抗真菌肽MAF-1A体外对近平滑念珠菌生物膜形成能力的影响、与生物膜结合及抑菌情况观察

Effects of antifungal peptide MAF-1A on biofilm formation,binding to biofilm,and antifungal ability of Candida parapsilosis

目的观察不同浓度抗真菌肽MAF-1A处理的近平滑念珠菌F1356生物膜形成能力及生物膜细胞活性。方法取F1356菌体适量分5份,其中4份分别加入终浓度为0.3、0.6、0.9、1.2 mg/mL的MAF-1A,等量无菌水处理为空白对照,另取适量F1356分5份,其中4份分别加入0.3、0.6、0.9、1.2 mg/mL的氟康唑(FLC),设置无菌水处理为空白对照,经培养24 h时观察F1356生物膜形成能力(OD490nm值),倒置显微镜下观察F1356生物膜中的菌体密度及膜致密结构。体外培养F1356使其形成生物膜结构,加入0.6 mg/mL的FITC-MAF-1A培养,分别于培养1、3、6、12、24 h采用荧光标记技术观察MAF-1A与F1356生物膜结合情况。将F1356菌体分为5份,其中4份分别加入终浓度分别为0.3、0.6、0.9、1.2 mg/mL的MAF-1A,加入无菌水者为空白对照,培养24 h后通过XTT细胞活性检测技术检测F1356生物膜细胞活性(OD450nm值)。结果与空白对照比较,0.6、0.9、1.2 mg/mL的MAF-1A处理的F1356生物膜形成能力降低(P≤0.05),镜下可见F1356生物膜中菌体密度减少,膜致密结构被破坏;与空白对照比较,0.6、0.9、1.2 mg/mL的FLC处理后F1356生物膜形成能力降低(P≤0.05),镜下可见0.6、0.9、1.2 mg/mL的FLC处理的F1356生物膜中菌体密度减少,膜致密结构被破坏。随培养时间延长,镜下可见黄绿色荧光信号逐渐增多,培养24 h时可观察到生物膜内细胞存在大量黄绿色荧光信号。随MAF-1A浓度增加,F1356生物膜细胞活性逐渐降低(P均≤0.05)。与对照组比较,0.3、0.6、0.9、1.2 mg/mL FLC处理的F1356生物膜活性降低(P均≤0.05),与0.3 mg/mL FLC比较,0.9、1.2 mg/mL FLC处理的F1356生物膜活性升高(P均≤0.05)。结论MAF-1A可抑制F1356的生物膜形成,且MAF-1A可与F1356生物膜结合进入生物膜细胞内聚集,2MIC浓度(0.6 mg/mL)即可抑制F1356生物膜的细胞活性。

Objective To observe the biofilm formation ability and cell activity of Candida parapsilosis F1356 treated with different concentrations of antifungal peptide MAF-1A.Methods F1356 were randomly divided into five groups,four of which were added with 0.3,0.6,0.9,1.2 mg/mLMAF-1A,and the left one was added with sterile water as the negative control group.Another 5 cases of F1356 were selected,four of which were added with 0.3,0.6,0.9,and 1.2 mg/mL fluconazole(FLC),and the left one was treated with sterile water as the positive control group.F1356 biofilm formation(OD 490nm value)was observed after 24-hour incubation,and the cell density,biofilm structure were observed under an inverted microscope.F1356 was cultured in vitro,and its biofilm structure was formed,which was then cultured with 0.6 mg/mL FITC-MAF-1A.At 1,3,6,12,and 24 h after culture,we used fluorescence labeling technology to observe binding between MAF-1A and F1356 biofilm.F1356 were divided into 5 groups,four of which were treated with 0.3,0.6,0.9,1.2 mg/mL MAF-1A,and the rest one with sterile water as control group.The activity of F1356 biofilm cells(OD 450nm)was detected by XTT assay after 24-hour culture.Results Compared with the control group,MAF-1A(0.6,0.9,and 1.2 mg/mL)reduced biofilm formation ability(P≤0.05);under the inverted microscope,the density of cells in the F1356 biofilm was reduced,and the dense structure of the film was destroyed.The changes in the FLC were the same as the above.Over the culture time,the yellow-green fluorescent signal gradually increased,and a large number of fluorescent signals were observed in the biofilm at 24 h.With the increase of MAF-1A concentrations,the cell vitality of F1356 biofilm was gradually reduced(P≤0.05).Compared with the control group,the cell activity of F1356 biofilm treated with 0.3,0.6,0.9,and 1.2 mg/mL FLC(P≤0.05).The activity of F1356 biofilms treated with 0.9 and 1.2 mg/mL FLC increased as compared with that of F1356 biofilm treated with 0.3 mg/mL FLC(all P≤0.05).Conclusion MAF-1A can inhibit the biofilm formation of F1356,and MAF-1A can combine with F1356 biofilm and enter the biofilm cell to aggregate,and 2MIC(0.6 mg/mL)can inhibit the cell activity of F1356 biofilm.