Effect Of Flavaspidic Acid BB Combined With Antibiotics On Staphylococcus Epidermidis Biofilm

ObjectiveStaphylococcus epidermidis(SE)is a common conditional pathogen in clinic.It is easy to adhere to the non-biological surface and form biofilm,causing biofilm related infection.At present,large doses of antibiotics are often used in clinical treatment of biofilm associated infections,which is expensive and easy to aggravate the drug resistance of bacteria.Many clinical studies have found that traditional Chinese medicine combined with antibiotics has a significant improvement in the treatment of drug-resistant bacteria infection.In addition to the direct effect of traditional Chinese medicine on drug-resistant bacteria to improve their sensitivity to antibiotics,some traditional Chinese medicine also has a specific role for bacterial biofilm,thus weakening the resistance of bacteria to antibiotics.Therefore,it is a new idea to search for active ingredients of traditional Chinese medicine combined with the clinical antibiotics to inhibit the formation of biofilm.Dryopteris fragrans(L.)Schott(D.fragrans)is a kind of folk medicine commonly used in the treatment of various dermatological diseases.Our previous study found that Flavaspidic acid BB could inhibit the biofilm formation of Staphylococcus epidermidis.However,we found that the antibacterial effect of Flavaspidic acid BB on the positive drug sensitive strains was not as good as that of the positive drug,but it had significant antibacterial effect on the positive drug resistant strains.In addition,some literatures have shown that the inhibitory effect of traditional Chinese medicine and its active monomers on bacterial biofilm is enhanced after combined with antibiotics,reducing the dosage of antibiotics and reversing the resistance of bacteria to antibiotics.Therefore,the purpose of this study is to investigate the antibacterial activity and biofilm formation of Staphylococcus epidermidis by the combination of Flavaspidic acid BB and antibiotics,and to explore whether the combination can reverse the antibiotic resistance of bacteria,or achieve the effect of reducing and increasing efficiency,so as to provide a new strategy for the clinical prevention and treatment of Staphylococcus epidermidis biofilm-associated infection,and to develop Flavaspidic acid BB as a new antibacterial agent.Furthermore,it can provide experimental data and theoretical basis for the development of Flavaspidic acid BB as a biofilm inhibitor.Methods1.Screening of antibacterial activity: The combined antimicrobial susceptibility against Staphylococcus epidermidis was evaluated with fractional inhibitory concentration idex(FICI)of Flavaspidic acid BB with mupirocin and erythromycin by chessboard dilution method.2.Determination of time-kill curve: The dynamic bactericidal effect of the combination of Flavaspidic acid BB and mupirocin on SE was evaluated by time-kill curve method in vitro.3.Construction of biofilm model in vitro: The growth dynamic curve and growth matrix curve of Staphylococcus epidermidis were determined by XTT experiment and semi quantitative adhesion experiment,and the microstructure of biofilm was observed by scanning electron microscope(SEM).4.Study on the effect of Flavaspidic acid BB combined antibiotics on the biofilm formation of Staphylococcus epidermidis: The in vitro model of Staphylococcus epidermidis biofilm was established,the minimum biofilm inhibitory concentration(MBIC)of Flavaspidic acid BB combined antibiotics on Staphylococcus epidermidis was determined,and the drug concentration combination was used to intervene Staphylococcus epidermidis at different stages(6h,24 h and 48h).XTT test was used to detect the metabolic activity of bacteria in the biofilm,semi quantitative adhesion test was used to detect the formation of biofilm matrix,and scanning electron microscope(SEM)was used to observe the changes of biofilm morphology.5.Detecting the expression of biofilm formation related genes by q RT-PCR method: The expression of biofilm formation related genes sar A,atl E,aa P,agr A were detected by q RT-PCR,so as to find the possible molecular mechanism of the combination.Results1.When Flavaspidic acid BB was combined with mupirocin,the FICI values were 0.2969 ± 0.027 ～ 0.4750 ± 0.050,all < 0.5,which showed synergistic effect.When Flavaspidic acid BB was combined with erythromycin,the FICI values were 0.5063 ± 0.000 ～ 0.7500 ± 0.050,which was in the range of 0.5-1,showing additive effect.2.According to the time-kill curve method,the combination of Flavaspidic acid BB and antibiotics can effectively inhibit and kill the SE.The results showed that the logarithm of the relative number of viable bacteria decreased with the increase of the concentration of the combination,and the killing effect was in a concentration-dependent manner.3.Construction of biofilm in vitro model: The growth dynamic curve and growth matrix curve of Staphylococcus epidermidis were determined by XTT experiment and semi quantitative adhesion experiment to determine the time period of adhesion,aggregation and maturation of Staphylococcus epidermidis biofilm;the observation of scanning electron microscope(SEM)showed that the biofilm in vitro model had been constructed.4.The effect of Flavaspidic acid BB,mupirocin and erythromycin on the biofilm formation of Staphylococcus epidermidis:(1)The optimal combination concentration of Flavaspidic acid BB and mupirocin against Staphylococcus epidermidis(SE04)was determined by micro chessboard dilution method,which was 20μg/ml Flavaspidic acid BB and 640μg/ml mupirocin,20μg/ml BB and 20μg/ml erythromycin.The best combination concentration of f Flavaspidic acid BB and mupirocin against another strain of Staphylococcus epidermidis(SE08)was 20μg/mlFlavaspidic acid BB and 5μg/ml mupirocin,20μg/ml Flavaspidic acid BB and 0.04μg/ml erythromycin.(2)The results of XTT test,semi quantitative adhesion test and scanning electron microscope showed that for drug-resistant Staphylococcus epidermidis(SE04):a.The drug combination group(Flavaspidic acid BB + mupirocin)could effectively reduce the metabolic activity of bacteria and the quality of biofilm substrate at the adhesion,aggregation and maturation stages of biofilm formation,with significant difference(P < 0.001).Compared with the single drug group,the effect of combination group was better than that of single drug group(P < 0.05).b.The drug combination group(Flavaspidic acid BB + erythromycin)could effectively reduce the metabolic activity of bacteria and the formation of biofilm substrate quality at the adhesion,aggregation and maturation stages of biofilm formation,with significant difference(P < 0.001).Compared with the single drug group,the effect of combination group was better than that of single drug group(P < 0.05).c.Scanning electron microscopy showed that the drug combination group could significantly reduce the amount of bacteria,reduce the adhesion between bacteria and the formation of extracellular polymeric substances(EPS).For the sensitive strain of Staphylococcus epidermidis(SE08):a.The drug combination group(Flavaspidic acid BB + mupirocin)could effectively reduce the metabolic activity of bacteria and the formation of biofilm substrate quality at the adhesion,aggregation and maturation stages of biofilm formation,with significant difference(P < 0.0001).Compared with the single drug groups,in the adhesion stage,the combined drug group was better than the Flavaspidic acid BB group(P < 0.01)and mupirocin group(P > 0.05),and the effect in the aggregation and mature stage was better than that in the single drug group(P < 0.05).b.The drug combination group(Flavaspidic acid BB + erythromycin)could effectively reduce the metabolic activity of bacteria and the formation of biofilm substrate quality at the adhesion,aggregation and maturation stages of biofilm formation,with significant difference(P<0.001).Compared with the single drug groups,in the adhesion stage,the combined drug group was better than the erythromycin group(P<0.01)and Flavaspidic acid BB group(P>0.05),and the effect in the aggregation and mature stage was better than that in the single drug group(P<0.05).c.Scanning electron microscopy showed that the drug combination group could effectively reduce the amount of bacteria in biofilm,reduce the aggregation of bacteria and the formation of extracellular polymeric substances(EPS).4.The expression of biofilm formation related genes aap、atl E、sar A and agr A was detected by q RT-PCR:(1)The expression of biofilm formation genes related of Staphylococcus epidermidis(SE04)by combination of Flavaspidic acid BB and mupirocin.a.At the adhesion stage of SE04 biofilm formation(6h),compared with the growth control group,the drug combination group could significantly down regulate the expression of aap and sar A(P < 0.0001),and up regulate the expression of atl E and agr A(P > 0.05 and P < 0.0001).b.At the aggregation stage of SE04 biofilm formation(24h),compared with the growth control group,the drug combination group could down regulate the expression of aap and sar A(P < 0.05 and P > 0.05),and up regulate the expression of atl E and agr A(P > 0.05).c.At the mature stage of SE04 biofilm formation(48h),compared with the growth control group,the drug combination group could down regulate the expression of aap,sar A and atl E(P < 0.0001,P < 0.001 and P > 0.05),and up regulate the expression of agr A(P > 0.05).(2)The expression of biofilm formation genes related of Staphylococcus epidermidis(SE04)by combination of Flavaspidic acid BB and erythromycin.a.At the adhesion stage of SE04 biofilm formation(6h),compared with the growth control group,the drug combination group could significantly down regulate the expression of aap and sar A(P < 0.0001 and P<0.001),and up regulate the expression of atl E and agr A(P>0.05 and P<0.0001).b.At the aggregation stage of SE04 biofilm formation(24h),compared with the growth control group,the drug combination group could down regulate the expression of aap and sar A(P < 0.01 and P > 0.05),and up regulate the expression of atl E and agr A(P < 0.001 and P < 0.0001).c.At the mature stage of SE04 biofilm formation(48h),compared with the growth control group,the drug combination group could down regulate the expression of aap and sar A(P < 0.0001 and P > 0.05),and up regulate the expression of atl E and agr A(P > 0.05 and P < 0.0001).(3)The expression of biofilm formation genes related of Staphylococcus epidermidis(SE08)combination of Flavaspidic acid BB and mupirocin.a.At the adhesion stage of SE08 biofilm formation(6h),compared with the growth control group,the drug combination group could significantly down regulate the expression of aap and sar A(P < 0.0001 and P > 0.05),and up regulate the expression of atl E and agr A(P > 0.05 and P <0.01).b.At the aggregation stage of se08 biofilm formation(24h),compared with the growth control group,the drug combination group could down regulate the expression of aap,atl E,sar A and agr A(P < 0.0001,P > 0.05,P< 0.001 and P > 0.05).c.At the mature stage of SE08 biofilm formation(48h),compared with the growth control group,the drug combination group could down regulate the expression of aap,sar A and atl E(P < 0.0001,P < 0.0001 and P < 0.001),and up regulate the expression of agr A(P > 0.05).(4)The expression of biofilm formation genes related of Staphylococcus epidermidis(SE08)combination of Flavaspidic acid BB and erythromycin.a.At the adhesion stage of SE08 biofilm formation(6h),compared with the growth control group,the drug combination group could significantly down regulate the expression of aap,sar A and atl E(P < 0.0001,P < 0.01 and P > 0.05),and up regulate the expression of agr A(P < 0.001).b.At the aggregation stage of SE08 biofilm formation(24h),compared with the growth control group,the drug combination group could down regulate the expression of aap,sar A and atl E(P < 0.0001,P < 0.001 and P >0.05),and up regulate the expression of agr A(P > 0.05).c.At the mature stage of SE08 biofilm formation(48h),compared with the growth control group,the drug combination group could down regulate the expression of aap,sar A,atl E and agr A(P < 0.0001,P < 0.01,P < 0.001 and P > 0.05).Conclusion(1)The combination of Flavaspidic acid BB and mupirocin has good antibacterial effect on Staphylococcus epidermidis,showing synergistic effect;The combination of Flavaspidic acid BB and erythromycin has good antibacterial effect on Staphylococcus epidermidis,showing additive effect.(2)The combination of Flavaspidic acid BB and mupirocin can effectively inhibit and kill the floating Staphylococcus epidermidis.The dynamic bactericidal effect of the combination of Flavaspidic acid BB and mupirocin is good,which can reduce the viable bacteria at least 3Log10CFU/m L,and the combination of the two drugs showed a synergistic effect.The combination of Flavaspidic acid BB and erythromycin could effectively inhibit and kill the floating Staphylococcus epidermidis.The dynamic bactericidal effect of the combination of Flavaspidic acid BB and erythromycin was good,which could reduce the viable bacteria quantity by at least 1-2 Log10 CFU/m L,and the combination of the two drugs showed additive effect.(3)The results showed that Flavaspidic acid BB combined with antibiotics could inhibit the biofilm formation of drug-resistant Staphylococcus epidermidis(SE04)to a certain extent,and the effect was better than that of each single drug group,indicating that Flavaspidic acid BB could enhance the inhibition of antibiotics on biofilm and reduce the antibiotic resistance of Staphylococcus epidermidis to a certain extent.The results showed that Flavaspidic acid BB combined with antibiotics could inhibit the biofilm formation of drug-resistant Staphylococcus epidermidis(SE08)in different stages,and could effectively reduce the metabolic activity of bacteria and the formation of biofilm base mass,and the effect was better than that of single drug administration groups,indicating that Flavaspidic acid BB could enhance the inhibition of antibiotics on biofilm and reduce the use of antibiotics The results showed that the amount of water used in the process was reduced and the efficiency was increased.(4)The results showed that Flavaspidic acid BB combined with antibiotics inhibited the expression of aap and sar A genes in different stages of biofilm formation of drug-resistant Staphylococcus epidermidis(SE04),resulting in the reduction of bacterial aggregation and adhesion,and the reduction of extracellular polysaccharide adhesin(PIA),which reduced the secretion of EPS in biofilm,thus reducing the formation of biofilm.The combination of the two drugs can up regulate the expression of Agra gene at different stages of biofilm formation,thus inhibiting the further formation of biofilm.In addition,the expression of atl E in different stages of biofilm formation of drug-resistant staphylococcus epidermidis(SE04)is different in drug combination group,and the specific reasons need to be further studied and explored.The results showed that Flavaspidic acid BB combined with antibiotics inhibited the expression of aap,sar A and atl E genes in different stages of biofilm formation of sensitive Staphylococcus epidermidis(SE08),resulting in the decrease of bacterial adhesion to the abiotic surface,the decrease of aggregation and adhesion,the decrease of extracellular polysaccharide adhesin(PIA),and the decrease of EPS secretion Biofilm formation.In the drug combination group,the expression of agr A gene was up-regulated in the adhesion and aggregation stages of biofilm formation,indicating that agr A may negatively regulate the biofilm formation.