| In this paper,Litsea cubeba essential oil is taken as the research object,and the particle size and stability of essential oil emulsion are taken as the indexes to prepare dilutable Litsea cubeba essential oil nanoemulsion.Through bacterial isolation and identification,drug sensitivity test,bacterial pathogenicity test,drug resistance gene and virulence gene detection,suitable multi-drug resistant Escherichia coli was screened out,and the antibacterial mechanism of litsea cubeba essential oil nanoemulsion on it was explored.The antibacterial mechanism of litsea cubeba essential oil nanoemulsion on multidrug-resistant Escherichia coli was further studied by transcriptome technology.Through the mouse disease model test,the full lethal dose concentration and the half lethal dose concentration are obtained,and through the clinical observation,autopsy results and blood routine test results of the test mice,the preventive effects of litsea cubeba essential oil nanoemulsion on the animal model of multi-drug resistant Escherichia coli infection are preliminarily explored.The specific test results are as follows:1?The results show that the litsea cubeba essential oil nanoemulsion prepared with mixed surfactant(Tween 80:absolute ethanol=2:1)is11.73%(m/m),litsea cubeba essential oil is 10.65%(m/m),sterile pure water is77.62%(m/m)has an average particle size of 49.23±1.23 nm(n=5),a polydispersity coefficient(PDI)of 0.153±0.003,and is still in a stable state after being left for 90 d.2?A total of 15 strains of Escherichia coli were isolated in this experiment.Through comprehensive analysis of bacterial drug resistance,drug resistance genes,virulence genes and mouse virulence test results,a multi-drug resistant pathogenic Escherichia coli containing 6 drug resistance genes such as tet A,bla CTX-M-U,bla CTX-M-9,mcr-1,Sul1,fos A3 and 9 virulence genes such as fim C,omp A,Iss2,yijp,ibe B,ibe A,mat,sod A,R1 and the like has been screened,and the disease course of the mouse is relatively long.3?The transmission electron microscope results showed that litsea cubeba essential oil nanoemulsion had destructive effect on the cell membrane of multidrug-resistant Escherichia coli.Qualitative and quantitative analysis of intracellular biomacromolecules and enzymes during litsea cubeba essential oil nanoemulsion treatment shows that it can lead to leakage of intracellular macromolecules,at the same time,it can inhibit the energy metabolism of Escherichia coli,reduce the activity of key mediating enzymes in the energy metabolism process,and make the thallus have no ability to absorb nutrients from the outside.4?Transcriptome study of Escherichia coli before and after the effect of Litsea cubeba essential oil nanoemulsion,683 differential genes were found,among which 367 were up-regulated and 316 were down-regulated.The analysis of GO enrichment showed that the up-regulated genes were mainly those related to ribosomal degradation,such as dea D,pcn B and Dna K.Down-regulated genes are mainly genes related to membrane receptors and energy metabolism,such as mcp,ula B,Nap AB and other genes.The enrichment analysis of the KEGG signaling pathway revealed that the differentially expressed genes were mainly concentrated in the phosphotransferase system(PTS),nitrogen metabolism,microbial metabolism in different environments,bacterial chemotaxis,ribonucleic acid degradation and other signaling pathways,and the gene expression of various enzymes and proteins in the coding material metabolism pathway and energy metabolism pathway was down-regulated.5?The total lethal dose of bacteria was 7.5×10~8CFU/m L,and the median lethal dose was 3.8×10~8CFU/m L.Anatomy of the mice after the attack showed that the liver of the mice in the model group showed obvious pathological changes.The liver of control group mice had no obvious pathological changes.Litsea cubeba essential oil nanoemulsion can increase the number of white blood cells and platelets to a certain extent,enhance the body's resistance,delay the development of the disease course,and has a certain preventive effect on Escherichia coli diseases. |
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