Research On A Broad-Spectrum Antimicrobial Polyguanidine Against Multi-Drug Resistant Pathogens

The discovery of natural antibiotics such as penicillin has opened up the golden era of antibiotic development.Due to overuse and misuse of antibiotics,many bacteria have acquired antibiotic multi-drug resistance through gene mutation and horizontal gene transfer.Infections caused by multi-drug resistant bacteria are gradually increasing,and have become a challen ging global healthcare problem.The discovery of antibacterial compounds of natural origin cannot meet the current demand for new antibiotics.Therefore,we need to find effectiv e strategies to deal with this urgent problem,and the development of new antibacterial drugs is one of the effective strategies.Cationic antibacterial polymers have the advantages of broad-spectrum antibacterial activity,rapid sterilization,and low resistance rate,and therefore are potential candidates of future antibacterial compounds.Cationic antibacterial polymers feature an amphiphilic structure with cation and hydrophobic domains,with the cationic charge interacting electrostatically with the negatively charged phospholipids and the hydrophobic chain further inserted into the lipid bilayer and disrupt membrane integrity via hole-forming mechanism.However,due to the similarity between bacterial cell membranes and mammalian cell membranes,cationic oligomers are relatively toxic to mammalian cells.As a cationic group with delocalized charge,guanidine has been proved to have considerable antibacterial potential in previous studies,and its toxicity is lower than quantinary ammonium ion with localized charges.Therefore,polyguanidine is a potential antimicrobial polymer with low toxicity.In the current work,we reported the research of poly（p-phenylenedimethyl octamethylene diguanidine）（PPODG）composed of hydrophobic long carbon chain and cationic guanidine group against multi-drug resistant pathogens.The main contents are as follows:（1）The determination of antibacterial activity,bactericidal activity and biocompatibility of PPODG.The experiment measured the antibacterial activity of PPODG against ESKAPE pathogens,and found that it has good antibacterial effect against multiple strains of multi-resistant pathogenic bacteria,especially the polymyxin-resistant Klebsiella pneumoniae QXM1.The sterilization kinetics experiment of PPODG on Staphylococcus aureus and Acinetobacter baumannii proved that PPODG can be quickly sterili zed within half an hour,which reflects its high sterilization efficiency.In the biological toxicity experiment,we ob served that PPODG has low toxicity to mammalian cells（HEK293 and 3T 3）and Caenorhabditis elegans,and PPODG has no obvious hemolytic eff ect on sheep red blood cells at a concentration of 32μg/m L,which reflects its good biocompatibility.In the bacterial resistance development experiment,we found that E.coli did not develop resistance to PPODG within 20 days,indicating that PPODG is no t easy to induce E.coli to develop resistance.（2）Using PPODG as a sensitizer,174 FDA-approved antibiotics were used for sensitization screening in Acinetobacter baumannii and polymyxin-resistant Klebsiella pneumoniae（QXM1）.In the experiment using Acinetobacter baumannii as the antibacterial object,we screened 29 hits,and in the experiment using polymyxin-resistant Klebsiella pneumoniae as the antibacterial object,we screened 13hits.In the presence of low concentration of PPODG,the hits can effectively inhibit the pathogenic bacteria under the condition of lower self-inhibitory concentration.In the standard checkerboard test of some outstanding hits and PPODG,we f urther found that there is indeed a synergistic bactericidal effect between the two,that is,FICi is less than or equal to 0.5.Immediately,we selected doxycycline and PPODG as the representative drug combination in Acinetobacter baumannii to do the bactericidal kinetics.The results show that the combination of the two drugs can achieve rapid and effective sterilization.（3）In the research of the antibacterial mechanism of PPODG,this project focused on the effects of compounds on bacterial cell membrane s.First,after treating E.coli with a series of PPODG concentrations,the poten tial of the membrane on the bacterial surface measured by the instrument gradually changed from negative to positive.This phenomenon is consistent with the assumption that th e cationic compounds interfere with the cell membrane.Di SC₃（5）depolarization experiment was used to examine the effect of PPODG on the permeability of intact E.coli inner membrane.We found that as the PPODG concentration increased,the fluorescence of E.coli increased,proving that the membrane potential was destroyed.At the same time,experiments using propidium iodide to test the permeability of cell membranes reached a similar conclusion,that is,as the concentration of PPODG increased,the membrane permeability of bacteria became larger.Scanning electron microscope characterized the bacterial surface morphology of E.coli and S.aureus on the membrane surface of bacteria treated with PPOD G at twice the MIC concentration,we found that the membrane surface of the bacteria appeared obvious holes.This series of mechanism research experiments proves that PPODG interferes with and destroys bacterial cell membranes.In addition to the above effects on the bacterial cell membrane of PPODG,we used flow cytometry to measure the change of ROS accumulation in E.coli after PPODG treatment.The results showed that there was ROS accumulation in the bacteria,indicating that PPODG had effects on the stimulating effect that destroys its homeostasis.（4）In this study,the co-culture model and the nematode model were used to evaluate the sterilization effect of PPODG in a complex environment.In the co-culture model of bacteria and cells,PPODG can effect ively and quickly kill S.aureus in contaminated 3T3 cells,proving that PPODG can still be effectively sterilized in the presence of mammalian cells.In the nematode infection model,PPODG can effectively rescue C.elegans infected with clinical multi-resistant Staphylococcus aureus or Acinetobacter baumannii.The above experiments prove that PPODG still has highly effective bactericidal acti vity in more complicated co-culture and nematode infection models.