Complex interplay between composted manure application, metal contamination, and antibiotic resistance genes profile under tropical field conditions

Manure applications in agricultural soils are a major driver of antibiotic resistance gene (ARG) dissemination, yet long-term effects of composted manure applications under tropical real field conditions remain unclear. This study assessed how successive composted manure applications influence soil physicochemical attributes, bacteriome and resistome profiles in the Brazilian Cerrado, including one site with naturally high heavy metal content. Across all sites, multidrug resistance genes were most abundant, followed by macrolide-lincosamide-streptogramin (MLS), tetracycline, β-lactam and glycopeptides resistance, aligning with predominance of Actinomycetota and Pseudomonadota as key ARG hosts. Manure increased soil pH and available phosphorus (P), with pH significantly shaping bacterial communities and pH and P the resistome in uncontaminated sites (2 and 3). However, in the metal-rich site (1), Cu was the dominant driver. Manure increased ARG richness and changed resistome structure but did not affect clinically relevant genes or resistome diversity. Metal resistance genes (MRGs), particularly for Cu and Zn, strongly influenced resistome dynamics, highlighting co-selection. Integrons integrase genes (intl) abundance increased in metal-depleted but not in metal-rich soils. While composting appears to mitigate ARG spread, particularly for clinically relevant genes, the high antibiotic use in livestock, large manure volumes, and potential for ARG persistence in tropical soils highlight the need for further research on manure treatment strategies and ARG fate in these environments. Environmental Implication. Our study highlights the environmental risks of antibiotic resistance gene (ARG) dissemination in tropical agricultural soils, emphasizing the role of manure application and heavy metal contamination in shaping soil resistome. While composted manure increased bacterial diversity and ARG richness, it did not significantly impact clinically relevant genes and resistome diversity, suggesting that composting may help mitigate ARG spread but does not eliminate it. Metals were the dominant drivers of ARG selection in the contaminated site, underscoring the role of co-selection mechanisms in maintaining resistance. However, manure applications increased integrons abundance, raising concerns about horizontal gene transfer and potential ARG proliferation into pathogens. These findings stress the urgent need for improved manure management policies in Brazil, where high antibiotic use in livestock and large manure volumes pose significant environmental and public health risks. Developing sustainable manure treatment strategies and monitoring ARG persistence are essential to limit antibiotic resistance proliferation in tropical agricultural ecosystems.