Study On The Activity Of Tea Tree Oil Against Staphylococcus Aureus

Staphylococcus aureus(S. aureus) is one of the most important pathogen of bacterial food poisoning, and in China and most countries, S. aureus has become one of the pathogenic bacteria in legal inspection and quarantine of import and export food. The important bacteria pollution monitoring in food product such as raw milk,cooked meat products in bulk, raw vegetables, meat and aquatic products found that the highest rate of pollution in bacteria is S. aureus. S. aureus can secrete enterotoxin,which is the main factor of food poisoning, and in the food processing environment, S.aureus can exist in the form of biofilm, cause food cross contamination or foodborne disease transmission, and threaten food security. Based on these serious phenomena,the development of strategies to effectively suppress the growth and exoprotein production of S. aureus in foods, thereby improving shelf-life and food quality, is of great interest. In recent years, interest has grown in natural medicinal products,essential oils and other botanicals because of their effective antimicrobial properties against a broad spectrum of food-related pathogenic microorganisms. In our study, tea trea oil(TTO), an essential oil distilled from Melaleuca alternifolia, is composed of terpene hydrocarbons and has broad-spectrum antimicrobial activity.The objectives of this study were to evaluate the inhibitory effects and mechanism of TTO on S. aureus in planktonic and biofilm.The minimum inhibition concentrations and the minimum bactericidal concentrations of TTO against all tested strains in suspension cultures ranged from 1 to 2 mg/m L and from 2 to 4 mg/m L,respectively; the minimum biofilm inhibition concentrations and the minimum biofilm bactericidal concentrations for TTO were 2 to 4 mg/m L and 32 to 64 mg/m L,respectively. Thus, this finding demonstrates that TTO had effectiver antibacterial activity against planktonic S. aureus than biofilm S. aureus. The growth curves of planktonic S. aureus and biofilm S. aureus dynamically show the antibacterial effects in different concentrations of TTO against planktonic S. aureus and biofilm S. aureus.The results of scanning electron microscopy, crystal violet staining assay and confocal laser scanning microscope(CLSM) verify the inhibition effect of TTO in a dose-dependent manner on planktonic S. aureus and biofilm S. aureus, respectively.The present study demonstrats that TTO effectively decreased the formation of S.aureus biofilms by affecting polysaccharide intercellular adhesin(PIA) expression and extracellulargenomic DNA(e DNA) release, and with the increase of drug concentrations of TTO, the amounts of PIA and e DNA gradually reduce in a dose-dependent manner. The study of extracellular virulence proteins produced by S.aureus reveals that the bacterial culture supernatants treated with subinhibitory concentrations of TTO can markedly inhibits hemolysis. In addition, the Western blot assay identifies that the production of α-hemolysin was inhibited by TTO in subinhibitory concentrations. The real-time RT-PCR results revealed that the transcription of ica A, sar A, cid A and hla were down regulated after S. aureus was exposed to TTO.To further investigate the inhibitory mechanism of TTO against S. aureus,Illumina RNA-seq technology was conducted to catalog global gene expression profiling of S.aureus treated with TTO. The RNA sequence analysis of TTO against S.aureus planktonic cells and biofilm revealed that 715 and 304 genes were differentially regulated(greater than a 2- or less than-2-fold change), respectively. Of these, the expression levels of 421 and 200 genes were markedly elevated in planktonic cells and biofilm, respectively, and 294 and 294 genes were significantly decreased. The known genes differentially expressed from RNA-seq were also annotated through GO classification and KEGG pathway analysis. The real-time RT-PCR was conducted to validate RNA sequence data, and the results of the genes expression including ica R, ssp A, lrg A, fnb B, lyt M, glp T, msm X, agr A, sar A and cid A are in accordance with the results of the real-time RT-PCR.To our knowledge, this transcriptome analysis provides the first insights into the transcriptional response of S. aureus to TTO challenge and enables exploration of the mechanisms of TTO antimicrobial activity toward S. aureus. The findings suggest that TTO regulates multiple desirable targets which could be further explored in the development of new food preservatives to avoid the contamination of S. aureus in food processing and storage.