家蝇抗菌肽MDC协同庆大霉素抗普通变形杆菌作用研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-0245

摘要/Abstract

摘要：

以普通变形杆菌（Bacillus proteus vulgaris）为研究对象,探讨家蝇抗菌肽（Musca domestical cecropin,MDC）和庆大霉素（Gentamicin）的体外联合抗菌效果及协同抗菌机制。采用微量肉汤稀释法、棋盘法、结晶紫染色等方法研究两药的协同抗菌作用和抗生物膜作用;通过荧光染色、流式细胞术（FCM）、透射电子显微镜（TEM）观察两药联用的抗菌机制。家蝇抗菌肽MDC与庆大霉素的FIC为0.313,表明两者具有较好的协同抗菌作用;结晶紫染色结果显示两药对细菌初始生物膜、成熟生物膜和胞外蛋白质的影响也有一定的协同作用;荧光和流式细胞术结果显示药物可以有效杀伤细菌,使细菌死亡数目上升;透射电镜观察到给药后大部分细菌细胞壁破损,细胞膜不完整,胞质内容物坏死溶解导致细胞肿胀破裂。家蝇抗菌肽MDC和庆大霉素具有协同抗普通变形杆菌作用,且两药的作用机制相辅相成。

关键词: 普通变形杆菌, 家蝇抗菌肽, 庆大霉素, 协同抗菌

Abstract:

The combined antibacterial action and the synergistic antibacterial mechanism of Musca domestica cecropin（MDC）and gentamicin on Bacillus proteus vulgaris were investigated. Micro broth dilution,chessboard,and crystal violet staining were used to study the synergistic antibacterial and antibiofilm effects of the two drugs. Fluorescence staining,flow cytometry and transmission electron microscopy（TEM）were employed to observe the antibacterial mechanism of the combination of the two drugs. The fractional inhibitory concentration of MDC and gentamicin was 0.313,indicating that the 2 drugs presented promising synergistic antibacterial effect. According to the results of crystal violet staining,the two drugs demonstrated a synergistic effect on the initial biofilm,mature biofilm and extracellular protein of the Bacillus proteus vulgaris. The results of fluorescence and flow cytometry showed that the two drugs efficiently killed bacteria and increased the number of bacterial deaths. TEM observation revealed that most bacterial cell walls were damaged,their cell membranes were incomplete,and intracellular necrosis caused cell swelling and rupture. The MDC and gentamicin act synergistically against Bacillus proteus vulgari,and the mechanism of the two drugs is complementary.

Key words: Bacillus proteus vulgaris, Musca domestica cecropin, gentamicin, synergistic antibacterial action

陈晴汝, 桂水清, 卢雪梅. 家蝇抗菌肽MDC协同庆大霉素抗普通变形杆菌作用研究[J]. 生物技术通报, 2020, 36(11): 85-93.

CHEN Qing-ru, GUI Shui-qing, LU Xue-mei. Synergistic Effect of Musca domestica Cecropin and Gentamicin Against Bacillus proteus vulgaris[J]. Biotechnology Bulletin, 2020, 36(11): 85-93.

图/表 11

图1 MDC对普通变形杆菌的MBC测定

图2 庆大霉素对普通变形杆菌的MBC测定

表1 MDC和庆大霉素对普通变形杆菌的MIC、MBC和FIC

抑菌物质	普通变形杆菌
抑菌物质	MIC/（μg·mL^-1）	MBC/（μg·mL^-1）	FIC（联合用药）
MDC	> 128	> 1 024	0.313
庆大霉素	32	32	0.313

图3 普通变形杆菌生物膜结晶紫染色结果图中箭头表示细菌与细菌共聚;A：药物MDC组;B：药物庆大霉素组;C：两药联合组;D：空白对照组;1-3则对应不同药物浓度

图4 药物对普通变形杆菌初始生物膜的影响 *：与空白对照组相比,** P < 0.05,***P < 0.001

图5 药物对普通变形杆菌成熟生物膜的影响 *：与空白对照组相比,***P < 0.001

图6 普通变形杆菌胞外蛋白质标准曲线

图7 普通变形杆菌胞外蛋白质浓度 *：与对照组相比,***P < 0.001

图8 普通变形杆菌荧光染色结果 A：SYTO9空白对照组;B：SYTO9 MDC组（128 μg/mL）;C：SYTO9庆大霉素组（32 μg/mL）;D：SYTO9联用组（128 μg/mL MDC 和32 μg/mL庆大霉素）;E：PI空白对照组;F：PI MDC组（128 μg/mL）;G：PI庆大霉素组（32 μg/mL）;H：PI联用组（128 μg/mL MDC 和32 μg/mL庆大霉素）

图9 流式细胞术检测PI染色细菌给药后死亡状况 A：空白对照组;B：药物MDC组;C：药物庆大霉素组;D：两药联合组 * ：与空白对照组相比,* P < 0.05,***P < 0.001

图10 透射电镜下普通变形杆菌的形态结构 A：空白对照组;B：抗菌肽MDC组;C：庆大霉素组;D：协同组

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