两种人工合成抗菌肽对表皮葡萄球菌浮游菌及其生物膜的作用

Anti-planktonic and anti-biofilm effects of two synthetic anti-microbial peptides against Staphylococcus epidermidis

目的:表皮葡萄球菌是院内感染常见的革兰氏阳性球菌之一,它亦黏附于医疗器械表面导致生物膜相关感染。本研究拟探讨两种人工合成的抗菌肽GH12和SAAP-148对表皮葡萄球菌浮游菌生长及其生物膜的影响。方法:通过微量肉汤稀释法检测抗菌肽GH12和SAAP-148的最低抑菌浓度和最低杀菌浓度;利用生物膜成膜能力阳性标准菌株在微孔板中构建生物膜,分别检测这两种抗菌肽对表皮葡萄球菌生物膜的抑制和分散作用;在盖玻片上构建表皮葡萄球菌生物膜,通过荧光染料SYTO9染色和激光共聚焦显微镜观察,检测两种抗菌肽对表皮葡萄球菌生物膜三维结构的破坏作用;通过流式细胞仪检测抗菌肽对表皮葡萄球菌细胞膜的破坏作用;采用实时反转录聚合酶链反应分析表皮葡萄球菌黏附相关基因icaA和icaD的表达情况。结果:GH12和SAAP-148对表皮葡萄球菌的最低抑菌浓度分别为8和16μg/mL,最低杀菌浓度均为64μg/mL;GH12和SAAP-148的质量浓度分别为8μg/mL(t=7.193,P<0.05)和16μg/mL(t=7.808,P<0.05)时均可显著抑制表皮葡萄球菌RP62A生物膜的形成,且均呈现出一定的剂量依赖性;GH12和SAAP-148的质量浓度分别为16μg/mL(t=5.369,P<0.05)和32μg/mL(t=4.474,P<0.05)时可显著分散RP62A已形成的生物膜,亦呈现剂量依赖性;通过激光共聚焦显微镜观察,GH12和SAAP-148可显著破坏生物膜的三维结构,使生物膜的总量显著减少;流式细胞仪检测发现最低抑菌浓度的GH12和SAAP-148还能显著破坏表皮葡萄球菌的细胞膜;1×最低抑菌浓度的抗菌肽能够显著抑制表皮葡萄球菌黏附相关基因icaA和icaD的表达。结论:抗菌肽GH12和SAAP-148通过破坏细胞膜对表皮葡萄球菌浮游菌生长具有显著的抑制作用,并能通过抑制黏附相关基因抑制和分散其生物膜。

Objective:Staphylococcus epidermidis is one of the most common Gram-positive cocci in nosocomial infection,which could adhere to the surface of medical apparatus and causes biofilm-related infections.In the present study,we aim to explore the antimicrobial effects of GH12 and SAAP-148 against Staphylococcus epidermidis.Methods:Micro-dilution methods were used to detect the minimal inhibitory/bactericidal concentration of peptides on Staphylococcus epidermidis.Biofilm formation positive type strain was used to determine the antibiofilm effects of the peptides.Biofilms were built on the cover slides and fluorescent dye SYTO9 and laser confocal microscope were used to observe the effects of peptides on the three-dimensional structure of Staphylococcus epidermidis biofilms.The cell membrane permeability of Staphylococcus epidermidis was detected by flow cytometry.Expressions of icaA and icaD genes were analyzed by realtime reverse transcription PCR.Results:The minimal inhibitory concentrations of GH12 and SAAP-148 against Staphylococcus epidermidis were 8 and 16μg/mL,respectively,and the minimal bactericidal concentration was 64μg/mL.GH12 and SAAP-148 significantly inhibited the biofilm formation of Staphylococcus epidermidis at the concentration of 8μg/mL(t=7.193,P<0.05)and 16μg/mL(t=7.808,P<0.05),respectively.Similarly,the GH12 and SAAP-148 significantly eradicated the pre-formed biofilms at the concentration of 16μg/mL(t=5.369,P<0.05)and 32μg/mL(t=4.474,P<0.05)in a dose-response manner,respectively.Meanwhile,the two peptides broke the structure of biofims and reduce the total biomass.GH12 and SAAP-148 at the concentration of minimal inhibitory concentration significantly disrupted the cell membrane of Staphylococcus epidermidis.The expressions of icaA and icaD genes were significantly inhibited by antimicrobial peptides at the 1×minimal inhibitory concentration.Conclusion:GH12 and SAAP-148 show significantly antimicrobial and anti-biofilm effects against Staphylococcus epidermidis by disruption of cell membrane and inhibition of icaA and icaD gene expression.