Correcting the on-plate conjugation artifact reveals limited antibiotic stimulation of plasmid dissemination.

The global spread of antibiotic-resistant bacteria (ARB) poses a major threat to public health, with wastewater frequently regarded as a hotspot for conjugation-mediated horizontal gene transfer. In this study, we employed a conjugation model and hybrid whole-genome sequencing to characterize the transfer of plasmid pRK2013 between Escherichia coli strains. Hybrid assembly revealed that conjugation frequently resulted in partial plasmid acquisition and chromosomal integration rather than stable episomal maintenance. Furthermore, we demonstrate that conventional plate-based enumeration is prone to methodological artifacts that confound true liquid-phase conjugation estimates. To address this, we disentangled transconjugants generated during liquid mating from those formed during selective agar screening, and quantified the systematic overestimation using a regression-based simulation informed by donor-to-recipient ratios. Accounting for this overestimation revealed that sub-inhibitory concentrations of antibiotics common in wastewater do not broadly stimulate plasmid transfer, contrary to common belief. Rather, levofloxacin at 32-256 μg/L imposed a modest but significant reduction in LB at 37 °C, while having negligible effects under non-growth conditions. Collectively, our findings call for a re-evaluation of conjugation quantification methods and demonstrate that population sizes and physiological state, rather than concentrations of antibiotics and other contaminants, are primary drivers of conjugation-mediated ARG dissemination in wastewater.