Therapeutic Effect Of Eugenol On Subcutaneous Abscess Infected By Staphylococcus Aureus

Staphylococcus aureus(S.aureus)is a common zoonotic pathogen,that is widely found in nature and often causes skin and soft tissue infections in humans and animals.It is a common pathogenic bacterium of body surface soft tissue infections in livestock farms.Staphylococcus aureus can colonize the wound and form a biofilm on the skin surface,specifically inhibiting the wound healing mechanism.The treatment of surface soft tissue infection requires the use of antibiotics,but the long-term use and abuse of antibiotics make the infection of drugresistant bacteria increase day by day.For example,as a drug-resistant strain,Methicillinresistant Staphylococcus aureus(MRSA)is characterized by high mortality,fulminant and invasiveness after infection.Due to the decreasing efficacy of antibiotics and the continuous emergence of drug-resistant strains,there is an urgent need for new strategies to treat drugresistant infections,so the search for effective drugs against resistant bacteria is critical.Eugenol is a phenolic aromatic compound mainly extracted from clove oil,which has antibacterial,antiviral,antifungal,anticancer,anti-inflammatory and antioxidant biological properties.There have been preliminary studies on the bacteriostatic mechanism of eugenol on S.aureus in vitro,but few studies on the therapeutic effect of Dantimol on subcutaneous abscess caused by clinical infection of Staphylococcus aureus.In this paper,we studied the inhibitory effect of eugenol on MRSA and S.aureus ATCC29213 strains of S.aureus biofilm and adhesion invasion,and the regulatory effect on the inflammation caused by S.aureus through in vitro and in vivo experiments.1 Effect of eugenol on cell membrane integrity and biofilm of S.aureusMicrobroth dilution method and AGAR diffusion method was used to determine the MIC and MBC of eugenol against S.aureus phenol.The growth curve of eugenol on S.aureus,AKP activity,bacterial nucleic acid protein leakage,bacterial ultrastructure,PI and NPN uptake,and the effects of eugenol on the formation and maturation of S.aureus ATCC29213 and MRSA biofilms were then detected.The effects of eugenol exposure on expression levels of biofilm and adhesion and invasion-related virulence factors were also examined.The results showed that the MIC and MBC of eugenol against S.aureus ATCC29213 and MRSA were 0.25 mg/mL and 0.5 mg/mL,0.125 mg/mL and 0.25 mg/mL,respectively.Eugenol can increase the concentration of nucleic acid and protein and the activity of AKP in bacterial culture medium.SEM and TEM showed thallus morphology,contents leakage and membrane rupture.The PI and NPN tests showed that eugenol promoted the uptake of PI and NPN by bacteria.Crystal violet staining showed that eugenol could inhibit the generation of S.aureus ATCC29213 and MRSA strains,and had a clear effect on mature biofilms.In addition,eugenol significantly decreased the mRNA expression of biofilm related genes agrA,sarA,cidA and icaA and adhesion factors clfA,clfB,fnbA and fnbB(P<0.001,P<0.01);Compared with S.aureus ATCC29213,eugenol showed more obvious inhibitory effect on MRSA.2 Eugenol antibacterial,anti-inflammatory and maintenance of tight junctions in vitroThe effects of eugenol on the activity of mouse skin fibroblasts(MSF)and mouse mononuclear macrophage leukemia cells(RAW264.7)and cell migration and proliferation were investigated by CCK8 test and scratch healing test.The effects of MRSA and S.aureus ATCC29213 on the activity,adhesion and invasion of MSF and RAW264.7 cells were detected by adhesion and invasion tests.The model of S.aureus infection and cell inflammation induced by teichoic acid(LTA)was established.The effects of eugenol on inflammatory factors,TLR2/MyD88/NF-κB/NLRP3 signaling pathway and Claudin-1 expression in inflammatory cells were investigated by Western blot(WB)and laser confocal microscopy(LSCM).The results showed that eugenol could significantly promote cell growth and migration healing.The results showed that eugenol could inhibit the expression of TLR2 and MyD88,reduce the phosphorylation of p65 and IκBa,inhibit the activation of NLRP3 and the expression of IL-1β,IL-6 and TNF-α.The adhesion and invasion of MRSA and S.aureus ATCC29213 were significantly inhibited,with the inhibition rate of adhesion reaching 65%76%and the inhibition rate of invasion reaching 70%-81%.In addition,eugenol reduced the loss of RAW264.7 and MSF cells Claudin-1 from inflammatory stimuli compared with inflammatory controls.3 The therapeutic effect of eugenol on subcutaneous abscess induced by Staphylococcus aureusThe subcutaneous abscess model of BALB/C mice was constructed with S.aureus ATCC29213 and MRSA,respectively,to explore the therapeutic effect of eugenol on subcutaneous abscess.HE staining and Masson staining were used to observe the effects of eugenol on subcutaneous tissue lesions and collagen fibre.The effects of eugenol on inflammatory cytokines,TLR2/MyD88/NF-κB/NLRP3 signaling pathway and Claudin-1 expression in inflammatory cells were investigated by Western blotting(WB)and laser confocal microscopy(LSCM).The results showed that both S.aureus ATCC29213 and MRSA infection could cause local back abscess in mice.The abscess caused by S.aureus ATCC29213 was large and irregular,and the abscess caused by MRSA was relatively regular.Histopathological observation showed more inflammatory cell infiltration and bleeding,collagen fiber fracture and hyperplasia of granulation tissue.The results of different concentrations of eugenol showed that 1%and 2.5%eugenol had the effect of eliminating pus and healing subcutaneous abscess,and 2.5%was better.The bacterial load test of skin showed that 2.5%eugenol could significantly reduce the bacterial load of skin.Pathological observation and Masson staining showed that inflammation was improved and collagen increased.LSCM observation showed that the expression of Claudin-1 was significantly reduced in infected skin tissues,and the loss of Claudin-1 was inhibited by eugenol treatment.WB analysis showed that eugenol could inhibit the activation of TLR2/MyD88/NFκB/NLRP3 signaling pathway,and significantly inhibit the expression of TNF-α,IL-1β and IL-6 inflammatory factors.These results indicate that eugenol can damage the structure of S.aureus,change the permeability of cell membrane,not only inhibit the formation of biofilms,but also have clearance effect on mature biofilms,and inhibit the transcription and expression of biofilms and adhesion related virulence factors.In subcutaneous abscess models,eugenol can promote abscess recovery by reducing skin inflammation and improving Claudin-1 expression.