Metagenomics Analysis of Antibiotic Resistance Gene Co-selection by Heavy Metals and BTEX in Landfill Soils

• Shotgun metagenomics uncovers microbial diversity and ARGs in landfill soils. • High prevalence of efflux pumps and antibiotic inactivation genes detected. • Heavy metal contamination (Ni, Pd, Pt) linked to industrial and traffic sources. • Toluene is the most prevalent BTEX compound, with significant spatial variations. • Positive correlation between bacterial abundance and heavy metals at specific sites Landfills are significant sources of environmental pollution, releasing heavy metals, volatile organic compounds (VOCs), and antibiotic resistance genes (ARGs) into surrounding soils. This study examined microbial communities, ARGs, and chemical contaminants in soils from seven landfill sites in Gilan, Mazandaran, and Golestan Provinces, northern Iran. Shotgun metagenomic sequencing, inductively coupled plasma (ICP) analysis for heavy metals, and gas chromatography–mass spectrometry (GC–MS) for benzene, toluene, ethylbenzene, and xylenes (BTEX) compounds were applied. Proteobacteria dominated most sites, while Firmicutes prevailed at one site, reflecting site-specific conditions. Bacterial communities showed a small core microbiome (7.5%) and high site specificity (42.5%). ARG profiling revealed efflux pumps (40%) as the most abundant mechanism, followed by antibiotic-inactivating enzymes (23%) and two-component systems (22%). While chromium and zinc levels were acceptable, nickel, palladium, and platinum indicated localized anthropogenic pollution. Toluene was the predominant BTEX compound. Network analyses revealed extensive ARG–mobile element associations, including 122 significant ARG–insertion sequence and 12,297 ARG–integron correlations, highlighting mobilization of clinically relevant genes such as mcr-9.1 and aadA7 . Co-selection networks of 141 ARGs and 578 metal resistance genes showed strong modularity (0.54) and key hub ARGs ( qnr, mexY, msbA ; degree = 70), with β-lactamase ACT-77 linked to zinc/copper resistance (r = −0.99). Correlations between bacterial abundance and pollutants suggested co-selection processes. These findings reveal complex interactions among microbial communities, ARGs, and pollutants, providing insights for improved landfill management and reduced environmental and health risks.