Hormetic Effect of Pyroligneous Acids on Conjugative Transfer of Plasmid-mediated Multi-antibiotic Resistance Genes within Bacterial Genus

Spread of antibiotic resistance genes (ARGs) by conjugation poses great challenges to public health. Application of pyroligneous acids (PA) as soil amendments has been evidenced as a practical strategy to remediate pollution of ARGs in soils. However, little is known about PA effects on horizontal gene transfer (HGT) of ARGs by conjugation. This study investigated the effects of a woody waste-derived PA prepared at 450 degrees C and its three distillation components (F1, F2, and F3) at different temperatures (98, 130, and 220 degrees C) on conjugative transfer of plasmid RP4 within Escherichia coli. PA at relatively high amount (40-100 mu L) in a 30-mL mating system inhibited conjugation by 74-85%, following an order of PA > F3 approximate to F2 approximate to F1, proving the hypothesis that PA amendments may mitigate soil ARG pollution by inhibiting HGT. The bacteriostasis caused by antibacterial components of PA, including acids, phenols, and alcohols, as well as its acidity (pH 2.81) contributed to the inhibited conjugation. However, a relatively low amount (10-20 mu L) of PA in the same mating system enhanced ARG transfer by 26-47%, following an order of PA > F3 approximate to F2 > F1. The opposite effect at low amount is mainly attributed to the increased intracellular reactive oxygen species production, enhanced cell membrane permeability, increased extracellular polymeric substance contents, and reduced cell surface charge. Our findings highlight the hormesis (low-amount promotion and high-amount inhibition) of PA amendments on ARG conjugation and provide evidence for selecting an appropriate amount of PA amendment to control the dissemination of soil ARGs. Moreover, the promoted conjugation also triggers questions regarding the potential risks of soil amendments (e.g., PA) in the spread of ARGs via HGT.