The Mechanism For The Bactericidal Activity Of Artemisia Argyi Essential Oil Against Pathogenic Foodborne Microorganisms And Its Application On Fresh-cut Vegetables

The food industry has an inherent need for the use of antimicrobials to overcome the challenges with the quality and safety of food.However,the misuse of antimicrobials has led to agricultural microbial resistance(AMR),which is a bane to food security and also predisposing humans to significant health threats through the consumption of contaminated foods.Thus,the growing call for “naturally and ecofriendly” products has steered up vast recommendations for the utilization of natural antimicrobials over chemical antimicrobials for their safety and effectiveness.Fortunately,the combination of two or more natural antimicrobials have been suggested as viable means for the optimization of the efficacy and efficiency of existing antimicrobials in what has been called the “synergistic antimicrobial” effect(SAE).Additionally,the introduction of appropriate enhancers such as polymers,and osmotic stress(salinity)have been shown to enhance antimicrobial efficacy by improving antimicrobial absorption,allowing targeted antimicrobial delivery,and improving biofilm permeation while reducing the possibility of causing further bacterial resistance.The plant kingdom is the major source of various medicines and most antimicrobials.One of the significant medicinal plants,Artemisia argyi H.Lév.& Vaniot(A.argyi),from genus Artemisia,tribe Anthemideae,is presently the focus of phytochemical research due to its biological and chemical potential for the production of essential oil(EO).However,studies exploring the bactericidal activities of essential oils have traditionally assumed and focused on their major components as the key determinants and carriers of their biological activities.Little attention is usually paid to the minor components.Suffice to say antimicrobial effectiveness is usually achieved at higher concentrations.However,the mechanisms underlying the antimicrobial effectiveness of the synergistic interaction of EO components remain opaque.Moreover,several components of EOs,especially constituents accounting for minor proportion by volume,are often neglected in studies exploring its antimicrobial potency.Thus,using Artemisia argyi essential oil as a case study,this study sort to explore the effective methods for enhancing the bactericidal effectiveness of cineole,component of Artemisia argyi essential oil,on pathogenic microbe Escherichia coli O157:H7.First,the study explored the antimicrobial potency of the synergistic interaction between the main constituents of A.argyi plant,cineole,and other known antimicrobial agents such as carvacrol,thymol,on the pathogenic food microorganism E.coli O157:H7.The findings revealed that out of all the antimicrobial agents tested,the antimicrobial activity of cineole was the least effective with a higher minimum inhibitory concentration(MIC)value of 32 μg/m L.Combining cineole with other natural agents,a synergistic effect was observed only with the combination of cineole and carvacrol(CN/CR),which significantly inhibited E.coli O157:H7 at a fractional inhibition concentration index(FICI)of 0.14 at FIC 0.02 and 0.13 for CN and CR respectively via destruction of bacterial cell structure leading to the leakage of intracellular matter.CN/CR synergy had a great interaction at molecular levels and remarkably hindered biofilm formation(98.42%),exopolysaccharides production(80.21%),and bacteria’s movement.Mechanistically,CN/CR repressed E.coli O157:H7 transcriptional genes including shiga-like toxin gene(stx1),flagella gene(flh D),intimin(eae),and quorum sensing gene(lux S).Moreover,(?MIC+?MIC)CN/CR produced a generally acceptable sensory score upon hindering E.coli O157:H7 when applied on freshly cut cucumbers.The strong CN/CR antimicrobial synergistic efficacy might be due to the strong hydrophobic bonds formed by both CN and CR at similar binding sites with E.coli O157:H7.Subsequently,the effectiveness of osmotic pressure to enhance the bactericidal potency of cineole was investigated.It was found that the MIC of cineole(32 μg/m L)was improved with varying degrees on Na Cl-induced osmotic pressure(CN-S)at MIC values of 16,4,and 2μg/m L for 1,3,and 5% CN-S.The results showed that Na Cl-induced osmotic pressure conferred additional effective influence for cineole in suppressing E.coli O157:H7 biofilm growth(99.98%)when compared to 1×MIC CN(63.25%).The incorporation of 5% CN-S was able to alter the production of exopolysaccharides(86.95%),swimming and swarming motilities(87.20% and 98.90% respectively),hydration,and cell membrane integrity within E.coli O157:H7 cells compared to 1×MIC CN which could only alter exopolysaccharides production,swimming and swarming motilities by 31.45%,25.79% and 15.42%,respectively.The primary cell attachment,aggregation,and colony formation were considerably disrupted,and the expression of specific virulence genes flh D,eae,stx2,stx1,as well as TA system antitoxins hip B,and maz E were downregulated.Furthermore,evaluating the mechanism through which CN-S suppresses E.coli O157:H7,it was revealed that aside the virulence genes,CN-S could downregulate curli(csg D),T3SS(esp A)and oxidative stress(rpo S,sod B)genes,that contributes to the adhesion,proliferation,biofilm formation and existence of E.coli O157:H7.Importantly,after storage at 4°C for 48 hours,the amount of internalized microbial cells in fresh cut cucumbers treated with the 5% CN-S reduced to 1.32 log CFU/g.Thus,this section of the study offers valuable insights into the mechanism by which osmotic pressure increases the bactericidal activities of natural antimicrobials,especially against biofilm development.Finally,to identify potential new antimicrobial components,all known AAEO constituents were explored and compared for their antimicrobial efficacy via a high throughput screening method.Intriguingly,it was observed that the minor constituents,which are usually neglected,piperitol,and alloaromadendrene,demonstrated a superior inhibitory capacity on the studied microorganisms including E.coli O157:H7 and S.aureus,compared to the well-known major constituents,cineole.Furthermore,in-vitro validation studies indicated that piperitol is capable of effectively preventing the growth of E.coli O157:H7 comparable to the major component,cineole.To the best of our knowledge,this is a first study to confirm the antimicrobial efficacy of the CN/CR synergy,which could serve as a substitute natural food sanitizer on fresh produce.Also,this work has revealed that osmotic stress enhances the antimicrobial activity of cineole by downregulating Shiga-like toxins,virulence,oxidative stress,and TA system genes which are part of critical indicators for adhesion,proliferation,and biofilm formation of microorganisms including E.coli O157:H7.Significantly,the study provides the experimental basis that minor components alone could be critical determinants of the bactericidal activity of essential oils and plant extracts and as such should not be overlooked.