Significant generational effects of tetracyclines upon the promoting plasmid-mediated conjugative transfer between typical wastewater bacteria and its mechanisms.

The generational effects of antibiotics are well-documented within the field of pharmacology, but their specific influence on the enhancement of plasmid-mediated conjugative transfer remains insufficiently understood, although plasmid-mediated conjugative transfer represents a crucial process for the horizontal dissemination of antibiotic resistance genes (ARGs). We pioneeringly investigate the effects of eight tetracyclines (TCs) across three generations on the conjugative transfer of RP4 plasmid, utilizing Escherichia coli strains isolated from typical wastewater. Results indicate that TCs enhance plasmid conjugative transfer, exhibiting significant generational effects. Average hormetic zone for conjugative transfer (ΔAR) induced by 1st-generation TCs is 4.46 to 5.95 times greater than that induced by 2nd- and 3rd-generation TCs, respectively. Furthermore, this intriguing phenomenon is unrelated to TCs' degradation characteristics (P > 0.05), but is closely associated with bacterial biochemical responses (P < 0.05), including reactive oxygen species (ROS), cell membrane permeability, and ATP synthesis. Moreover, mechanistic results reveal that key structural parameters of TCs significantly impact their interactions with the essential protein UspD, which regulates biochemical processes and relevant genes on RP4 plasmid in donor bacteria, thereby substantially enhancing the plasmid conjugative transfer frequency and exhibiting significant generational effects (P < 0.001). Our findings shed light on the generational effects of TCs on plasmid conjugative transfer in wastewater treatment systems, offering theoretical supports for ARGs dissemination control.