Pharmacodynamic Evaluation Of Thymol Against Staphylococcus Aureus Biofilm Infections

Methicillin-resistant Staphylococcus aureus(MRSA)is a common human pathogen,resulting biofilm-associated infections.In this study,we selected thymol which can inhibit the formation of biofilm and eliminate mature biofilms by crystal violet staining method in vitro,and explored the effect of thymol which enhanced the efficiency of vancomycin in a mouse infection model.This study is divided into the following three parts:1.Antibacterial mechanism of thymol on methicillin-resistant Staphylococcus aureus(MRSA)The inhibitory effect of thymol on MRSA(TCH1516)was determined via the micro-broth dilution method and the colony-counting method.Solution conductivity,DNA exosmosis amount,SDS-PAGE and transmission electron microscope(TEM)were detected to investigate the effect of thymol on cell membrane,cell wall,soluble protein synthesis and microscopic structures.Both MIC and MBC values of thymol against TCH1516 were 256 μg/m L.After treatment with 2 MIC thymol for 1 h,compared with control group,the conductivity improved 18.08 ± 1.80%.The content of DNA was increased by 123.40 ± 8.06 ng/μL than that in the control group.Compared with control group,after treatment with 2 MIC thymol for 24 h,the protein content decreased by 68.20±0.15%.According to TEM,thymol may have played antibacterial role by destroying cell membrane and cell wall,interfering binary fission.Thymol could damage the cell membrane,interfering the metabolism of protein,and binary fission.2.Effect of thymol on TCH1516 biofilms The abilities of thymol to inhibit biofilm formation,to eliminate mature biofilms,PIA(polysaccharide intracellular adhesion)synthesis,the release of extracellular DNA(eDNA)and the change of sar A,ica A and cid A transcriptional level were assessed by CV staining,SEM,congo red plate method,spectrophotometric and RT-PCR.The results showed that thymol could inhibit bacterial biofilm formation and remove the mature biofilms.As the thymol concentration increases,there is a gradual decrease in the synthesis of PIA,which indicates that thymol inhibits the synthesis of PIA in MRSA biofilms.Thymol could inhibit the release of eDNA from MRSA biofilms.After treatment with 32 μg/m L thymol for 24 h,release of eDNA decreased by 49.43± 12.55%,compared with control group.Afer treatment for 24 h,32 μg/m L thymol decreased the transcript levels of sar A,cid A and ica A by 94.01 ± 2.84%,45.01 ±4.31%,and 30.85 ± 17.83%,respectively,when compared with those in the untreated group.The results showed that thymol had significant inhibitory and removal effect on bacterial biofilms.The mechanism may be that it inhibits the synthesis of PIA and the release of eDNA by reducing the transcriptional levels of ica A,sar A and cid A genes.3.Thymol enhances clearance of MRSA biofilms in a mouse peritoneal implant infection model.30 female mice were randomly divided into control group,thymol group,vancomycin group,low-dose combination treatment group,middle-dose combination treatment group and high-dose combination treatment group.Using the infusion tube as the carrier,the mice biofilm infection models were established,and then the corresponding drugs were injected into models according to the above groups.After24 hours,the number of viable bacteria on the carrier surface were counted,the morphological changes of biofilms on the carrier surface were observed by scanning electron microscope(SEM).The white blood cell count and the levels of inflammatory factors(TNF-α and IL-6)were measured,and the histopathological changes were observed.The results showed that after 24 h of treatment,the colony count on the carrier surface of drug treatment group was significantly less than that of the control group,and there was almost no bacterial growth in the high dose group;under SEM,biofilms decreased significantly in combination treatment group.The results of pathological sections showed that there were many inflammatory cells in control group,but no inflammatory cells were observed in high-dose combination treatment group.The white blood cell count in the drug alone groups or combined groups decreased,compared with control group.In high-dose combination treatment group the white blood cell count returned to the normal range and the content of TNF-αdecreased by 22.57 ± 8.81%,and the content of IL-6 increased by 26.96 ± 9.95%,compared with control group.The results showed that thymol had a removal effect on MRSA biofilms in mice,and the combined application of thymol and vancomycin could significantly enhance its bactericidal effect on biofilms,and it had certain therapeutic effects.In summary,this study shows that:(1)the antibacterial mechanism of thymol is to inhibit the growth of TCH1516 by changing the permeability of cell membrane,destroying the integrity of cell wall and interfering the metabolism of protein,and binary fission in bacteria.(2)thymol may inhibit the formation of biofilms and eliminate mature biofilms by inhibiting the transcriptional levels of ica A,cid A and sar A,reducing the synthesis of PIA and the release of eDNA.(3)thymol can enhance the effect of vancomycin on MRSA biofilm infection.