枯草芽孢桿菌分離株及其表面活性素對葡萄球菌的抗菌機制

Antibacterial effect of surfactin produced by Bacillus subtilis isolates on Staphylococcus

黃柏晨(Huang Po-Chen)

指導教授 ： 陳又嘉

國立屏東科技大學/農學院/生物科技系/碩士(2022年)

https://doi.org/10.6346/NPUST202200062

摘要

從土壤中所分離出的1-2、1-4、3-3菌株對金黃色葡萄球菌(Staphylococcus aureus)以及中間葡萄球菌 (Staphylococcus intermedius)表現出優秀的抗菌能力。再經過革蘭氏染色、內孢子染色、碳水化合物利用分析、16S rRNA核醣體基因鑑定比對後，判斷此三株分離株均為枯草芽孢桿菌(Bacillus subtilis)，枯草芽孢桿菌的抗菌效果主要歸功於表面活性素(surfactin)的生產。在本研究中透過將分離株的培養上清液沉澱純化後再經高效液相層析（high performance liquid chromatography，HPLC）、三(羥甲基)甲基甘氨酸-十二烷基硫酸鈉聚丙烯酰胺凝膠電泳(Tricine sodium dodecyl sulphate–polyacrylamide gel electrophoresis，Tricine SDS PAGE)分析，確定了分離株1-2、1-4具有生產表面活性素的能力，並且在300mL的LB medium中培養48hr後總產量分別為23.77mg及17.21mg，並且二株分離株生產表面活性素的能力會因為在培養基當中額外添加L-谷胺酸 (L-Glutamate)而提升約1.6倍。值得一提的是雖然分離株1-2具有更高的表面活性素產量但是在相同濃度下分離株1-4所生產的表面活性素對金黃色葡萄球菌及中間葡萄球菌的抗菌作用更為明顯。而3-3菌株則僅能生產微量的表面活性素因此對金黃色葡萄球菌以及中間葡萄球菌的抗菌作用均不明顯。表面活性素的抗菌機制涉及破壞細胞膜結構從而使膜內外的滲透壓失衡進而發揮抗菌效果，在本研究中透過sytox green染色、胞外蛋白外流分析從而驗證該現象，在含有sytox green染劑的LB medium中經過1-2、1-4所生產的表面活性素處理的金黃色葡萄球菌以及中間葡萄球菌相較於控制組出現螢光單位大幅度上升的現象，而經過液相層析法-質譜聯用(Liquid chromatography–mass spectrometry，LC-MS/MS)的分析結果也顯示出經過表面活性素處理後的金黃色葡萄球菌及中間葡萄球菌的樣品有鑑定出胞內蛋白的現象。而分離株1-2與1-4所生產的表面活性素能夠與抗生素apramycin達到最佳的協同效果，兩者的共同處理可以在對抗金黃色葡萄球菌以及中間葡萄球菌時大幅度的降低apramycin的使用濃度(MIC/10)。綜合以上實驗結果顯示出B. subtilis分離株1-2、 1-4可以透過生產表面活性素造成金黃色葡萄球菌及中間葡萄球菌的細胞膜損傷從而發揮抗菌效果，並且具有與抗生素進行協同作用，進而降低抗生素使用濃度的潛力。

關鍵字

枯草芽孢桿菌 ； 細胞膜損傷 ； 表面活性素 ； 協同作用

並列摘要

The 1-2, 1-4, 3-3 strains isolated from soil showed strong antibacterial activity against S.aureus and S.intermedius. After Gram staining, endospore staining, qualitative analysis of carbohydrates, and 16S rRNA ribosomal gene identification, it was confirmed the three isolates were Bacillus subtilis. The antibacterial effect of B.subtilis was mainly attributed to the production of surfactin. In this study, by precipitating and purifying the culture supernatant of the isolates and then analyzing by high performance liquid chromatography (HPLC)and Tricine sodium dodecyl sulphate–polyacrylamide gel electrophoresis(Tricine SDS PAGE), it was confirmed that isolates 1-2 and 1-4 had the ability to produce surfactin, and they were able to produce surfactin in 300 mL of the total yields were 23.77 mg and 17.21 mg after culturing in LB medium for 48 hours, and the ability of the two isolates to produce surfactin was increased by about 1.6 times due to the addition of L-Glutamate to the medium. It is worth mentioning that although isolates 1-2 had higher surfactin production, the surfactin produced by isolates 1-4 had better antibacterial effects on S.aureus and S.intermedius at the same concentration. However, isolates 3-3 can only produce a small amount of surfactin, so the antibacterial effect on S.aureus and S.intermedius is not obvious. The antibacterial mechanism of surfactin involves the destruction of the cell membrane structure to unbalance the osmotic pressure inside and outside the membrane to exert an antibacterial effect. In this study, this phenomenon was verified by sytox green staining and extracellular protein efflux analysis. Compared with the control group, the 1-2, 1-4 surfactin-treated group showed a significant increase in the fluorescence unit, and the analysis results of liquid chromatography-mass spectrometry (LC-MS/MS) also showed that the surfactin-treated of S.aureus and S.intermedius could identify intracellular proteins. The surfactin produced by isolates 1-2 and 1-4 can achieve the best synergistic effect with the apramycin, the co-treatment of them can greatly reduce the level of apramycin concentration(MIC/10) against S.aureus and S.intermedius. These experimental results show that B. subtilis isolates 1-2 and 1-4 can cause the cell membrane damage of S.aureus and S.intermedius through the production of surfactin, thereby exerting an antibacterial effect, And it has the potential to synergize with antibiotics, thereby reducing the concentration of antibiotics used.

並列關鍵字

Bacillus subtilis ； cell membrane damage ； surfactin ； synergy

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