Single-cell analysis reveals antibiotic affects conjugative transfer by modulating bacterial growth rather than conjugation efficiency

Antibiotic resistance genes (ARGs) pose a significant threat to human health and the environment. Quantifying the efficiency of horizontal gene transfer (HGT) is challenging due to diverse biological and environmental influences. Single-cell level approaches are well-suited for investigating conjugative transfer, given its reliance on cell-to-cell contact nature and its capacity to offer insights into population-level responses. This study introduces a self-developed system for automated time-lapse image acquisition and analysis. Using a custom dual-chamber microfluidic chip and Python-based image analysis pipeline, we dynamically quantify the ARGs conjugation efficiency at single-cell level. By combining experiments with individual-based modelling, we isolate the effects of subinhibitory antibiotic concentrations on conjugation efficiency from those related to bacterial growth dynamics. No significant variation in Escherichia coli conjugation efficiency was observed across kanamycin concentrations (0 to 50 mg l⁻¹). Moreover, recipient cells with higher growth rates show a greater propensity for plasmid acquisition, suggesting the physiological state of cells pre-conjugation influences their susceptibility to gene transfer. Our methodology eliminates population growth bias, revealing the intrinsic nature of conjugation efficiency. This approach advances our understanding of the factors influencing HGT efficiency and holds promise for studying other microbial interactions. Synopsis: This study employs single-cell analysis to reveal that subinhibitory concentrations of antibiotics affect the conjugative transfer of antibiotic resistance genes by modulating bacterial growth rate rather than conjugation efficiency.