| Enterococcus spp.are resident bacteria in the human and animal intestines,and some strains were once widely used as probiotics.Recent studies have found that many Enterococcus are opportunistic pathogens and can cause infections such as endocarditis,bacteremia,and urinary tract infections.In addition,because of their multiple drug resistance,Enterococcus has become an important pathogen of hospital infection,which has prolonged the hospital stay of patients,increased the cost of treatment and increased the risk of treatment failure and patient death.Over the past 20 years,vancomycin-resistant Enterococcus has caused high mortality rates and posed a serious threat to health care.Linezolid(LNZ)is the last line of defense for vancomycin-resistant Enterococcus.However,with the discovery of linezolid-resistant Enterococcus in 2001 and gradually increasing reports of increasing detection rates of drug-resistant bacteria,linezolid-resistant Enterococcus has become an urgent problem.In recent years,reports of Enterococcus infections in animals have been increasing,and linezolid-resistant Enterococcus has also been detected in animals and can be transmitted to humans through multiple pathways.However,sufficient attention has not been paid to drug-resistant Enterococcus in veterinary clinics.In an environment where the development of new drugs is not progressing well,strategies to explore drug combinations against multi-drug resistant bacteria are being explored,and this study attempts to investigate their effects and mechanisms against linezolid-resistant Enterococcus faecium using rhamnolipids(RLS)in combination with linezolid.Currently,Pseudomonas aeruginosa,which is mainly used for RLS production,is an opportunistic pathogen and a potential threat in industrial production,and the yield of rhamnolipid-producing strains is low.So,in this study,it was first conducted to screen for RLS-producing strains.In this study,a total of 139 samples of different species were collected from different regions,and six strains of bacteria were screened by oil drainage circle assay,CTAB-MB agarose gel plate assay and hemolysis assay,and 16 S r RNA as well as the gyr B gene were sequenced to identify the strain species.The products of the six strains were extracted using extraction methods and further purified using column chromatography,high performance liquid chromatography and thin layer chromatography.The study initially determined the synergistic antibacterial effect of LNZ and rhamnolipid by checkerboard assay and bacterial growth curve assay,and then observed the ability of synergistic antibacterial effect of LNZ and RLS by time-dependent killing curve and combined disk test,followed by anti-biofilm assay to detect the inhibition ability of LNZ and RLS on Enterococcus biofilm.To investigate the mechanism of the synergistic antibacterial effect of LNZ and RLS,we used molecular docking method to predict the binding ability of RLS to proteins encoding drug resistance genes of Enterococcus,and detected the effect of both drugs on the expression of drug resistance genes by q PCR assay.Finally,we established a mouse Enterococcus infection model,explore the antibacterial ability of LNZ and RLS in vivo,detected the weight change,feces,colon and kidney bacterial load,kidney and colon pathological changes,and observed and recorded the clinical symptoms of mice.The main results were as follows:(1)Six rhamnolipid-producing strains were screened,strains 37296 and 37299 were Bacillus velezensis,and strains 37278,38463,38465 and 37471 were Bacillus subtilis.(2)The combination of linezolid and rhamnolipid had synergistic anti-Enterococcus effect with FICI index between 0.25 and 0.5.(3)Bacterial growth curves showed that rhamnolipid concentrations between 0 μg/m L and 64μg/m L did not inhibit the growth of Enterococcus.(4)The time-dependent killing curve showed that rhamnolipids increased the activity of linezolid in killing Enterococcus.(5)The combined disk test showed that the synergistic inhibition effect of linezolid and rhamnolipid was correlated with the concentration of rhamnolipid.(6)The combination of linezolid and rhamnolipid has the ability to inhibit the production of biofilm by Enterococcus.(7)Rhamnolipids bind well to the proteins encoding esp,optr A and poxt A resistance genes.(8)The combination of linezolid and rhamnolipids inhibited the expression of the drug resistance gene poxt A.(9)By establishing a mouse model of Enterococcus infection,the following main results were obtained.a The combined use of linezolid and rhamnolipid treated mice with Enterococcus infection resulting in weight loss,loss of appetite and reduced activity.b The results of bacterial load in feces,colon and kidney of mice showed that linezolid and rhamnolipid could promote the excretion of Enterococcus from the body of mice.c The pathological results showed that linezolid and rhamnolipids have a significant effect on the treatment of colon epithelial tissue edema,damage,and renal tubular epithelial cell degeneration.d The combination of the two drugs inhibited the proliferation of Enterococcus in the kidney of mice.The above results indicate that Bacillus velezensis has been found to produce RLS for the first time in this study,and it has the potential to become a strain for safe production of RLS in industry.We have demonstrated the synergistic antibacterial effect of RLS and LNZ in vitro and in vivo experiments,and explored the mechanism of the synergistic antibacterial effect of RLS and LNZ in vitro.It has contributed to the prolongation of the service life of linezolid and provided a new scheme for the treatment of drug-resistant Enterococcus infection. |
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