Enhanced plasmid-mediated conjugative transfer of resistance genes across bacterial species promoted by graphene oxide

The rapid proliferation of antibiotic resistance genes (ARGs) in the environment has become a major concern. However, the effect of graphene oxide (GO), a newly recognized pollutant, on the transfer of ARGs among bacteria remains poorly understood. In this study, we investigated the influence of GO at concentrations ranging from 25 to 400 mg L-1 on the frequency of conjugative transfer of ARGs mediated by the RP4 plasmid. Our results demonstrated a significant increase in the transfer of ARGs between Gram-negative Escherichia coli DH5 alpha and Gram-positive Bacillus subtilis WB600, compared to the control without GO. Experimental validation through PCR and gel electrophoresis confirmed the successful transfer of the RP4 plasmid between the two bacterial strains. Additionally, GO treatment led to a significant decrease in cell viability for both donor and recipient bacteria, with their activities reaching approximately 42% and 45% of the control, respectively. Microscopic observations and physiological analyses demonstrated that GO induced oxidative stress, resulting in blurred or damaged cell membrane edges, increased membrane permeability, and enhanced adhesion and contact between bacterial cells. These factors likely contributed to the heightened frequency of RP4 plasmid transfer facilitated by GO. Transcriptomic analysis uncovered notable changes in gene expression in response to GO treatment. In both bacteria, genes such as ompA, ompW, and bamC associated with the cell membrane; oxyR, gor, and ahpC involved in oxidative stress; atpA, atpB, and atpE related to ATP generation regulation; as well as metastasis-related genes like korA, korB, and trbB were found to be altered. Overall, this study provides evidence that GO has the potential to promote the transfer of ARGs among diverse bacteria, highlighting the ecological risks and potential hazards associated with its presence. We analyzed the plasmid-mediated transfer frequency between Gram-negative and Gram-positive bacteria at different GO concentrations to unveil its impact on conjugative transfer of ARGs.