黄芩苷联合万古霉素对金黄色葡萄球菌生物膜的体外影响

In vitro effect of baicalin combined with vancomycin on Staphylococcus aureus biofilm

目的通过建立体外金黄色葡萄球菌(SAU)生物膜(BF)模型,研究黄芩活性成分-黄芩苷(BC)对BF的影响及其与万古霉素(VAN)的协同杀菌效果。方法选取临床分离并且能够稳定形成BF的SAU 17564,采用TSB-G系统,复制体外BF模型,试管二倍稀释法测定药物的最低抑菌浓度,连续稀释法进行活菌计数,激光共聚焦显微镜(CLSM)结合BF定量软件COMSTAT对BF结构进行分析。结果建模3d,BF经256μg/ml BC组、4μg/ml VAN组作用后BF内活菌数分别为(7.91±0.21)、(7.95±0.19)log10 CFU/ml,与空白对照组相比差异无统计学意义;256μg/ml BC+4μg/ml VAN组的活菌数为(7.56±0.28)log10 CFU/ml,与空白对照组相比差异有统计学意义(P<0.05);建模7d,BF经4μg/ml VAN组、512μg/ml BC组、256μg/ml BC组、128μg/ml BC组及128μg/ml BC+4μg/ml VAN组作用后,BF内活菌数分别为(8.12±0.18)、(8.12±0.10)、(8.11±0.11)、(8.22±0.12)、(8.21±0.16)log10 CFU/ml,与空白对照组相比差异无统计学意义;512μg/ml BC+4μg/mlVAN组、256μg/ml BC+4μg/mlVAN组活菌数分别为(7.47±0.16)、(7.52±0.17)log10 CFU/ml,与空白对照组相比P<0.05;建模3d或7d的BF经CLSM观察及COMSTAT分析,256μg/ml BC+4μg/ml VAN组的生物量均比空白对照组、256μg/ml BC组及4μg/mlVAN组明显减少(P<0.05)。结论黄芩苷能破坏SAU已形成的3d及7dBF,增强VAN对BF内SAU的清除作用。

OBJECTIVE To establish the in vitro model of Staphylococcus aureus biofilm so as to observe the effect of baicalin （the active ingredient of scutellaria ）on the biofilm and the synergistic bactericidal effect of its combina- tion with vancomycin. METHODS The clinical isolates of S. aureus 17546 which could form biofilm stably were en- rolled in the study, then the in vitro S. aureus biofilm model was duplicated with the use of TSB-G system; the minimum inhibitory concentrations （MICs） were determined by using doubling dilution; the bacterial colony counts were calculated by serial dilution method; the structure of the biofilm was analyzed with the use of confoeal laser scanning microscope （CLSM） combined with COMSTAT software. RESULTS Three clays after the formation of biofim, the bacterial colony counts were （7.91±0.21） log^10 CFU/ml in the EC group after being reacted with 256 μg/ ml of baiealin, （7.95 ±0.19）log^10 CFU/ml in the VAN group after being reacted with 4 μg/ml of vancomycin, as com- pared with the control group, the difference was not statistically significant; the bacterial colony counts of the 256μg/ml BC plus 4 μg/ml VAN group were （7.56 ±0.28）log^10 CFU/ml, as compared with the control group, the difference wasstatistically significant （P〈 0.05）. Seven days after the formation＇ of biofilm, the bacterial colony counts were （8. 12 ± 0. 18） log^10 CFU/ml in the 4 μg/ml VAN group, （8.12 ±0.10） log^10 CFU/ml in the 512 μg/ml BC group, （8.11 ±0.11） log^10CFU/ml in the 256 μg/ml 13（2 group, （8.22±0.12） logt^10 CFU/ml in the 128 μg/ml BC group, （8.21 ±0.16）1og^10 CFU/mI in the 128 μg/ml BC plus 4μg/ml VAN group, as compared with the control group, the differ- ence was not statistically significant. The bacterial colony counts were （7.47 ±0.16） log^10 CFU/ml in the 512 μg/ml BC plus 4μg/ml VAN group, （7.52 ±0.17）log^10 CFU/ml in the 256 μg/ml BC plus 4μg/ml VAN group, as compared with the control group, the difference was statistically significant（P〈0.05）. The analysis of 3 or 7-day formation of bio- film with CLSM combined with COMSTAT showed that the biomass of the 256 μg/ml 13（2 plus 4 μg/ml VAN group were significantly reduced as compared with the control group, 256 μg/ml BC group, or 4μg/ml VAN group （P〈 0.05）. CONCLUSION Baiealin can destroy the biofilms of S. aureus that have formed for 3 or 7 days and can en- hance the effect of vancomycin on removal of the S. aureus in the biofilms.