Mapping the underlying drivers of resistome risk across diverse environments.

Background: Understanding the drivers of antimicrobial resistance (AMR) across the One Health spectrum is crucial for controlling its spread. The MetaCompare framework, which assesses "resistome risk" based on antibiotic resistance gene (ARG) co-occurrence patterns on metagenomic contigs, has been expanded to distinguish between "ecological resistome risk" (ERR) and "human health resistome risk" (HHRR) scores across anthropogenic gradients. However, comprehensive surveys are still needed to untangle the biological (e.g., ARG relative abundance), ecological (e.g., taxonomic diversity), and technical (e.g., coverage) factors influencing these risk scores. Here, we analyzed 1,326 metagenomes from 12 key environments using the MetaCompare 2.0 pipeline to map global ERR and HHRR landscapes, identifying significant factors modulating risk scores through network analysis, machine learning, and multivariate regression models. Results: ERR and HHRR scores varied significantly across environments and were highly correlated (ρ = 0.73, p < 2e-16), indicating shared underlying drivers. Transient environments closely linked to human activity, such as wastewaters and the human gut, produced the highest ERR and HHRR scores, while stable environments like sediments, soils, and activated sludge yielded the lowest. These patterns corresponded directly with taxonomic diversity, where more diverse ecosystems exhibited lower risk scores, supporting the hypothesis that niche occupation may act as an ecological barrier to ARG invasion. In contrast, scores were positively correlated with Conclusions: This study demonstrated how the MetaCompare 2.0 pipeline can effectively disentangle complex relationships between ARG abundance, composition, and environmental context. Although robust across diverse environments, the framework's ability to detect ARGs and their co-occurrences may be limited in high-diversity, low-coverage samples, such as soils and sediments. Finally, we provide a series of recommendations for appropriate use cases for MetaCompare 2.0.