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 (amp^R) in both extracellular (e-) and intracellular (i-) forms. The second-order rate constant (k_O3) 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-amp^R 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-amp^R 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.

在 pH 7 的磷酸盐缓冲溶液中研究了臭氧 (O ₃ ) 和游离有效氯 (FAC)对抗生素抗性基因 (ARG) 的降解和失活动力学，其中O ₃（在叔丁醇存在下）和 pH FAC 为 6.8 或 8.1。我们在细胞外 (e-) 和细胞内 (i-) 两种形式中都使用了质粒 (pUC19) 编码的氨苄青霉素抗性基因 ( amp ^R )。2686 个碱基对 (bp) 长的 e-pUC19 降解为 O ₃的二级速率常数 ( k _O3 )，由定量聚合酶链反应测定确定，计算为 ~2 × 10 ⁵ M ^{− 1} s ⁻¹. 与 e-pUC19的降解速率常数相比，用各种受体大肠杆菌菌株测量的 O ₃对 e-pUC19 的灭活速率常数在 2 倍以内。i-pUC19 的降解/失活动力学与 e-pUC19 的相似，表明细胞成分对i-pUC19 的O ₃反应性影响很小。对于 FAC，e-pUC19 的降解和失活率在叔丁醇存在下降低，这意味着直接参与 FAC 以及一些自由基（例如，^· OH）反应。e- amp ^R的降解率直接 FAC 反应的片段可以通过先前报道的两步顺序反应模型来解释，其中速率常数随 e- amp ^R线性增加段长度。e-pUC19 在暴露于 FAC 期间的失活速率常数对于不同的受体菌株可变高达 4.3，揭示了 DNA 修复在观察到的失活效率中的作用。由于 pH 依赖性 FAC 形态变化，e-pUC19 在 pH 6.8 下的降解/失活比在 pH 8.1 下显着更快，而 i-pUC19 动力学对 pH 的依赖性要小得多，表明细胞内质粒 DNA 与 FAC 的反应发生在细胞质 p​​H 下(~7.5)。我们的结果可用于预测和/或测量通过臭氧化和氯化水处理质粒编码的 ARG 的降解/失活效率。