Degradation and deactivation of plasmid-encoded antibiotic resistance genes during exposure to ozone and chlorine
Degradation and deactivation of plasmid-encoded antibiotic resistance genes during exposure to ozone and chlorine
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暴露于臭氧和氯期间质粒编码的抗生素抗性基因的降解和失活
DOI：
10.1016/j.watres.2021.117408
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发表时间：
2021
期刊：
影响因子：
12.8
通讯作者：
Lee, Yunho
中科院分区：
文献类型：
--
作者：
Yoon, Younggun;He, Huan;Dodd, Michael C.;Lee, Yunho
在pH为7的磷酸盐缓冲溶液中，研究了臭氧和游离有效氯对抗生素耐药基因ARG的降解和失活动力学。我们使用了胞外(e-)和胞内(i-)两种形式的氨苄西林耐药基因(AMPR)编码的质粒(PUC19)。定量聚合酶链式反应测定了2 686个碱基对的e-PUC19降解为O_3的二级速率常数KO_3=~2×10~5M−_1S_−_1。与e-pUC19的降解速率常数相比，秋水仙素的降解速率常数在2倍以内。I-pUC19的降解/失活动力学与e-pUC19相似，表明细胞组分对I-pUC19的臭氧反应性影响很小。对于FAC，叔丁醇的存在降低了e-pUC19的降解和失活速率，这表明FAC参与了直接的FAC以及一些自由基(如·OH)反应。E-AmpR片段直接FAC反应的降解速率可以用先前报道的两步连续反应模型来解释，在该模型中，速率常数随着e-AmpR片段长度的线性增加而增加。E-pUC19在FAC作用下的失活速率常数对于不同的受体菌株来说差异高达4.3倍，揭示了DNA修复在观察到的失活效率中的作用。E-pUC19在pH 6.8时的降解/失活明显快于pH为8.1的FAC形态变化，而i-pUC19的降解/失活动力学对pH的依赖性要小得多，说明胞内DNA与FAC的反应发生在细胞质内pH(~7.5)。我们的结果对于预测和/或测量臭氧和氯化处理水处理后的质粒编码的Args的降解/失活效率很有用。
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 oftert‑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 × 105M−1s−1. The deactivation rate constants of e-pUC19 by O3measured with various recipientE. colistrains 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 O3reactivity toward i-pUC19. For FAC, the degradation and deactivation rates of e-pUC19 were decreased in the presence oftert‑butanol, implying involvement of direct FAC as well as some radical (e.g.,•OH) reactions. The degradation rates of e-ampRsegments 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-ampRsegment 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.