Font Size: a A A

Inhibition Of Carvacrol Against The Mono/Dual-Biofilm Of Pseudomonas Fluorescens Based On Quorum Sensing

Posted on:2021-01-26Degree:MasterType:Thesis
Country:ChinaCandidate:Y Y WangFull Text:PDF
GTID:2370330623458898Subject:Food Science and Engineering
Abstract/Summary:PDF Full Text Request
Quorum sensing is a form of bacterial communication that regulates the secretion of biofilms,spoilage enzymes and virulence factors.Biofilm is a structured aggregation of bacteria that the bacteria on its surface and embedded in the living body from the polymeric material in the cells.Pseudomonas fluorescens is an important psychrophilic spoilage bacteria and strong biofilms forming strain,and Staphylococcus aureus is a common pathogen of aquatic products in China,whose biofilms are widely concerned.Plant essential oils?EOs?are also well known as antibacterial agents and are often used as food preservatives to control food spoilage and foodborne pathogenic bacteria based on their growth inhibiting properties.In this study,the spoilage trains P.fluorescens,A.salmonicida and the pathogenic bacteri a S.aureus isolated from refrigated large yellow croaker were studied.Screening the structura l analogs of cinnamaldehyde and carvacrol from 10 essential oils obtained carvacrol with th e best antibacterial and anti-biofilm ability.the changes of the anti-biofilm ability and AHLs s ecretion of carvacrol against P.fluorescens wild strain and?LuxI were compared.The effect s of carvacrol on differentially expressed genes and metabolic pathways of P.fluorescens wer e analyzed by transcriptomics sequencing and molecular docking.Additionally,the interactio n between P.fluorescens and S.aureus in fish juice based on AI-2,and the resistance of mi xed biofilm for carvacrol.It provides a technical basis for the biofilm of food-borne microorg anisms,and the application of plant essential oils in the control of microbology in aquatic pro duct to preservation and processing environments.The main results are as the following:1.The results showed that 10 kinds of carvacrol and cinnamaldehyde structural analogues can significantly inhibit P.fluorescens,Aeromonas salmonicida and S.aureus growth and biofilm formation.The antibacterial activities of EOs was tightly related to their different structure.The antibacterial activity of carvacrol structural analogs is higher than that of cinnamaldehyde structural analogs and is related to benzene rings,aldehyde groups and hydroxyl groups which are on the benzene ring.At the sub-inhibitory concentration,EOs significantly inhibited the biofilm,polysaccharide production,adhesion and motility of the three strains?P<0.05?.Anti-biofilm of EOs component was positively correlated with the decrease of AHLs activity.Carvacrol is the most effective bacteriostatic and biofilm-inhibiting ability.2.Carvacrol has the same and antibacteria aganist four P.fluorescens isolates.The effects of carvacrol on the biofilms,extracellular proteases and AHLs signaling molecules of two P.fluorescens PFuk4,PF07?WT?and their?LuxI were compared.Two?LuxI presented the less biofilm and protease compared to WT.It was found that the inhibition of biofilm and spoilage ability in?LuxI treated by carvacrol was significantly lower than that in WT.For biofilms,the inhibitory rates of PFuk4 and PF07 by 0.4 mM carvacrol were 35.8%and 25.4%,while those of two?LuxI were only 7.9%and 21.8%?P<0.05?.The inhibitory difference of biofilm thickness for carvacrol aganist the WT and?LuxI was confirmed by observation of confocal laser scanning microscope?CLSM?.For the extracellular protease,the inhibitory rate of two wild strains were 15.6%and 11.5%,respectively,whereas that of two?LuxI were only7.8%and 7.6%.Additionally.the sub-inhibitory concentration of carvacrol was significantly reduced in the type and content of AHLs secreted by P.fluorescens The C4-HSL activity in two P.fluorescens reduced by 68.8%and 94.8%supplemented with carvacrol at 0.4 mM,and?LuxI only contained trace AHLs.By using docking analysis,carvacrol could reduce activation energy and bind to active amino acid of LuxR to comeptively combine LuxR protein with C4-HSL.Transcriptomic analysis revealed that carvacrol affected various biofilm-related genes,including up-regulation of polysaccharide metabolism,pst system,and PhoB/U two-component system,and the increase of latter two systems were associated with a decrease in intracellular c-di-GMP which result in inhibition of biofilm formation.Carvacrol also significantly down regulated its motility and spoilage-related genes such as flagella synthesis,bacterial tropism,decarboxylase forming biogenic amines and protease.Meanwhile,carvacrol significantly down-regulated the gene encoding LuxR protein as a receptor of quorum sensing.Thus it was indicated that carvacrol interacted with the target protein LuxR to interrupt the LuxI/LuxR pathway regulating biofilm and spoilage.3.The interaction of biofilm formation and the resistance of carvacrol in the co-culture of P.fluorescens and S.aureus were explored.The results showed that P.fluorescens inhibited S.aureus in co-culture to be the main planktonic and biofilm cell both in TSB and fish juice.Compared to the single biofilm,the co-culture formed thicker and denser mixed biofilm with more EPS.Moreover,S.aureus is more sensitive to carvacrol than P.fluorescens,whereas the co-culture enhances resistance to carvacrol.The inhibitory rates of 0.4 mM carvacrol aganist single biofilm of P.fluorescens and S.aureus were 21.6%and 41.8%,while those of the dual biofilm at two initial inoculation?104:104?104:102?were 13.4%and 18.6%.By CLSM and SEM observation,carvacrol could affect the structure of single and dual bofilm,reduced the thickness of the perfomed biofilm and increased the heterogeneity of biofilm.Furthmore,the EPS in the single biofilm disappeared,while a small amount of EPS distributed in cell aggregation in mixed biofilm.Carvacrol greatly repressed the protease activity,TVB-N and hemolytic activity in fish juice under mono or co-culture?P<0.05?,however,co-culture had the high resistance to carvacrol.Compared with the single culture,the AI-2 activity significantly increased in the supernatant of co-culture,and the AHLs secretion apparently decreased.It was found that DPD?AI-2 premise?could stimulate a significant increase in mixed biofilms,supplementing with two exogenous DPD and C4-HSL.Thus,the above results indicated that carvacrol exhibited the highest antibacterial and anti-biofilm activity in ten EOs.Carvacrol at sub-MIC significantly decreased the biofilm formation,motility and protease activity of P.fluorescens by binding or repressing LuxR protein with carvacrol.It was revealed that flagellar assembly and chemotaxis,glucose metabolism,proteases and amino acid decarboxylases,and oxidative phosphorylation were disrupted by interfering with the LuxI/LuxR quorum sensing pathway in the addition of carvacrol.It was revealed that P.fluorescens co-cultured with S.aureus had a competitive advantage,and,the increase of biofilm and EPS in the dual biofilm were mediated by AI-2signaling,resulting in the higher resistance to carvacrol.
Keywords/Search Tags:biofilm, pseudomonas fluorescens, carvacrol, quorum sensing, luxR, staphylococcus aureus
PDF Full Text Request
Related items