Associations between antibiotic and metal resistance genes in geothermal springs

Metals, particularly toxic heavy metal(loid) pollutants, have been widely reported to facilitate the co-selection of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) in contaminated environments. However, in natural geogenically metal-rich systems such as geothermal springs-often considered pristine environments analogous to early Earth-the occurrence of ARGs and their associations with MRGs remain poorly understood. Here, we investigated ARGs and MRGs distribution patterns in China's largest geothermal field using metagenomic and metatranscriptomic analyses. ARGs were detected in all studied geothermal springs, with total abundances ranging from 52.80 to 668.12 TPM. Macrolide-lincosamide-streptogramin (MLS), bacitracin, and rifamycin resistance genes were the most abundant ARGs. Significant associations between ARGs and MRGs were observed across the geothermal springs, as evidenced by Mantel tests and Procrustes analysis. For instance, bacitracin (R = 0.96, P = 1.06E-05) and rifamycin (R = 0.95, P = 3.41E-05) resistance genes exhibited strong positive correlations with arsenic (As) resistance genes. Metagenome-assembled genomes (MAGs) analyses further identified putative key drivers mediating the linkage between ARGs and MRGs (e.g., Thiomonas and Tepidimonas). Metatranscriptomic data confirmed the active transcription of ARGs, MRGs, and mobile genetic elements (MGEs). Collectively, this study provides a systematic understanding of the distribution patterns of ARGs in pristine geothermal springs and highlights their associations with MRGs.