| Quorum-sensing（QS）is a bacterial self-regulatory mechanism,depending on the density of the colony,which regulates the expression of the corresponding proteins at the genetic level,realizing the exchange of information within the colony,between the colony or with the host,and regulating the behavioral activities of the colony.Biofilm is one of the main forms of bacterial QS regulation.Biofilm formation is an important self-defense mechanism of bacteria,the presence of biofilm reduces the effectiveness of antibiotics and fungicides,also affects the pathogenicity of bacteria.The development of QS interfering drugs is undoubtedly an effective means of treating bacterial infections and reducing antibiotic resistance.This experiment focused on the inhibitory effects of1,8-cineole on E.coli biofilm and QS,the main inhibitory gene of 1,8-cineole was obtained by analyzing the genes expression,and the deletion strain of this gene was constructed to demonstrate that 1,8-cineole exerts its anti-QS effect mainly by inhibiting the expression of this gene,and to investigate the effect of the deletion of this gene on bacterial QS and virulence.Eventually,the therapeutic effect of 1,8-cineole on E.coli diarrhea and the effect of deletion of this gene on bacterial pathogenicity were observed through animal experiments.Through the above in vivo and in vitro experiments,we investigated the antibacterial and anti-QS activities of 1,8-cineole and elucidated the anti-QS mechanism of 1,8-cineole against E.coli,providing a theoretical basis for the development and application of 1,8-cineole as an anti-colony sensing inhibitor.The main research contents and results are as follows:1 In vitro anti-E.coli activity of eucalyptolE.coli O101（CVCC3749）was used as the experimental strain to study the in vitro anti-E.coli activity of 1,8-cineole.Measuring the minimum inhibitory concentration and minimum bactericidal concentration of 1,8-cineole against E.coli.The bactericidal curves of 1/8MIC-1/2MIC concentrations of 1,8-cineole against E.coli were plotted,and measured the changes of OD260,OD280,conductivity,bacterial hydrophobicity and important physiological metabolic enzyme activities in the bacterial solution after the effect of different concentrations of 1,8-cineole and studied the effects on bacterial protein synthesis ability.The results showed that:（1）The minimum inhibitory concentration（MIC）of 1,8-cineole against E.coli was 6.2μg/m L and minimum bactericidal concentration（MBC）was 12.4μg/m L,and there was no inhibition of E.coli growth when the concentration of 1,8-cineole was less than the MIC.（2）After treatment with 1,8-cineole at MIC and 2 MIC concentrations,the hydrophobicity of E.coli increased significantly,resulting in aggregation and growth inhibition.The permeability of E.coli cell membrane was significantly increased,causing the effluxof electrolytes and macromolecules from the bacterium.（3）The protein synthesis capacity of E.coli was significantly reduced after 1,8-cineole treatment,while the activities of key enzymes（Alkaline phosphatase,β-Galactosidase）used in bacterial physiological metabolism were inhibited.The above experimental results indicated that 1,8-cineole has good in vitro anti-E.coli activity,and exerts antibacterial effects by affecting bacterial hydrophobicity,cell membrane permeability,bacterial protein synthesis ability and the physiological metabolism of related enzymes.2 Effect of 1,8-cineole on quorum sensing and biofilm of Escherichia coliE.coli were co-cultured with non-inhibitory concentrations of 1,8-cineole to investigate the effects on bacterial biofilm formation capacity,motility,and adhesion.The effects of 1,8-cineole on the microstructure of E.coli biofilms were observed by light microscopy and laser confocal microscopy（CLSM）.The expression of bacterial QS and virulence genes after the effect of non-inhibitory concentrations of 1,8-cineole were determined by real-time fluorescence quantitative PCR and found the main inhibitory genes of 1,8-cineole.The results showed that:（1）E.coli could secrete AI-2 signaling molecules,which were most active during the logarithmic phase of bacterial growth.（2）The ability of E.coli biofilm formation was reduced by 61.7%after the inhibition of1,8-cineole in non-inhibitory concentrations,and bacterial adhesion and motility were significantly inhibited.（3）In the light microscopy and CLSM field of view,E.coli lost its normal morphology after 1,8-cineole treatment,the fluorescence region reduction and the fluorescence intensity was weakened,the thickness of the biofilm was reduced,and the originally tight biofilm structure became sparse and affected the biofilm structure.（4）1,8-cineole inhibited the expression of both population sensing genes and virulence genes in E.coli.The gene expression inhibition rates of 1/2 MIC 1,8-cineole treatment for 18 h were as follows:luxS 65%,lsr B 47.9%,mtn 25.4%,csg A 55.3%,csg B 72.4%,fim B31.6%,fim E 40.2%,rpo S 28.9%.The above experimental results indicated that1,8-cineole had significant anti-QS activity,which could inhibit the biofilm formation ability and motility of E.coli and disrupt the biofilm structure;among the selected QS genes,luxS gene was most significantly inhibited.3 Effect of luxS gene deletion on QS and biofilm in E.coliPrevious studies have shown that the gene most significantly repressed by1,8-cineole is the luxS gene.In this chapter,we constructed the luxS gene deletion strain and investigated the effects on E.coli growth,adhesion,biofilm formation ability,biofilm microstructure,motility and virulence genes expression in the absence of luxS gene.We also investigated whether 1,8-cineole also had a significant inhibitory effect on luxS gene deletion E.coli under the same treatment conditions to determine whether 1,8-cineole exerts its inhibitory effect on bacterial QS and biofilm mainly by suppressing the expression of luxS gene.The results showed that:（1）The deletion of the luxS gene did not affect the normal growth of E.coli.（2）luxS gene deletion caused significant inhibition of both adhesion and motility of E.coli,60%reduction of biofilm formation ability,significant disruption of biofilm microstructure,and different degrees of inhibition of bacterial QS and virulence genes expression.（3）There was no further significant inhibition of biofilm formation ability,biofilm microstructure and motility,and no further significant reduction in the expression of QS and virulence genes by1,8-cineole in luxS gene deletion E.coli.The above results showed that the deletion of luxS gene had different effects on E.coli adhesion,biofilm formation,biofilm microstructure,motility and virulence gene expression,and 1,8-cineole exerted its inhibitory effects on E.coli QS and biofilm mainly by affecting the expression of luxS gene.4 Therapeutic effect of 1,8-cineole on E.coli diarrhea and effect of luxS gene deletion on pathogenicity of E.coliSeventy Kunming mice were randomly divided into seven groups:control group,model group,berberine group（45 mg/kg）,the luxS gene deletion group,1,8-cineole high-dose group（12 mg/10 g）、medium-dose group（6 mg/10 g）and low-dose group（3mg/10 g）.Except for the normal group,mice in each group were given an intraperitoneal injection with the optimal diarrheal concentration of E.coli suspension（0.1 m L/10 g）determined during the pre-experiment.After intraperitoneal injection for 6 h,mice in the berberine group,1,8-cineole high-dose group,medium-dose group and low-dose group were gavaged in the morning and evening for 5 days according to their respective dosages,and the body weight and diarrhea of the mice were recorded,spleen index of mice was measured,the amount of different leukocytes in each group of mice was by blood routine test,the concentration of cytokines（IL-1β,IL-6 and TNF-α）in blood was assessed by ELISA,the pathological changes in the duodenal intestine of mice were finally observed by hematoxylin-eosin staining.The results showed that:（1）The optimal diarrhea-causing concentration of E.coli was 1.5×109 CFU/m L.（2）After1,8-cineole treatment,the body weight of mice increased faster,the body leukocytes decreased,the concentration of IL-1β,IL-6 and TNF-αpro-inflammatory factors decreased（P<0.05）,the spleen index was not significantly different from that of the control group,and the pathological changes in the duodenum of mice were significantly improved,and the best effect was obtained with the medium dose of 1,8-cineole treatment.（3）The deletion of luxS gene reduced the pathogenicity of E.coli.luxS gene deletion reduced leukocytes in mice by 52.3%,and the concentrations of IL-1β,IL-6 and TNF-αpro-inflammatory factors were reduced by 15.5%,27.2%and 25.9%,respectively,and the effects on the spleen and duodenum were diminished.The above experimental results indicated that 1,8-cineole had better efficacy on E.coli-induced diarrhea in mice,and the deletion of luxS gene could reduce the pathogenicity of E.coli.In conclusion,this experiment investigated the antibacterial and anti-QS activity of1,8-cineole through in vivo and in vitro experiments,and the anti-QS mechanism was also elucidated.In vitro experiments showed that 1,8-cineole had strong in vitro anti-E.coli activity and could exert anti-QS effects by inhibiting the expression of luxS gene.1,8-cineole reduced the biofilm formation ability of E.coli after the action of 1,8-cineole,disrupted the biofilm structure,and inhibited both adhesion and motility;The results of in vivo experiments showed that 1,8-cineole had better therapeutic effects on E.coli diarrhea in mice.1,8-cineole treatment reduced leukocytes,IL-1β,IL-6 and TNF-αpro-inflammatory factor concentrations in mice（P<0.05）,spleen index did not differ significantly from the control group,and duodenal pathological changes in mice were significantly improved,while luxS gene deletion reduced the pathogenicity of E.coli.This study elucidated the mechanism of 1,8-cineole against colony sensing in E.coli and provided a theoretical basis for the development and application of 1,8-cineole as an anti-colony sensing inhibitor. |
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