Tracking clinically critical antibiotic-resistant bacteria and resistance genes in biogas slurry-amended soil: occurrence, enrichment and vertical distribution.

Clinically critical antibiotic-resistant bacteria (ARB) and resistance genes (ARGs) pose a significant public health threat due to their clinical significance, pathogenic potential, and high mobility. Biogas slurry application introduces diverse ARB and ARGs into agricultural soils, progressively increasing microbial resistance to critically important antibiotics. However, previous studies have mainly focused on veterinary-associated ARB and ARGs, with limited attention to clinically critical ones. Here, we tracked clinically critical ARB and ARGs in biogas slurry-amended soils using a self-developed selective screening and target gene identification approach, and identified the key drivers of their enrichment and vertical distribution under biogas slurry application. ARB resistant to cefoperazone, levofloxacin, vancomycin, and polymyxin B were widely detected, together with clinically critical ARGs, including fosB1, floR, tet(G), aadA2, sul1, oqxA, oqxB, and fosA2. Biogas slurry application significantly enriched these ARB and ARGs and redistributed them into deeper soil layers. These patterns were primarily associated with shifts in ARG-associated bacterial communities, particularly bacteria belonging to genera of Arenimonas, Candidatus, Solibacter, Flavisolibacter, Iamia, Methylocystis, Nitrosospira, Reyranella, and Sphingomonas. In addition, co-enrichment of antibiotics and MGEs, induced by biogas slurry application, further shaped their vertical distribution patterns in the soil profile. These findings provide insights for optimizing manure recycling and mitigating antimicrobial resistance risks in agricultural systems.