Degradation and deactivation kinetics of plasmid-encoded extracellular antibiotic resistance gene during exposure to singlet oxygen in water

Abstract

Antibiotic resistance genes (ARGs) are widespread in aquatic environments, which may promote the dissemination of antibiotic resistance among bacterial populations through natural transformation process. ARGs in sunlit surface waters can be degraded by photochemically-produced reactive oxygen species (ROS), but the role of ROS has been poorly understood. This study investigated the degradation and deactivation of an extracellular ampicillin resistance gene (ampR) encoded in pUC19 plasmid by single oxygen (1O2) as a representative ROS. pUC19 was treated by 1O2 produced by rose bengal photo-sensitization at different exposure levels. The ampR concentration, measured by quantitative polymerase chain reaction (qPCR), increased initially, and then gradually decreased with increasing 1O2 exposure to 3.810-6 Ms. The initial increase of the ampR concentration could be explained by 1O2-induced plasmid conformation change from supercoiled to nicked circular/linear forms with the latter showing enhanced qPCR amplification efficiency. The degradation rates of ampR increased with increasing target amplicon length (215, 422, 636, and 849 bps), from which a second-order rate constant of 8.1(0.4)106 M-1 s-1 was calculated for the reaction of 1O2 with pUC19, based on an assumption of equal sensitivity of DNA damage across the entire plasmid. The deactivation rate of pUC19, measured by a gene transformation assay using Escherichia coli DH5 as recipient cells, was 3.7(0.3)106 M-1 s-1. The ~2-fold slower rate of the gene deactivation compared to the pUC19 degradation might be due to a repair of 1O2-induced DNA damage in the recipient cells. The obtained kinetic information from this work can be useful for evaluating or predicting the removal efficiency of plasmid-encoded ARGs in sunlit surface waters or 1O2 based water treatment processes.