Establishment Of In Vitro Bacterial Biofilm Model And In Vitro Eradication Effect Of Recombinant Lysostaphin On Biofilm Of Staphylococcus

A bacterial biofilm is a communities of cells encased in a film structure that adhere to a surface or damaged tissue for adaptation to the natural environment, The bacteria in biofilm could resist the foreign and body's immune attack because of the protect effect of the film, it's difficult to dissolved by body's antibodies or swallowed by macrophages is the major reason for persistent infections. The bacterial biofilm is widespread in nature, any bacteria could form biofilm as long as conditions permit, staphylococci which is the major reason leads to persistent infections in clinical is one of the strains more tends to form biofilm. Because of the special structure and characters, the bacteria in biofilm could resist the sterilization of most antibiotics, so, how to eliminate biofilm has gained renewed interest in recently years, lysostaphy was paid attention as an agent against staphylococcus especially S. aureus in the 1960s and 1970s. In this study, the in vitro biofilm model was studied, and the kinds of affecting factors to biofilm formation was evaluated, based on this, evaluated the in vitro eradication effect of recombinant lysostaphin (rLspn) on biofilm of staphylococcus.A. The set up of in vitro biofilm modelIn this part of the experiment, we focused on the establish of the in vitro biofilm model, contrasted the traditional plate method with the slide method, in the traditional plate method, The absorbtance of bioflims can be detected with crystal violet staining method, the final result is not stable, in the slide method, the biofilms can be analyzed quantitatively with the ultrasound-viable bacterial counting method, the final result is stable, based on this, used the slide method to cultivate biofilm in vitro, the result showed that, the ultrasound-viable bacterial counting result used silicon rubber slide in several groups of experimental were higher than used glasses, so the silicon rubber is the better material, the ultrasound-viable bacterial counting result used TSB in several groups of experimental were higher than used nutrient broth, so TSB is better for cultivate biofilm in vitro, finally, in order to investigate different points in time and temperature on the formation of biofilm that cultivated by silicon rubber slide and TSB, the ultrasound-viable bacterial counting method was employed to measure the viable bacterial counts of biofilm, the scanning electron microscopy (SEM) technique was employed to observe the conformation of bacterial biofilm, both of them could reflect the influence of biofilm in different points of time and temperature, as well as the development of biofilm, the result showed that, 37℃is better for biofilm than 22℃, the biofilm continued growth in groups of different time (24hr, 48hr, 72hr), it's mature after 72hr.B. In vitro eradication effect of recombinant lysostaphin on biofilm of staphylococcusNorvancomycin (NVAN) has often been used against S. aureus in clinical, contrast with NVAN to investigate the disruptive effect of recombinant lysostaphin (rLspn) treatment on staphylococcus biofilm in vitro, staphylococcus epidermidis strain ATCC12228, a non-biofilm forming strain used as a negative control, other strains have ability to form biofilm, the concentration of two drugs was MIC～100MIC, but for staphylococcus epidermidis strain ATCC12228 the concentration of two drugs was 1/10MIC～10MIC, get the silicon rubber slide of the two S. aureus biofilm with various concentrations of drugs in groups of different time (2hr, 4hr, 8hr, 24hr), the ultrasound-viable bacterial counting method was employed to measure the viable bacterial counts of biofilm for reflect the effect of drugs, the conformation of biofilm after with rLspn MIC, NVAN 100MIC and without drugs was observed by SEM, get the silicon rubber slide of staphylococcus epidermidis 3-26 biofilm with various concentrations of drugs after 24hr incubation, the other treatment was same as S. aureus, the conformation of staphylococcus epidermidis ATCC12228 with rLspn MIC and NVAN MIC was observed by SEM, the other treatment was same as staphylococcus epidermidis 3-26, rLspn had better effect on S. aureus biofilm, the viable bacterial counts after treated with various concentrations of rLspn was dropped through the time points, the SEM image treated with rLspn MIC was obviously different with control, NVAN had poor effect on S. aureus biofilm, the viable bacterial counts after treated with various concentrations of NVAN had no significant fluctuations, the SEM image treated with NVAN 100MIC had no significant difference with control, rLspn had a certain effect on staphylococcus epidermidis 3-26 biofilm, NVAN had poor effect on staphylococcus epidermidis 3-26 biofilm, the result was coincide with the SEM observation, both of the two drugs had better effect on staphylococcus epidermidis ATCC12228, although the concentration was in a lower grade, the viable bacterial counts of biofilm corresponding lower, the effect of rLspn was slightly better than NVAN, the result was supported by the corresponding SEM observation.