超声/过氧化氢联合对水中四环素抗性基因的去除研究
Removal of tetracycline resistance genes in water by ultrasound/hydrogen peroxide
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摘要:
背景 四环素抗性基因是新型环境污染物抗生素抗性基因(ARGs)中最具有代表性的一种,常在抗性菌(ARB)中存在,广泛分布于水环境中,对人类健康和生态环境的稳定造成了巨大的潜在危害。
目的 探索新的、有效去除水环境中四环素抗性基因的方法,分析四环素抗性基因去除效果的影响因素,为环境中ARGs的去除方法提供参考。
方法 构建含有
tetA 、tetR 这两种四环素抗性基因的大肠杆菌(E.coli ),并将其接种在无菌水中(D 600=1.0),分别用超声、过氧化氢(又称双氧水,H2O2)及超声/H2O2联合对两种抗性E.coli 的抗性基因进行去除。超声处理设为3个组,分别为20次(C20)、40次(C40)、80次(C80),0次(C0)作为对照组;H2O2处理设为3个组,分别为10、20、60 mg·L-1,以不加H2O2作为对照组;超声/H2O2联合处理为两种单独处理组的9种组合形式,对于有H2O2参与的处理组,设30 min(T30)、60 min(T60)、120 min(T120)、240 min(T240)、360 min(T360)5个时间点采样,以0 min(T0)作为对照组。每种处理条件的样本量设为3个,通过处理前后目标tetA 、tetR 数量级的减少量,即处理后密度(ρ )与初始密度(ρ 0)比值的常用对数值lg(ρ /ρ 0)来评估每种方法的去除效果。结果 超声单独作用于抗性
E.coli 时发现随着超声次数的增加,tetA 、tetR 下降的数量越多,但总的下降幅度不大:C80时,tetA 、tetR 分别下降0.62、0.17个数量级,且超声对tetA 的去除效率明显高于tetR (P < 0.05)。在电镜下观察超声处理后抗性细菌表面产生球形凸起,部分细菌结构破碎。H2O2单独作用于抗性E.coli 时发现H2O2剂量与处理时间之间具有交互作用(P < 0.05);不同H2O2剂量之间差异有统计学意义(P < 0.05),H2O2对抗性E.coli 中的tetA 、tetR 的去除效果呈现出低浓度要优于高浓度的现象。H2O2对tetA 的去除作用随着时间的累积十分明显,最高可降低0.51个数量级(10 mg·L-1 H2O2,360 min);而其对tetR 却呈现出反向的增加作用,最高可增加0.45个数量级(60 mg·L-1 H2O2,240 min)。超声、H2O2联合作用于抗性E.coli 时发现超声处理与H2O2剂量之间具有交互作用(P < 0.001),呈现出超声次数越多,H2O2剂量越低,去除效果越好的趋势:tetA (C80,10 mg·L-1 H2O2,30 min)、tetR (C40,10 mg·L-1 H2O2,120 min)最高分别减少1.15、0.56个数量级。超声、H2O2共同作用30 min后,tetA 、tetR 的数量随时间的累积变化并不明显(P > 0.05),但不同剂量的H2O2对两种抗性基因的去除效果差异有统计学意义(P < 0.001)。结论 超声处理可对大肠杆菌造成机械损伤,导致大肠杆菌破碎、内容物流出。H2O2对水溶液中的四环素抗性基因
tetA 、tetR 有明显的去除作用,对E.coli 内的tetA 有较为明显的去除效果,对tetR 有反向增加的趋势。与单一方法相比,超声联合H2O2可实现低浓度H2O2对抗性E.coli 内tetA 、tetR 的有效去除。Abstract:Background Tetracycline resistance genes are the most representative new environmentallypolluting antibiotic resistance genes (ARGs), mostly in the form of antibiotic resistant bacteria (ARB), and are widely distributed in the water environment, posing potential harms to human health and ecological environment stability.
Objective This study explores a new and effective method for removing tetracycline resistance genes in the water environment, and analyzes the factors affecting the removal, in order to provide reference for the removal of ARGs in the environment.
Methods Escherichia coli (E.coli ) containing tetracycline resistance genestetA andtetR was constructed and inoculated in sterile water (D 600=1.0). The two resistance genes were removed by ultrasound, H2O2, and ultrasound/H2O2, respectively. Three groups of ultrasonic treatment were set: 20 times (C20), 40 times (C40), and 80 times (C80), taking 0 times (C0) as the control group. Three groups of H2O2 treatment were set: 10, 20, and 60 mg·L-1, with 0 mg·L-1 H2O2 as the control group. Ultrasound/H2O2 combined treatment was divided into nine combinations of the two separate treatments. For the H2O2 treatment groups, samples were collected at five time points: 30min (T30), 60 min (T60), 120 min (T120), 240 min (T240), and 360 min (T360), and 0 min (T0) was used as the control group. The sample size of each treatment was 3. The removal effect of each treatment was evaluated by the reduction of the magnitude order of targettetA andtetR before and after treatmentlogarithm base 10 of the ratio of the concentration after (ρ ) to before (ρ 0).Results When resistant
E.coli was treated by ultrasound alone, it was found that with increasing number of repetitions, moretetA andtetR decreased, but the overall decrease was not significant: When ultrasound was administered 80 times,tetA andtetR decreased by 0.62 and 0.17 orders of magnitude respectively, and the removal efficiency of ultrasound ontetA was significantly higher than that ontetR (P < 0.05). Under electron microscope, the surface of resistant bacteria showed spherical protuberance and some bacterial structures were broken after the ultrasound treatment. When H2O2 acted on resistantE.coli alone, there was a significant interaction between H2O2 dose and treatment time (P < 0.05), and a significant difference among different H2O2 doses (P < 0.05). The removal effect of lowconcentration H2O2 ontetA andtetR in resistantE.coli was better than that of high-concentration H2O2. H2O2 removedtetA over time by up to 0.51 orders of magnitude (10 mg·L-1 H2O2, 360 min), but increasedtetR by up to 0.45 orders of magnitude (60 mg·L-1 H2O2, 240 min). When the resistantE.coli was treated with ultrasound and H2O2 combination, there was a significant interaction between ultrasound treatment repetition and H2O2 dose (P < 0.001). Higher ultrasound repetitions and lower H2O2 doses were associated with better removal effects:tetA (C80, 10 mg·L-1 H2O2, 30 min) andtetR (C40, 10 mg·L-1 H2O2, 120 min) decreased by 1.15 and 0.56 orders of magnitude, respectively. After the combined treatment for 30 min, the changes oftetA andtetR were not significant over time (P > 0.05). However, different doses of H2O2 showed significant differences in the removal effect on the two resistance genes (P < 0.001).Conclusion Ultrasonic treatment can cause mechanical damage to
E.coli , resulting inE.coli fragmentation and entocyte outflow. H2O2 has an obvious removal effect on tetracycline resistance genestetA andtetR in sterile water, as well as an obvious removal effect ontetA and a reverse increasing effect ontetR inE.coli . Compared with single treatment, ultrasound combined with low-concentration H2O2 can effectively removetetA andtetR in resistantE.coli with a higher efficiency.