Study On The Effects And Mechanisms Of Environmental Nano-pollutants On The Horizontal Transfer Of Antibiotic Resistance Genes

Plasmid-mediated horizontal transfer of antibiotic resistance genes（ARGs）poses a major threat to global public health and has become a hot spot in the environmental field.In recent studies,ARGs have been frequently detected in various environmental media,which have a significant impact on human health.ARGs mainly use plasmids as carrier vectors,and the protein channels encoded by specific genes undergo horizontal gene transfer between bacteria of the same or even different species,which leads to the spread of resistance genes in the environment.Nano pollutants are ubiquitous in the environment,and mainly include natural and artificial nanoparticles.Natural nanocolloids（NCs）refer to particles with a particle size of 1~1000 nm.Compared with large-scale colloidal substances,NCs smaller than 100 nm have high reactivity and adsorption performance,which can affect a variety of environmental pollution The migration and transformation behavior of objects.Therefore,it is worth studying whether NCs ubiquitous in the environment can transfer ARGs horizontally.At the same time,single-layer molybdenum disulfide（MoS₂）is a new class of graphene-like two-dimensional nanomaterials.Its wide application in the environmental field will inevitably be released into the environment.However,whether MoS₂ enters the environment will affect the ARGs The spread of transmission is unclear.In this study,the water environment was used as the medium to enrich the NCs in the natural water environment,and the water microscopic system was constructed to explore the impact of NCs on the plasmid-mediated horizontal conjugation and transfer of ARGs in the water environment and the mechanism;the soil was used as the medium to research the influence of MoS₂ exposure on the abundance of ARGs in the soil environment;further construct a soil microenvironment system,in-depth analysis of MoS₂ on the horizontal transfer of ARGs mediated by the RP4 plasmid in the soil environment The influence and molecular mechanism.（1）In the water environment,the exposure of NCs inhibits the horizontal conjugative transfer of RP4 plasmid,and as the concentration of NCs increases（1 mg/L,5 mg/L,10 mg/L）,the number of conjugants is 7.8× 103,3.9×103,1.2×103,and the splice frequency multiples were 2.141,4.395 and 13.91 times that of the control group,respectively.The permeability of cell membrane is reduced but the change is not obvious;the content of reactive oxygen free radicals（ROS）of NCs（5 mg/L,10 mg/L）is significantly reduced by 10 times and 31.6 times;when NCs（10 mg/L）,the content of reactive oxygen radicals（ROS）is significantly reduced;The level of glutathione（GSH）was reduced by 2.32 times;there was no significant change in superoxide dismutase（SOD）;the concentration of extracellularly secreted polysaccharides was reduced by 29.3%;bacterial adhesion was reduced by 50%;ATP levels were reduced by 40.2%.Metabolomics results show that NCs can significantly inhibit microbial fatty acid metabolism（hexadecenoic acid and hexadecanoic acid,etc.）and amino acid metabolism（glutamic acid,alanine,aspartic acid,lysine,arginine,etc.）,Proline process,inhibit the synthesis of ROS and SOD-related metabolites;when NCs are exposed,the TCA pathway is significantly down-regulated,and ATP synthesis is blocked.Transcriptomics results showed that when 10 mg/L NCs were exposed,bacterial membrane protein expression genes（ycg I,art M,sap C,ugp A,ycj O,cys U,pro W,his P）were significantly down-regulated,hindering the interaction between the donor and recipient bacteria.At the same time,lipopolysaccharide metabolism genes（waa G,lpx T）and adhesion protein genes（yad K,yad N,yde R,yfc V）are also significantly down-regulated,which inhibits the synthesis of extracellular polymer（EPS）,resulting in a decrease in bacterial adhesion,thereby reducing Horizontal conjugative transfer of RP4 plasmid.（2）The results of in-situ soil exposure showed that after 45 days of exposure to 25 mg/kg MoS₂,the abundance of bla TEM,and sul 2 genes increased significantly,which was about 3.2 times and 1.5 times higher than that of the blank.At the same time,the abundance of genes erm F,erm B located on the chromosome did not change significantly.The above research results show that MoS₂ can significantly promote the horizontal transfer of ARGs mediated by gene movement elements.In the aseptic soil conjugative transfer system,the conjugative transfer frequency of MoS₂ at different concentrations（5,10,25 mg/kg）was 1.54 times,1.71 times,and 1.42 times that of the blank treatment group,respectively.It shows that MoS₂ can significantly promote the horizontal transfer of resistance plasmids.（3）In vitro experiments found that MoS₂（5mg/L,10 mg/L and 25 mg/L）increased the number of zygotes by about 1.56-1.67 times,the permeability of the bacterial cell membrane did not change significantly,and the bacterial adhesion increased by 1.3-1.6 times,while the ROS level was significantly reduced by 42.5%.MoS₂ in the treatment group（10 and 25 mg/L）can increase the ATP level by 40.6% and 23.2%,respectively.Transcriptomics results show that MoS₂ promotes the synthesis of ATP during energy metabolism by up-regulating the expression of metabolic genes such as glc F,glx R,ttd B,and glc E;at the same time,MoS₂ up-regulates cell secreted protein-related genes（gsp C,fli P,gsp H）The expression of bacterial EPS is promoted,enhances the cell adhesion,thereby promoting the horizontal conjugative transfer of plasmid RP4 in the simulated soil environment.