| Enterococci is a conditional pathogen that lives in human and animal intestines,which can cause serious abdominal infection,urinary tract infection and blood infection.The drug resistance of enterococci is increasing day by day,and the infection is spread through food and the environment.It has become the main pathogen of nosocomial infection.The formation of biofilm is one of the important reasons to enhance the drug resistance of enterococci.Finding new drugs to eliminate biofilm has become a new hot spot in the treatment of multidrug-resistant enterococci.ObjectiveThis study is to clarify the relationship among dibenzlurea,Enterococci faecium cell wall peptidoglycan hydrolase,and biofilm.It is to provide a new theoretical basis for the study of peptidoglycan hydrolase inhibitors in Enterococcus faecium,and can provide a new idea for the clinical treatment of multi-drug resistant bacteria.Methods1.Synthesis and antibacterial screening of compounds: 12 diphenylurea derivatives were chemically synthesized.Gram-positive bacteria,Gram-negative bacteria and Candida albicans were selected for antibacterial screening,and the MIC was determined.Cytotoxicity test was used to determine the cytotoxicity the human hepatocellular carcinoma cell line,and the diphenylurea compound with the best activity and low toxicity was screened out.2.Screening the strains with strong and weak film-forming ability of clinical isolates: The film-forming ability of 9 strains of Enterococcus faecium was detected by the CV method.3.Detection of the activity of diphenylurea compound against biofilm: the minimum biofilm clearance concentration(MBEC)of compound ZJ-2 was measured by CV and XTT,the clearance of biofilm in the presence of compound ZJ-2 was observed by SEM,and the effect of compound ZJ-2 on the cell wall of enterococcus faecium was observed by TEM.4.Drug resistance induction experiment of compound ZJ-2: The drug resistance tendency of compound ZJ-2 was studied by broth dilution continuous passage resistance induction experiment.5.Detection of peptidoglycan hydrolase and genes related to biofilm aggregation:q RT-PCR test was used to observe the regulatory ability of compound ZJ-2 on peptidoglycan hydrolase and genes related to biofilm adhesion and aggregation in Enterococcus faecium.6.Experimental study on inhibiting inflammatory factors IL-6 and TNF-α in the intestinal model: A simulated intestinal model in vitro was established to detect the levels of cytokines IL-6 and TNF-α in Caco-2 cells induced by Enterococcus faecium,and the expression levels of inflammatory factors in positive control and compound ZJ-2 were observed.7.In vivo antimicrobial studies for the treatment of the compound ZJ-2:mice peritonitis/septicemia model induced by Enterococcus faecium.Compounds ZJ-2,vancomycin and linezolid were administered as subcutaneous treatment 2 hours later.Retroorbital plexus venous blood was taken at different periods to test for bacterial load and blood inflammatory cytokine expression.Pathological changes in mouse liver tissue were observed to evaluate the antibacterial effect of compound ZJ-2 in vivo.Results1.Diphenylurea derivatives all showed good narrow-band antibacterial activity,which was only effective against Gram-positive bacteria.Among them,compound ZJ-2had the best antibacterial activity against drug-resistant Enterococcus faecium(MIC was 0.78 μmol/L),and had little influence on the survival of Hep G-2 and low toxicity.2.Enterococcus faecium strain E3101 with high biofilm formation capacity was selected,and it was all resistant to erythromycin,chloramphenicol and gentamicin.3.The results of induced resistance experiment showed that compound ZJ-2 no longer changed after enterococcus faecium passage 18,and compound ZJ-2 showed a lower tendency to induce resistance to enterococcus faecium.4.The MBEC of compound ZJ-2 against Enterococcus faecium E3101 is0.78-1.56 μmol/L,which is better than linezolid.XTT reduction experiment showed that the metabolic activity of bacteria decreased in a dose-dependent manner.SEM showed that compound ZJ-2 could inhibit and remove the biofilm of Enterococcus faecium,and TEM showed that compound ZJ-2 could destroy the cell wall,and the contents of the cell overflowed,causing the bacteria to die.5.Under the action of compound ZJ-2,the gene expression levels of Enterococcus faecium E3101 peptidoglycan hydrolases sag A and atl A decreased significantly to 17.0% and 28.5% respectively,and the expression levels of biofilm aggregation-related genes agg and esp decreased by 82.0% and 81.9% respectively.6.In vitro simulated gut model,the inflammatory factors IL-6 and TNF-α acted after a dose-dependent decline after using the compound ZJ-2.It shows that the compound ZJ-2 inhibited the inflammatory response caused by E.faecium and effectively protected the integrity of the intestinal epithelium.7.The experimental data of the mouse abdominal infection model show that compound ZJ-2 can significantly reduce the load of E3101 and the level of cytokines in the blood of mice,and alleviate the cell necrosis and inflammatory cell infiltration in the liver tissue of mice.The efficacy is superior to the clinical antibiotic linezolid.ConclusionThis study clarified the mechanism of action of the peptidoglycan hydrolase inhibitor ZJ-2 targeting E.faecium,providing a new idea for the development of biofilm clearance drugs and a new strategy for the clinical combination treatment of E.faecium infection.Figure [14] table [6] reference [52]... |
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