| The overuse of antibiotics eventually leads to the detection of antibiotic resistant bacteria（ARB）and antibiotic resistance genes（ARGs）in various environmental matrices,including air,soil,lake water,groundwater,wastewater and even drinking water.ARBs and ARGs as a class of emerging pollutants are highly diffuse and persistent.ARGs can be transfer to other microorganisms by MGEs（plasmids,transposons,integrons and bacteriophages）,resulting in the dissemination of ARGs.Some traditional disinfection methods,such as chlorine disinfection,ozone oxidation,and Fenton oxidation,have been applied to remove ARB and ARGs.However,there is a risk of high dose of disinfectants and promotion of horizontal transfer of ARGs.Recently,discharge plasma is to apply a certain voltage to the anode and cathode electrodes to ionize the air to form physical and chemical effects such as collection of high-energy electrons,reactive oxygen species（ROS）,and ultraviolet radiation,has received significant attention in various bacteria inactivation in water environment,due to its the advantages of high efficiency and no need for the addition of external chemicals.Unlike ordinary bacteria,the ARGs carried by ARB can still be spread among microorganisms through gene transfer although the ARB is inactivated,including from dead cells to living cells.Information regarding the mechanism of discharge plasma on the removal efficacy and regulatory factors of ARB and ARGs are still unclear.In this study,elimination of ARB and the associated ARGs in aqueous solutions were explored using a novel strategy,namely discharge plasma,and focusing on the removal efficiency of ARB and ARGs and the potential of ARGs horizontal transfer resistance by discharge plasma.Main results for this study are as follows:1.The removal effect and mechanism of discharge plasma on AR E.coli were evaluated from the effects of electrical and carrier gas parameters on AR E.coli removal efficiency.Highly effective AR E.coli inactivation performance was obtained.Approximately 6.6-log AR E.coli was inactivated within 10 min plasma treatment at 11 k V,and the antibiotic resistance to tested antibiotics（tetracycline,gentamicin,and amoxicillin）significantly decreased by 87.5%,93.8%and 96.8%,respectively.The qualitative and semi-quantitative studies of active substances showed that·OH,1O2,and O2·-contributed to AR E.coli elimination.The reaction rate constants of AR E.coli removal were decreased by 41.8%,53.4%and 37.8%after the addition of ROS inhibitors mannitol,L-His and SOD,respectively,indicating that compared with·OH and O2·-,1O2 was significantly reduced in AR E.coli removal process plays a stronger role.In addition,ROS attacks led to destruction of cell membrane,structures of proteins,and destroy of nucleotide bases of DNA,ultimately leading to efficient removal of AR E.coli.2.A discharge plasma was developed to remove AR E.coli strains carrying amoxicillin resistance gene（bla TEM-1）,tetracycline resistance genes（tet C and tet W）,gentamicin resistance gene（aac（3）-II）,and integron gene int I1,and the removal mechanism was discussed by the structure and function of DNA and ARGs.The experimental results showed that the elimination efficiencies of tet C,tet W,bla TEM-1,aac（3）-II,and integron int I1 reached 2.28-log,2.88-log,5.19-log,5.71-log,and 5.46-log after 10 min plasma treatment.These elimination performances were positively related to the plasma intensity,duration time,and air flow rate,and the removal effect of ARGs was the best after 10 min of 11 k V 400 L h-1discharge treatment.Analysis of electron spin resonance spectroscopy,reverse transcriptase-polymerase chain reaction,UV-visible spectroscopy,Fourier transform infrared spectroscopy,high performance liquid chromatography and electrophoresis gel was found that·OH and 1O2destroyed the structural integrity of ARGs during the discharge plasma treatment,making DNA decomposed into nucleotide bases,and finally leading to the removal of ARGs.3.Simultaneous removal of ARB and ARGs in water by a discharge plasma oxidation was investigated,focusing on evaluating the impacts of inorganic ions on the inactivation performance of ARB and the ARGs profiles.The experimental results demonstrated that CO32-consumes the·OH produced by the discharge plasma,resulting in the ARB removal rate dropping to 50.8%.The presence of NO3-,Cu2+,and Fe2+promoted the generation of·OH in the discharge plasma system,resulting in an increase in the removal efficiency of ARB by36.1%,37.0%and 38.9%,respectively;whereas SO42-did not have distinct effect of AR E.coli removal efficiency.At the same time,the presence of NO3-,Cu2+,and Fe2+also promoted the cell membrane structure,enzyme activity,and intracellular reactive oxygen species accumulation in the presence of these inorganic ions were consistent with the removal rules of AR E.coli and ARGs.4.The inhibition mechanisms on conjugative transfer of ARGs by a RP4 plasmid with two different Escherichia coli（E.coli）strains were investigated under discharge plasma exposure.The experimental results demonstrated that the conjugate transfer of ARGs significantly inhibited by the discharge plasma treatment.Compared with the control,the conjugative transfer frequency significantly decreased from 4.08×10-5 to 1.2×10-8 after 10 min of treatment at 11 k V,and the genes were significantly down-regulated by 63.5%.The conjugation transfer frequencies of ARGs were significantly increased after adding the three ROS traps（P<0.05）,and the order was SOD<L-His<mannitol.Compared with 1O2 and O2·-,·OH was more likely to attack ARGs,thereby inhibiting the conjugate transfer frequency.The expression of genes related to oxidative stress response,cell membrane permeability,cell contact,and intracellular energy driving force was significantly inhibited by discharge plasma treatment.Finally,the expression of conjugative metastasis-related genes was significantly down-regulated,and the conjugative transfer of ARGs was inhibited.In conclusion,highly effective AR E.coli and ARGs removal performance was obtained,and significantly reduce the resistance and horizontal gene transfer,thereby effectively controlling the risk of antibiotic resistance transmission. |
