Antimicrobial resistance is a critical global health concern, causing significant mortality. Horizontal gene transfer (HGT), primarily conjugation, plays a major role in disseminating antibiotic resistance genes (ARGs). While antibiotic use promotes HGT, the impact of non-antibiotic pharmaceuticals, which constitute 95% of the pharmaceutical market, remains largely unknown. Previous research indicated that some non-antibiotic drugs exhibit antibiotic-like effects and contribute to antibiotic resistance emergence. However, their role in promoting conjugation between different bacterial populations (intra- and intergenerational) was not fully understood. This study aimed to investigate the effect of various commonly used non-antibiotic pharmaceuticals on the conjugative transfer of plasmid-borne ARGs. The researchers used two conjugation models, one environmentally relevant and the other clinically relevant, employing different bacterial strains and plasmids. The selected pharmaceuticals included NSAIDs (ibuprofen, naproxen, diclofenac), gemfibrozil, propranolol, and iodipamide, all widely consumed and present in various environments. The study's importance lies in understanding how these ubiquitous pharmaceuticals might contribute to the spread of antibiotic resistance, posing a significant threat to public health.

The introduction adequately reviews existing literature on antibiotic resistance, horizontal gene transfer (especially conjugation), and the effects of antibiotics at sub-inhibitory concentrations on HGT. It mentions previous work showing that some non-antibiotic pharmaceuticals may have antibiotic-like effects and can promote the transfer of antibiotic resistance through natural transformation in a single bacterial population. However, it highlights the gap in knowledge regarding the effect of these pharmaceuticals on conjugation between different bacterial populations and the underlying mechanisms involved. This positions the current study within the existing literature by focusing on a previously unexplored aspect of non-antibiotic pharmaceutical effects on antibiotic resistance dissemination.

The study employed two conjugation models: Model-1 (environmentally relevant) and Model-2 (clinically relevant). Model-1 used *Escherichia coli* K-12 LE392 (donor, carrying plasmid RP4) and *Pseudomonas putida* KT2440 (recipient). Model-2 used *E. coli* MG1655 (donor, with plasmid pSM198A) and *E. coli* J53 (recipient). The researchers exposed these mating pairs to different concentrations of six non-antibiotic pharmaceuticals: ibuprofen, naproxen, diclofenac, gemfibrozil, propranolol, and iodipamide. The concentrations were chosen to represent both clinically relevant and environmentally relevant levels. Conjugation was assessed by enumerating transconjugants on selective media. The study also included various techniques to investigate the mechanisms underlying the observed effects. These include: transmission electron microscopy (TEM) to visualize changes in bacterial cell morphology; flow cytometry to measure reactive oxygen species (ROS) generation and cell membrane permeability; RNA sequencing to analyze changes in gene expression; and proteomic analysis to identify changes in protein expression. Statistical analysis, including t-tests, linear regressions, and Pearson correlation, was performed to determine the significance of the findings. Anaerobic conditions were also used to investigate the role of ROS in the conjugation process. A ROS scavenger (thiourea) was employed to further explore the mechanisms of action.

The study found that several non-antibiotic pharmaceuticals significantly accelerated the conjugative transfer of ARGs in both Model-1 and Model-2. Ibuprofen, naproxen, and gemfibrozil consistently increased the number of transconjugants and the conjugation transfer ratio at all tested concentrations. Diclofenac and propranolol showed increased transfer at higher concentrations. Iodipamide did not show a significant effect. The increase in conjugation was concentration-dependent for most drugs. TEM observations revealed changes in bacterial cell morphology after exposure to the pharmaceuticals. Flow cytometry showed increased ROS generation and cell membrane permeability in both donor and recipient cells exposed to the pharmaceuticals, suggesting these changes play a role in facilitating conjugation. RNA sequencing and proteomic analyses revealed that the pharmaceuticals induced changes in gene expression, including upregulation of genes involved in the SOS response and efflux pumps, similar to what is observed under antibiotic stress. These results indicate that the pharmaceuticals enhance bacterial conjugation by increasing ROS production, membrane permeability and inducing cellular stress responses, creating an environment favorable for HGT.

The findings demonstrate that commonly used non-antibiotic pharmaceuticals can significantly enhance the dissemination of antibiotic resistance through bacterial conjugation. The observed effects are likely due to a combination of factors, including increased ROS generation, altered cell membrane permeability, and activation of cellular stress responses. These findings are significant because they highlight a previously underestimated factor contributing to the spread of antibiotic resistance. The widespread use of these pharmaceuticals means that their impact on the dissemination of ARGs could be substantial. Further research should focus on investigating the long-term consequences of this phenomenon, considering the interplay between multiple pharmaceuticals, and exploring strategies to mitigate the risk of accelerated antibiotic resistance spread.

This study provides strong evidence that several non-antibiotic pharmaceuticals can promote the horizontal transfer of antibiotic resistance genes via conjugation. The mechanisms likely involve increased ROS production, membrane permeability changes, and stress response activation. These findings underscore the need for further research into the impact of these drugs on the spread of antibiotic resistance and the development of strategies to minimize this risk. Future work should examine the ecological implications of these findings, and explore potential mitigation strategies.

The study focused on specific bacterial strains and plasmids, which may limit the generalizability of the findings. While the concentrations used were environmentally and clinically relevant, further research is needed to determine the real-world impact of these pharmaceuticals in more complex environments. The study primarily focused on conjugation, while other HGT mechanisms were not investigated.