Antibacterial mechanism of thymol to methicillin-resistant Staphylococcus aureus
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摘要:目的
探讨百里香酚对耐甲氧西林金黄色葡萄球菌(MRSA)的抑菌作用机制，为开发高效低毒的抗MRSA药物提供可靠的理论依据。
方法采用微量肉汤稀释法和菌落计数法测定百里香酚对MRSA标准菌株USA300的最低抑菌浓度(MIC)和最低杀菌浓度(MBC)；通过测定菌液电导率和DNA含量确定百里香酚对USA300细胞膜的影响；通过SDS-PAGE检测百里香酚对USA300可溶性蛋白质代谢的影响；利用透射电镜观察经百里香酚处理后USA300菌体细胞的超微结构；通过结晶紫染色法测定百里香酚对USA300生物被膜形成的影响。
结果百里香酚对USA300具有一定的抑制作用，其MIC和MBC均为256 mg·L–1。与对照组相比，512 mg·L–1百里香酚作用菌体1 h后，菌液电导率增加18.08%±1.80%，DNA外渗量增加(123.40±8.06) mg·L–1，作用24 h后，菌体可溶性蛋白质含量比对照组降低68.20%±0.15%。百里香酚对USA300细胞壁和细胞膜具有破坏作用并干扰其正常的二分裂，亚抑菌浓度下对生物被膜的形成具有一定抑制作用。
结论百里香酚通过改变菌体细胞膜通透性，干扰蛋白质代谢和正常的二分裂来抑制细菌的生长，亚抑菌浓度下能够在不影响细菌生长的前提下抑制其生物被膜的形成。百里香酚具有开发为抗MRSA药物的潜力。
Abstract:ObjectiveTo investigate the antibacterial mechanism of thymol to methicillin-resistant Staphylococcus aureus(MRSA), and provide a reliable theoretical basis for the exploitation of high efficient and low toxic anti-MRSA medicine.
MethodThe minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of thymol to MRSA standard strain USA300 were determined via microbroth dilution method and colony counting method. Bacterium solution conductivity and DNA amount were detected to investigate the effect of thymol on USA300 cell membrane. SDS-PAGE was used to detect the effect of thymol on USA300 soluble protein synthesis. The ultra structure of USA300 cell treated by thymol was observed through transmission electron microscope. The effect of thymol on USA300 biofilm formation was investigated via crystal violet staining method.
ResultThymol had definite inhibitory activity on USA300, both MIC and MBC of thymol to USA300 were 256 mg·L–1. Compared with the control group, after treating the strain with 512 mg·L–1 thymol for 1 h, the conductivity of bacterium solution was improved by 18.08%±1.80%, and DNA exosmosis amount increased by (123.40±8.06) mg·L–1; after treating for 24 h, the soluble protein content of USA300 was reduced 68.20%±0.15%. Thymol destroyed cytoderm and cytomembrane of USA300, disturbed its normal binary fission, and inhibited biofilm formation at subinhibitory concentration.
ConclusionThymol can inhibit bacterium growth by changing its cytomembrane permeability and disturbing protein synthesis and normal binary fission, and depress biofilm formation at subinhibitory concentration in the premise of not affecting bacterium growth. Thymol has the potential of developing as anti-MRSA medicine.
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图 1 不同质量浓度的百里香酚对USA300在BHI液体培养基中生长的影响
Figure 1. Effects of different mass concentrations of thymol on USA300 growth in BHI liquid medium
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图 2 512 mg·L–1百里香酚对USA300菌液电导率的影响
“**”表示相同培养时间的对照组和512 mg·L–1百里香酚处理组间差异极显著(P<0.01，t检验)
Figure 2. Effect of 512 mg·L–1 thymol on conductivity of USA300 bacterium solution
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图 3 512 mg·L–1百里香酚对USA300 DNA外渗量的影响
“**”表示相同作用时间的对照组和512 mg·L–1百里香酚处理组间差异极显著(P<0.01，t检验)
Figure 3. Effect of 512 mg·L–1 thymol on DNA exosmosis amount of USA300
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图 4 512 mg·L–1百里香酚对USA300可溶性蛋白质代谢的影响
M：标准分子量蛋白Marker；1：16 h对照组；2：16 h 512 mg·L–1 百里香酚处理组；3：24 h对照组；4：24 h 512 mg·L–1百里香酚处理组
Figure 4. Effect of 512 mg·L–1 thymol on soluble protein synthesis of USA300
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图 5 512 mg·L–1 百里香酚不同作用时间对USA300菌体细胞超微结构的影响
A1：4 h 百里香酚处理组；A2：4 h对照组；B1：16 h 百里香酚处理组；B2：16 h对照组；C1：24 h 百里香酚处理组；C2：24 h对照组
Figure 5. Effects of different treating time of 512 mg·L–1 thymol on ultra structures of USA300 cells
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