Diversity of Pharmaceuticals Enhances Antibiotic Resistance in the Invertebrate Gut via Biofilm-Mediated Mechanisms.

The environmental accumulation of non-antibiotic pharmaceuticals is an emerging driver of antibiotic resistance. While individual compounds are known to shape the soil resistome, and contaminant diversity also plays a role, the impact of pharmaceutical diversity on the gut resistome of soil invertebrates remains unclear. Here, we combined metagenomics and metaproteomics to examine the collembolan gut and soil resistome across a gradient of pharmaceutical diversity under diurnal warming. Increasing pharmaceutical diversity at a constant total concentration significantly enriched antibiotic resistance genes (ARGs) in the gut microbiome, with no comparable effect in surrounding soils. This enrichment was mainly driven by multidrug resistance associated with efflux activity and biofilm-related processes, accompanied by increases in ARG-carrying taxa such as Gordonia and Ochrobactrum. Notably, Ochrobactrum encoded biofilm-related aryl polyene pathways. In vitro experiments confirmed that biofilm formation promotes resistance through coordinated cellular responses. Metaproteomic data indicated that Ochrobactrum initiates early biofilm formation by recruiting extracellular matrix producers such as Bacillus and Pseudomonas. Diurnal warming modulated these responses, indicating an interaction between chemical diversity and climate stress. These findings identify pharmaceutical diversity as an independent driver of ARG enrichment in host-associated microbiomes and establish chemical complexity as a key factor in assessing the ecological risks of pharmaceutical pollution.