Combinations Of DihydrotanshinoneⅠwith Ampicillin Impact The Biofilm Forming Of Staphylococcus Epidermidis And The Antibiotic Resistance

ObjectiveStaphylococcus epidermidis(S.epidermidis)exists on the skin and mucosal,and get to the deep tissues forming biofilms caused by implant operation.The biofilms will cause the related infections.Once the biofilms formed,the biofilms will increase tolerance up to 100-1000 times higher concentrations of antibiotics than planktonic cells.So biofilms are difficult to treat.On the other hand,the changes of nutrients,oxygen concentration,and osmotic pressure inside biofilms will activate the resistance genes,which might increase the resistance.Recently,biofilm-associated infection is always treated by large doses of antibiotics.The overuse of antibiotics may cause the development of resistance and also cost a lot.So there is a current need to find some more effective and cheap drugs to cure the infection caused by S.epidermidis biofilms.Salvia miltiorrhiza is a kind of Sichuan-specific medicinal herbs,and its active ingredient Tanshinone barely has toxicity and exerts anti-biofilm action in S.epidermidis.In our previous research,Tanshinone could impact the biofilms at adhesion,accumulation and mature phases,but with the higher concentration(32μg/ml,16 times higher of MIC).There were some researches that Traditional Chinese medicine and the effective ingredients could enhance the effects of antibiotics.And there were still no report about Tanshinone combined antibiotics impacting S.epidermidis biofilms.Therefore,in this study we would like to find out the effects and mechanism of Tanshinone combined antibiotics affecting the S.epidermidis biofilms,and invest the effects on the mecA of MRSE which could form the biofilms.The research was like to figure out the feasibility of the combination using in the infection caused by S.epidermidis biofilms and the reverse of MRSE antibiotic resistance,and provide experimental evidence of tanshinone taking part of the prevention and treatment in S.epidermidis biofilm-related and MRSE infections.Methods1.The minimal inhibitory concentration(MIC)and the minimal biofilm inhibitory concentration(MBIC)of cryptotanshinone,dihydrotanshinoneⅠ,ampicillin and oxacillin were detected.Detect the fractional inhibitory concentration index(FICI)of tanshinone combined with ampicillin or oxacillin on SE1457.Then choose the most effective combination.2.Generated vitro biofilm model,according the biofilm growth curve of S.epidermidis biofilms,the main three phases were chosen as the culture time.The biofilms were under the intervention of the combined drμgs at the minimal FICI.Semi-quantitative adhesion assay was used to detect the foundation quality,XTT test was used to detect the bacterial metabolism,SEM was used to dected the micromorphology of S.epidermidis biofilms.Also research the time effect and phenotypic influence of combined drugs on S.epidermidis biofilm formation.Realtime fluorescent quantitative PCR was used to detect the expressions of biofilmrelated genes(icaA,atlE,aap,agrA and stk)with the combination of tanshinone and β-lactam antibiotics,to find out mechanism of the combination affecting the forming of biofilm.3.The biofilms formed by MRSE(SE21021 、 SE26508 、 SE30291)were generated in vitro,and were treated with the combined drμgs at the minimal FICI.Moreover,real-time fluorescent quantitative PCR and ELISA were respectively used to detect the expression of mecA gene and PBP2 a protein.Hence,explore the reverse on MRSE resistance by combined drugs.Results1.The most effective combination on S.epidermidis was dihydrotanshinoneⅠ and ampicillin.At the biofilm state,the concentration of lowest index was 2μg/ml ampicillin combined with 2μg/ml dihydrotanshinoneⅠ.2.At the attachment phase,there is an obvious effect of 2μg/ml ampicillin combined with 2μg/ml dihydrotanshinoneⅠon decresing the biofilm matrix,inhibiting the metabolism inside bioflim,and ruining the structure of biofilm.The inhibition was better than 2μg/ml ampicillin and 2μg/ml dihydrotanshinoneⅠ used alone.In addition,the inhibition showed the best after effect 18 h.The expressions of biofilm-related genes were downregulated by the combined drugs.At accumulation phase,2μg/ml ampicillin combined with 2μg/ml dihydrotanshinoneⅠ could decrese the biofilm matrixes,inhibit the metabolism inside bioflim,and ruin the biofilm structure.The inhibition was more effective with the time progress and still better than the two drugs used alone.However,the expressions of genes were not downregulated by the combination.And at the mature phase,there is no distinct effect of the combination.Even the expressions of genes were upregulated by the combination.3.As for SE21021,the 2μg/ml ampicillin combined with 2μg/ml dihydrotanshinoneⅠ downregulated the expression of mecA,and the downregulation was more obvious than dihydrotanshinoneⅠ alone.And the combination also inhibited the expression of PBP2 a,and the inhibition was better than ampicillin alone.As for SE26508,each drug promoted the expression of mecA,but the expression of mecA treated with combination was lower than the others.However,the combination reduced the PBP2 a,but not well as ampicillin and dihydrotanshinoneⅠalone.For SE30291,the combination inhibited the expressions of mecA and PBP2 a.Conclusion1.Before the biofilms growing to the mature phase,the 2μg/ml ampicillin combined with 2μg/ml dihydrotanshinoneⅠ could inhibit the forming of biofilm,and better than the two drugs using alone.If the biofilms growed into the mature stage,it was hard to be erased by the combination.So the early intervening of drugs is the effective method to cure and prevent the S.epidermidis biofilm forming.2.2μg/ml ampicillin combined with 2μg/ml dihydrotanshinoneⅠcould decrease the expressions of mecA and PBP2 a in some MRSE strains.But the experimental strains were limit,so the conclusion need to be confirmed by increasing the amount of experimental strains and detecting the resistance phenotype.