Gut microbiota restructuring during diet-induced obesity: a dense longitudinal study reveals phasic perturbation, network hubs, and temporally ordered taxonomic shifts

Diet-induced obesity is associated with profound alterations in gut microbial communities, yet the temporal dynamics of these changes, including the sequence, rate, and phase-specificity of taxonomic shifts, remain incompletely characterized. Mice were assigned to normal diet (ND, n = 19, 4–5/cage) or high-fat diet (HFD, n = 20, 5/cage), with 468 fecal samples collected at 12 timepoints over 70 days (D1, D3, D5, D7, D14, D21, D28, D35, D42, D56, D63, D70), encompassing acute, early, mid, and late phases. Microbial composition was profiled using 16S rRNA V3-V4 gene sequencing. Alpha and beta diversity were assessed, and differential abundance analysis was performed using ANCOM-BC2 and MaAsLin3 to ensure robust identification of diet-responsive taxa. Co-occurrence network analysis, community state modeling, indicator species analysis, temporal cross-correlation, and PICRUSt2 function prediction were employed to characterize ecological dynamics. HFD feeding significantly reduced alpha diversity (Shannon index: HFD 4.58 ± 0.53 vs. ND 5.03 ± 0.34, p < 0.001) and altered community composition (PERMANOVA diet R² = 0.177, p = 0.001). Five genera were consistently identified as differentially abundant by both analytical methods: Lactococcus (enriched in HFD, Log2FC = 6.31), Taurinivorans (enriched, Log2FC = 4.41), Duncaniella (depleted, Log2FC = -3.22), Lepagella (depleted, Log2FC = -2.82), and CAG-873 (depleted, Log2FC = -2.71), with large effect sizes (|Cohen’s d| = 1.22–2.24). Per-mouse correlations confirmed significant associations with body weight (|ρ| = 0.67–0.82, all p.adj < 0.001). Four of five genera ranked among the top 10 network hubs by degree centrality. Temporal analysis revealed distinct phases of microbial reorganization, with Lactococcus expanding rapidly during the acute phase while Duncaniella showed progressive depletion. Cross-correlation lag analysis demonstrated that Duncaniella depletion preceded weight gain (peak at lag − 2, r = -0.31, modest effect), whereas Taurinivorans enrichment followed weight gain (peak at lag + 1, r = + 0.31, modest effect). Community state analysis identified two distinct configurations, with HFD mice exhibiting greater instability (15.8% vs. 7.1% transition rates). Indicator species analysis identified 23 genera significantly associated with specific temporal phases, with Lactococcus as the strongest chronic-phase indicator (IndVal = 0.924). PICRUSt2 functional prediction revealed profound metabolic reprogramming, with 342 pathways significantly altered (63% of tested), including enrichment of simple carbohydrate degradation and depletion of complex polysaccharide utilization, consistent with the taxonomic shifts observed. This study delineates the temporal progression of HFD-induced gut microbiota community restructuring, revealing a multi-phasic model in which rapid, taxon-specific responses in the acute phase are followed by progressive compositional alteration and chronic instability. Analysis distinguishes Duncaniella depletion as a potential temporal precedence of weight gain, while Taurinivorans enrichment appears secondary to metabolic deterioration. These findings identify specific microbial targets, characterize critical temporal windows for intervention.