Study On Antibacterial Activity Of Novel Antimicrobial Peptides Against MRSA And Application In Prevention Of Implant Infection After Spinal Surgery

Objective:Select 2 kinds of antimicrobial peptides through in vitro antibacterial ability and hemolysis test and study further the antibacterial mechanism of these two peptides.To establish an animal model of postoperative spinal implant infection in mice,and evaluate their efficacy in preventing postoperative spinal implant infection in mice.Methods:(1)Minimal inhibitory concentration(MIC):broth dilution method was used to determine the MIC of 5 antimicrobial peptides(W1,W2,W3,W4,W5),vancomycin,ampicillin,colistin,ciprofloxacin against common bacteria after spinal surgery.(2)Hemolysis experiment:the 4%erythrocyte suspension was prepared and incubated with different concentrations of antimicrobial peptides at 37℃for 1h.The absorbance was determined to calculate the hemolysis fraction of antimicrobial peptides.(3)The damage of antimicrobial peptides to methicillin-resistant Staphylococcus aureus(MRSA)biofilms was observed by scanning electron microscopy(SEM):two antimicrobial peptides(W4 and W5)were screened by MIC and hemolysis test,and the screened antimicrobial peptides were co-incubated with mature biofilms.The damage of antimicrobial peptides(W4,W5),vancomycin and ampicillin to MRSA mature biofilm was observed under SEM.(4)The damage of antimicrobial peptides to MRSA morphology was observed by scanning SEM:antimicrobial peptides W4 and W5 were incubated with MRSA bacteria at MIC.Scanning electron microscope was used to observe the damage of antibacterial peptides to the morphology of bacteria.(5)A mouse model of postoperative spinal implant infection was established and antimicrobial peptides were injected into the surgical site.One week after modeling,the surgical site infection was evaluated by visual observation.The plate counting method was used to analyze the antibacterial effect of the antimicrobial peptides.HE pathological staining was used to observe the tissue inflammation at the surgical site.Results:(1)MIC:five antibacterial peptides(W1,W2,W3,W4,W5)showed strong antibacterial effects on common gram-positive and negative bacteria in spinal postoperative infection.When the concentration of MRSA bacterial solution was 1×10~5CFU/m L,both W4 and W5 had strong antibacterial effects on MRSA,and their MIC were 10μg/μL.(2)Hemolysis experiment:When the antimicrobial peptides reached 1000μg/m L,the hemolysis fraction of W1 and W2 was about 10%.W3,W4 and W5 have the lowest hemolysis fraction,about 5%,which is relatively safe.When the concentration was250μg/m L,the hemolysis fraction of all antimicrobial peptides did not exceed 1%.(3)SEM was used to observe the damage of antimicrobial peptides to MRSA biofilm:Antimicrobial peptides W4 and W5 can destroy MRSA mature biofilm effectively.The destructive effect of the antimicrobial peptides was comparable to that of vancomycin and significantly stronger than that of ampicillin.(4)SEM was used to observe the damage of antibacterial peptides on the morphology of MRSA bacteria:MRSA bacteria were treated with antimicrobial peptides W4 and W5 at MIC.The experimental results showed that MRSA bacterial membrane appeared serious collapse and damage,and even appeared the leakage of bacterial contents.(5)In vivo studies,one week after modeling,obvious swelling and severe suppurative infection occurred at the surgical site in the control group and the ampicillin group.At the same time,plate counts of the spine and implants at the surgical site also showed high levels of bacteria.HE staining showed extensive inflammatory cell infiltration.The above results comprehensively proved that serious bacterial infection occurred in the surgical site of the control group and the ampicillin group.However,in the antibacterial peptide group(W4,W5)and vancomycin group,the above situation was significantly alleviated.And the antimicrobial peptide group and vancomycin group showed similar therapeutic effects.Conclusions:Five novel antimicrobial peptides(W1,W2,W3,W4,W5)showed good antibacterial activity and low hemolytic activity in vitro,among which W4 and W5showed significant antibacterial effect and low hemolytic activity.In vivo experiments have proved that W4 and W5 can effectively prevent implant infection after spinal surgery,which provides a new strategy for preventing implant infection after spinal surgery.