Reversing the paradigm: Oxygen-dependent switch of nTiO₂ from promoting to suppressing ARG conjugation

The environmental spread of antibiotic resistance genes (ARGs) poses a significant public health threat. While nano-titanium dioxide (nTiO 2 ) is known to promote ARG transfer, its role in prevalent anaerobic environments remains poorly understood. Therefore, we comprehensively evaluated the influence of nTiO 2 exposure on the plasmid-mediated ARGs conjugation under aerobic/anaerobic conditions. Under aerobic conditions, nTiO 2 significantly promoted the ARGs dissemination through both intra-genus ( E. coli to E. coli ) and inter-genus ( E. coli to P. denitrificans ) transfer. Reactive oxygen species (ROS) was determined as the decisive factor driving aerobic conjugation, which increased membrane permeability and downregulated conjugative pathways. Addition of ROS scavenger thiourea effectively abolished the stimulatory effect of nTiO₂ on conjugative transfer. However, nTiO 2 treatment significantly inhibited conjugative ARG transfer under anaerobic conditions. The dose-dependent inhibition was confirmed in anaerobic denitrification system. Exogenous H 2 O 2 switched the inhibitory effect of nTiO₂ on anaerobic conjugation. Physical barrier formed by nTiO₂ adsorption might contribute to inhibited conjugative transfer of ARGs. Therefore, this study defines the distinct roles of the inhibitory physical effect caused by the adsorption barrier and promotive chemical effect induced by ROS in the nTiO₂-mediated ARG conjugation, which contributes to a deeper understanding of the environmental propagation mechanisms of ARGs under nanomaterial exposure.