Preliminary Study On The Effect Of Protamine Intervention On Staphylococcus Aureus Biofilm

Protamine is a natural bacterial inhibitor which has great potential development and application in medicine and food field. It attracts more and more attention in recent years. Through building staphylococcus aureus biofilm, the object of this study is to research the effect of protamine on staphylococcus aureus biofilm, and to explore its effect on the synthesis of PIA and secretion of eDNA. It can provide a new approach for preventing the staphylococcus aureus safety concerns in food and pharmaceutical field, and lay a foundation to further study about the mechanism of action of protamine. This paper had the following results:1. S.aureus was biofilm positive strains tested by Congo red agar. Then Staphylococcus aureus were grown to biofilms on the glass. The biofilm was observed by silver staining. Viable bacterial counts were determined by serial dilution. The conclusions were as follows:With the increase of culturing days, number of living bacterium in the biofilm increased continuously. It reached maximum at the3rd days, and then significantly deceased. There was no significant difference between colony counts during4to6days. The living bacterium number in biofilm begun to decline after6days. The number of living bacterium in biofilm changes was not consistent with the quantity of biofilm according to the results of crystal violet staining and silver staining.2. From the two aspects of protamine added before and after cultivating S.aureus, the effect of protamine on the number of living bacterium and biofilm morphology was discussed. The results were as follows:The MIC of protamine to s.aureus was125μg/mL. The MBC of protamine to s.aureus was1000μg/mL. The number of living bacterium in biofilm of each day fell sharply with4MIC concentration of protamine initially. It could not be seen basically clouds of biofilm onto the glass slide. However, the number of living bacterium on slides was zero with1MBC concentration (1000μg/mL) initially. It had different degrees of bactericidal effect on mature biofilm bacteria for protamine, and there was concentration dependence relationship. The effects of sterilization and removal of biofilm related to incubation time and the concentration of protamine. The5days biofilm was more sensitive to protamine. Protamine with4MIC could sterilize all bacterium in biofilm. Compared to protamine added after biofilm formation, protamine added initally could better control the biofilm formation. 3. The results showed that protamine could obviously inhibit adhesion bacterium of S.aureus biofilm to produce extracellular polysaccharide which was marked with fluorescent whitening agent. The effect of inhibitory on extracellular polysaccharide was obvious with higher concentration of protamine which was added initially. When the biofilm has formed already and then was intervened by protamine, it needed a higher concentration protamine (>4MIC), so that it could inhibit the adhesion bacterium to produce extracellular polysaccharide significantly. It showed that protamine added in initial and after biofilm had been formed all could significantly reduce the S.aureus to produce water soluble and water insoluble extracellular polysaccharide ability by anthrone method. The study also found that adhesion bacterium could synthesize more water soluble extracellular polysaccharide early in the biofilm formation and synthesize more water insoluble extracellular polysaccharide if biofilm was been continued to develope.4. The amount of PIA expression was significantly suppressed both in the culture medium and biofilm by Dot blotting with protamine (>1MIC) in initial. The inhibition effect of the high concentration of protamine (4MIC) was more obvious.Through the extraction of eDNA and using fluorescence spectrophotometry to study the effect of protamine on eDNA release, the results showed that addition protamine in initial, eDNA release significantly suppressed. The higher the concentration of protamine, the smaller quantity of the eDNA release.