medication, including antibiotics, for 2 months prior to recruitment were allowed and no previous gastrointestinal surgery, discounting appendectomy or chronic illness requiring medical treatment. During the intervention the participants were given metformin up to 1 g twice daily. Participants were examined five times in the fasting state with blood sampling and recording of gastrointestinal symptoms. Examinations took place at Frederiksberg Hospital, Denmark before and after the pre-intervention period, halfway through and immediately after the end of intervention and after the wash-out period. Faecal samples were collected at nine evenly distributed time points, and bacterial DNA was extracted and subjected to 16S rRNA gene amplicon sequencing in order to evaluate gut microbiota composition. Subjective gastrointestinal symptoms were reported at each visit. RESULTS: Data from participants who completed visit 1 (n=23) are included in analyses. For the primary outcome the relative abundance of 11 bacterial genera significantly changed during the intervention but returned to baseline levels after treatment cessation. In line with previous reports, we observed a reduced abundance of Intestinibacter spp. and Clostridium spp., as well as an increased abundance of Escherichia/Shigella spp. and Bilophila wadsworthia. The relative abundance at baseline of 12 bacterial genera predicted self-reported gastrointestinal adverse effects. CONCLUSIONS/INTERPRETATION: Intake of metformin changes the gut microbiota composition in normoglycaemic young men. The microbiota changes induced by metformin extend and validate previous reports in individuals with type 2 diabetes. Secondary analyses suggest that pre-treatment gut microbiota composition may be a determinant for development of gastrointestinal adverse effects following metformin intake. These results require further investigation and replication in larger prospective studies. TRIAL REGISTRATION: Clinicaltrialsregister.eu 2015-000199-86 and ClinicalTrials.gov NCT02546050 FUNDING: This project was funded by Danish Diabetes Association and The Novo Nordisk Foundation.

The effect of drinking water pH on the human gut microbiota and glucose regulation: results of a randomized controlled cross-over intervention.

Studies in rodent models have shown that alterations in drinking water pH affect both the composition of the gut microbiota and host glucose regulation. To explore a potential impact of electrochemically reduced alkaline (pH ≈ 9) versus neutral (pH ≈ 7) drinking water (2 L/day) on human intestinal microbiota and host glucose metabolism we conducted a randomized, non-blinded, cross-over study (two 2-week intervention periods, separated by a 3-week wash-out) in 29 healthy, non-smoking Danish men, aged 18 to 35 years, with a body mass index between 20.0 to 27.0 kg m-2. Volunteers were ineligible if they had previously had abdominal surgery, had not been weight stabile for at least two months, had received antibiotic treatment within 2 months, or had a habitual consumption of caloric or artificially sweetened beverages in excess of 1 L/week or an average intake of alcohol in excess of 7 units/week. Microbial DNA was extracted from faecal samples collected at four time points, before and after each intervention, and subjected to 16S rRNA gene amplicon sequencing (Illumina MiSeq, V4 region). Glycaemic regulation was evaluated by means of an oral glucose tolerance test.No differential effect of alkaline versus neutral drinking water was observed for the primary outcome, overall gut microbiota diversity as represented by Shannon's index. Similarly, neither a differential effect on microbiota richness or community structure was observed. Nor did we observe a differential effect on the abundance of individual operational taxonomic units (OTUs) or genera. However, analyses of within period effects revealed a significant (false discovery rate ≤5%) increase in the relative abundance of 9 OTUs assigned to order Clostridiales, family Ruminococcaceae, genus Bacteroides, and species Prevotella copri, indicating a potential effect of quantitative or qualitative changes in habitual drinking habits. An increase in the concentration of plasma glucose at 30 minutes and the incremental area under the curve of plasma glucose from 0 30 and 0 120 minutes, respectively, was observed when comparing the alkaline to the neutral intervention. However, results did not withstand correction for multiplicity. In contrast to what has been reported in rodents, a change in drinking water pH had no impact on the composition of the gut microbiota or glucose regulation in young male adults. The study is registered at www.clinicaltrials.gov (NCT02917616).