Comparative Metagenomics of Gut Microbiota Across Diets in Urban Populations

Thesis Abstract

In urban populations, dietary patterns shape gut microbial ecology with implications for metabolic health and disease risk, yet comparative metagenomic analyses across diverse dietary regimes remain underexplored in metropolitan contexts. This study aims to elucidate how habitual diets influence gut microbiota composition, functional potential, and microbial community dynamics using whole-metagenome shotgun sequencing. The specific objectives are (1) to compare taxonomic profiles and functional pathways of gut microbiota among omnivorous, vegetarian/plant-based, and high-fat low-carbohydrate dietary groups; (2) to assess associations between dietary variables (macronutrient ratios, fiber intake, and processed-food consumption) and microbial diversity metrics (alpha and beta diversity) using multivariate regression; (3) to identify diet-linked metagenomic functional modules, such as short-chain fatty acid production pathways and bile salt hydrolase activities; (4) to evaluate temporal stability and inter-individual variability within dietary groups through cross-sectional sampling and, where feasible, short-term (three-month) follow-up subsets; and (5) to test theoretical predictions derived from the Community Assembly Theory and the Diet-Microbiome Interaction framework regarding niche specialization and functional redundancy. A cross-sectional design will be employed. The population comprises adults aged 25–55 residing in three major urban centers, with balanced representation of sex, BMI categories, and socio-economic status. A stratified random sampling approach will recruit 900 participants distributed equally across the three dietary groups (n=300 per group). Fecal samples will be collected using standardized collection kits and stored at ?80°C until processing. Metagenomic DNA will be extracted with validated protocols, and library preparation will precede sequencing on the Illumina NovaSeq platform to yield 150 bp paired-end reads with an average depth of 10 Gbp per sample. Alongside sequencing, a structured dietary intake questionnaire and 7-day food diaries will quantify macro- and micronutrient intake, fiber types, fermented foods, and ultra-processed food exposure. Additional covariates include age, sex, BMI, antibiotic use within six months, physical activity, and smoking status. Bioinformatic analyses will begin with quality control and host-microbial read separation, followed by taxonomic profiling via MetaPhlAn3 and functional profiling through HUMAnN3 to derive pathway abundance and gene family counts. Alpha diversity metrics (Shannon, Simpson, and Pielou’s evenness) and beta diversity via Bray-Curtis and Jaccard distances will be computed. Differential abundance analyses will employ DESeq2 for taxa and HUMAnN3-derived pathways, controlling for covariates. Multivariate associations between diet variables and microbiome features will be tested using PERMANOVA and redundancy analysis (RDA). Regression models will examine the relationship between fiber intake and butyrate-producing pathway abundance, while structural equation modeling will explore mediation effects of microbial functions on diet-health associations (e.g., adiposity, fasting glucose). Subgroup analyses will assess sex- and BMI-stratified patterns. The study will adhere to ethical standards, obtaining institutional review board approval, informed consent, and data protection measures; de-identified data will be stored securely. Expected findings include distinct taxonomic assemblages and functional repertoires linked to diet type, with plant-based diets associated with enrichment of fiber-degrading taxa and SCFA biosynthesis pathways, omnivorous diets showing higher taxa richness but more variable functional profiles, and high-fat low-carbohydrate diets displaying shifts toward bile-acid metabolism-related pathways. Diet-driven variations in microbial community structure are anticipated to correlate with fiber intake and carbohydrate quality more strongly than with total caloric intake, while functional redundancy is expected to buffer some taxonomic variability. The study will contribute to knowledge by clarifying how urban dietary patterns shape gut microbiome structure and function at a metagenomic level, informing targeted dietary interventions to optimize microbiome-mediated health benefits in diverse city populations. The main conclusions will emphasize diet-specific microbiome signatures and their potential roles in metabolic health, supporting the refinement of dietary guidelines that consider microbiome-mediated mechanisms. Recommendations will include longitudinal follow-up studies to evaluate causal links, integration of metabolomic data to validate functional outputs, and policy-oriented guidance for urban nutrition programs that promote dietary patterns fostering beneficial gut microbiota configurations.