Effects of inhibitory peptide of Staphylococcus epidermidis biofilm on adhesion and biofilm formation of this bacterium
-
摘要: 目的 研究表皮葡萄球菌生物膜抑制肽(简称抑制肽)对表皮葡萄球菌早期黏附及生物膜形成的影响。 方法 采用多肽合成仪合成抑制肽,其纯度为96.8%、相对分子质量为874.4。(1)采用终浓度为1~256 μg/mL的抑制肽培养表皮葡萄球菌ATCC 35984(下同)菌液,不含菌液的M-H肉汤为空白对照,观察抑制肽对该菌的MIC(样本数为3)。(2)采用含终浓度为16、32、64、128、256 μg/mL抑制肽的胰蛋白胨大豆肉汤(TSB)培养液培养表皮葡萄球菌菌液(设为相应浓度抑制肽组),以TSB培养基培养前述菌液作为阴性对照组,从培养即刻起每小时观察该菌的生长情况(结果以吸光度值表示),绘制该菌培养24 h内的生长曲线(每组各时相点样本数为3)。(3)采用含终浓度为16、32、64、128、256 μg/mL抑制肽的TSB培养液培养表皮葡萄球菌菌液(设为相应浓度抑制肽组),以TSB培养基培养前述菌液作为阴性对照组,培养4 h检测该菌黏附情况,培养20 h检测该菌生物膜的形成情况(结果均以吸光度值表示,样本数均为10)。(4)采用含128 μg/mL抑制肽的TSB培养液培养表皮葡萄球菌菌液(设为128 μg/mL抑制肽组),以TSB培养基培养前述菌液作为阴性对照组,培养24 h后采用激光扫描共聚焦显微镜观察该菌的黏附与生物膜形成情况(样本数为3)。对数据行单因素方差分析、LSD检验、Dunnett T3检验。 结果 (1)抑制肽对表皮葡萄球菌的MIC大于256 μg/mL。(2)与阴性对照组比较,各浓度抑制肽组表皮葡萄球菌培养24 h内的生长曲线无明显变化。(3)256、128、64、32 μg/mL抑制肽组培养4 h表皮葡萄球菌的黏附情况分别为0.20±0.04、0.27±0.03、0.35±0.04、0.40±0.04,明显少于阴性对照组(0.53±0.10,
P <0.05或P <0.01);16 μg/mL抑制肽组培养4 h该菌的黏附情况(0.47±0.09)与阴性对照组相近(P >0.05)。256、128、64 μg/mL抑制肽组培养20 h的表皮葡萄球菌生物膜形成情况分别为0.49±0.10、0.68±0.06、0.93±0.13,明显少于阴性对照组(1.21±0.18,P <0.05或P <0.01);32、16 μg/mL抑制肽组培养20 h该菌生物膜形成情况分别为1.18±0.22、1.15±0.26,与阴性对照组相近(P 值均大于0.05)。(4)激光扫描共聚焦显微镜显示,培养24 h后阴性对照组黏附的表皮葡萄球菌较多,形成的生物膜结构致密;128 μg/mL抑制肽组黏附的表皮葡萄球菌较少,形成的生物膜结构较为疏松。 结论 抑制肽能显著抑制表皮葡萄球菌早期黏附及生物膜形成,但其结构需要进一步修饰。Abstract: Objective To study the effects of inhibitory peptide ofStaphylococcus epidermidis (SE) biofilm (briefly referred to as inhibitory peptide) on adhesion and biofilm formation of SE at early stage. Methods By using peptide synthesizer, the inhibitory peptide was synthesized with purity of 96.8% and relative molecular mass of 874.4. (1) Solution of SE ATCC 35984 (the same below) was cultivated with inhibitory peptide in the final concentrations of 1–256 μg/mL, and the M-H broth without bacteria solution was used as blank control. The MIC of the inhibitory peptide against SE was determined (n =3). (2) Solution of SE was cultivated with trypticase soy broth (TSB) culture solution containing inhibitory peptide in the final concentrations of 16, 32, 64, 128, and 256 μg/mL (set as inhibitory peptide groups in corresponding concentration), and solution of SE being cultivated with TSB culture medium was used as negative control group. Growth of SE was observed every one hour from immediately after cultivation (denoted as absorbance value), and the growth curve of SE during the 24 hours of cultivation was drawn, with 3 samples in each group at each time point. (3) Solution of SE was cultivated with TSB culture solution containing inhibitory peptide in the final concentrations of 16, 32, 64, 128, and 256 μg/mL (set as inhibitory peptide groups in corresponding concentration), and solution of SE being cultivated with TSB culture medium was used as negative control group. Adhesive property of SE was observed after cultivation for 4 hours (denoted as absorbance value,n =10); biofilm formation of SE was observed after cultivation for 20 hours (denoted as absorbance value,n =10). (4) Solution of SE was cultivated with TSB culture solution containing inhibitory peptide in the final concentration of 128 μg/mL (set as 128 μg/mL inhibitory peptide group), and solution of SE being cultivated with TSB culture medium was used as negative control group. Adhesive property of SE and its biofilm formation were observed with confocal laser scanning microscope (CLSM), and the sample numbers were both 3. Data were processed with one-way analysis of variance, LSD test, and Dunnett T3 test. Results (1) The MIC of inhibitory peptide against SE exceeded 256 μg/mL. (2) There was no significant difference in the growth curve of SE between inhibitory peptide groups in different concentrations and negative control group. (3) After 4 hours of cultivation, the absorbance values of adhesive property of SE in 256, 128, 64, and 32 μg/mL inhibitory peptide groups were respectively 0.20±0.04, 0.27±0.03, 0.35±0.04, and 0.40±0.04, which were significantly lower than the absorbance value in negative control group (0.53±0.10,P <0.05 orP <0.01); the absorbance value of adhesive property of SE in 16 μg/mL inhibitory peptide group was 0.47±0.09, which was close to the absorbance value in negative control group (P >0.05). After 20 hours of cultivation, the absorbance values of biofilm formation of SE in 256, 128, and 64 μg/mL inhibitory peptide groups were respectively 0.49±0.10, 0.68±0.06, and 0.93±0.13, which were significantly less than the absorbance value in negative control group (1.21±0.18,P <0.05 orP <0.01); the absorbance values of biofilm formation in 32 and 16 μg/mL inhibitory peptide groups were respectively 1.18±0.22 and 1.15±0.26, which were close to the absorbance value in negative control group (withP values above 0.05). (4) CLSM showed that more adhering bacteria and compact structure of biofilm were observed in negative control group, but less adhering bacteria and loose structure of biofilm were observed in 128 μg/mL inhibitory peptide group. Conclusions The inhibitory peptide can inhibit adhesion and biofilm formation of SE at early stage, but its structure still needs to be further modified.-
Key words:
- Staphylococcus epidermidis /
- Biofilms /
- Inhibitory peptide
点击查看大图
计量
- 文章访问数: 15
- HTML全文浏览量: 3
- PDF下载量: 2
- 被引次数: 0