Preliminary Effects And Mechanisms Of Intervention Of Tanshinone On Staphylococcus Epidermidis Biofilm Formation

ObjectiveStaphylococcus epidermidis is the fourth nosocomial infection pathogens, and biofilm is the major causative agent. To treat these infections, a large amount of antibiotics has been used, which easily causes the drug-resistance. Traditional chinese medicine, unlike the antibiotics, is a predominance of new dr?g exploiture in our country. This study plans to select the sensitive monomer of tanshinone, and using it to explore the effects and molecular mechanisms of intervention on Staphylococcus epidermidis biofilm formation. Then provide experimental basis for clinical selection of tanshinone for the prevention and treatment in Staphylococcus epidermidis biofilm infections.Method1.Detect the minimal inhibitory concentration (MIC) and the minimal biofilm inhibitory concentration (MBIC) of tanshinone and its main monomer component on biofilm-resistant Staphylococcus epidermidis, then screen the better monomer of tanshinone.2.Construct vitro biofilm model, and draw biofilm growth curve of Staphylococcus epidermidis by semi-quantitative adhesion assay and XTT test, which determine the biofilm adhesion, aggregation, mature stage. Detect biofilm foundation quality, bacterial metabolism, the micromorphology of Staphylococcus epidermidis at different stages under the intervention of the dr?gs at the 10MIC above or 1/2MIC by semi-quantitative adhesion assay, XTT test, SEM respectively, thus researching the phenotypic influence of dr?gs on Staphylococcus epidermidis biofilm formation.3.Choose vancomycin as the positive control. Detect the expression of key genes (icaA?atlE?aap?luxS) in biofilm formation at the concentration of 10MIC or 1/2MIC by real-time fluorescent quantitative PCR, in order to research the relationship among genes, dr?gs and biofilm. Also find out mechanism of the monomer impacting the expression of key genes during biofilm formation.Resultl.The monomer of tanshinone which has strong inhibition of biofilm is cryptotanshinone. And the MIC of cryptotanshinone is 2 ?g/mL, while the MBIC is 32 ?g/mL2.According to the biofilm growth curve, we discover that bacteria attachment phase is at 6h, the accumulation phase is at 24h, and the mature phase is at 48h. For SE1457 and clinical strains at the attachment phase, there is an obvious effect on impacting the formation of biofilm matrix, killing the bacteria in the bioflim, and destroying the microstructure with vancomycin at 32 ?g/mL and cryptotanshinone at both 128 ?g/mL and 32 ?g/mL. Difference is statistically significant (P<0.05). And the inhibition effect of cryptotanshinone at 128 ?g/mL is better than 32 ?g/mL, difference is statistically significant (P<0.05). At accumulation and mature phase, cryptotanshinone at 128 ?g/mL and vancomycin at 32 ?g/mL can obviously inhibit the formation of biofilm matrix, kill the bacteria in the bioflim and destroy the microstructure, and difference is statistically significant (P<0.05). But there is no distinct effect of cryptotanshinone at the concentration of 32 ?g/mL.3.For both SE1457 and clinical strains, the attachment ability can be impacted by vancomycin at 2 ?g/mL and cryptotanshinone at 1 ?g/mL. So can the biofilm matrix and metabolism inside biofilm be inhibited either, and difference is statistically significant (P< 0.05). The effect of cryptotanshinone at 1 ?g/mL is better than vancomycin at 2 ?g/mL, and difference is statistically significant (P<0.05).4.At the attachment phases and accumulation phases of both SE1457 and clinical strains, expression of key genes (icaA?atlE?aap?luxS) in biofilm formation can be depressed by vancomycin at 32 ?g/mL and cryptotanshinone at 128 ?g/mL, and difference is statistically significant (P<0.05).But at the mature phase, only cryptotanshinone at 128 ?g/mL can depress the expression of key genes (icaA?atlE? aap?luxS), and difference is statistically significant (P<0.05). While there is no effect on the expression of those genes by busing vancomycin at 32 ?g/mL and cryptotanshinone at 32 ?g/mL (P>0.05)5.For both SE1457 and clinical strains, cryptotanshinone at 1 ?g/mL can obviously restrain the ability of attachment and suppress the expression of key genes (icaA?atlE? aap?luxS) at the planktonic phases, and difference is statistically significant (P< 0.05).Even the depression is stronger than vancomycin at 32 ?g/mL, and difference is statistically significant (P<0.05).Conclusion1.Cryptotanshinone over 10MIC can eliminate biofilm through impacting the formation of biofilm matrix, killing the bacteria inside biofilm and destroying the complete structure at every phase. And the mechanism might be via depression of key genes (icaA?atE?aap?luxS) to affect the protein which is important while biofilm formation. The elimination depends on the dosage reaction. With development of biofilm, only the higher dose of cryptotanshinone can get rid of it.2.The effect of cryptotanshinone at sub-MIC (1/2MIC) is better than vancomycin at sub-MIC at the attachment phase, which reveals there is some value of cryptotanshinone in preventing the infection caused by Staphylococcus epidermidis biofilm.