The overlooked risk of horizontal transfer of plasmid-borne antibiotic resistance genes induced by synthetic phenolic antioxidants

Plasmid-borne conjugation transfer of antibiotic resistance genes (ARGs) triggered by non-antibiotic stresses has attracted widespread attention, known to motivate conjugation through well-recognized reactive oxygen species and SOS response. However, a notable knowledge gap remains on the potential risks of reductive compounds, such as synthetic phenolic antioxidants (SPAs), in facilitating horizontal gene transfer by the other mechanisms beyond intracellular ROS. Therefore, intragenus and wastewater indigenous microbiota conjugation models were established to examine conjugative transfer frequency of RP4 plasmid under exposure of four extensively detected SPAs. The mechanisms were elucidated utilizing fluorescence detection, RT-qPCR, and transcriptomic analysis with 3-tert-butyl-4-hydroxyanisole (BHA) serving as a representative SPA. Results demonstrated that conjugation transfer frequencies of both models were significantly promoted without triggering SOS responses under exposure to high doses of BHA. Furthermore, BHA exposure benefited conjugation progress through improving membrane permeability of donors and ameliorating cellular energy supply. In addition, BHA exposure activated the RP4-encoded transfer apparatus by regulating the expression of associated genes. This study highlighted and provided a stark reminder about the potential horizontal gene transfer risks posed by SPAs exposure, which were regarded as a neglected driver in the dissemination of ARGs.