The Inhibiting Effects Of Sulfonated Chitosan On Bacteria And Its Biofilm

Biofilms are three-dimensional bacterial communities attached to surfaces,either living or abiotic,surrounded by an extracellular matrix,which has been characterized as the dominant living-state of bacteria in the nature.It is easy to cause a wide range of chronic,relapsing infectious disease,and the bacteria in biofilms were 1000 times more resistant to the action of antimicrobials than planktonic bacteria.Biofilms bring a serious risk in medical and food safety,because it is difficult to be eradicated by ordinary antibiotics and disinfectants,once they are formed.Therefore,it is of great significance to develop natural and safe anti-biofilm agents.Chitosan,the N-deacetylated derivative of chitin,which is the second most abundant natural organic-compound found in nature after cellulose.Chitosan has been found to be nontoxic,biodegradable and biocompatible in addition to having antimicrobial characteristics.However,the solubility of chitosan greatly limits its wide application.In this paper,molecular modification of chitosan was carried out,and sulfonate group was introduced into 2-NH2 of chitosan through nucleophilic substitution reaction,aiming at improving its solubility and antimicrobial activity,and in-depth study its biofilm inhibition performance against P.aeruginosa,E.coli and S.aureus.The specific research contents and results are as follows:(1)The water-soluble sulfonated chitosan was prepared and characterized by FT-IR,NMR,GPC,EA and TGA.(2)The in vitro antimicrobial activities of water-soluble chitosan(WCS)and sulfonated chitosan(SCS)against Pseudomonas aeruginosa(P.aeruginosa),Escherichia coli(E.coli)and Staphylococcus aureus(S.aureus)were investigated.Results showed that the minimum inhibitory concentration(MIC)of WCS and SCS against P.aeruginosa,E.coli and S.aureus were 1,1 and 2 mg/mL respectively.The minimal bactericidal concentration(MBC)of WCS were 16,8 and 32 mg/mL respectively,and the MBC of SCS were 8,4 and 16 mg/mL respectively.It can be seen that SCS has better bactericidal performance than WCS.From the time-kill curves of WCS and SCS acting on P.aeruginosa,E.coli and S.aureus,the MIC of WCS and SCS can significantly inhibit the growth of these three test bacteria.When the concentration of the sample reaches 2MIC and 4MIC,WCS and SCS play a bactericidal action.(3)The effects of different time on the biofilm formation of P.aeruginosa,E.coli and S.aureus were analyzed.The results showed that the biofilm formation ability of the three test bacteria was the highest when the culture time was 2 days.WCS and SCS inhibited the biofilm formation of P.aeruginosa,E.coli and S.aureus cultured in 2 days:the live bacteria count,secretion of exopolysaccharide and metabolic activity in biofilms of the three tested bacteria were significantly decreased after the treatment with SCS and WCS.Among them,SCS inhibited the biofilms of the three tested bacteria more strongly than WCS,and it was related to the concentration.The biofilm was observed by confocal laser scanning microscope(CLSM),it can be clearly found that there are thick and evenly distributed green cluster with multiple layers of active cell in the control biofilm,indicating extensive biofilm formation on coverslips surfaces.The CLSM images treated with 1/2MIC and MIC concentration of WCS and SCS can be seen:the green fluorescence intensity was significantly decreased,the reddish brown fluorescing intensity was increased,and the structure of the biofilm was clearly destroyed.Further observation by scanning electron microscopy(SEM)showed that compared with the control group,the bacterial biofilm treated by WCS and SCS at 1/2MIC was severely damaged,the extracellular secretion was greatly reduced,and the morphology of the enveloped bacteria was sag,twisted and broken,and the complete cell morphology is lost.The experimental results showed that WCS and SCS had better inhibiting the formation of the three tested bacterial biofilms,while SCS had stronger bacteriostatic and inhibiting biofilm formation than WCS.