低频超声对金黄色葡萄球菌生物膜的促渗作用研究

Effect of Low-Frequency Ultrasound on Permeation of Staphylococcus Aureus Biofilm

目的:探讨低频超声对血卟啉单甲醚(Hematoporphyrin monomerthyl ether,HMME)在金黄色葡萄球菌生物膜中渗透效果的影响。方法:将直径25 mm,孔径0.22μm的微孔滤膜平铺于LB琼脂培养基表面,再将纯种的金黄色葡萄球菌(ATCC6538)菌液,通过均匀涂布接种到滤膜表面,37℃恒温培养48 h获得实验所需的生物膜模型。将培养所得生物膜样本随机分为空白对照组,单独超声组,单独药物组和实验组,其中实验组按超声强度(0.5 w/cm^2或1 w/cm^2)和超声作用时间(1 min、2 min或3 min)不同又分为实验(A、B、C、D、E、F)组。所用渗透药物为20μg/m L的HMME溶液。对照组使用等量的生理盐水孵育,孵育时间为3 min,单独超声组也用生理盐水孵育并加0.5 W/cm^2的超声作用3 min。HMME在405 mm激发光下有特征性光谱,通过测量光谱曲线下积分面积,可间接反映生物膜中HMME渗入量。结果:实验A组(0.5 w/cm^2,1 min)的荧光光谱曲线下积分面积显著高于单独药物组(P<0.05)。声强相同时,随作用时间增加,所得光谱曲线下积分面积显著增加(P<0.01或P<0.05);作用时间相同时,声强越大,光谱曲线下积分面积亦越大(P<0.01)。结论:低频超声可以显著增加HMME渗透进入金黄色葡萄球菌生物膜的量,且渗透效果与超声作用时间和超声声强正相关。

Objective： To investigate the effect of low-frequency ultrasound on permeation of Hematoporphyrin monomerthyl ether （HMME） in Staphylococcus aureus biofilm. Methods： Flatting microporous membrane （Ф 6 mm） on LB agar surface, Staphylococcus aureus bacteria （ATCC6538） were inoculated onto the surface of membrane. To obtain the required biofilm model, the bacteria was cultured for 48 h under the condition of constant temperature（37 ℃）. The cultivated biofilm samples were randomly divided into 4 groups, control group, single ultrasound group, single drug group and the experimental group （ultrasound ＋ drug group）. Furthermore, the experimental group was subdivided into different experiment （A, B, C, D, E, F） groups based on the different ultrasonic intensity （0.5 w/cm2 or 1 w/cm2） and ultrasonic action time （1 min, 2 min or 3 min）. The medications used were 20 μg/mL HMME solution. The same amount of normal saline was used in the control group and incubated for 3 min. The individual ultrasonic group was incubated with normal saline and added 0.5 w/cm2 ultrasonic treatment for 3 min. We tested the characteristic specmtm of HMME was tested which was excited by 405 nm laser in each sample, then measured the integral size of area of each spectrum under the curve and compared the size of the areas which would indirectly reflect the amount of permeated HMME. Results： The area of fluorescence integral under the curve in ultrasound ＋ drug group was significantly higher than that of single drug group （P〈0.05）.When the ultrasonic intensity was same, the integral size of area under the curve was increased significantly with the increase of reaction time （P〈0.01 or P〈0.05 ）. When the time was same, the ultrasonic intensity was greater, then the integral area under the curve was bigger （P〈0.01）. Conclusions： Low frequency ultrasound can significantly increase the quantity of HMME which penetrates into the Staphylococcus aureus biofilm. The effect of permeation is positively correlated with both the ultrasonic intensity and the time of ultrasonic treatment.