Polystyrene and its dissolved organic matter accelerate antibiotic resistance gene dissemination in anaerobic digestion by posing resistance pressure on functional microorganisms

Microplastics (MPs) in sludge can release dissolved organic matter (MP-DOM) and affects the dissemination of co-existing antibiotic resistance genes (ARGs). However, the contributions and mechanisms of MPs themselves and released MP-DOM on ARGs dissemination during anaerobic digestion (AD) remain unclear. This study investigated the dissemination potential of ARGs under MP and MP-DOM treatment by combining metagenomic sequencing with partial least squares structural equation modeling (PLS-SEM). Results showed that both treatments increased the abundances of ARGs, virulence factor genes and mobile genetic elements, with 1.25-1.79 fold enhancement relative to control, highlighting ARGs dissemination in AD. Notably, MP-DOM exhibited a stronger enhancement than MPs. The enhancement was mainly ascribed to the resistance pressure on functional microbial communities becoming ARGs hosts by increasing membrane permeability and type IV secretion system activation through reactive oxygen species generation, as evidenced by the 108.2 % and 120.5 % increases in VirD4 expression under MPs and MP-DOM, respectively. Different from the induced selection pressure of MPs on ARGs dissemination, MP-DOM mainly exerted a strongly biological effect by stimulating stress-adaptive carbohydrate metabolism (glycolysis and pentose phosphate) within hydrolytic microorganisms (Clostridium and Defluviicoccus) in the microbially-driven AD environment. This study identifies a dominant factor yet previously overlooked driver of MP-DOM that amplifies ARGs dissemination in sludge AD, providing new insights into the risks of plastic-derived contaminants.