Effect And Primary Mechanisms Of Artesunate Against Biofilm Of Pseudomonas Aeruginosa

Objective: To investigate whether artesunate could influence the biofilm formation and the biofilm structure of P.aeruginosa wild type PAO1 in vitro,and to discuss its mechanism.Methods: Spectrophotometry was used to detect the effect of artesunate(AS)on P.aeruginosa growth curve.After crystal violet dyeing,a microplate reader was used to detect the OD 570 nm values and observe biofilm biomass at 24 h.After 3 days,the biofilm treated with different concentrations of artesunate was used to calculate the viable count by plate dilution counting.Features of Bio-volume,average thickness,roughness coefficient and average diffusion distance were quantitatively analyzed by confocal laser scanning microscope combined with COMSTAT software.Plate dilution counting method was used to observe the effect of artesunate combined with sub-inhibitory concentration of ciprofloxacin(1/2MIC,1/4MIC)on the number of viable cells in biofilm to observe whether they had synergistic effect.In addition,Twitching motility assays was used to detect the effect of artesunate on the distance of twitch movement of P.aeruginosa.Ferrozine assay was used to measure the effect of artesunate on Fe(II)in P.aeruginosa biofilms.Finally,the effect of artesunate combined with Fe(II)on the number of viable cells and structure in P.aeruginosa biofilms.was observed.Results: Artesunate had no inhibitory effect on planktonic bacteria.3 day biofilms were treated with different concentrations of artesunate for 12 h,Compared with control group,the 512μg/mL group and 1024μg/mL group were significantly different(p<0.05).Similary,biofilm biomass at 24 h in 512μg/mL artesunate group and 1024μg/mL artesunate group were significantly decreased compared with control group(p<0.05).Compared with the control group,the values of biomass,average thickness and average diffusion distance significantly decreased in artesunate group(p<0.05),but the crude coefficient increased in artesunate group(p<0.05).Artesunate has no synergistic effect with sub-inhibitory concentrations of ciprofloxacin.In addition,compared with the control group,the distance of twitch movement of P.aeruginosa in the artesunate group increased,and the content of Fe(II)in the biofilm decreased.Compared with the artesunate group,the number of viable cells in the biofilm was increased in artesunate combined with Fe(II)group,but was lower than that of the control group.Compared with the artesunate group,the values of biomass,average thickness and average diffusion distance significantly increased in artesunate group(p<0.05),the crude coefficient decreased in artesunate combine with Fe(II)group(p<0.05),but there was no statistical difference compared with the control group.Conclusion: In this study,artesunate could interfere with the formation of P.aeruginosa biofilms by stimulating the twitching motion of P.aeruginosa.And destroy the structure of mature biofilm by reducing Fe(Ⅱ)in biofilms.