Fig 1.
Study design depicting sample collection in both cohorts.
Samples were coded as follows: M0 –before starting metformin treatment, M24h – 24 hours after first metformin dose, and M7d – 7 days after the first intake of metformin. T2D –type 2 diabetes.
Table 1.
Characteristics of the analyzed cohorts.
Fig 2.
Diversity characteristics of analyzed samples.
(A) Beta diversity characterizing and comparing samples before metformin therapy between healthy individuals (H) and OPTIMED cohort patients (T2D). Ellipses represent the 95% confidence interval surrounding each group of samples. Different symbols represent the participants of the study. Red circles correspond to healthy individuals while green triangles represent type 2 diabetes patients. (B) Alpha diversity calculated in all analyzed time points. Groups marked as follows: H–healthy individuals; T2D – type 2 diabetes patients. Samples: M0—before starting metformin treatment; M24h – 24 hours after the first intake of metformin; M7d –after 7 days treatment with metformin. Violin plot representing the effective number of species combines boxplots, depicting the median value and interquartile ranges, with Kernel density plots.
Fig 3.
GraPhlAn cladogram for taxonomic composition changes during a week-long metformin administration.
(A) healthy individuals, (B) type 2 diabetes patients. Samples are marked as follows: M0—before starting metformin treatment (blue); M7d –after 7 days treatment with metformin (yellow). Colors of nodes and shading indicate the microbial lineages that are enriched within corresponding samples. Only differentially abundant taxa at the genus or higher taxonomic ranks are indicated. For detailed results in lower taxonomical levels, see S1 and S2 Figs.
Fig 4.
Differences in gut microbiome taxonomic composition between patients with severe side effects (SE_1) and no or mild reported side effects (SE_0) in both cohorts at all analyzed time points.
(A) Healthy individuals at M0, (B) healthy individuals at M7d, (C) OPTIMED cohort at M0, (D) OPTIMED cohort at M7d. Taxa enriched in patients with severe side effects are indicated with yellow color, and taxa enriched in patients with no or mild side effects are in blue. Samples: M0 – before starting metformin treatment; M7d –after 7 days treatment with metformin.
Table 2.
Characteristics of OPTIMED cohort’s subgroups divided by response to metformin therapy during the first three months of therapy.
Fig 5.
Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) of OPTIMED cohort subgroups at M0 time point.
(A) Sample plot depicting the first two sPLS-DA components with 95% confidence level ellipse plots. The background coloring describes the predicted area for each class, defined as the 2D surface where all points are predicted to be of the same class. Samples and subgroups coded as follows: R–Responders, orange triangles; NR–Non-Responders, blue circles. (B) VIP (variable importance projection) score dot-chart classified by sPLS-DA. Depicted taxonomic groups with VIP≥1.5 in the first component. (C) The contribution of each taxonomic group on the first and second components, the length of the bar represents the importance of each feature to the component (importance from bottom to top). Colors indicate the patient subgroup (NR (blue) vs R (orange)) in which the taxonomic group is most abundant.
Fig 6.
Cellular function enrichment analysis comparing functional profiles before and after 7 days long metformin therapy both in the OPTIMED cohort and in healthy individuals.
Respectively, H_M0 (blue) and T2D_M0 (yellow) results represent data on pathways and functions reduced during metformin therapy and H_M7d (red) and T2D_M7d (green) – pathways and functions increased, only significant (p<0.05) enrichment categories are shown. Enrichment results are depicted in the following set of functional panels: (A) Biosynthesis, (B) Degradation, (C) Energy, (D) Other pathways, (E) Central dogma, (F) Cell exterior. Groups marked as follows: H–healthy individuals; T2D – type 2 diabetes patients. Samples: M0—before starting metformin treatment; M7d –after 7 days treatment with metformin. Pathway abbreviations: (A) AA Syn – amino acid biosynthesis; Nucleo Syn – nucleoside and nucleotide biosynthesis; FA/Lip Syn–fatty acid and lipid biosynthesis; Amine Syn–amine and polyamine biosynthesis; Carbo Syn – carbohydrate biosynthesis; Sec Metab Syn–secondary metabolite biosynthesis; Cofactor Syn–cofactor, carrier, and vitamin biosynthesis; Cell-Struct Syn–cell structure biosynthesis; Metab Reg Syn–metabolic regulator biosynthesis. (B) AA Deg–amino acid degradation; Nucleo Deg–nucleoside and nucleotide degradation; FA/Lip Deg–fatty acid and lipid degradation; Amine Deg–amine and polyamine degradation; Carbo Deg –carbohydrates and carboxylates degradation; Sec Metab Deg–secondary metabolite degradation; Alcochol Deg–alcohol degradation; Aromatic Deg–aromatic compound degradation. (D) C1 Util–C1 compound utilization and assimilation; Inorganic Nutr – inorganic nutrient metabolism; Act/Inact/Inter—Activation/Inactivation/Interconversion. (E) Prot Metab–protein metabolism. (F) Cell Wall Gen–cell wall biogenesis/organization proteins; LPS Metab – Lipopolysaccharide Metabolism Proteins; Plasma Mem–plasma membrane proteins; Periplasm–periplasmic proteins.