Metagenomic Comparison of Bat Colony Resistomes Across Anthropogenic and Pristine Habitats

Background/Objectives: The mammalian microbiota constitutes a reservoir of antimicrobial resistance genes (ARGs), which can be shaped by environmental and anthropogenic factors. Although bat-associated bacteria have been reported to harbor diverse ARGs globally, the ecological and evolutionary determinants driving this diversity remain unclear. Methods: To characterize ARG diversity in wildlife exposed to contrasting levels of human influence, we analyzed homologs of resistance mechanisms from the Comprehensive Antibiotic Resistance Database in shotgun metagenomes of bat guano. Samples were collected from a colony exposed to continuous anthropogenic activity in Spain (Salamanca) and from a wild, non-impacted bat community in China (Guangdong). Metagenomic analyses revealed marked differences in taxonomic and resistome composition between sites. Results: Salamanca samples contained numerous hospital-associated genera (e.g., Mycobacterium, Staphylococcus, Corynebacterium), while Guangdong was dominated by Lactococcus, Aeromonas, and Stenotrophomonas. β-lactamases and MurA transferase homologs were the most abundant ARGs in both datasets, yet Salamanca exhibited higher richness and functional diversity (median Shannon index = 1.5; Simpson = 0.8) than Guangdong (Shannon = 1.1; Simpson = 0.66). Salamanca also showed enrichment of clinically relevant ARGs, including qacG, emrR, bacA, and acrB, conferring resistance to antibiotics critical for human medicine. In contrast, Guangdong exhibited a more restricted resistome dominated by β-lactamase and MurA homologs. Beta diversity analysis confirmed significant compositional differences between resistomes (PERMANOVA, R2 = 0.019, F = 1.33, p = 0.001), indicating ecological rather than stochastic structuring. Conclusions: These findings suggest that anthropogenic exposure enhances the diversity and evenness of resistance mechanisms within bat-associated microbiomes, potentially increasing their role as reservoirs of antimicrobial resistance.