Fulvic acid in humic substances accelerates dissemination of antibiotic resistance via conjugative transfer.

Humic substances (HS) are widely used as soil amendments to improve soil fertility and support sustainable agriculture, yet the differential roles of their major fractions, fulvic acid (FA) and humic acid (HA), in plasmid-mediated dissemination of antibiotic resistance genes (ARGs) remain unclear. In this study, Escherichia coli (E. coli) HB101 carrying the RP4 plasmid and E. coli NK5449 were used as donor and recipient, respectively, to compare the effects of HA and FA on ARGs conjugative transfer. Both HA and FA significantly promoted RP4 transfer, with 5 g/L FA increasing conjugative transfer frequency (CTF) by 6.29-fold and regulating conjugation-related genes. Compared with HA, FA caused greater membrane disruption, probably due to its smaller molecular size and higher solubility. FA also induced ROS accumulation, increasing extracellular polymeric substances (EPS)-mediated cell contact. Transcriptomic analysis suggested that FA repressed lipopolysaccharide (LPS) biosynthesis-related pathways and increased ATP supply for conjugation. Partial least squares path modeling (PLS-PM) identified cell contact as the strongest direct driver of CTF, while the SOS response had the highest total effect. Notably, FA application below 40 kg/ha in protected vegetable systems may provide a low-exposure reference for subsequent soil validation to balance agronomic benefits and ARGs dissemination risk. This study reveals the mechanisms by which FA-rich HS facilitate plasmid-mediated ARGs dissemination and highlights FA as a potential risk factor in agricultural applications, offering guidance for the safer use of FA-containing products.