| The growing antimicrobial resistance of bacteria has become the focus of global attention,which not only affects the clinical treatment of humans and animals,but also poses a huge threat to food safety,people’s life and health and public health security.However,in stark contrast to the rapid growth of drug resistance,the development of new antibiotics and drugs is seriously lagging behind.Antimicrobial peptides are considered to be the most promising alternatives to antibiotics due to their multi-target and non-specific bactericidal effects,as well as the characteristics of not easily causing bacterial resistance.In this study,we screened and modified the antimicrobial peptides in the antimicrobial peptide database for the pathogenic bacteria of typical Gram-positive bacteria and Gram-negative bacteria,and obtained an antimicrobial peptide RW with broad-spectrum antibacterial activity and low toxicity.-BP100-4D,and the antibacterial effect and safety of the antimicrobial peptide RW-BP100-4D were evaluated experimentally.In this study,21 antimicrobial peptides with better antibacterial effects were screened from the database of antimicrobial peptides(DBAASP).The minimum inhibitory concentrations(MIC)of the above antimicrobial peptides against typical gram-positive bacteria Staphylococcus aureus ATCC29213 and typical gram-negative bacteria Escherichia coli ATCC25922 were tested by microbroth dilution method.The results showed that the antimicrobial peptide RW-BP100 had the lowest MIC against Staphylococcus aureus ATCC29213 and Escherichia coli ATCC25922(MIC=12μg/m L).In order to obtain antimicrobial peptides with better antibacterial activity,RW-BP100 was modified to obtain a series of derivatives,among which RW-BP100-4D had the best antibacterial effect,and its MICs against Staphylococcus aureus ATCC29213 and Escherichia coli ATCC25922 were respectively It is 3 μg/m L and 6 μg/m L,and the bactericidal effect is good.An experimental study on the bactericidal effect of the antimicrobial peptide RW-BP100-4D was carried out.The bactericidal effect of RW-BP100-4D against various Gram-positive bacteria,Gram-negative bacteria and fungi was tested by micro-broth dilution method,and its MIC and minimum bactericidal concentration(MBC)were determined.The results showed that the MIC range for the target bacteria was 3-12μg/m L,and the MBC range was 6-50 μg/m L,indicating that the antimicrobial peptide RW-BP100-4D was a highly active and broad-spectrum bactericidal antimicrobial peptide.The bactericidal activity of antimicrobial peptide RW-BP100-4D on Staphylococcus aureus ATCC29213 biofilm was studied by in vitro bacterial biofilm experiments.,and can effectively remove the mature biofilm formed by Staphylococcus aureus ATCC29213,and its anti-biofilm activity is positively correlated with the application concentration.We also further investigated the bactericidal activity of the antimicrobial peptide RW-BP100-4D against conditioned intracellular bacteria.The antimicrobial peptide RW-BP100-4D can effectively kill Staphylococcus aureus ATCC29213 and Salmonella entering the cow mammary epithelial cell MAC-T.ATCC13076,the sterilization rate exceeds 99.9%.At the same time,we investigated the safety of antimicrobial peptide RW-BP100-4D application.Hemolysis and cytotoxicity experiments showed that when the antimicrobial peptide concentration was ≤100 μg/m L,the hemolysis rate of sheep erythrocytes was < 4%,and the survival rate of MAC-T cells and mouse monocyte-macrophages(RAW264.7)was greater than 99%.The stability test of antimicrobial peptide RW-BP100-4D shows that antimicrobial peptide RW-BP100-4D is relatively stable at 40-80℃,p H=2-7,and is more sensitive to protease in alkaline environment,and its degradation rate is greater than 90%.The bactericidal effect of antimicrobial peptide RW-BP100-4D on the contamination of various food-borne pathogens in chicken was evaluated by chicken sterilization model.Antibacterial peptide RW-BP100-4D has a good killing effect on single bacteria and multiple bacteria contaminated on the chicken surface,and the sterilization rate is more than 90%.The therapeutic effect of antimicrobial peptide RW-BP100-4D on local bacterial infection and systemic bacterial infection in mice was evaluated by mouse skin infection model and bacteremia model.The results showed that in the mouse skin infection model,antimicrobial peptide RW-BP100-4D-BP100-4D can kill 99.99% of methicillin-resistant Staphylococcus aureus(MRSA)strains,and the bactericidal efficiency is significantly higher than the antibiotic ampicillin.In the mouse bacteremia model,it was found that the antimicrobial peptide RW-BP100-4D could not effectively kill the bacteria in most of the organs of the mouse,indicating that it has a poor therapeutic effect on the systemic infection of the mouse.In conclusion,a short peptide RW-BP100-4D with broad-spectrum antibacterial activity was screened and transformed in this study.RW-BP100-4D has small molecular weight,low cytotoxicity and basically no hemolysis,so it has high safety;RW-BP100-4D can effectively inhibit the contamination of food-borne pathogens and spoilage bacteria on the surface of food,and it can cause skin infections in mice.MRSA strains have good killing effect,so they have good application potential in the prevention and control of food contamination or infection caused by the above pathogens. |
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