没食子提取物对金黄色葡萄球菌生物膜的抗菌机制

Antimicrobial mechanism of Turkish gall extract against Staphylococcus aureus biofilms

摘要:

背景 金黄色葡萄球菌(Staphylococcus aureus)是危害人体健康的常见有害菌，在生长过程中极易黏附在非生物或生物表面形成细菌生物膜，临床使用过程中极易形成耐药性，给临床治疗带来极大挑战。目的 研究没食子提取物对金黄色葡萄球菌ATCC 6538的抗菌机制，为植物源抑菌剂的进一步开发利用。方法 采用刚果红平板测试法和结晶紫染色法建立金黄色葡萄球菌生物膜体外模型；利用96孔板微量倍比稀释法测定没食子提取物对金黄色葡萄球菌生物膜的最小抑制浓度(minimum inhibitory concentration, MIC)、最小杀菌浓度(minimum bactericidal concentration, MBC)及生长曲线；通过测定胞外成分泄漏、AKP活性、ATP含量、细胞活力研究了没食子提取物对生物膜内细胞膜、细胞壁结构及能量代谢的影响；通过DNA电泳、测定细菌DNA和蛋白质浓度，探究没食子提取物对细菌生物大分子的影响；同时，通过扫描电镜和透射电镜观察没食子提取物对成熟生物膜的清除作用，阐明没食子提取物对金黄色葡萄球菌生物膜抗菌机制的多样性。结果 没食子提取物对金黄色葡萄球菌的MIC及MBC分别为56.25 μg/mL和112.50 μg/mL，没食子提取物对金黄色葡萄球菌生物膜有明显抑制作用，并且随着没食子提取物浓度增大而加强，它破坏了细菌的细胞结构，使得菌体发生明显褶皱和破损，导致糖、蛋白质等物质泄漏，降低能量代谢及细胞活力，抑制生物膜的合成，破坏成熟生物膜成分，从而达到抑制细菌生长的目的。结论 没食子提取物能够从多方面抑制金黄色葡萄球菌生物膜的形成，有望为植物源天然产物的进一步应用提供理论基础，为临床防治金黄色葡萄球菌生物膜提供理论依据。

Abstract:

Background Staphylococcus aureus is a common harmful bacterium that endangers human health. It is readily to attach both non-biological and biological surfaces to form biofilms during its growth. Consequently, S. aureus is prone to developing drug resistance in clinical practice, which poses a challenge to clinical treatment.Objective To decipher the mechanism of Turkish gall extract in inhibiting S. aureus ATCC 6538 for the development of plant-derived bacteriostatic agents.Methods The in vitro model of the S. aureus biofilm was established by Congo red plate and crystal violet staining. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Turkish gall extract against S. aureus biofilms and the biofilm growth curve were determined by the 96-well plate micro-dilution method. The effects of Turkish gall extract on the cell membrane integrity, cell wall structure, and energy metabolism of viable bacteria in the biofilm were assessed based on the leakage of extracellular components, alkaline phosphatase (AKP) activity, adenosine triphosphate (ATP) content, and cell viability. Additionally, the impact of Turkish gall extract on bacterial biomacromolecules was investigated via DNA electrophoresis and quantification of bacterial DNA and protein concentrations. Concurrently, the performance of Turkish gall extract in eradicating mature biofilms was evaluated by scanning electron microscopy and transmission electron microscopy, on the basis of which the diverse inhibitory mechanisms of Turkish gall extract against S. aureus biofilms were deciphered.Results The MIC and MBC of Turkish gall extract against S. aureus were 56.25 μg/mL and 112.5 μg/mL, respectively. The Turkish gall extract inhibited the biofilm of S. aureus in a concentration-dependent manner. It destroyed the cell structure, causing obvious wrinkles and breakage of the bacteria and the leakage of intracellular components such as sugars and proteins, which diminished energy metabolism and overall cell viability. The extract inhibited the biofilm formation and destroyed mature biofilm components, hence inhibiting bacterial growth.Conclusion Turkish gall extract demonstrates the ability to inhibit the biofilm formation of S. aureus through multiple mechanisms. This study provides a theoretical basis for the further application of plant-derived natural products and clinical prevention and treatment of S. aureus biofilms.