Effect And Mechanism Study Of Geraniol On Methicillin-Resistant Staphylococcus Aureus Biofilm

Methicillin-resistant Streptococcus aureus（MRSA）is one of the most representative drug-resistant bacteria in the world,and it is also a common pathogen of zoonosis.In animal husbandry,MRSA often causes wound infection,cow mastitis and endometritis,which seriously affects the development of the agricultural economy and public health safety.As MRSA often causes complex infections and high drug resistance,there is an urgent need for new anti-MRSA infection programs.More and more studies have shown that biofilm plays an important role in the process of MRSA infection,which makes it more difficult to treat the infection caused by it.This study focused on the inhibitory effect of geraniol on the in vitro activity and biofilm of MRSA strain USA300.The main contents and results are as follows:（1）Preliminary study on the in vitro antibacterial mechanism of geraniol on USA300The double dilution method and colony counting method showed that the minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of geraniol to USA300 in vitro were 512μg/m L and 1024μg/m L,respectively.Compared with the control group,2×MIC geraniol reduced the number of USA300 cells by 73.53±2.53%（p<0.01）after 12 h.Through DISC₃（5）staining,it was found that compared with the control group,the fluorescence intensity of DISC₃（5）increased by 109.15±44.45（A.U.）（p<0.01）in18 min after the addition of 2×MIC geraniol,indicating that the proton driving force（proton motive force,PMF）of the cell membrane was destroyed and depolarization occurred.Through PI staining and Luardan probe staining,it was found that PI fluorescence intensity and Luardan GP respectively increased by 58.31±6.48（A.U.）（p<0.01）and 0.35±0.06（p<0.01）after treatment with 2×MIC geraniol for 30 min.Indicating that geraniol has a significant effect on the cell membrane and membrane fluidity of USA300.The results of the transmission electron microscope showed that geraniol could affect the normal morphology of USA300.（2）Inhibitory effect and mechanism of geraniol on USA300 biofilm in vitroThe results of crystal violet staining showed that geraniol could significantly inhibit the biofilm formation of USA300 and scavenge the mature biofilm in vitro.The Congo red plate method showed that geraniol significantly inhibited the synthesis of intercellular adhesion polysaccharides（PIA）of USA300.The results of spectrophotometry showed that compared with the control group,geraniol reduced extracellular DNA（eDNA）release by 57.10%±4.59%（p<0.01）and staphyloxanthin synthesis by 70.7%±12.34%（p<0.05）.Finally,through the RT-qPCR technique,it was found that 256μg/m L geraniol could significantly inhibit the expression of genes related to biofilm and staphyloxanthin synthesis.Compared with the control group,the transcription levels of sarA,icaA,atl A and crtM decreased by85.03%±14.40%（p<0.01）,79.78%±4.63%（p<0.01）,93.45%±1.18%（p<0.01）and93.45%±1.18%（p<0.01）,respectively.Geraniol has a significant inhibitory effect on the formation of USA300 biofilm and a significant scavenging effect on mature biofilm.The inhibition effect of geraniol on the formation of USA300 biofilm and the clearance of mature biofilm are significant,and its mechanism of action may be to downregulate the transcription level of sarA,inhibit the synthesis of PIA,and affect eDNA Release.Geraniol simultaneously inhibits the biosynthesis of staphyloxanthin by reducing the transcriptional level of crtM.（3）Therapeutic effect of geraniol combined with vancomycin on biofilm infection in miceConstruction of PVC tube attached by USA300 biofilm as implant in vitro.The implants were placed into 42 female mice in the left groin to construct infection models.Mice were randomly divided into a control group,geraniol group,vancomycin group,combination low-dose group（10 mg/kg geraniol+40 mg/kg vancomycin）,medium-dose group（20 mg/kg geraniol+40 mg/kg vancomycin）and high-dose group（40 mg/kg geraniol+40 mg/kg vancomycin）,and the corresponding drugs were injected according to the group.After 24 h the first dose of treatment,the surface colony counting and scanning electron microscopy of the carrier were performed to observe the biofilm coverage and morphology of the surface.Mouse blood is subjected to white blood cell count and TNF-αand IL-6 level determination.The results showed that the number of viable bacteria on the carrier of the combined medium and high dose group was significantly reduced,and the biofilm structure was significantly destroyed.The number of leukocytes in all dosing groups was reduced to normal levels,and TNF-αand IL-6 levels in the combination high-dose group decreased by 46.33%±7.45%（p<0.01）and 41.01%±7.90%（p<0.01）,respectively.The results showed that geraniol had a certain effect on destroying the structure of biofilm in mice,and its combination with vancomycin could enhance its bactericidal effect on bacteria in biocoating in mice and its therapeutic effect on inflammation.In summary,this study shows that:（1）The in vitro bacteriostatic mechanism of geraniol is:inhibiting the normal growth of USA300 by destroying the normal physiological structure of bacteria and changing cell membrane permeability and fluidity.（2）The sub-inhibitory concentration geraniol inhibits bacterial biofilm formation by downregulating the transcription level of sarA,and biosynthesis of staphyloxanthin by downregulating the transcription of crtM.（3）The therapeutic effect of geraniol combined with vancomycin on biofilm infection in mice was better than that of their individual use.Geraniol has anti-MRSA and biofilm activity in vitro,and geraniol combined with vancomycin has a good therapeutic effect in vivo,geraniol has the potential to treat MRSA biofilm infection with vancomycin,which provides theoretical support for clinical treatment of MRSA and biofilm infection.