Primary Research On The Mechanism Of Ambroxol Against Pseudomonas Aeruginosa Biofilm

PartⅠEffects of ambroxol on permeability of ciprofloxacin across Pseudomonas aeruginosa biofilm【Objective】To investigate the effect of ambroxol on the permeability of ciprofloxacin across the bacterial biofilm of Pseudomonas aeruginosa.【Methods】In vitro model of P. aeruginosa biofim was established using 0.22μm pore diameter medical micropore film. The permeability of ciprofloxacin across the bacterial biofilm was measured by High Performance Liquid Chromatography (HPLC).The effects of 2mg/ml and 0.75mg/ml ambroxol on the model were also measured by HPLC.【Results】After 7 days'continuous cultivation, P. aeruginosa could establish compact and well-distributed biofilm on the surface of micropore film. Ciprofloxacin could slowly penetrate through the biofilm and the equilibration was achieved in 8h. When combined with 2mg/ml ambroxol,the permeation of ciprofloxacin(10μg/ml,20μg/ml和50μg/ml)is 6.369±0.023μg/ml,12.61±0.019μg/ml,27.63±0.014μg/ml respectively ,significantly increased compared to NS control group. 0.75mg/ml ambroxol group showed the same tendency,but not so obviously .【Conclusions】In vitro model of P. aeruginosa biofilm can be successfully established using medical micropore film as a supporter; ambroxol can significantly promote the permeability of ciprofloxacin across the bacterial biofilm.PartⅡEffects 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).【Method】Multifunction fluorometer was used to measure fluorescence of green fluorescent protein in pGFPuv transformated PAO1 in 96-well plate. The adhesion ratio(%) of PAO1 was calculated to determine the effects of ambroxol on bacterial biofilm adhesion. The tetramethylrhodamine 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 have 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 tetramethylrhodamine conjugated WGA stained. Quantification of EPS show that total EPS(μg)/dry weight of bacterial(g) decreased from 523.76±10.12 to 477.82±7.90 after ambroxol intervention,t=8.76,P<0.05.【Conclusions】Ambroxol could decrease the adhesion and EPS produce ability of PAO1 significantly.PartⅢEffects of ambroxol on Pseudomonas aeruginosa biofilm forming process and quantification of biofilm structure【Objective】To investigate the effect of ambroxol on PAO1 biofilm forming process by Confocal Laser Scanning Microscopy(CLSM). To quantify the BF structure with Image Structure Analyer(ISA) software.【Methods】In vitro model of 1d,3d,6d PAO1 biofilm was established.The effects of normal saline,ambroxol 0.75mg/ml and 2mg/ml on BF were monitored by CLSM respectively, based on fluorophores from PAO1 with nucleic acid SYTO9/PI stained. Quantification of the effect could be calculated by ISA software.【Results】1.The PAO1 biofilm forming process was investigated successfully by CLSM after stained with the nucleic acid SYTO9/PI,and also demonstrated heterogeneous distribution of live/dead bacteria in biofilm. 2.The quantitative data from ISA software showed:①NS control group: the thickness was 12.68±1.71μm,24.67±2.11μm,29.77±2.11μm at 1d,3d,6d respectively, showed increased tendency; the areal porosity(AP) was 0.89±0.02,0.83±0.02,0.74±0.01 respectively, showed decreased tendency ; the Average Diffusion Distance(ADD) was 1.00±0.003 ,1.03±0.001 , 1.04±0.002 respectively, increased slightly; the Textual Entropy(TE) was 2.68±0.48,3.63±0.53,5.87±0.32 respectively, showed increased tendency.②6d BF model: After administration of ambroxol 2 mg/ml, BF thickness decreased from 29.77±2.11μm to 11.01±2.71μm,t=21.15,p<0.05;AP increased from 0.74±0.01 to 0.86±0.03 ,t=-14.7,p<0.05;TE decreased from 5.87±0.32 to 5.21±0.61 ,t=3.71,p<0.05;All the differences are significant comparing to control group. The effect of ambroxol 0.75mg/ml has the same tendency as ambroxol 2mg/ml group,but not so obviously.③After administration of ambroxol,the 1d,3d BF showed the same tendency as 6d model.【Conclusions】1.ISA software can quantify the structure of PA01 biofilm at different stage. 2.Ambroxol can affect PAO1 biofilm at different time stage; the effect of 2mg/ml ambroxol was more observe than 0.75mg/ml ambroxol group.