Comprehensive sequence analysis reveals the vast β-lactam resistome

Most antibiotic resistance genes (ARGs) evolved in environmental microbes long before humanity's antibiotic breakthrough, and widespread antibiotic use accelerated the dissemination of ARGs among clinical pathogens. Although environmental ARG classes, such as β-lactamases, are widely discussed, their presence and distribution remain underexplored, with existing studies often lacking the scalability and taxonomic resolution required for comprehensive analysis. In this study, we present a comprehensive map of the β-lactamase resistome using an integrated bioinformatics and experimental workflow. We identify a vast reservoir of putative β-lactamase sequences spanning all four β-lactamase classes—more than 12-fold the number listed in current ARG databases. We generated a β-lactamase taxonomy-environment map by identifying ~ 108,000 putative β-lactamases across diverse bacterial phyla and environmental ecosystems. Notably, the relative frequency of putative β-lactamases was only ~3.9-fold lower than that of the essential recA gene across diverse environmental ecosystems, with signals of relative enrichment observed in wastewater and plant-associated samples. The enrichment in plant-associated and wastewater samples suggests that these environments may represent important reservoirs of putative β-lactamases. We identify taxonomic and environmental contexts that may represent candidate reservoirs of close homologs of clinically relevant β-lactamases, extending beyond wastewater to plant-associated, terrestrial, and aquatic settings. Overall, we present a comprehensive workflow and reusable mapping tool to identify and classify the β-lactamase resistome, revealing its diversity, distribution, and a largely unexplored reservoir with potential for mobilization into clinical pathogens.