Antibiotic resistance is a major global health threat, with horizontal gene transfer (HGT) significantly contributing to its spread. Transformation, a type of HGT, involves the uptake of exogenous DNA by competent bacteria. This process is influenced by various stressors, including antibiotic exposure. Antidepressants, widely consumed globally, are increasingly detected in the environment and are not fully metabolized, leading to exposure of the human gut microbiome and the wider environment. Previous research has shown that fluoxetine can induce ROS production and subsequent antibiotic resistance in *E. coli*. This study aimed to investigate whether commonly-prescribed antidepressants can promote HGT of ARGs via transformation.

The literature extensively documents the global threat of antibiotic resistance and the significant role of HGT, particularly transformation, in its dissemination. Transformation is a chromosomally-encoded process that enables bacteria to acquire exogenous genetic material, including ARGs, even after the death of ARB. Stress conditions, such as antibiotic exposure, can enhance transformation rates. While the impact of antibiotics on HGT is well-established, the role of non-antibiotic pharmaceuticals, such as antidepressants, remains largely unexplored. One study highlighted fluoxetine's ability to trigger ROS overproduction and subsequent mutagenesis, leading to multi-drug resistance. However, the effects of various antidepressants on ARG dissemination via transformation hadn't been comprehensively studied.

This study used a transformation assay with *Acinetobacter baylyi* ADP1 (a naturally competent bacterium) as the recipient and plasmid pWHP126 (carrying tetracycline and ampicillin resistance genes) as the donor. Six commonly prescribed antidepressants (duloxetine, sertraline, escitalopram, fluoxetine, bupropion, and agomelatine) were tested at clinically relevant concentrations. Transformation ratios were quantified by counting transformants on selective media. The mechanisms were investigated using assays under aerobic and anaerobic conditions, with and without ROS scavengers. Whole-genome RNA sequencing and proteomic analyses were performed to assess transcriptional and translational responses to antidepressant exposure. Minimum inhibitory concentrations (MICs), ROS production, and cell membrane permeability were also measured.

Four antidepressants (duloxetine, sertraline, fluoxetine, and bupropion) significantly enhanced the transformation ratio of *A. baylyi* ADP1 in a dose-dependent manner. This effect was associated with increased ROS production, confirmed by flow cytometry and gene expression analysis. The antidepressants stimulated transcription and translation of genes involved in the antioxidant response (e.g., superoxide dismutase), Fe-S cluster, and bacterioferritin. Furthermore, the antidepressants increased cell membrane permeability, as demonstrated by flow cytometry and gene expression analysis of porin component genes and outer membrane protein assembly-related genes. Anaerobic conditions and the addition of a ROS scavenger significantly reduced both ROS production and the enhanced transformation rate, indicating a causal link between ROS overproduction and increased transformation. The study also showed increased transcription and translation of competence, SOS response, and ATP synthesis-related genes under antidepressant exposure. A comFEBC-knockout mutant showed significantly reduced transformation rates, highlighting the importance of the competence machinery.

The findings demonstrate that some commonly prescribed antidepressants can significantly enhance the horizontal transfer of ARGs via transformation in a naturally competent bacterium. This enhancement is strongly linked to antidepressant-induced oxidative stress, which leads to increased membrane permeability and upregulation of genes involved in competence and stress response. The results suggest a novel mechanism by which non-antibiotic drugs can contribute to the spread of antibiotic resistance. The observed increase in transformation is not merely a consequence of increased membrane permeability but is also related to the activation of the competence system itself. The clinical and environmental implications are significant given the widespread use of antidepressants and their persistence in the environment.

This study reveals a previously unrecognized mechanism by which antidepressants can contribute to the spread of antibiotic resistance. The findings emphasize the need to consider the potential impact of non-antibiotic pharmaceuticals on the dissemination of ARGs. Further research should investigate the effects of other pharmaceuticals and environmental factors on bacterial transformation, and explore strategies to mitigate the risk of antibiotic resistance spread.

The study focused on a single bacterial species (*A. baylyi* ADP1) and a limited number of antidepressants. The generalizability of these findings to other bacterial species and antidepressants needs further investigation. The in-vitro model might not completely reflect the complexities of the in-vivo environment. The study did not examine the long-term effects of chronic antidepressant exposure on the spread of antibiotic resistance.