Diversity and abundance of antimicrobial resistance genes in manure from pig farms with varying antibiotic use: a long-read metagenomic sequencing approach

Livestock production contributes to the emergence and spread of antimicrobial resistance (AMR), with pig farming accounting for a large share of veterinary antibiotic use. Manure application to fields can release drug-resistant bacteria and AMR genes into the environment, creating potential transmission routes to humans. Mobile genetic elements such as plasmids and transposons facilitate horizontal transfer of AMR genes between bacteria, including pathogens. However, quantitative data on the manure resistome and its links to antibiotic use remain limited. Shotgun metagenomics provides broad insights into microbiota and AMR composition, with long-read sequencing offering improved resolution of the genomic context of AMR genes. Here, we applied long-read shotgun metagenomics to investigate the diversity, abundance, and mobility potential of AMR genes in 24 manure samples from 14 Swiss pig farms with documented antibiotic use. Across 24 manure samples, 225 distinct AMR genes were detected, with tetracycline resistance genes being most prevalent. Manure samples from farms reporting the highest recent antibiotic use contained greater AMR gene abundance and richness. Metagenomic assemblies revealed that 77% of AMR genes with resolved flanking regions were located near transposases, recombinases, integrases, or relaxases, suggesting high transfer potential. The tigecycline resistance gene tet(X6) and related variants were identified in 21 of 24 samples, frequently embedded within mobile genetic elements. Two samples contained complete gene clusters of the vancomycin resistance determinant vanB, one of which was part of the conjugative transposon Tn1549. In one sample, a single highly abundant plasmid encoding beta-lactam and aminoglycoside resistance accounted for 42% of the total AMR gene load. Pig manure is a reservoir of diverse and mobile AMR genes, including those conferring resistance to critically important antibiotics. Long-read metagenomics adds valuable genomic context, supporting AMR monitoring and risk assessment within a One Health framework.