| Objective:This study aimed to investigate the antibacterial effect of eugenol(EG),a natural plant extract,on clinically isolated carbapenem-resistant Klebsiella pneumoniae(CRKP).To evaluate its ability to clear the bacterial biofilm,observe the morphological changes of the cell membrane and biofilm after the action of EG,detect the leakage of substances in bacteria after the action of EG,and explore the possible antibacterial mechanism of EG on CRKP.New alternative treatment strategies are provided to clear CRKP and its associated infections.Methods:Collect the CRKP detected by the Laboratory of the Affiliated Hospital of Qingdao University;The inhibitory activity of EG against CRKP was detected by the agar plate diffusion method and bacterial growth inhibition curve test.The clearing ability of EG to CRKP biofilm was further studied by using confocal laser scanning microscopy(CLSM).Scanning electron microscope(SEM)and transmission electron microscope(TEM)was used to observe the effect of EG on the morphology and structure of CRKP bacterial cell membrane;Utilize β-Galactosidase test,protein leakage test,and DNA leakage test were used to measure the leakage level of substances in bacteria after the structure of bacterial cell membrane was damaged;The level of reactive oxygen species(ROS)production in bacteria after different concentrations of EG treatment was measured using the reactive oxygen species detection kit.The action mechanism of EG on CRKP was explored through the determination of thiobarbituric acid(TBA)and reduced glutathione(GSH).All data are expressed in (3±SD.The statistical analysis was carried out using Graph Pad PRISM 8.0.All data were determined by t-test to determine the difference between groups.The error bar represents the SD of three experiments in each group.The asterisk represents the significant difference in data when * p<0.05,* * p<0.01,* * * p<0.001.Results:The results of the agar plate diffusion experiment and growth curve experiment in the antibacterial activity detection of EG showed that different concentrations of EG showed good antibacterial effects on CRKP.0.5mg/m L EG killed over 85% of CRKP,and when the concentration of EG was 1.0mg/m L,almost 100% of CRKP was eradicated,indicating that EG had excellent antibacterial activity(P<0.001),and the antibacterial activity of EG on CRKP was concentration-dependent;Within 24 hours,different concentrations of EG showed a sustained bacteriostatic effect,and the effect became more obvious with the increase of EG concentration.When the concentration of EG is 1.0mg/m L,almost no bacteria survive(100%);CLSM observation of the biofilm experiment showed that EG significantly decreased the average thickness of CRKP biofilm in a concentrationdependent manner(P<0.001);The results of scanning electron microscopy(SEM)and transmission electron microscopy(TEM)showed that EG changed the morphology and structure of CRKP,including the damage of bacterial cell membrane and the leakage of substances in bacteria,and the more the damage of bacterial membrane structure and the leakage of substances in bacteria increased with the increase of EG concentration;ROS detection kit detected that the intracellular fluorescence intensity of CRKP increased significantly(P<0.001).The results of the thiobarbituric acid(TBA)assay and reduced glutathione(GSH)assay showed that EG could perform antibacterial activity against CRKP by inducing high ROS expression and GSH reduction,thus destroying bacterial membrane and causing bacterial death(P<0.001).Conclusion:The natural plant extract EG has an obvious bacteriostatic effect on CRKP and has good scavenging ability on its biofilm.The experiment found that EG can cause damage to bacterial membrane structure and the leakage of substances in bacteria,and lead to bacterial death by mediating high ROS expression and GSH reduction.Therefore,EG has a good application prospect as an alternative treatment strategy to eliminate CRKP. |
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