A total of 21 samples were obtained, including 14 human fecal samples, 3 avian fecal samples, and scant environmental samples (soil, drinking water, riverbed sediment).  The study utilized shotgun metagenomics combined with 16S rRNA sequencing. Data processing tools such as QIIME 2.0, MetaPhlAn, ShortBRED, and ResistoXplorer were employed, ensuring a robust computational pipeline for taxonomic and functional analyses.

The analysis uncovered 72 virulence factor genes and 53 distinct ARG subtypes. Notably, the poultry samples demonstrated the highest load of ARGs, supporting the hypothesis that animal reservoirs significantly contribute to the environmental resistome. Furthermore, the detection of horizontal gene transfer phenomena underlines the complex genetic exchanges that promote the spread of resistance across species boundaries.

The identification of bacteria such as Prevotella, Escherichia, Campylobacter, and Shigella, along with specific phages, adds further complexity to the ecosystem analyzed. The integration of metagenomics with network analysis (using tools like Gephi) provides a systems-level understanding, although the inherent limitation of metagenomic approaches in assigning specific functions to individual species remains a challenge.

This paper contributes significantly to our understanding of the environmental and cross-species transmission of ARGs and pathogens. While the methodology is sound and leverages advanced metagenomic tools, future studies with expanded sampling and integrated multi-omics approaches will be essential to advance these preliminary findings. The One Health approach underscored here provides an essential framework for future research and policy design in the mitigation of antimicrobial resistance.