Research On The Mechanism Of Peptide Drμgs In Subinhibitory Concentration To Promote Conjμgative Transfer Of Plasmid

Antimicrobial resistance of bacteria seriously threatens the health of humans and animals.In particular,the horizontal spread of plasmid-mediated antimicrobial resistance has expanded the scope of harm.The conjugative transfer of plasmids can be promoted or inhibited by certain substances.Among them,the substances which can facilitate plasmid transfer have the characteristics of improving the permeability of the cell membrane.Since the antibacterial mechanism of colistin and vancomycin is to affect the permeability of the cell membrane.we predict that the peptide drugs may affect the conjugative transfer of plasmids,and further affecting the resistance genes spread.Therefore,we established a conjugative transfer model of plasmids,and explored the effects of these two drugs on conjugative transfer.At the same time,we used wild-type plasmids to verify the function of the drugs in promoting plasmid transfer.Finally,we explored the underlying mechanism for the effects of colistin and vancomycin on plasmids transfer.Using E.coli J53 and E.coli DH5α to build the plasmid conjugative transfer model.Then the model was treated by colistin and vancomycin in different sub-MIC concentrations.When the colistin concentration was 1/8 MIC,the conjugation frequency of the plasmid reached 9.84×10-4,which was 38 times higher than the antibiotic-free group.when the vancomycin concentration was 1/4 MIC,the conjugation frequency of the plasmid was increased 3 folds,reach to 8.67×10-5.These data indicated that sub-MIC concentrations of colistin and vancomycin can promote the conjugative transfer of RP4-7 plasmid.To understand the clinical impact of drugs on conjugative transfer,the effects of drugs on the conjugative transfer of wild-type plasmid IncI2 plasmid(carrying mcr-1 gene)and IncX3 plasmid(carrying blaNDM-5 gene)were determined.The results showed that when the concentration of colistin was 1/4MIC and 1/8MIC,the conjugation frequency of IncI2 plasmid and IncX3 plasmid was significantly increased,especially the conjugation frequency of IncI2 plasmid was increased by more than 10 times.Vancomycin could not affect the transfer of IncX3 plasmid,but the conjugation frequency of IncI2 plasmid increased by nearly 10 times at a concentration of 1/8 MIC.To initially explore the mechanism of colistin and vancomycin affecting plasmid transfer.Firstly,we found the bacteria surface was locally damaged after treated with drugs through scanning electron microscopy.Using fluorescent probes PI and NPN to detect the permeability of the cell membrane,it was found that the permeability of the bacterial cell membrane increased under the action of the drug.Secondly,the changes of ROS in bacterial cells after drug action were also measured,it was found that the vancomycin caused an increase in ROS content in the recipient bacteria(E.coli J53)at 1/4 MIC.Finally,we used qPCR technology to analyze the changes in the related genes expression.The ompC and ompF genes encode outer membrane porins,which are involved in the transport of osmotic substances.The expression of OmpF increases under hypotonic conditions,but the opposite is true for OmpC.The expression of ompC and ompF were relatively down-regulated and up-regulated,respectively,which was consistent with the increase in cell membrane permeability detected by fluorescent probes,and both indicated an increase in cell membrane permeability.At the same time,both the trbBp gene that regulates the formation of connections between bacteria and the global regulation genes(korA,korB,trbA)that inhibits the expression of the trbBp gene are determined.Real-time fluorescence quantitative results showed that the expression of korA,korB,trbA genes were down-regulated,and the expression of trbBp showed an up-regulated trend.Therefore,it shows that the drug inhibits the expression of the global regulatory gene,thereby promoting the expression of the trbBp genes.In summary,colistin and vancomycin can promote plasmid transfer at sub-MIC concentrations.The main reason is that the drugs increase the permeability of cell membrane.The expression of genes related to plasmid transfer were changed.Drugs promoted the expression of genes related to the formation of conjugative pairing relationships between bacteria.This study proves the promoting effect of colistin and vancomycin on plasmid transfer.This study proved that the sub-MIC concentrations of colistin and vancomycin can promote the transfer of plasmids between E.coli.It is suggested that sub-MIC peptide drugs not only enrich the corresponding drug resistance genes,but also accelerate the spread of other drug resistance genes by promoting the conjugative transfer of plasmids.The results provide corresponding data for the risk assessment of related drugs.At the same time,it reminds us that we should strengthen the control of corresponding drugs and monitor residues to avoid the spread of drug resistance.