黄芩素协同万古霉素对耐甲氧西林金黄色葡萄球菌早期生物被膜的研究

In vitro effect of baicalein in combination with vancomycin on methicillin-resistant Staphylococcus aureus early biofilm

目的通过建立体外耐甲氧西林金黄色葡萄球菌（MRsA）早期生物被膜（BF）模型，研究黄芩素对BF的影响及其联合万古霉素（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MRSA17546，采用胰蛋白胨大豆肉汤添加0．5％葡萄糖为培养基，复制体外早期BF模型，实验分为空白对照组、阳性对照组[16mg／L克拉霉素（CLR）组、4mg／LVAN组、16mg／L CLR＋4mg／LVAN组]、实验组（32mg／L黄芩素组、32mg／L黄芩素＋4mg／I．VAN组）。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，扫描电镜观察BF的形态结构。结果早期BF经VAN、cI．R及黄芩素作用后BF内活菌数分别为（7．95±0．19）、（8．03±0．23）、（7．95±0．18）log10 cfu／ml，与空白对照组[（7．99±0．25）log10 cfu／m1]相比差异无统计学意义（P〉0．05）。16mg／L CLR＋4mg／L VAN组、32mg／L 黄芩素＋4mg／LVAN组活菌数为（7．71±0．25）、（7．29±0．16）log10 cfu／ml，低于空白对照组（P〈O．01），且32mg／L 黄芩素＋4mg／L VAN组比16mg／L CLR＋4mg／L VAN组活菌数进一步减少（Pd0．05）。经扫描电镜观察，可见黄芩素组载体上的BF比空白对照组减少，VAN组BF内细菌数未见明显减少，而黄芩素＋VAN组BF内的细菌数明显比空白对照组减少。结论黄芩素能破坏MRSA已形成的早期BF，增强VAN对BF内MRSA的清除作用，且黄芩素破坏BF及其协同杀菌作用强于cI。R。

Objective To investigate the effect of baicalein in combination with vancomycin （VAN） on methicillin resistant Staphylococcus aureus （MRSA） early biofilm. Methods Clinical isolates of MRSA 17546 which could form biofilm stably,were cultured in TSBG medium for biofilm formation in three days. MIC was measured by doubling dilution. Viable bacterial counts were determined by serial dilution. Biofilm was observed by scanning electron microscope （SEM）. Results After the early biofim was reacted with 4 mg/L VAN, 16 mg/L clarithromycin （CLR） or 32 mg/L baiealein for 12 hours,the biofilm bacterial counts were （7.95 ± 0.19）, （8.03±0.23）, （7.95 ±0.18） log10 cfu/ml, there was no statistical significance compared to the control group （7.99±0.25） log10 cfu/ml, P 〉0.051. The counts of the CLR plus VAN group and the baicalein plus VAN group were （7.71 ± 0.25）, （7.29 ± O. 16） log10 cfu/ml,which were significantly less than that of the control group （ P 〈0.01）, and the counts ofthe baicalein plus VAN group was less than that of the CLR plus VAN group （ P d0.05）. Besides,the biofilm of the baicalein group was less than the control group, the bacteria counts of the VAN group showed no decrease, and the baicalein plus vancomyein group showed obvious decrease compare to the control group observed by SEM. Conclusions Baicalein can destroy the early biofilrn of MRSA in vitro and enhance susceptibility of VAN to MRSA within biofilm.