Invasion dynamics of antimicrobial-resistant E. coli in river biofilms: impacts on the resistome, microbiomes, and horizontal gene transfer.

River biofilms are frequently exposed to invasion by antibiotic-resistant bacteria (ARB) due to episodic or chronic wastewater inputs, yet the ecological processes governing the fate of invaders and their resistance plasmids remain poorly understood. We experimentally exposed river-grown biofilms from sites differing in microbial diversity and wastewater impact to a genetically tagged ARB Escherichia coli carrying a transferable IncPα plasmid with the nptII resistance gene. Over two weeks, we tracked invader and plasmid dynamics using qPCR and plasmid-to-genome ratios as a proxy for horizontal gene transfer (HGT), complemented by 16S rRNA gene sequencing and metagenomics. Both quantification approaches yielded consistent results: the invader transiently established in all biofilms, peaking within 48 h and declining to near-background levels after 14 days. Decreasing plasmid-to-genome ratios indicated limited HGT and progressive plasmid loss. Biofilms impacted by wastewater showed slower declines, suggesting greater plasmid persistence in disturbed environments and increased abundance of specific indigenous antimicrobial resistance genes of public health concern. While the overall resistome exhibited short-lived shifts, and indigenous resistomes remained largely stable. These findings demonstrate that invader-biofilm interactions are dynamic and shaped by community context, supporting the One Health framework and highlighting how environmental conditions modulate antimicrobial resistance risks in freshwater ecosystems.