DNaseⅠ对痤疮丙酸杆菌生物膜的抑制效应研究

Inhibitory effect of deoxyribonucleaseⅠagainst Cutibacterium acnes biofilms

目的探讨DNaseⅠ对痤疮丙酸杆菌生物膜的抑制效应。方法构建痤疮丙酸杆菌生物膜并给予DNaseⅠ处理,分为不同DNaseⅠ浓度组(0、5、10、20 U/ml)。采用四甲基氮盐法检测生物膜活力,结晶紫半定量法检测生物膜含量,激光共聚焦显微镜观察生物膜结构及活菌/死菌比值。组间差异比较采用单因素方差分析。结果5、10、20 U/ml的DNaseⅠ处理后痤疮丙酸杆菌生物膜活力值分别为1.882±0.421、1.653±0.287、1.473±0.154,与阴性对照组(2.668±0.245)相比存在显著抑制,且DNaseⅠ浓度越高抑制作用越显著(F=9.68,P=0.005)。5、10、20 U/ml DNaseⅠ组生物膜含量值分别为1.039±0.003、0.489±0.079、0.147±0.034,与阴性对照组(1.359±0.071)相比显著降低,且DNaseⅠ浓度越高生物膜含量越低(F=174.40,P<0.001)。激光共聚焦显微镜观察结果显示,与阴性对照组相比,5、10、20 U/ml DNaseⅠ组生物膜结构被破坏,且DNaseⅠ浓度越高生物膜结构破坏越严重。5、10、20 U/ml DNaseⅠ组痤疮丙酸杆菌生物膜活菌/死菌比值分别为2.303±0.457、1.534±0.526、1.263±0.354,比阴性对照组(4.475±0.146)显著降低,且浓度越高生物膜活菌/死菌比值越低(F=56.75,P<0.0001)。结论DNaseⅠ对痤疮丙酸杆菌生物膜的结构有破坏作用,对其活力有抑制作用。

Objective To investigate the inhibitory effect of deoxyribonucleaseⅠ(DNaseⅠ)on Cutibacterium acnes biofilms.Methods Cutibacterium acnes biofilms were constructed,and then were divided into 4 groups(negative control group,5,10 and 20 U/ml DNaseⅠgroups)to be treated with DNaseⅠat different concentrations of 0,5,10 and 20 U/ml respectively.The biofilm viability was evaluated by tetrazolium salt colorimetric assay,the biofilm content was determined by crystal violet staining-based semi-quantitative analysis,the biofilm structure was observed by confocal laser scanning microscopy,and the live/dead bacteria ratio was calculated.One-way analysis of variance was used to analyze differences between groups.Results After the treatment with DNaseⅠ,the biofilm viability was significantly inhibited in the 5,10 and 20 U/ml DNaseⅠgroups(1.882±0.421,1.653±0.287,1.473±0.154,respectively)compared with the negative control group(2.668±0.245),and the inhibitory effect was gradually enhanced with the increase in concentrations of DNaseⅠ(F=9.68,P=0.005).Crystal violet semi-quantitative analysis showed that the biofilm content was also significantly lower in the 5,10 and 20 U/ml DNaseⅠgroups(1.039±0.003,0.489±0.079,0.147±0.034,respectively)than in the negative control group(1.359±0.071),and the higher the DNaseⅠconcentration,the lower the biofilm content(F=174.40,P<0.001).Confocal laser scanning microscopy showed that the biofilm structure was destroyed in the 5,10 and 20 U/ml DNaseⅠgroups compared with the negative control group,and the higher the DNaseⅠconcentration,the more severe the destruction of biofilm structure.Additionally,the live/dead bacteria ratio was significantly lower in the 5,10 and 20 U/ml DNaseⅠgroups(2.303±0.457,1.534±0.526,1.263±0.354,respectively)than in the negative control group(4.475±0.146),and the ratio decreased with the increase in concentrations of DNaseⅠ(F=56.75,P<0.0001).Conclusion DNaseⅠhad a destructive effect on the structure of Cutibacterium acnes biofilms,and could inhibit their viability.