Degradation and deactivation of plasmid-encoded antibiotic resistance genes during exposure to ozone and chlorine

Degradation and deactivation kinetics of an antibiotic resistance gene (ARG) by ozone (O3) and free available chlorine (FAC) were investigated in phosphate-buffered solutions at pH 7 for O3 (in the presence of tert‑butanol), and pH 6.8 or 8.1 for FAC. We used a plasmid (pUC19)-encoded ampicillin resistance gene (ampR) in both extracellular (e-) and intracellular (i-) forms. The second-order rate constant (kO3) for degradation of 2686 base pair (bp) long e-pUC19 toward O3, which was determined by quantitative polymerase chain reaction assay, was calculated to be ~2 × 105 M−1s−1. The deactivation rate constants of e-pUC19 by O3 measured with various recipient E. coli strains were within a factor of 2 compared with the degradation rate constant for e-pUC19. The degradation/deactivation kinetics of i-pUC19 were similar to those of e-pUC19, indicating only a minor influence of cellular components on O3 reactivity toward i-pUC19. For FAC, the degradation and deactivation rates of e-pUC19 were decreased in the presence of tert‑butanol, implying involvement of direct FAC as well as some radical (e.g., •OH) reactions. The degradation rates of e-ampR segments by direct FAC reaction could be explained by a previously-reported two-step sequential reaction model, in which the rate constants increased linearly with e-ampR segment length. The deactivation rate constants of e-pUC19 during exposure to FAC were variable by a factor of up to 4.3 for the different recipient strains, revealing the role of DNA repair in the observed deactivation efficiencies. The degradation/deactivation of e-pUC19 were significantly faster at pH 6.8 than at pH 8.1 owing to pH-dependent FAC speciation variation, whereas i-pUC19 kinetics exhibited much smaller dependence on pH, demonstrating intracellular plasmid DNA reactions with FAC occurred at cytoplasmic pH (~7.5). Our results are useful for predicting and/or measuring the degradation/deactivation efficiency of plasmid-encoded ARGs by water treatment with ozonation and chlorination.