Long-Term Application of Fermented Fertilizer Attenuates the Accumulation of Antibiotic Resistance Genes in Aquaculture Sediment

Aquaculture sediments are increasingly recognized as important reservoirs of antibiotic resistance genes (ARGs). Although thermophilic fermentation is widely used to reduce ARGs and pathogens in manure, most biosafety assessments stop at the fertilizer product itself, leaving unresolved whether these benefits persist after application to aquaculture sediments. Here, we compared inorganic fertilizer (IF), raw manure (RM), and fermented fertilizer (FF) to test whether fermentation confers sustained biosafety benefits in aquaculture pond sediments. After a 6-month co-culture period, sediment samples were analyzed using shotgun metagenomic sequencing, ARG and mobile genetic element (MGE) profiling, antibiotic residue determination, and network analyses. Long-term fertilization significantly altered sediment physicochemical properties, microbial community composition, and resistome structure. Among the three groups, the RM exhibited the highest total ARG abundance and the greatest number of unique ARG subtypes, with significant enrichment of multidrug resistance genes as well as pathogen-, disease-, and host-associated mobile genetic elements (MGEs). In contrast, the FF group showed the lowest total ARG abundance and fewest unique ARG subtypes, along with suppression of pathogen-associated MGEs, indicating that FF can effectively reduce the risk of ARG dissemination. However, the potential impact of residual antibiotics still warrants attention. Redundancy analysis showed that TC and TN primarily explained bacteriome and resistome variation under RM, whereas pH, EC, AP, and AK were more strongly associated with FF. Co-occurrence analysis further suggested that fertilizer-driven microbial community shifts may regulate ARG persistence and potential cross-ecosystem dissemination. Overall, fermented fertilizer attenuated, but did not eliminate, manure-derived resistance risks in aquaculture sediments. These findings support fermented fertilizer as a safer management option than raw manure and highlight the need for integrated risk assessment combining ARGs, MGEs, microbial hosts, and antibiotic residues.