Inhibition Mechanism Of Litsea Cubeba Essential Oil On Staphylococcus Aureus And Its Biofilm

Staphylococcus aureus(S.aureus)is one kind of the most common food-borne pathogenic bacteria,causing both animal and human diseases.What's important,it can form biofilm after infection,which will significantly increase the risk of food contamination.As a result,this study was to investigate the relevant antibacterial and anti-biofilm mechanism against S.aureus using litsea cubeba essential oil(LC-EO)as a natural antibacterial agent.Firstly,the antibacterial mechanism of LC-EO treatment on S.aureus cells was analyzed through several ways,including cell membrane permeability comparative experiments,determination of major intracellular enzyme activities and respiratory metabolism inhibition experiment.Meanwhile,the inhibition mechanism of LC-EO treatment on S.aureus biofilm was studied.Firstly,the effect of LC-EO treatment on extracellular proteins,polysaccharides and eDNA was tested.What's more,the influence of LC-EO treatment on transcriptional level of related regulation genes in biofilm formation,including several key genes in ica operon,main regulation gene about biofilm formation(sarA),and stress response factor?~B regulation gene(sigB)was also elucidated in this study.The main results are as follows:(1)The minimal inhibitory concentration(MIC)and minimal bactericidal concentration(MBC)of LC-EO against S.aureus was 0.5 mg/mL and 1.0 mg/mL,respectively.And 99.999%of sterilizing rate was realized after the LC-EO(MIC)treatment for 4 h.The cell surface hydrophobicity added,and cell membrane permeability was increased,leading to intracellular substances leakage after the LC-EO treatment.Furthermore,the LC-EO treatment could inhibit S.aureus intracellular ATP,DNA and soluble protein content,three key enzyme(alkaline phosphatase,ATPase and?-galactosidase)activity,and hexose monophophate pathway(HMP).Furthermore,the key enzyme(G6PDH)activity in HMP would be significantly inhibited after the LC-EO treatment for 4 h.(2)The ability to form biofilm of the experimental strain(S.aureus ATCC 29213)was tested using Congo Red plates method.The minimal biofilm inhibitory concentration(MBIC)and minimal biofilm extermination concentration(MBEC)of LC-EO against S.aureus biofilm was 1 mg/mL and 2 mg/mL,respectively.The results of silver staining and resazurin test demonstrated that the LC-EO treatment could destroy biofilm and further break interior cells structure.(3)The results of crystal violet staining and colony-counting experiment illustrated that with the increase of the concentration of LC-EO,the sterilizing rate of biofilm was improved.And 99.99%of sterilizing rate was realized after the LC-EO(4mg/mL)treatment for 8 h.The LSCM and SEM images further verified the destructive effect of LC-EO on complex structure of S.aureus biofilm and cells.(4)The bacterial adhesion experiment demonstrated that LC-EO could affect initial adhesion of S.aureus to inhibit biofilm formation.The hydrolysis experiment result showed that S.aureus biofilm is mainly constituted by protein,followed by extracellular polysaccharide and eDNA.And the results of ultraviolet spectroscopy showed that the LC-EO treatment could inhibit extracellular polysaccharides and proteins synthesis,but had no influence on eDNA.(5)After the treatment of LC-EO(MBIC),the transcriptional levels of icaR and sigB genes were increased by 4.13 and 5.8 times,respectively,while the transcriptional levels of icaA,icaD and sarA genes were decreased by 58.9%,90.3%and 68.4%,respectively.However,the transcriptional levels of icaC and icaB genes appeared no significant change after the LC-EO treatment.