Study On Antibacterial And Virulence Regulation Mechanism Of Clove Essential Oil Against Staphylocossus Aureus

Staphylococcus aureus(S.aureus)is a facultative anaerobic gram-positive bacterium widely distributed in natural environment.As one of the most common foodborne pathogens,S.aureus is very likely to contaminate food during the processing,transportation,and storage stages,resulting in food poisoning and other foodborne diseases.The main pathogenic mechanism of S.aureus is closely related to its production of a variety of staphylococcal enterotoxins(SEs)with high temperature resistance,low p H resistance,salt resistance and other characteristics.SEs cause a variety of foodborne diseases by interfering with intestinal nerve signals.Traditional high temperature sterilization can not be applied to all ingredients,and will lead to the destruction of food nutrition and flavor.The increasing use of chemical antibacterials and antibiotics not only poses a threat to human health,but also leads to the development of multidrug-resistant strains.In contrast,natural products with low toxicity and high antibacterial activity have great application potential as a new generation of food antibacterial agents.Among them,plant essential oils are highly favored due to their broad-spectrum antibacterial activity,low susceptibility to drug resistance,and safe edible properties.In this study,clove essential oil(CEO)extracted from natural plant clove and its main component eugenol were selected as the research object to investigate the antibacterial effect of CEO against S.aureus,and preliminarily revealed the antibacterial mechanism of CEO against S.aureus.On this basis,this study further investigated the inhibition and eradication effect of CEO on S.aureus biofilm,and preliminarily revealed its mechanism.Based on transcriptome technology and phosphorylated proteomics technology,this study further explored the mechanism of eugenol action on S.aureus and its biofilm from the level of gene and protein.In addition,the influence of eugenol on the accessory gene regulator(agr)system of S.aureus was investigated and the inhibitory effect of eugenol on the expression level of virulence gene and extracellular virulence protein secretion of S.aureus was further investigated.Finally,in order to verify the applicability of CEO in food,this study took raw pork,raw beef and raw chicken as food models to investigate the inhibitory effect of CEO on S.aureus and its enterotoxin in raw meat.The main contents and conclusions of this study are as follows:(1)CEO has significant antibacterial activity against S.aureus,and its antibacterial mechanism is multifaceted.First of all,CEO can destroy the cell wall of S.aureus and change the permeability of cell membrane,leading to the leakage of intracellular macromolecules such as alkaline phosphatase(AKP),nucleic acid and protein,resulting in the rupture and death of S.aureus cells.Secondly,CEO can interfere with the respiratory metabolism of S.aureus,inhibit the production of energy in embdenmeyerhof-parnas(EMP)and tricarboxylic acid cycle(TCA)of S.aureus,so as to inhibit the growth of bacteria.Third,eugenol,the principal component of CEO,may bind to DNA of S.aureus and interfere with DNA replication,transcription and repair,thus inhibiting bacterial growth.(2)CEO can significantly inhibit and eradicate S.aureus biofilm.The mechanism of action mainly lies in: in the adhesion stage of the initial formation of biofilm,CEO can inhibit the expression level of staphylococcal accessory regulator A(sar A),reduce the expression of cell wall-anchored(CWA),and thus reduce the cell adhesion;during the biofilm aggregation stage,the CEO can inhibit the synthesis of polysaccharide intercellular adhesion(PIA)and reduce the secretion of extracellular polymeric substances(EPS)such as extracellular proteins,thereby inhibiting the formation of S.aureus biofilm;in the mature stage of biofilm,the CEO can reduce the metabolic capacity of bacteria within the S.aureus biofilm,thereby inhibiting the sustained development of the biofilm;during the biofilm dissemination stage,CEO can inhibit the diffusion process of S.aureus biofilm by interfering with the agr system and reducing the secretion of phenol-soluble modulins(PSMs).(3)CEO can interfere with the agr system of S.aureus and significantly inhibit the expression level of virulence genes.Eugenol,the principal component of CEO,can significantly reduce the phosphorylation level of Agr A,so as to interfere with the normal signal transduction of agr system,resulting in the decreased expression level of S.aureus virulence factor.Eugenol also inhibited the expression of rec A,thus significantly downregulating the expression level of sea.Meanwhile,through the detection of the hemolytic activity of S.aureus,the detection of total enterotoxin of S.aureus and western blot analysis,it was proved that CEO had significant inhibition on the secretion of extracellular toxins such as α-hemolysin,enterotoxin A and enterotoxin B of S.aureus.(4)CEO has application potential in storage and preservation of meat products.While maintaining the color and texture of raw meat such as pork,beef and chicken,CEO not only has a good antibacterial effect on S.aureus in raw meat,but also can effectively inhibit the secretion of S.aureus enterotoxin in raw meat.This study provides experimental basis and useful reference for the application of CEO in food.