Study On Bacteriostatic Effect And Mechanism Of Antibacterial Peptides Combined With Antibacterial Peptides On Candida Albicans

Candida is a kind of fungi,also known as Candida,which is the most common conditional pathogen among fungi.These pathogens are mainly distributed in vagina,mouth,skin,digestive tract and other human organs in a symbiotic state in human body.Candida can cause skin-surface infections and,in more serious cases,systemic infections.Studies have shown that candidiasis albicans plays a dominant role in fungal infectious diseases,and it is on the rise year by year.In addition to controlling systemic infection and reducing mortality,it is urgent to find an effective antifungal treatment with low cost and strong effect to improve superficial infection.In organisms,Antimicrobial Peptides（AMPs）is an important part of innate immune response,and it is a kind of polypeptide with natural immunity,which has the efficacy of sterilizing,inactivating,removing viruses,living bacteria and other microorganisms.Up to now,more than 2000 kinds of AMPs have been found,which indicates that AMPs play a very important role in the immune response of organisms.Some literatures and clinical experiments have verified the synergistic effect of AMPs combined with antibiotics on Candida.However,the experimental scope of AMPs is limited,the application degree is low,and the research on AMPs drug resistance is less.Therefore,considering many disadvantages of traditional antifungal drugs,combined with a large number of studies,we selected AMPs and traditional antifungal drugs which have a certain literature basis and are widely used for synergistic research,and carried out Fractional inhibitory concentration index（FICI）to screen AMPs and antifungal drugs with synergistic effect,in order to provide more support and help for the treatment of candidiasis.In recent years,Candida parapsilosis（C.parapsilosis）has been used as indicator bacteria in many related literatures,and compared with Candida albicans（C.albicans）to verify the effect of AMPs on C.albicans.Therefore,this study draws lessons from previous research results,taking C.albicans as working bacteria and setting C.parapsilosis as positive control,fully verifying the inhibitory effect of AMPs combined with drugs on C.albicans,and enhancing the experimental persuasiveness.In the experiment,different AMPs,including AP16,AP16-K,KK-20,Mt6-21DLeu and Hst-5,were tested by Minimum Bactericidal Concentration（MBC）and Minimal Inhibitory Concentration（MIC）.The results show that the MIC_C.albicansand MBC_C.albicans of Hst-5are 32±10μg/m L and 64±10μg/m L respectively,and the MIC_{C.parapsilosis} and MBC_{C.parapsilosis} are 16±10μg/m L and 64±10μg/m L respectively.The results showed that Hst-5 had obvious antibacterial and bactericidal effects on C.albicans and C.parapsilosis.At the same time,the antibacterial effects of five AMPs combined with six antifungal drugs ICZ,Am B,5-FC,VCZ,FCZ and PCZ on fungi were determined by chessboard method.The results showed that the FICI values of Hst-5-Am B on the inhibition of C.albicans and C.parapsilosis were 0.2±0.1 and 0.3±0.1 respectively,which indicated that the combined action of Hst-5-Am B had obvious synergistic effect（FICI<0.5）.In order to further verify the synergistic bactericidal effect of Hst-5&Am B on fungi,20 clinical isolates of C.albicans and C.parapsilosis were selected as test tool strains by using chessboard method again.The results showed that the FICI index of Hst-5&Am B combined with C.albicans and C.parapsilosis was less than0.5,which showed obvious synergistic effect;In addition,the combined action can obviously reduce the MIC value of Hst-5 and Am B,and improve the sterilization ability.In addition,the synergistic effect of Hst-5-Am B on the growth inhibition of C.albicans and C.parapsilosis was confirmed in germ tube and mycelium inhibition experiments.In order to fully understand the antifungal mechanism of Hst-5 combined with Am B,the permeability of cell membrane phosphorus（Pi）,the level of reactive oxygen species（ROS）and the degree of lipid peroxidation were studied.The results showed that the average concentration of Pi in culture medium was 0.093±0.001μg/m L and0.090±0.002μg/m L when the antimicrobial peptide Hst-5 alone was used to treat C.albicans or C.parapsilosis,respectively,which did not affect the membrane permeability of fungi.When Hst-5-Am B was combined with C.albicans or C.parapsilosis,the average concentration of Pi was 0.263±0.010μg/m L and 0.242±0.010μg/m L,respectively,which showed stronger permeability to cell membrane than that of Am B alone（P<0.05）.The ROS values of C.albicans and C.parapsilosis increased by about 50 when Am B acted alone;The ROS values of C.albicans and C.parapsilosis increased by about 200 when Hst-5-Am B was combined with Hst-5（p<0.05）.Am B alone could induce lipid peroxidation damage in the cell membranes of C.albicans and C.parapsilosis,while Hst-5-Am B combined could aggravate the increase of ROS in C.albicans and C.parapsilosis and increase the degree of lipid peroxidation damage.The results indicated that Hst-5-Am B had synergistic bactericidal effect on C.albicans and C.parapsilosis through oxidative damage.In general,the antibacterial activities of five AMPs,AP16,AP16-K,KK-20,Mt6-21DLeu and Hst-5,were screened separately,and it was found that Hst-5 had a positive effect on the inhibition of C.albicans.It was found that Hst-5 and Am B had synergistic inhibitory effect on C.albicans.In addition,it is elucidated that Hst-5 synergistic with Am B inhibits C.albicans through the principle of peroxidation damage.This study provides strong support for the better clinical application of AMPs,and also provides a research basis for finding efficient,convenient and less side-effect treatment methods.