Effect Of Tebuconazole On Plasmid Mediated Horizontal Transfer Of Antibiotic Resistance Genes And Its Mechanism

The development of bacterial antibiotic resistance is a serious threat to global human health.The horizontal transfer of antibiotic resistance genes（ARGs）is considered to be the main factor in the spread of antibiotic resistance.ARGs can spread in the environment with the help of mobile genetic elements（MGEs）.Fungicides are one of the important means to prevent and control crop diseases in agricultural production.What impact will the residual fungicides in the environment have on the spread and diffusion of ARGs?At present,there are few relevant reports,and further research is needed.In this study,the effect of tebuconazole on the abundance of ARGs and MGEs in soil was discussed through indoor simulated soil experiment.It was found that tebuconazole could increase the abundance of ARGs and MGEs.On this basis,the homologous conjugation transfer system of E.coli HB101 containing plasmid RP4 as donor bacteria（with Tet,Kan and Amp resistance）and E.coli NK5449 as recipient bacteria（with Rif resistance）was constructed.The effect and mechanism of tebuconazole on the horizontal transfer of ARGs were discussed at the cellular level,physiological and biochemical level and gene level.The research results are expected to provide theoretical support for the environmental risk assessment of fungicides and the control of ARGs environmental transmission,which has important practical significance for ensuring human health and ecological environment safety.（1）Effects of tebuconazole on ARGs and MGEs in soil.After tebuconazole treatment for7 days,the relative abundances of aad A2-02,aad A2-03 and tet T increased compared with the background soil,indicating that tebuconazole produced selective pressure on ARGs in soil,thus affecting the abundance of ARGs;Correlation analysis shows that int I1 has a significant positive correlation with aad A2-03,tet R,sul1 and sul2,indicating that there is a close correlation between ARGs abundance and MGEs abundance,which further showed that gene horizontal transfer was the main reason for tebuconazole to promote ARGs migration and diffusion in soil.（2）Effect of tebuconazole on plasmid RP4 conjugation transfer in homologous conjugation system.Tebuconazole exposure induced an increase in the frequency of conjugation and transfer of resistant plasmid RP4,indicating that tebuconazole promoted the conjugation and transfer of ARGs in the set concentration range（0.01-50 mg/L）,and the highest frequency of conjugation and transfer of RP4 plasmid was 8.68E-06 at 10 mg/L,which was 7.93 times higher than that of the control group,and the lowest frequency of conjugation and transfer of RP4 plasmid was 1.24E-06 at 0.1 mg/L,which was 1.13 times higher than that of the control group;The number of donor bacteria was positively correlated with the frequency of conjugation transfer（R~2=0.7124）,indicating that the concentration of donor and recipient bacteria was a very important factor in the process of conjugation transfer;Under tebuconazole stress,there is a significant positive correlation between the conjugation transfer frequency of plasmid RP4 in the conjugation system and the expression of trf Ap gene（r=0.91）,indicating that regulating the expression of conjugation regulatory gene is an important way for tebuconazole to affect the horizontal transfer of ARGs.（3）Effect of tebuconazole on cell membrane permeability of donor/recipient bacteria.Scanning electron microscopy showed that holes were formed on the surface of most bacterial cell membranes and the permeability of bacterial cell membranes increased under tebuconazole exposure;Flow cytometry analysis showed that the cell membrane permeability of donor/recipient bacteria increased,and the cell membrane permeability was the largest at50 mg/L,but the frequency of conjugation and transfer decreased,which may be the fatal cell membrane damage of high concentration tebuconazole to donor/recipient bacteria;Under tebuconazole stress,there is a positive correlation between the conjugation transfer frequency of plasmid RP4 in the conjugation system and the expression level of omp A gene（r=0.76）.The changes of the expression levels of porin genes omp A,omp F and omp C may indicate the change degree of cell membrane and the enhancement of cell membrane permeability,which is conducive to the conjugation transfer of RP4.（4）Effect of tebuconazole on stress response of donor/recipient bacteria.Tebuconazole exposure induced bacteria to produce oxidative stress and SOS response,increased the ability of donor/recipient bacteria to inhibit hydroxyl free radicals,enhanced the activities of CAT and SOD,increased the content of reduced glutathione（GSH）,and up-regulated the m RNA expression of some genes involved in oxidative stress and SOS response,which promoted the conjugation and transfer of ARGs;The ability of receptor bacteria to inhibit hydroxyl radical,CAT and SOD activities and GSH content were positively correlated with the frequency of conjugation transfer,indicating that the conjugation transfer of PR4 was mainly related to the recipient bacteria.