S.Suis Biofilm Treaded By Rhubarb Water Extracts And Construction Of Emodin Nanoparticles

Streptococcus suis type 2(SS2)with biofilm formation ability is a important pathogen of zoonotic infectious diseases.Biofilm can reduce the penetration of antimicrobial agents and increase protection against the host immune system.Bacterial biofilms play a important role in persistent infections,which are rarely eradicated with antimicrobial therapy.So it has important significance to develop a new drug which can reduce biofilm formation and eradicate bacteria persistent infection.In our study,effect of rheum water extracts on biofilm formation was determined and the proteomics was briefly discussed to explain the drug intervention mechanism of biofilm formation.The results demonstrated that the 1/2 MIC rheum water extracts could obviously reduce the biofilm formation after incubated 48 h.The iTRAQ technique was used to detect the proteomics of SS2 biofilm.1501 proteins was determined,13.19%proteins changed(P<0.1)and 5.6%proteins change significantly(P<0.05).About 16 proteins was analyzed in extracellular matrix and only 1 proteins was down-regulated.305 proteins was analyzed in cell membrane,36 proteins was down-regulated and 14 proteins was up-regulated.303 proteins was analyzed in cell cytoplasm,9 proteins was down-regulated and 37 proteins was up-regulated.64 ribosomal proteins was analyzed and 30 proteins was up-regulated.The proteins in different cell parts was analyzed from different molecular function,such as the proteins with catalytic activity,the proteins with metal ion binding,the proteins with transporter activity,the proteins with signal transducer activity,the proteins with nucleotide binding and so on.The proteins which participates different biological process were also studied,such as the proteins with metabolic process,the proteins with transporter activity,the proteins with cell communication,the proteins with cell organization and biogenesis,the proteins with cell division and so on.In our research,the gelatin was choosed to synthesize the polymer of Gelatin-CD with the β-cyclodextrin.The chemical structure of the polymer was identified by IR and 1H-NMR.The cyclodextrin content was determined by UV-Vis method,and it is 15%～18%.The critical micelle concentration(CMC)of self-synthetic polymer was determined by pyrene and the CMC of the polymer were about 0.65 μg/mL.The hemolytic activity of the polymer was detect,there was no hemolysis phenomenon with 2 mg/mL polymer.The emodin nanoparticles were prepared by probe ultrasonication method with Gelatin-CD and emodin.The differents influences on the emodin nanoparticles preparation was investigated,such as the different stir time,the different ultraphonic time and so on.The mean diameters of the emodin nanoparticles was 174 nm and Zeta potential was-4.64 mv,the encapsulation efficiency was 78.52%.Transmission electron micrographs was used to observe the images of emodin nanoparticles which is spindlel shape and has a narrow size distribution.DSC results showed that emodin was in the nanoparticles core with an amorphous form.The emodin nanoparticles showed a sustained release profile,which can maintain the stability of emodin and avoid drug leakage in physiologicalstate.The emodin nanoparticles adhesion and infiltration for the SS2 biofilm was researched in our syudy.Confocal laser scanning microscopy(CLSM)was used to observe the adhesion and infiltration of the emodin nanoparticles for the SS2 biofilm,and the results showed that the preparation which was gathered on the biofilm is better than emodin after 15 min or 30 min.And the fluorescence intensity of nanoparticles was significantly stronger than emodin after cultivating 1 h、3 h or 5 h.The result suggests that emodin nanoparticles have stronger permeability for streptococcus suis biofilm than emodin.In addition,the biofilm eliminate experiment and colony forming unit(CFU)enumeration were also used in indirect investigating the biofilm infiltration of nanoparticles,the experimental results show that 12.5 mu g/mL nanoparticles and 50 mu g/mL emodin can eliminate biofilm,and the number of living bacterium in nanoparticles group was 327,the living bacterium in emodin group was 220000,the results showed that the nanoparticles is more easy to enter the biofilm and kill the bacteria.The experiment of the SS2 biofilm elimination and CFU enumeration were consistent with the measurement results of CLSM.The effect of the emodin nanoparticles on SS2 biofilm formation was determined,and the result indicated that the emodin nanoparticles group has a remarkable effect on SS2 biofilm than the emodin group.The emodin nanoparticles with 0.78 μg/mL could inhibit biofilm formation after incubated 48 h.The result showed that the biofilm formation is inhibited with time-dependent and dose-dependent.