Divergent mechanisms of active antibiotic resistance gene enrichment in soil driven by pesticide diversity

Antimicrobial resistance is an escalating global threat, with soils serving as reservoirs and conduits for the dissemination of antibiotic resistance genes (ARGs). Pesticide use in agriculture contributes to ARG proliferation, and ~60% of agricultural soils contain multiple pesticide residues. However, how pesticide diversity influences ARG dynamics in active microbial populations (active ARGs) remains unclear. Here, we evaluate the effects of pesticide diversity on active soil ARGs through a long-term field experiment integrating bioorthogonal non-canonical amino acid tagging (BONCAT), fluorescence-activated cell sorting (FACS), and metagenomics. We show that both low and high pesticide diversity significantly increase active ARG abundance relative to untreated control, whereas total ARG levels remain largely unchanged. The underlying mechanisms differ with pesticide diversity. At low diversity, active ARG co-selection via efflux pumps in Acinetobacter baumannii is a prominent mechanism. At high diversity, elevated reactive oxygen species and SOS responses promote horizontal gene transfer of active ARGs, as validated by culture experiments. These findings demonstrate that increasing pesticide diversity accelerates the emergence and dissemination of active ARGs, highlighting the need for integrated pesticide management strategies that consider both application intensity and diversity to mitigate resistance risks under the One Health framework. Here, the authors conduct a long-term field experiment to show that applying diverse pesticide mixtures increases active antibiotic resistance genes in soils. Low pesticide diversity co-selects for specific resistance genes, whereas high diversity accelerates spread between bacteria via horizontal gene transfer.