Inactivation efficiency of plasmid-encoded antibiotic resistance genes during water treatment with chlorine, UV, and UV/H2O2

Abstract

This study assessed the inactivation efficiency of plasmid-encoded antibiotic resistance genes (ARGs) both in extracellular form (e-ARG) and present within Escherichia coli (intracellular form, i-ARG) during water treatment with chlorine, UV (254 nm), and UV/H₂O₂. A quantitative real-time PCR (qPCR) method was used to quantify the ARG damage to amp^R (850 bp) and kan^R (806 bp) amplicons, both of which are located in the pUC4K plasmid. The plate count and flow cytometry methods were also used to determine the bacterial inactivation parameters, such as culturability and membrane damage, respectively. In the first part of the study, the kinetics of E. coli inactivation and ARG damage were determined in phosphate buffered solutions. The ARG damage occurred much more slowly than E. coli inactivation in all cases. To achieve 4-log reduction of ARG concentration at pH 7, the required chlorine exposure and UV fluence were 33-72 (mg × min)/L for chlorine and 50-130 mJ/cm² for UV and UV/H₂O₂. After increasing pH from 7 to 8, the rates of ARG damage decreased for chlorine, while they did not vary for UV and UV/H₂O₂. The i-ARGs mostly showed lower rates of damage compared to the e-ARGs due to the protective roles of cellular components against oxidants and UV. The contribution of OH radicals to i-ARG damage was negligible in UV/H₂O₂ due to significant OH radical scavenging by cellular components. In all cases, the ARG damage rates were similar for amp^R versus kan^R, except for the chlorination of e-ARGs, in which the damage to amp^R occurred faster than that to kan^R. Chlorine and UV dose-dependent ARG inactivation levels determined in a wastewater effluent matrix could be reasonably explained by the kinetic data obtained from the phosphate buffered solutions and the expected oxidant (chlorine and OH radicals) demands by water matrix components. These results can be useful in optimizing chlorine and UV-based disinfection systems to achieve ARG inactivation.

Keywords: Antibiotic resistance; Antibiotic resistance genes; Chlorine; Disinfection; Hydroxyl radical; UV.