From stimulation to attenuation: exposure mode-dependent conjugative transfer of antibiotic resistance gene under tetrabromobisphenol A-induced stress

Non-antibiotic pollutants can transiently intensify plasmid-mediated dissemination of antibiotic resistance genes (ARGs), but long-term effects remain unclear. Here, we investigated how exposure mode (i.e., single versus multiple exposure) differentially shaped RP4-mediated conjugative dissemination of ARGs under environmentally relevant concentrations of tetrabromobisphenol A (TBBPA). Single TBBPA exposure increased conjugation frequency 8.5-13.2-fold, coincident with elevated intracellular reactive oxygen species (ROS), greater adenosine triphosphate (ATP) availability, and higher membrane permeability. In contrast, repeated TBBPA exposure attenuated these acute physiological responses and abolished the single-exposure-induced enhancement of conjugation, thereby restoring transfer frequencies to levels not statistically distinguishable from the unexposed control. Transcriptomics revealed that single exposure upregulated stress-response, energy-metabolism, and signaling genes conducive to RP4 conjugation, whereas multiple exposures elicited repression of these pathways, shifting cells toward a low-energy, low-permeability state. Metabolomics corroborated that a single exposure transiently enriched metabolites associated with membrane dynamics and stress mitigation, whereas multiple exposures broadly depleted cofactors and energy-related metabolites. Additional pollutant and sediment-derived recipient assays supported the broader phenotypic occurrence of acute stimulation followed by repeated-exposure attenuation. Overall, these findings show that TBBPA exposure modes can shape RP4-mediated ARG transfer in donor-recipient model systems, providing new insights into the environmental risks posed by non-antibiotic pollutants.