氨溴索对铜绿假单胞菌生物膜早期黏附及胞外多糖的影响

目的研究氨溴索对铜绿假单胞菌PA01菌株BF早期黏附及胞外多糖复合物（Extracellular Pdymeric Substances，EPS）的影响。方法利用荧光多功能酶标仪检测各组不同时间点96孔板中pGFPuv转化PA01菌株的荧光强度，计算黏附率以表示干预对不同时间点细菌黏附的影响；利用罗丹明标记的麦胚凝集素（WGA）特异性结合细菌EPS，荧光显微镜下定性观察各组EPS的变化；利用硫酸-苯酚法定量各组细菌EPS的产量。结果8h组，氨溴索高浓度干预后细菌的黏附率由0．72±0．17下降至0．49±0．08，t=4．03，P〈0．05，与克拉霉素阳性对照组黏附率（0．50±0．06）相比，t=-1．19，P〉0．05；氨溴索低浓度干预后黏附率也有下降，但不及高浓度组明显；其余时间组趋势与8h组大致相似。罗丹明标记WGA可使胞外多糖显色，在荧光显微镜下观察可见氨溴索干预后EPS减少，稀薄；EPS定量实验，EPS总量（μg）／细菌干重（g）氨溴索干预组（477．82±7．90）较生理盐水对照组（523．76±10．12）有明显降低，t=8．76，P〈0．05。结论氨溴索可显著减少PA01菌株黏附及产EPS的能力。

Effects of ambroxol on bacterial biofilm adhesion and exopolysaccharides

Objective To investigate the effects of ambroxol on adhesion of bacterial biofilm at early stage and Extracellular Polymeric Substances （EPS）. Methods Multifunction fluorometer was used to measure fluorescence of green fluorescent protein in pGFPuv transformated PA01 in 96-well plate. The adhesion ratio（ % ） of PA01 was calculated to determine the effects of ambroxol on bacterial biofilm adhesion. The tetramethylrhedamine conjugated wheat germ agglutinin （WGA） was used to stain the EPS, in order to qualitation the EPS by fluorescence microscope; the phenol-sulfuric acid method was used to quantitate the EPS in each group. Results Eight hour group： after intervention with high dose of ambroxol, the adhesion ratio （ % ） was decreased from 0.72 ± 0.17 to 0.49 ± 0.08, t = 4.03, P 〈 0.05, and compared with clarithromycin positive control group （0.50 ± 0.06 ）, t = - 1.09, P 〉 0.05 ; Low dose of ambroxol could also affect the adhesion ratio, but not so obvious as high dose; Other time groups had the approximate tendency as 8 hour group; The EPS in biofilm was significantly decreased after intervention with ambroxol, which was visualized by fluorescence microscope, based on the tetramethylrhedamine conjugated WGA stained. Quantification of EPS showed that total EPS （μg）/dry weight of bacterial （g） decreased from 523. 76 ± 10. 12to477.82±7.90 after ambroxol intervention,t=8.76,P〈0.05. Conclusion Ambroxol could decrease the adhesion and EPS produce ability of PA01 significantly.