Metagenomic analysis of antibiotic resistance and pathogens in landfill leachates: Environmental implications

Landfill leachates, rich in antibiotics, antibiotic-resistant bacteria (ARB), and antibiotic resistance genes (ARGs), pose significant risks to human, animal, and environmental health. This study analyzed leachate samples from seven sites in Gilan, Mazandaran, and Golestan provinces, northern Iran, using metagenomic sequencing, Inductively Coupled Plasma analysis (ICP), and Gas Chromatography (GC)-based BTEX (Benzene,Toluene, Ethylbenzene and Xylene) quantification. ShortBRED (Short, Better Representative Extract Dataset) analysis revealed higher ARG abundance in GL2 and GL4 compared to GL1 and GL7 (p = 0.0056-0.0265), with Pseudomonadaceae, Moraxellaceae, and Enterobacteriaceae as dominant bacterial families. Statistical analysis revealed that metal contaminants had strong correlations with the microbial community structure. Sb and Zn concentrations were strongly negatively correlated with total ARGs abundance (Sb: r = -0.883, p = 0.013; Zn: r = -0.857, p = 0.024). Moreover, there was obvious intense niche partitioning, and major bacterial families showed highly significant correlations with specific metals; e.g., Enterobacteriaceae abundance was inversely correlated with copper (Cu; r = -0.89, p = 0.007) but positively correlated with zinc, antimony, and tin (r = 0.79-0.85, p < 0.05), suggesting metal-mediated selection and co-selection for resistance mechanisms. A PERMANOVA confirmed that spatial analysis was precluded by the sampling design (R² = 1.0, residual df = 0), limiting site comparisons to descriptive trends. A correlation between ARGs and elements was noted at GL7 (r = 0.476, p = 0.0251). BTEX levels varied significantly, with higher concentrations in GL2 vs. GL4 (p = 0.0482), GL5 vs. GL3 (p = 0.0090), GL5 vs. GL4 (p = 0.0044), and GL5 vs. GL7 (p = 0.0251), and benzene/toluene exceeding ethylbenzene (p = 0.0276, p = 0.0093) and xylene (p = 0.0038, p = 0.0012). These findings highlight landfill leachates as reservoirs of pathogenic ARB and ARGs, influenced by environmental and microbial factors. The study suggests leveraging leachate-associated bacteria for biochemical treatment of antibiotics and ARGs, and provides a basis for targeted surveillance and risk management to mitigate ARG dissemination and protect ecological/public health.