Analysis of antibiotic resistance genes in livestock manure and receiving environment reveals non-negligible risk from extracellular genes†

Antibiotic resistance genes (ARGs), extracellular and intracellular, collectively constitute the complete resistome within farming environments. However, a systematic analysis of extracellular ARGs (eARGs) and intracellular ARGs (iARGs) remains missing. This study characterized eARGs and iARGs in livestock manure and examined their effects on the agricultural soil receiving them. The findings indicated differences in DNA concentration and the ratios of iDNA and eDNA across various manures, with chicken manure demonstrating the highest eDNA levels (20.7–22.7%). Different ARG subtypes had distinct pollution levels in livestock manure. Generally, except for bla_TEM-1 and bla_OXA-1, ermC, ermB, and cfr, other ARGs were abundant in eDNA (beyond 10⁴ copies per g DW in each sample) and iDNA (beyond 10⁷ copies per g DW) of animal manure. The copy numbers of eARGs and iARGs differed in different manures, with swine manure having the highest, ranging from 6.08 × 10³ to 4.30 × 10⁸ and from 3.21 × 10⁷ to 9.51 × 10¹⁰ copies per g DW, respectively. Both iARGs and eARGs were more abundant in soil when manure was applied. The impacts of the various manures varied, with chicken manure having the most significant influence. Interestingly, several eARGs were much more abundant in soil than their intracellular counterparts, highlighting the need to regulate and manage both eARGs and iARGs.