The bacterial infection that involves antimicrobial resistance is a rising global threat to public health. Chlorine-based water disinfection processes can inactivate antibiotic resistant bacteria. However, at the same time, these processes may cause the release of antibiotic resistance genes into the water as free DNA, and consequently increase the risk to disseminate antibiotic resistance via natural transformation. Presently, little is known about the contribution of residual chlorine affecting the transformation of extracellular antibiotic resistance genes (ARGs). This study investigates whether chloramine and free chlorine promote the transformation of ARGs and how this may occur. We reveal that both chloramine and free chlorine, at practically relevant concentrations, significantly stimulated the transformation of plasmid-encoded ARGs by the recipient Acinetobacter baylyi ADP1, by up to a 10-fold increase. The underlying mechanisms underpinning the increased transformations were revealed. Disinfectant exposure induced a series of cell responses, including increased levels of reactive oxygen species (ROS), bacterial membrane damage, ROS-mediated DNA damage, and increased stress response. These effects thus culminated in the enhanced transformation of ARGs. This promoted transformation was observed when exposing disinfectant-pretreated A. baylyi to free plasmid. In contrast, after pretreating free plasmid with disinfectants, the transformation of ARGs decreased due to the damage of plasmid integrity. These findings provide important insight on the roles of disinfectants affecting the horizontal transfer of ARGs, which could be crucial in the management of antibiotic resistance in our water systems.

Antibiotic resistance is a serious global threat for public health. Considering the high abundance of cell-free DNA encoding antibiotic resistance genes (ARGs) in both clinical and environmental settings, natural transformation is an important horizontal gene transfer pathway to transmit antibiotic resistance. It is acknowledged that antibiotics are key drivers for disseminating antibiotic resistance, yet the contributions of non-antibiotic pharmaceuticals on transformation of ARGs are overlooked. In this study, we report that some commonly consumed non-antibiotic pharmaceuticals, at clinically and environmentally relevant concentrations, significantly facilitated the spread of antibiotic resistance through the uptake of exogenous ARGs. This included nonsteroidal anti-inflammatories, ibuprofen, naproxen, diclofenac, the lipid-lowering drug, gemfibrozil, and the β-blocker propranolol. Based on the results of flow cytometry, whole-genome RNA sequencing and proteomic analysis, the enhanced transformation of ARGs was affiliated with promoted bacterial competence, enhanced stress levels, over-produced reactive oxygen species and increased cell membrane permeability. In addition, a mathematical model was proposed and calibrated to predict the dynamics of transformation during exposure to non-antibiotic pharmaceuticals. Given the high consumption of non-antibiotic pharmaceuticals, these findings reveal new concerns regarding antibiotic resistance dissemination exacerbated by non-antibiotic pharmaceuticals.

Antibiotic resistance is a global threat to public health. The use of antibiotics at sub-inhibitory concentrations has been recognized as an important factor in disseminating antibiotic resistance via horizontal gene transfer. Although non-antibiotic, human-targeted pharmaceuticals are widely used by society (95% of the pharmaceuticals market), the potential contribution to the spread of antibiotic resistance is not clear. Here, we report that commonly consumed, non-antibiotic pharmaceuticals, including nonsteroidal anti-inflammatories (ibuprofen, naproxen, diclofenac), a lipid-lowering drug (gemfibrozil), and a β-blocker (propranolol), at clinically and environmentally relevant concentrations, significantly accelerated the dissemination of antibiotic resistance via plasmid-borne bacterial conjugation. Various indicators were used to study the bacterial response to these drugs, including monitoring reactive oxygen species (ROS) and cell membrane permeability by flow cytometry, cell arrangement, and whole-genome RNA and protein sequencing. Enhanced conjugation correlated well with increased production of ROS and cell membrane permeability. Additionally, these non-antibiotic pharmaceuticals induced responses similar to those detected when bacteria are exposed to antibiotics, such as inducing the SOS response and enhancing efflux pumps. The findings advance understanding of the transfer of antibiotic resistance genes, emphasizing the concern that non-antibiotic, human-targeted pharmaceuticals enhance the spread of antibiotic resistance among bacterial populations.