| Biofilms are structured, specialized communities of adherent microorganisms encased in a complex extrapolymeric substance matrix. Exopolysaccharide (EPS) is the main composement of biofilm matrix and it provides biofilm cells with a protected environment with microchannels for the flow of water and other essential nutrients. Numerous studies have shown that poly-β-(1, 6)-N-acetyl-D-glucosamine contribute to the surface attachment and intercellular adhesion in Staphylococcus aureus and Staphylococcus epidermidis biofilms. In the previous study, we found theβ-1,6-N-acetylglucosaminidase, Nag1, was an important biofilm-releasing enzyme, which degraded theβ-(1, 6)-linkage of N-acetylglucosamine residues. In this study we assessed the antibiofilms effects of Nag1 and evaluated the effects of immobilized Nag1.Different models were used for biofilm inhibition evaluation. In static microtiter plate, Nag1 had a broad spectrum on biofilm formations of 9 tested gram-positive strains. With a concentration of 0.3 U/mL, more than 80% of biofilm formation was inhibited and similar results were observed in dynamic flow cell chambers and polyvinyl echloride catheter. The inhibition rate for polyvinyl echloride discuses and lactoprene urethral catheter was 79% and 76%, respectively.Further evaluation indicated that Nag1 rapidly and efficiently detached S. aureus and S. epidermidis biofilms. With a concentration of 0.3 U/mL, more than 80% biofilms were removed in microtiter plates, while it was more than 90% for dynamic models. Nag1 also displayed the biofilm-releasing activity against S. epidermidis biofilms on polyvinyl chloride discuses and lactoprene urethral catheter with a rate of 79% and 76%, respectively.The minimum inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and minimum biofilm eliminating concentration (MBEC) results were compared to determine changes in the pattern of antibiotic sensitivity of bacteria from the planktonic to the biofilm phase of growth. In their biofilm state, bacteria are much less susceptible to antibiotics compared to their antibiotic susceptibility in the planktonic state. However, Nag1 could restore bacteria sensitivity to antibiotics.The S. aureus biofilm models in acute burn wound infections for in vivo evaluation of Nag1 antibiofilm effects were successfully constructed. Skins from the infections site were observed under confocal laser scanning microscope (CLSM). The results showed that biofilm treated with Nag1 was thickness and consisted of less biomass. Ampicillin coupled with Nag1 severely inhibited and disrupted the biofilms fromation. Taken together, Nag1 has a biofilm inhibition and detachment activity against S. aureus biofilm in thermally injured mice.In order to use Nag1 more effectively, the hydrogel nanoparticles of linoleic acid modified CMCS was prepared. Nag1 was then loaded on nanoparticles. Factors affecting the activity of the immobilized enzyme, including temperature, storage etc., were investigated in this study. The results showed that the stabilities of Nag1 for heat and storage were improved after immobilization on nanoparticles. After eight cycles of use, the immobilized enzyme retain on average around 63% of the initial activity. Nanoparticles appear to act as a powerful tool in enzyme immobilization because of the reuse of the immobilized enzyme and the enhancement of thermal stability in both the structure and the enzymatic activity of the protein. The results showed that the immobilized enzyme has a more effective biofilm inhibition and detachment activity against S. aureus and S. epidermidis biofilm.The effect of CMCS on biofilm formation was determined. Gram-positive and Gram-negative bacterial biofilm formations on microtiter plates and in dynamic conditions are prevented by CMCS. CMCS inhibits the adhesion of bacteria with an efficiency of >90%. It prevents Gram-positive bacterial biofilm formation at efficiencies of 63% and Gram-negative bacterial biofilm formation at efficiencies of 71% when CMCS is added at 1 h after biofilm initiation. The prevention of initial bacterial adherence and cell–cell interaction was ascribed to flocculation. The flocculation of Gram-positive and Gram-negative bacteria was 17% and 25% in the presence of CMCS, respectively. CMCS may neutralize bacterial surface charge and bridge the bacterial aggregates. CMCS may serve as an antibiofilm agent.In conclusion, biofilm formations are inhibited and detached efficiently by Nag1 in different models, particllarly in thermally injured mice. Nag1 can be loaded onto nanoparticles. The anti-biofilm activity was improved after immobilization on nanoparticles. Gram-positive and Gram-negative bacterial biofilm formations were significantly inhibited by CMCS, due to its effects on cells flocculation. This work has practical significance in finding new approaches to the treatment of bacterial biofilm... |
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