Non-antibiotic pharmaceuticals enhance the transmission of exogenous antibiotic resistance genes through bacterial transformation

Abstract

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.

Fig. 1. Transformation of free plasmid harbouring ARGs induced by non-antibiotic pharmaceuticals.

a Schematic of the experimental design. b Fold changes of transformation frequency under the exposure of various concentrations of non-antibiotic pharmaceuticals, relative to pharmaceutical-free solvents. c Electrophoresis of plasmid pWH1266 (lane 1 is the original plasmid, lanes 2–3 are plasmids extracted from transformants of control with water and ethanol as the solvent, and lanes 4–9 are plasmids extracted from transformants of the pharmaceutical-dosed groups). d Electrophoresis of plasmid PCR products for tetA gene (lane 1 is the original plasmid, lanes 2–3 are plasmids extracted from transformants of control with water and ethanol as solvent, and lanes 4–9 are plasmids extracted from transformants of the pharmaceutical-dosed groups). e Electrophoresis of PCR products generated for the bla gene from the extracted plasmids (lane 1 is from the original plasmid, lanes 2–3 are from plasmids extracted from transformants of controls with water and ethanol as solvent, and lanes 4–9 are from plasmids extracted from transformants of the pharmaceutical-exposed groups). Significant differences between non-antibiotic-dosed samples and the control were analysed by independent-sample t-test and corrected by Bonferroni correction method, *P^* < 0.05, **P* < 0.01, and ***P* < 0.001.

Fig. 2. The effect of non-antibiotic pharmaceuticals causing increased ROS generation and altering the cell membrane integrity in A. baylyi.

a Fold changes of ROS fluorescence intensity under the exposure of non-antibiotic pharmaceuticals. b Fold changes of ROS fluorescence intensity with the addition of the ROS scavenger thiourea (5 mg/L of non-antibiotic pharmaceuticals dosage). c Fold changes of transformation frequency with the addition of ROS scavenger thiourea and 5 mg/L of non-antibiotic pharmaceuticals. d Fold changes of fluorescence intensity on PI-stained cells under the exposure of non-antibiotic pharmaceuticals. Significant differences between non-antibiotic-dosed samples and the control were analysed by independent-sample t-test and corrected by Bonferroni correction method, *P* < 0.05, **P* < 0.01 and ***P* < 0.001. For figures b and c, significant differences between the groups with and without thiourea dosage were analysed by independent-sample t-test, *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 3. Genotypic RNA and protein analyses for A. baylyi under the exposure of non-antibiotic pharmaceuticals.

a Log₂ fold changes of key genes and proteins related to ROS generation. b Log₂ fold changes of key genes and proteins related to stress response. c Log₂ fold changes of key genes and proteins related to cell membrane status. d Fold changes of key genes and proteins related to bacterial competence. e Log₂ fold changes of key genes and proteins related to DNA repair and recombination. f Log₂ fold changes of key genes and proteins related to antibiotic effects.

Fig. 4. The simulated changes in the number of bacteria with the increase of transformation time.

a Variation trends of wild-type bacteria, transformant and free plasmid in the control group. b The number of transformants and the corresponding stability time (shown as pink circles) under the exposure of different non-antibiotic pharmaceuticals.

Fig. 5. The overall mechanisms explaining the roles of non-antibiotic pharmaceuticals causing enhanced transformation of exogenous ARGs.

a Non-antibiotic pharmaceuticals promote bacterial competence. b Non-antibiotic pharmaceuticals enhance stress levels. c Non-antibiotic pharmaceuticals induce over-production of ROS. d Non-antibiotic pharmaceuticals increase cell membrane permeability.