Longitudinal replicated metagenomic analysis of biosolids-amended soils reveals enrichment of ARGs, virulence factors, and ESKAPE pathogens.

Biosolids land application introduces antibiotic resistance genes (ARGs) and clinically relevant pathogens into agricultural soils, raising concerns about long-term environmental and public health impacts. Despite growing interest in biosolids reuse, there remains a critical need for replicated, longitudinal studies to assess how biosolids amendments shape soil microbiomes and resistomes during crop cultivation. In this replicated longitudinal greenhouse study, we used shotgun metagenomics to characterize the impact of biosolids amendment on the soil microbiome, resistome, virulence factors, and ESKAPE pathogens during carrot cultivation. Biosolids-amended soils exhibited increased richness of microbial genera (e.g., Rhodanobacter, Dyella, and Thermomonas), ARG subtypes (resistance to sulfonamide, tetracycline, fosmidomycin, and macrolides), and virulence factors compared to pristine controls. Notably, all six ESKAPE pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., were detected at elevated relative abundances (1.4- and 3.4-fold) in biosolids-amended soils and remained detectable throughout the 11-week cultivation period. Network analysis revealed statistically supported co-occurrences between microbial taxa and ARGs (with resistance to tetracyclines, beta-lactams, chloramphenicol, and multidrugs), suggesting possible host associations. These findings underscore the ecological and clinical relevance of biosolids amendment and highlight the need for integrated surveillance frameworks to mitigate antimicrobial resistance dissemination in agricultural environments.