Study On The Activity And Mechanism Of Action Of Totarol Against Staphylococcus Aureus

It has been reported that food contaminated with pathogenic bacteria such as Staphylococcus aureus(S.aureus)represents a serious health risk to human beings.S.aureus is mostly accountable for food-borne diseases and food poisoning,which are critically related to public health and this area has received increasing attention in recent years.In addition,S.aureus causes a wide variety of infections,which are also of major concern worldwide.S.aureus produces multiple virulence factors,resulting in food infection and poisoning.These virulence factors include hyaluronidases,proteases,coagulases,lipases,deoxyribonucleases and enterotoxins.Among the extracellular proteins produced by S.aureus that contribute to pathogenicity,the exotoxins ?-hemolysin,staphylococcal enterotoxin A(SEA)and staphylococcal enterotoxin B(SEB)are thought to be of major significance.Totarol,a traditional Chinese medicinal herb isolated from the sap of Podocarpus totara,is an antibacterial novel phenolic diterpenes and it has been revealed to inhibit the proliferation of several pathogens effectively.However,there are no reports on the effects of totarol on the production of ?-hemolysin,SEA or SEB secreted by S.aureus and its antibacterial mechanism is also still undefined.Therefore,in this study,the first aim was to evaluate the effects of totarol on these three exotoxins,and then the antibacterial activity and membrane-disruptive mechanism of totarol against S.aureus was also investigated.The results revealed that totarol inbihited the growth of S.aureus at a low dose,and it can inhibited the production of exotoxins,in addition,totarol exerted its bactericidal effects by damaging bacterial cell membranes,which led to rapid cell death.The MIC values of totarol against S.aureus are determined,we concluded that the MIC values of totarol against S.aureus strains were 2-4 ?g/m L.As for S.aureus ATCC 29213,the MIC value is 2 ?g/m L.In the time-kill curves assay,the results demonstrated that totarol inhibited bacteria growth in a dose-dependent manner in both laoboratory and food models.At each time point,totarol at 0.25?g/m L against S.aureus ATCC 29213 indicated no reduction in the number of viablecells,followed by 0.5 ?g/m L,which demonstrated a greater reduction,and at 1 ?g/m L,which indicated the greatest reduction in the number of viable cells.In the hemolysis assay,compared to the positive control,when cultured with 0.25,0.5 and 1 ?g/m L of totarol,the percentage of hemolysis was reduced to 95.6%,81.5% and 26.5%,respectively.A dose-dependent attenuation of hemolysis was observed in the tested strain.The results of western blor assay revealed that totarol at subinhibitory concentrations was effective at inhibiting ?-hemolysin,SEA and SEB secreted by ATCC 29213 in a dose-dependent manner.And as expected,totarol also markedly decreased the transcription of Sar A,agr A,hla,sea and seb in ATCC 29213 in a dose-dependent manner.Furthermore,the levels of TNF-? release were reduced when RAW 264.7 cells were cultured with supernatant from S.aureus treated with increasing subinhibitory concentrations of totarol.Cytoplasmic contents leakage is a classic indication of damage to the bacterial cytoplasmic membrane,which serves as a structural component.Therefore,the data of electron conductivity assay indicated that that varying degrees of increase for conductivity can be observed in the bacterial suspension after totarol treatment,and totarol had various degrees of effects on the integrities of the bacterial cell membranes.And we utilized totarol to induce the production of cytoplasmic ?-galactosidase in S.aureus.These results of SDS-PAGE assay and agarose gel electrophoresis assay indicated that totarol enable to cause irreversible damage to the cytoplasmic membranes,and decrease the content of cellular soluble proteins and nucleic acids by permeating and disrupting cell membranes,which led to cell death.Physical and morphological changes may encounter the cell wall surface deterioration of S.aureus when treated with a suitable antibacterial agent.The SEM assay showed when the bacterial cells were treated with totarol at different test time period,the cells became deformed,wrinkled,shriveled,irregular,pitted,adhesive to each other and some swelling cells were even broken,which may give rise to the leaching out of nutrient and genetic materials.The results in TEM assay,indicated that totarol caused significant damages to the cellular structures including cell membrane and cytoplasm region,which also eventually led to bacterial cells rapid lyses and death.From these data,our works provide not only additional evidences in support of totarol being regarded as a natural antibacterial agent but also fundamental understanding on the mode of antibacterial action.We propose that totarol couldpotentially be used as a promising natural compound in the food and pharmaceutical industries.