微泡超声增强庆大霉素对大肠杆菌抗菌作用的机制研究

目的 观察微泡超声对庆大霉素抑制大肠杆菌的增强效果,并探讨其可能机制.方法 配制1×107 CFU/ml大肠杆菌(ATCC 25922)悬液,分为空白对照组、微泡(Sonovue)组、超声组和微泡超声组,每组按照庆大霉素浓度进一步分为0、1、2 μg/ml三个浓度组(n=8).微泡浓度为体积分数10％.超声辐照强度为300 mW/cm2,频率为46.5 kHz,占空比为33％,辐照时间为12 h.平板计数法比较各组残留活菌密度,取对数(log10 CFU/ml)行统计学分析.每组另取少量菌液行透射电镜扫描,观察残留菌的微观形态.结果 未添加庆大霉素时,四组残留菌量的差异无统计学意义;庆大霉素1 μg/ml时,微泡超声组(5.44±0.49 log10 CFU/ml)与空白对照组(7.44±0.64 log10 CFU/ml)、微泡组(7.19±0.38 log10 CFU/ml)、超声组(6.86±0.29 log10 CFU/ml)的差异均有统计学意义;庆大霉素浓度为2μg/ml时,微泡超声组(2.87±0.28 logl0 CFU/ml)与空白对照组(4.45±0.43 log10 CFU/ml)、微泡组(4.33±0.40 log10 CFU/ml)、超声组(3.89±0.37 log10 CFU/ml)的差异仍均有统计学意义.上述药物浓度下超声组与空白对照组菌量的差异均无统计学意义.透射电镜扫描提示,与单纯超声辐照相比,微泡超声辐照可迸一步改变细菌胞膜的形态,胞膜皱褶更明显,存在较多不连续.结论 与单纯超声辐照相比,微泡超声可以进一步提高庆大霉素对大肠杆菌的抗菌效果,可能与微泡增强超声的“声孔效应”有关.

Enhanced antimicrobial effect of gentamicin against Escherichia coli through microbubble-mediated ultrasonic techniques

Objective To investigate effect of microbubble-mediated ultrasonic techniques on activity of gentamicin against E. coli, as well as the possible mechanism. Methods The planktonic E. coli were divided into control group, ultrasound group, microbubble group and microbubble-ultrasound group. There were 3 different concentrations of gentamicin (0, 1, 2 μg/ml) in each group. The concentration of the microbubbles was 10% (v/v). The parameters of ultrasound included irradiation intensity (300 mW/cm²), frequency (46.5 kHz), duty ratio (33%) and expose time (12 hours). Bacteria were cultured and detected by a semiquantitative plate assay. The numbers of CFU were calculated and statistically analyzed (log10 CFU/ml). Finally, the bacterial liquid was obtained and scanned by transmission electron microscope to observe the micromorphology structure of bacteria. Results In 0 μg/ml of gentamicin group, there was no statistical difference in amount of residual bacteria between 4 groups. In 1μg/ml of gentamicin group, the number of residual bacteria in microbubble-ultrasound group (5.44±0.49 log10 CFU/ml) was significantly smaller than those in microbubble group (7.19±0.38 log10 CFU/ml), control group (7.44±0.64 log10 CFU/ml) and ultrasound group (6.86±0.29 log10 CFU/ml). In 2 μg/ml of gentamicin group, the number of residual bacteria in microbubble-ultrasound group (2.87±0.28 log10 CFU/ml) was still smaller than those in microbubble group (4.33±0.40 log10 CFU/ml), control group (4.45±0.43 log10 CFU/ml) and ultrasound group (3.89±0.37 log10 CFU/ml). The electron micrographs showed the bacterial cytoplasmic membrane was more folded and discontinuous in microbubble-ultrasound group. Conclusion Compared with the single ultrasound, the microbubble-mediated ultrasonic techniques can further enhance the antibacterial effect of gentamicin against E. coli, and its mechanism is likely to be associated with "Sonoporation".