| Skin and soft tissue infection(SSTI)is a relatively common clinical infectious disease,and the main pathogen causing SSTI is Staphylococcus;In recent years,with the widespread use and even abuse of broad-spectrum antibacterial drugs in clinical practice,the Staphylococcus pathogens that cause SSTI have shown multiple drug resistance to clinically commonly used antibacterial drugs.The formation of biofilms is one of the main resistance mechanisms of bacteria,and biofilms can greatly enhance the adaptability and tolerance of bacteria to the environment and establish physical and chemical barriers against antibacterial drugs.Isoasipidin BB is a new phloroglucinol compound extracted and isolated from Dryopteris fragrans(L.)Schott.In the early stage,the antibacterial activity of isoasipidin BB was screened,and it was found that isoasipidin BB had different degrees of antibacterial activity against clinical strains of Staphylococcus,among which the antibacterial effect was the strongest against clinical strains of Staphylococcus epidermidis.Therefore,the purpose of this experiment is to expand the scope of antibacterial screening of isoasipidin BB against clinical strains of Staphylococcus epidermidis,and to select strains resistant to clinically commonly used antibacterial drugs,then we will explore the effect and mechanism ofisoasipidin BB on the biofilm of drug-resistant strains,and the above content isoasipidin BBdeveloped into a clinical treatment drug for SSTI caused by drug-resistant Staphylococcus epidermidis to provide experimental evidence.Main research content:(1)Screening of antibacterial activity of isoasipidin BB: according to the M07-A9 program of the Clinical and Laboratory Standards Institute(CLSI),erythromycin,mupirocin,and fusidic acid,which are commonly used clinically in the treatment of SSTI,are used as positive controls,and microdilution method was used to determine the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of isoasipidin BBagainst 15 clinically isolated strains of Staphylococcus epidermidis,and screening out strains that are sensitive to isoasipidin BB but resistant to positive drugs;The time-sterilization curve was drawn by the counting method of plate viable bacteria,and the in vitro antibacterial activity of isoasipidin BB was dynamically evaluated against resistant Staphylococcus epidermidis clinical isolates(SEP-05)sensitive to isoasipidin BB at different growth stages.(2)Establishment of in vitro biofilm of resistant Staphylococcus epidermidis: establish a biofilm model of resistant Staphylococcus epidermidis in vitro by using the guide piece static culture method and the microplate static culture method;the crystal violet semi-quantitative adhesion experiment and the XTT experiment were used to draw the in vitro growth curve of the biofilm,and the changes in the microscopic morphological structure of the biofilm at different stages were observed by Scanning Electron Microscope(SEM)to determine the difference in the biofilm in vitro,so as to determine the time point of the growth phase(adhesion,aggregation,maturity,and end of maturity).(3)The scavenging effect of isoasipidin BB on biofilm: through crystal violet semi-quantitative adhesion experiments,XTT experiments and SEM observations,with erythromycin as a positive control,the effect of different concentrations of isomerin BB on the biofilm of resistant Staphylococcus epidermidis at different growth stages(adhesion,aggregation and maturity)was studied.The total amount,the metabolic activity of the bacteria in the membrane and the influence of the microscopic morphological structure.(4)The effect of isoasipidin BB on the extracellular matrix content of biofilms: through the phenol-sulfuric acid method,BCA method and ultramicro ultraviolet-visible spectrophotometer Nano Drop one C determination,study the effect of different concentrations of isomemarin BB on the main components of extracellular matrix of resistant Staphylococcus epidermidis biofilm,extracellular polysaccharide and protein And e DNA content.(5)Preliminary mechanism of the effect of isoasipidin BB on the formation of biofilms: through the determination of carbon and hydrogen adsorption capacity,fibrinogen adhesion determination,soft agar plate analysis,Triton X-100 induced autolysis test,ATPase activity determination and XTT experiment to explore the effect of isoasipidin BB on surface hydrophobicity,fibrinogen adhesion,exercise capacity,autolysis rate and biofilm metabolic activity of drug-resistant Staphylococcus epidermidis;(6)The effect of isoasipidin BB on the expression of key genes in biofilm formation: through the fluorescence quantitative PCR technology,the expression levels of key genes(Aap,Atl E,Ica A,Lux S,Rec A)in the formation of biofilm formation of resistant Staphylococcus epidermidis after treatment with different concentrations of isoasipidin BB were explored.Main research results:(1)Screening of antibacterial activity of isoasipidin BB: the range of MIC for 15 strains of Staphylococcus epidermidis(SEP01-SEP15)is0.53-1.67μg/m L,and the range of MBC is 0.94-33.33μg/m L,and isoasipidin BB showed strong sensitivity to strains resistant to mupirocin,erythromycin and fusidic acid in the positive control,and its antibacterial activity against15 clinical strains was significantly better than that of the positive control Fusidic acid(P<0.01).The time-kill curve of isoasipidin BB on SEP-05 shows that isoasipidin BB has better antibacterial activity on SEP-05 at different growth stages than the positive control erythromycin(P<0.05),and the effect is consistent with time and dose dependent.(2)Establishment of in vitro biofilm of resistant Staphylococcus epidermidis: The biofilm formation process of SEP-05 is divided into four stages: 0-12 h biofilm grows slowly,which is the adhesion period;12-24 h biofilm grows most rapidly,which is the aggregation period;24-48 h basically does not grow,which is the mature period;48-72 h biofilm begins to decrease,which is the end of maturity.(3)The effect of isoasipidin BB on biofilm removal: MIC,2MIC,and 4MIC isoasipidin BB can remove the biofilm in the adhesion,aggregation and maturation stages of SEP-05.It can also inhibit the metabolic activity of bacteria in the biofilm and destroy the microscopic morphological structure of the biofilm.The effect is dose-dependent,and the effect is better than the positive control erythromycin(P<0.05).(4)The effect of isoasipidin BB on the extracellular matrix content of the biofilm: The results showed that the extracellular polysaccharide,protein and e DNA content in the extracellular matrix of the biofilm after treatment with different concentrations of isoasipidin BB decreased to varying degrees compared with the growth control group(P<0.05).It was dose-dependent,with the highest decrease of 45.21%,69.20%,and 58.10%respectively.(5)Preliminary mechanism of the effect of isoasipidin BB on the formation of biofilm: isoasipidin BB can inhibit the formation of biofilm by affecting the surface hydrophobicity,fibrinogen adhesion,exercise capacity,autolysis rate and biofilm metabolic activity of SEP-05(6)The effect of isoasipidin BB on the expression of key genes in biofilm formation: The results of fluorescence quantitative PCR show that isoasipidin BB can affect the key genes(Aap,Atl E,Ica A,Lux S,Rec A)in the formation of biofilm through SEP-05 Changes in expression levels,thereby inhibiting the formation of biofilms.In summary,(1)isoasipidin BB has good antibacterial activity against 15 clinical strains of Staphylococcus epidermidis,especially the strains resistant to the positive control drug show good sensitivity;(2)the mechanism of isoasipidin BB scavenging effect on different stages of drugresistant Staphylococcus epidermidis biofilms: affecting the production of extracellular matrix of biofilms,destroying key links in the process of its formation,and affecting the expression of key genes in the process of biofilm formation.The results of this study can provide theoretical support for the further development of isoasipidin BB into a new drug resistant to SSTI caused by drug-resistant Staphylococcus epidermidis. |
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