Mechanisms of 6PPD Affecting the Conjugative Transfer of Antibiotic Resistance Genes

¹ Sanya Oceanographic Institution, Ocean University of China, Sanya 572000, China
² Institute of Coastal Environmental Pollution Control, Ministry of Education Key Laboratory of Marine Environment and Ecology, College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China

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Abstract

Within the “One Health” framework, the dissemination of antibiotic resistance genes (ARGs) in agricultural soils has drawn considerable attention. A novel contaminant—the tire antioxidant additive N- (1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD)—can enter agricultural soils through tire wear particles and their leachates. As an emerging organic pollutant, the impact of 6PPD on horizontal gene transfer (HGT) of ARGs remains underexplored. To evaluate its effect on interspecies conjugation under conditions relevant to agricultural soil environments, an intergeneric mating system was established simulating oligotrophic soil conditions, employing Pseudomonas putida KT2440 as the donor and Escherichia coli K12 as the recipient (hereafter referred to as the donor and recipient strains, respectively). Using environmentally relevant concentrations (0.5~20 μg L⁻¹), this work systematically examined how 6PPD influences the RP4 plasmid-mediated conjugative transfer from P. Putida KT2440 to E. coli K12, and further elucidated the underlying molecular mechanism by quantifying intracellular reactive oxygen species (ROS) levels in both mating partners. The results demonstrate that 6PPD significantly promotes conjugation in a dose-dependent manner, an effect primarily achieved via the induction of intracellular ROS accumulation within the donor and recipient cells. It must be noted, however, that the present study has only established a correlative relationship between ROS buildup and enhanced conjugative transfer; a causal link still awaits confirmation through additional experiments, such as those employing ROS scavengers. Collectively, these findings offer experimental evidence for identifying and assessing the risk that tire-derived contaminants pose in driving the spread of environmental antibiotic resistance in soils..