Silver Nanoparticle On The Roleof Bacterial Mortality Of P.Aeruginosa Biofilms

Part 1Objective: To build the models of bacterial biofilms (BF) in vitro by simulating the microenvironment of bacterial growth in vivo and to settle the foundation for further study on the biological characteristic of BF.Methods: Aerosol method and shaking method were used respectively to simulate the micro-environment of BF formation in vivo. Pseudomonas aeruginosa (PAO1) were cultured for 3d, and then the BF formation were examined with confocal laser scanning microscopy (CLSM) after PAO1 being respectively stained by FITC-ConA and SYT09/PI. Meanwhile,The morphological characteristic of BF were examined by scanning electron microscopy (SEM).Results: Under micro-enviroment simulated by the aerosol method, Much platelike BF stained by FITC-ConA and cloud-wool-like BF stained by SYTO9/PI were observed under fluorescence microscope. By SEM, a large number of bacteria gathering into mass and the BF with a three-dimensional structure were observed. Under the micro-enviroment simulated by the shaking method, streamline-like BF were observed by the three aforesaid methods.Conclusion: The aerosol method and shaking method could be used to established the BF models respectively simulating the respiratory system, circulational system and urinary system in vivo. Part 2Objective: To analyze the effect of silver nanoparticle on the bacteria mortality and the spatial structure of P.aeruginosa biofilms.Methods: The ethylene-vinyl acetate (EVA) plastics in different concentration of silver nanoparticle were used as carriers for the bacteria adhesion and the BBF model in vitro were built by shanking method, simulating the micro-environment of P.aeruginosa in vivo.The bacteria in contral group were cultured for 3d and then respectively examined by SEM and by fluorescence microscope after being stained by FITC-ConA. The specimens on 0.5d,1d,2d,3d and 5d were respectively stained by SYTO9?PI and then were examined by CLSM to get the images of different levels.These images were analyzed by the sofeware named Image-Pro Plus 6.0 to compare bacterial mortality under the different interventions conditions,and the software named Image Structure Analyer (ISA) were used to get quantitative data on spatial structure of P.aeruginosa biofilms.Results:1. The treamline-like BF were detected on the 3-day-culturing control group without intervening by EVA by SEM and fluorescence microscope.2.With the Image Pro Plus 6.0,the P.aeruginosa mortality was significantly affected by concentration of silver ion and action time(F = 84.62,85.67, p<0.0001).The bacterial mortality of silver nanoparticle intervening group was significantly higher than control group at different time points(P<0.05).However the intervening group with different concentration of silver nanoparticle showed no significant difference on bacterial mortality(P>0.05). The bacterial mortality of BBF intervened with different concentration of silver nanoparticle (0.05% and 0.1%) in 1d, 2d and 3d was significant higher than those in 0.5d and 5d. The bacterial mortality on 0.1% silver nanoparticle EVA plastic in 2d was found to be the highest.3. By analyzed with ISA , the spatial structure of biofilm was significantly affected by the concentration of silver nanoparticle and the action time.The Areal Porosity(AP), Average Diffusion Distance, (ADD), Textural Entropy(TE) in silver nanoparticle intervening group were significantly higher than control group at different time points(P<0.05). However the intervening group with different concentration of silver nanoparticle showed no significant difference on aforesaid index(P>0.05).And these index showed unchanged with the culture continuing At the same time point, The AP in the silver nanoparticle intervening group is significantly higher than the control group ,while the ADD is significantly lower(P<0.05).The TE in all intervening group showed a peak value in 2d and at the same time ,the concentration of silver nanoparticle is higher ,the TE is lower.4. The mean thickness of P.aeruginosa in all intervening group showed a peak in 3d.The action time was demonstrated to have effect on BBF thickness(F = 985.81 ,p<0.001) while the concentration of silver nanoparticle was demonstrated to play on effect(F=2.11 , p>0.1).Conclusion:1. The shaking method could be used to established the BBF model in vitro.2. The bacterial mortality and the spatial structure of BBF are affected by silver nanoparticle.