Study On The Inhibitory Activity Of An Actinomycete Against Staph-ylococcus Epidermidis Biofilm Formation And Its Inhibitory Mechanism

Staphylococcus epidermidis is one of the important pathogenic bacteria causing subclinical mastitis in dairy cows.The biofilm structure formed by Staphylococcus epidermidis can enhance the adhesion and colonization ability of pathogenic bacteria on the surface of mammary epithelial cells effectively and cause the pathogen to release virulence factors continuously.The use of high-dose traditional antibiotics as the first choice for clinical treatment of drugs,which makes the biofilm for the pathogens within the membrane to provide protective barrier,hinder the drug and cell target binding,exacerbated the emergence of multiple drug-resistant strains,causing dairy cow mastitis attack and difficult to cure.It was very important to screen natural cpmpound that inhibit biofilm formation.Actinomycetes are a treasure trove of rich and diverse secondary metabolites.On the basis of previous studies,the actinomycete TRM 41337 fermentation broth has shown strong activity against Staphylococcus epidermidis ATCC 35984,so the study was carried out.(1)The fermentation conditions of actinomycete TRM 41337 were optimized.The effects of actinomycete TRM 41337 on the biofilm formation of Staphylococcus epidermidis ATCC 35984 were determined by single factor,Plackeett-Burman experiment and response surface design experiment,respectively.The culture medium of Staphylococcus epidermidis ATCC 35984 with the greatest effect on biofilm formation was determined as follows: starch 20 g / L,yeast leaching powder 1 g / L,dipotassium hydrogen phosphate 0.5 g / L,magnesium sulfate 0.38 g / L,pH 9.0.The optimum fermentation conditions were as follows: fermentation temperature 25 ?,shaking speed 180 r / min,loading volume 150 mL(500 mL shake flask),inoculation amount 10%,fermentation time 10 d.By using microtiter plate semi-quantitative tracking,the relative biofilm formation ability of the fermented medium was only 18.98%,and the activity of the biofilm was 10% lower than that of the pre-optimized culture medium.(2)The active protein was purified by column chromatography and identified by SDS-PAGE and LC-MS,respectively.Results showed that it was glycosidase related protein(transglycosylase-associated protein).In order to further explore the stability of the purified protein,the protein was treated by using different physical and chemical factors in this experiment.The results showed that its effect on the relative biofilm formation ability of Staphylococcus epidermidis ATCC 35984 was the most obvious,when the protein was alkaline.We also found that this protein was not sensitive to temperature and had high temperature resistance potential.At the same time,the influence of different physicochemical factors on the protein was not obvious.Therefore,the protein was found to be relatively stable.(3)The mechanism of actinomycete TRM 41337 inhibited Staphylococcus epidermidis biofilm was investigated by milk plate test,cell surface hydrophobicity test,extracellular DNA and extracellular polysaccharide detection gradually.The results showed that the biofilm formation ability of the active protein was 25.74% under alkaline condition.It was worth noting that the protein was not inactivated at high temperature(100 ?)and its relative biofilm formation ability was 59.04%.However,after thetreatment of protein through the rest of the physical and chemical factors,the impact of its activity was not obvious.The aim of this experiment is to study the TRM 41337 active substances inhibiting Staphylococcus epidermidis biofilm,determine its structure and types,reveal its inhibition mechanism,provide a theoretical basis for the anti biofilm formation of dairy cow mastitis and drug development.