MGE-associated ARGs exhibit higher expression efficiency than chromosomal non-MGE loci and predominantly contribute to resistance expression in pig farm wastewater.

Antibiotic resistance (AMR) in animal agriculture represents a critical One Health challenge, with pig farms serving as major reservoirs for antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). While MGEs are known drivers of ARG dissemination, their impact on actual expression of resistance in these complex microbial communities is poorly understood. This study aimed to quantify the contribution of MGE-associated ARGs to overall resistance expression and compare their transcriptional efficiency to chromosomally located ARGs in pig farm wastewater. Utilizing deep metagenomic and metatranscriptomic sequencing on wastewater from five typical Chinese pig farms, we comprehensively elucidated ARG presence, genetic context, host association, and transcriptional activity. We identified a vast dataset comprising 811 ARG types conferring resistance to 27 antibiotic classes, with 71.02 % actively transcribed, indicating significant resistance potential. MGEs were associated with 34.87 % of ARG-like ORFs. Crucially, MGE-associated ARGs were responsible for the majority (62.07 %) of total ARG transcript abundance, exhibiting an expression efficiency nearly 2.5 times higher than ARGs on chromosomal non-MGE loci. Taxonomic analysis revealed that these highly expressed MGE-associated ARGs were primarily hosted by genera known to include opportunistic pathogens, such as Enterococcus, Escherichia, and Klebsiella. Differential coverage binning further uncovered diverse draft genomes simultaneously harboring multiple highly expressed ARGs and MGEs, potentially contributing to the persistence and spread of highly resistant bacterial strains. Our findings underscore that MGEs not only propagate ARGs but critically enhance their expression, providing vital data for effective AMR surveillance and mitigation strategies within the One Health framework.