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tet A、tet R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(D600=1.0),分别用超声、过氧化氢(又称双氧水,H_(2)O_(2))及超声/H_(2)O_(2)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;H_(2)O_(2)处理设为3个组,分别为10、20、60 mg·L^(-1),以不加H_(2)O_(2)作为对照组;超声/H_(2)O_(2)联合处理为两种单独处理组的9种组合形式,对于有H_(2)O_(2)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tet A、tet R数量级的减少量,即处理后密度(ρ)与初始密度(ρ0)比值的常用对数值[lg(ρ/ρ0)]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,tet A、tet R下降的数量越多,但总的下降幅度不大:C80时,tet A、tet R分别下降0.62、0.17个数量级,且超声对tet A的去除效率明显高于tet R(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。H_(2)O_(2)单独作用于抗性E.coli时发现H_(2)O_(2)剂量与处理时间之间具有交互作用(P<0.05);不同H_(2)O_(2)剂量之间差异有统计学意义(P<0.05),H_(2)O_(2)对抗性E.coli中的tet A、tet R的去除效果呈现出低浓度要优于高浓度的现象。H_(2)O_(2)对tet A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) H_(2)O_(2),360 min);而其对tet R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) H_(2)O_(2),240 min)。超声、H_(2)O_(2)联合作用于抗性E.coli时发现超声处理与H_(2)O_(2)剂量之间具有交互作用(P<0.001),呈现出超声次数越多,H_(2)O_(2)剂量越低,去除效果越好的趋势:tet A(C80,10 mg·L^(-1) H_(2)O_(2),30 min)、tet R(C40,10 mg·L^(-1) H_(2)O_(2),120 min)最高分别减少1.15、0.56个数量级。超声、H_(2)O_(2)共同作用30 min后,tet A、tet R的数量随时间的累积变化并不明显(P>0.05),但不同剂量的H_(2)O_(2)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。H_(2)O_(2)对水溶液中的四环素抗性基因tet A、tet R有明显的去除作用,对E.coli内的tet A有较为明显的去除效果,对tet R有反向增加的趋势。与单一方法相比,超声联合H_(2)O_(2)可实现低浓度H_(2)O_(2)对抗性E.coli内tet A、tet R的有效去除。
[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 humanhealth 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 genes tet A and tet R was constructed and inoculated in sterile water(D600=1.0).The two resistance genes were removed by ultrasound,H_(2)O_(2),and ultrasound/H_(2)O_(2),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 H_(2)O_(2)treatment were set:10,20,and 60 mg·L^(-1),with 0 mg·L^(-1) H_(2)O_(2) as the control group.Ultrasound/H_(2)O_(2) combined treatment was divided into nine combinations of the two separate treatments.For the H_(2)O_(2) treatment groups,samples were collected at five time points:30 min(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 target tet A and tet Rbefore and after treatment[logarithm base 10 of the ratio of the concentration after(ρ)to before(ρ0)].[Results]When resistant E.coli was treated by ultrasound alone,it was found that with increasing number of repetitions,more tet A and tet R decreased,but the overall decrease was not significant:When ultrasound was administered 80 times,tet A and tet R decreased by 0.62and 0.17 orders of magnitude respectively,and the removal efficiency of ultrasound on tet A was significantly higher than that on tet R(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 H_(2)O_(2) acted on resistant E.coli alone,there was a significant interaction between H_(2)O_(2) dose and treatment time(P<0.05),and a significant difference among different H_(2)O_(2) doses(P<0.05).The removal effect of lowconcentration H_(2)O_(2) on tet A and tet R in resistant E.coli was better than that of high-concentration H_(2)O_(2).H_(2)O_(2) removed tet A over time by up to 0.51 orders of magnitude(10 mg·L^(-1) H_(2)O_(2),360 min),but increased tet R by up to 0.45 orders of magnitude(60 mg·L^(-1) H_(2)O_(2),240 min).When the resistant E.coli was treated with ultrasound and H_(2)O_(2) combination,there was a significant interaction between ultrasound treatment repetition and H_(2)O_(2) dose(P<0.001).Higher ultrasound repetitions and lower H_(2)O_(2) doses were associated with better removal effects:tet A(C80,10 mg·L^(-1) H_(2)O_(2),30 min)and tet R(C40,10 mg·L^(-1) H_(2)O_(2),120 min)decreased by 1.15 and 0.56 orders of magnitude,respectively.After the combined treatment for 30 min,the changes of tet A and tet R were not significant over time(P>0.05).However,different doses of H_(2)O_(2) 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 in E.coli fragmentation and entocyte outflow.H_(2)O_(2)has an obvious removal effect on tetracycline resistance genes tet A and tet R in sterile water,as well as an obvious removal effect on tet Aand a reverse increasing effect on tet R in E.coli.Compared with single treatment,ultrasound combined with low-concentration H_(2)O_(2) can effectively remove tet A and tet R in resistant E.coli with a higher efficiency.
作者
朱光平
陈日萍
薛晨晨
白洁
范宏亮
吴南翔
ZHU Guangping;CHEN Riping;XUE Chenchen;BAI Jie;FAN Hongliang;WU Nanxiang(Hangzhou Medical College,Hangzhou,Zhejiang 310013,China)
出处
《环境与职业医学》
CAS
CSCD
北大核心
2021年第7期775-780,共6页
Journal of Environmental and Occupational Medicine
基金
国家自然科学基金青年基金(21904118)
浙江省自然科学基金(LY17B050008)。
关键词
抗生素抗性基因
抗性菌
超声
H2O2
联合处理
去除方法
antibiotic resistance gene
resistant bacteria
ultrasound
H2O2
combined treatment
removal method
