Humic substances with different molecular weights independently increased antibiotic resistance in agricultural soils contaminated with sulfamethazine.

Humic substances (HS) are known to enhance soil structure, but their effects on the antibiotic resistance distribution in agricultural soils, especially under sulfonamide contamination, remain poorly understood. This study employed an indoor soil microcosm experiment combined with metagenomic sequencing to examine the effects of high molecular weight humic acid (HA) and low molecular weight fulvic acid (FA) on the dynamics of the antibiotic resistance in sulfamethazine (SM2) contaminated agricultural soil, with the aim of identifying key driving factors. The results revealed that both HA and FA, especially at 1 g/kg, increased the total abundance of antibiotic resistance genes (ARGs), including dominant genes, such as Sul1, Cmx, VanR, Sul2 and FloR. Additionally, HS application led to increased abundance of mobile genetic elements (MGEs) and potential ARG hosts, such as Actinobacteria. Notably, HA inhibited the growth of cultivable sulfonamide-resistant bacteria (SRB), while FA promoted their growth. However, the antibiotic resistance ratio of cultivable bacteria remained relatively high under both HS treatments, consistent with the elevated ARG abundance. This may be attributed to the enhanced competitiveness of Pseudomonas within the SRB community under HS exposure. Variance partitioning analysis (VPA) indicated that MGEs and microbial communities jointly contributed to ARG variation and were closely associated with the antibiotic resistome. This study provides new insights into the ecological risks associated with HS application in agricultural soils.