The Studies On Isolation, Purification And Antibiofilm Activities Of The Exopolysaccharide From Vibrio Sp.QY101

Bacterial biofilm are sessile communities of bacterial cells enclosed in a self-produced polymeric matrix, which is the physical barrier to antibiotics penetration. Bacterial exopolysaccharides (EPSs) have always been suggested to play crucial roles in the bacterial initial adhesion and the development of complex architecture in the later stages of bacterial biofilm formation. However, recently reports demonstrated that few bacterial EPSs not only did not take part in biofilm formation, but also inhibited self or other strains’biofilm formation, such as E. coli group II capsular polysaccharide. In the previous work of our lab, the crude extract of Vibrio sp.QY101 fermentation supernatant was found to have broad-spectrum biofilm inhibition activity, and the active component was demonstrated to be involved in polysaccharides. This paper aimed at isolating and purifying the exopolysaccharide, indentifying its characters, monosaccharides compositions, molecular weight and so one, and further investigating its antibiofilm activity and mechanism.Exopolysaccharide A101 with antibiofilm activity was purified from the culture supernatant of Vibrio sp.QY101 with ion-exchange chromatography and gel exclusion chromatography. IR, HPSEC combined with HPLC analysis indicated that the molecular weight of A101 was up to 546 KDa and the monosaccharide compositions were complex, but uronic acids, rhamnose and glucosamine were the main ones.Comparing A101 and the known Vibrio exopolysaccharides, A101 was demonstrated to be more similar to Vibrio vulnificus MO6-24 I group capsular polysaccharide (CPS) inhibiting biofilm formation of strain itself, in that both of them were acidic polysaccharides with negative charge. Further gene cloning and sequence alignment analysis suggested that the Vibrio I group polysaccharide transport protein (Wza) existed in QY101. These results suggested that A101 might belong to I group capsular polysaccharide.Static and dynamic models were used for biofilm evaluation. To observe biofilm in real-time manner, strain S.aureus RN6390-GFP constitutively expressing the green fluorescent protein (GFP) was constructed. Biofilm grown in the Flow-cell was observed with inverted fluorescence microscope and laser scanning confocal microscope. Results displayed that mature biofilm was formed after 24h incubation, and then maintained at a dynamic equilibrium steady state.In static conditions, A101 displayed a dose-dependent inhibitory effect on biofilm formation of P. aeruginosa FRD1 and S. aureus RN6390. In the presence of 100μg/mL A101, about 75% of biofilm formation of P. aeruginosa FRD1 was inhibited, and S. aureus RN6390 revealed >90% inhibition. Further investigation demonstrated that the antibiofilm activity of A101 was broad-spectrum. Among the tested strains, biofilms formation by 12 out of 15 Gram-negative strains (80%) and 7 out of 10 Gram-positive strains (70%) can be inhibited by A101. Furthermore, A101 exhibited prominent prevention effects (≥50%) on 12 out of 25 tested strains (48%). In Flow cell model, A101 exerted more powerful biofilm inhibition. In the presence of 100μg/mL A101, the biofilm biomass of P. aeruginosa FRD1 dropped to less than 5% of that grown in the absence of A101, while the same treatment resulted in a more than 99% decrease to the biofilm biomass of S. aureus RN6390. Mature biofilm disruption experiment was also carried out in Flow cell model. The results indicated that after 12h incubation with 100μg/mL A101, more than 85% of the mature biofilm of P. aeruginosa FRD1 was dispersed, while there was no effect on that of S. aureus RN6390 at all.The mechanism of A101 antibiofilm activity was primarily investigated. Firstly,the fully and complex structure of A101 were necessary for the antibiofilm activity, because the acid hydrolytes were inactive. Second, growth curve analysis revealed that both P. aeruginosa FRD1 and S. aureus RN6390 grew a bit faster in the presence of 100μg/mL A101, indicating that A101 was not bactericidal. Furthermore, the ability of cells adhered to surfaces analysis demonstrated that A101 inhibiting celll-surface interactions occurred on S. aureus RN6390, but not on P. aeruginosa FRD1. Moreover, the cells aggregates detection showed that A101 was able to interfere with the cell-cell interactions and inhibit multicellular aggregates formation of both P. aeruginosa FRD1and S. aureus RN6390. However, the preformed cells aggregates of P. aeruginosa FRD1, not S. aureus RN6390 were disrupted. Take together, the antibiofilm activity of A101 should involve the interference effects on cell-surface and cell-cell interactions of strains by its complex structures.In conclusion, in this study we showed that an exopolysaccharide A101 purified from the the marine Vibrio sp. QY101 fermentation supernatant exhibited both biofilm formation inhibition activity and pre-existing biofilm disruption activity. And the mechanism underlying the antibiofilm effect of A101 was preliminarily investigated. The study may be valuable in developing new drugs for biofilm-associated infections, and also promote a new recognition about the functions of bacterial EPSs.