The Effect And Mechanism Of Ambroxol Against Biofilm Of Mucoid Pseudomonas Aeruginosa

PART I EFFECT OF AMBROXOL ON THE STRUCTURE OF BIOFILM OF MUCOID PSEUDOMONAS AERUGINOSA【Objective】To establish the biofilm model of Pseudomonas aeruginosa in vitro and to observe the effect of ambroxol on the structure of Pseudomonas aeruginosa biofilm. Further to detect the effect between ambroxol and ciprofloxacin.【Methods】After 7-day culture of Pseudomonas aeruginosa with plate culture method ,mature BF formed. The stucture of the biofilm was detected by scanning electron microscope (SEM). The parameters of BF structure were analyzed through pictures from Confocal Laser Scanning Microscopy (CLSM) with Image Structure Analyer (ISA) software .Fluorescence intensity of the viable bacteria in biofilm treated by ambroxol alone and together with ciprofloxacin were determined with fluorescence microscope.【Result】After the treatment of ambroxol, biofilm was destroyed and the matrix outsides became thin and abnormal compared with the control by SEM, small quantity of bacteria dispersed. The results of ISA showed that after administration of ambroxol 2 mg/ml, BF thickness, Average Diffusion Distance (ADD),and Textual Entropy (TE) decreased from 39.22±2.01 ,1.08±0.001和6.25±0.22 to 10.09±2.32 , 1.00±0.002 and 5.19±0.41 respectively (p<0.05); Areal porosity (AP)increased from 0.70±0.03to 0.91±0.02 (p<0.05)。The effect of ambroxol 0.75mg/ml has the same tendency as ambroxol 2mg/ml group, but not so obvious.The changes of BF thickness, AP, ADD and TE are 20.10±2.86 ,0.82±0.04,1.03±0.004 and 5.36±0.34 respectively (p<0.05)。Further results of fluorescence microscope showed the viable bacteria in biofilm was reduced significantly (F=18, p<0.05) when ambroxol concentration is higher than 0.49mg/ml , and there is synergistic effect between ambroxol and ciprofloxacin (F=15.1, p<0.05), and the effect enhanced with the concentration of ambroxol increased.【Conclusion】Ambroxol can affect the biofilm structrue of Pseudomonas aeruginosa and enhance the bactericidal ability of antibiotics. PARTⅡEFFECT OF AMBROXOL ON ALGINATE OF PSEUDOMONAS AERUGINOSA BIOFILMS IN VITRO【Objective】To explore the effect of ambroxol on alginate which is the main component of biofilm (BF) formed by clinical isolate of Pseudomonas aeruginosa. Further to investigate the effect of ambroxol on the expression of important genes and the activity of key enzyme involved in synthesis of alginate, and the effect of ambroxol on the degradation of alginate.【Methods】Plate culture method was used to establish biofilm model of Pseudomonas aeruginosa in vitro. Mature BF formed after 7-day culture. Bacteria embedded in BF were isolated through vibration and collected for investigation. The effect of ambroxol on the contents of alginate was detected through Sulphuric acid-Oxybenzene Method. The mRNA expression of important genes involved in synthesis of alginate such as algD, algU, algR and mucA were detected by using RT-PCR; Activity of guanosine diphospho-D-mannose dehydrogenase(GMD) which is the key enzyme involved in synthesis of alginate is detected with spectrophotometer; Degradation of alginate were determined by the difference between the contents of alginate right after isolated and those have been placed for 1 hour.【Results】After the treatment of ambroxol with the concentration of 3.75mg/ml, the contents of alginate (mg/g) were decreased from 86.4024±0.8588 to 59.9199±0.5803( F=66.2 ,p<0.01 ); the mRNA expression of algD, algU, algR and mucA were changed from 1.2994±0.0173 ,1 .0488±0.0457 , 0.9888±0.0267,and 0.8731±0.0336 to 1.0253±0.0265, 0.9594±0.0106, 0.8536±0.0179,and 1.0770±0.0503 respectively(F= 91.9 , 41.1, 88.4 and 56.9, p <0.05).The activity of GMD decreased from 0.0989±0.0055 to 0.0558±0.0016( F= 121.2,p<0.01) .The degradation of alginate(△mg/g)changed from 1.4122±0.0073 to 1.4175±0.0019(F=21.8, p>0.05). The effect of ambroxol with the concentration of 1.875mg/ml had the same tendency as ambroxol with the concentration of 3.75mg/ml, but not so obvious.【Conclusions】Ambroxol can decrease the contents of alginate , influence the mRNA expression of algD, algU, algR and mucA, and decrease the activity of GMD. But it had no effect on degradation of alginate. PARTⅢTHE INHIBITING EFFECT OF AMBROXOL ON THE TRANSFORMATION FROM WILD TO MUCOID TYPE OF PSEUDOMONAS AERUGINOSA WITH THE STIMULATION OF HYDROGEN PEROXIDE IN VITRO【Objective】To explore the inhibiting effect of ambroxol on the transformation from wild to mucoid type of Pseudomonas aeruginosa with the stimulation of hydrogen peroxide in vitro.【Methods】Wild type of Pseudomonas aeruginosa PAO1 was stimulated by hydrogen peroxide alone, hydrogen peroxide and ambroxol respectively to investigate the change of appearance, detect mutation of mucA by gene sequencing, and measure the content of alginate.【Results】With the continuous stimulation of hydrogen peroxide, PAO1 converted into mucoid phnotype. The appearance changed from dry and regular colony to moist, large and irregular. Mutation was found in some sites of mucA gene which was detected by gene sequencing. Moreover the contents of alginate increased from undetecable to 69.62260±0.7652mg/g. However with the addition of hydrogen peroxide combined with ambroxol, no change was found in appearance of PAO1 as well as gene mutation mucA. The contents of alginate were still undetectable.【Conclusions】Ambroxol can antagonize the effect of hydrogen peroxide to inhibit the conversion from wild to mucoid type of Pseudomonas aeruginosa. PART IV THE EFFECT OF AMBROXOL ON TUBE-ASSOCIATED LUNG INFECTION OF MUCOID PSEUDOMONAS AERUGINOSA BIOFILMS IN VIVO【Objective】To establish murine model of tube–associated lung infection caused by Pseudomonas aeruginosa with biofilms ( BF ) formation, further to explore the effect of ambroxol on the mucoid Pseudomonas aeruginosa biofilms in vivo and effect of ambroxol on lung infection caused by biofilms.【Methods】Plate culture method was used to establish biofilm model of Pseudomonas aeruginosa in vitro. Tube with biofilm formation was planted into trachea and murine model of tube–associated lung infection caused by Pseudomonas aeruginosa with biofilms formation was established. Rats were allocated into 4 groups randomly: Group 1, rats with biofilm-covered tube intubation were given ambroxol; Group 2, rats with biofilm-covered tube intubation were given saline as control; Group 3, rats with sterile tube intubation were given saline as control; Group 4, rats without any operation as normal control. The rats were sacrificed on the 4th and 7th day after intubation respectively. Tubes were drawn out to investigate the structure of BF with scanning electron microscopy and detect the content of alginate and bacterial count. Meantime macroscopic lung pathology and histopathological changes were investigated and anti-alginate IgG, IFN- and IL-10 in blood plasma were determined using enzyme-linked immunosorbent assay (ELISA).【Results】Murine model of tube–associated lung infection caused by Pseudomonas aeruginosa with biofilms formation was successfully established. The structure of BF was destroyed. Bacterial count in BF of Group 1 and Group 2 were 8.3494±0.025 lgCFU VS 8.5669±0.11 lgCFU and 8.1527±0.015 lgCFU VS 8.5009±0.013 lgCFU on day 4 and day 7 after infection respectively (F=87.6 and F=507.5, p<0.05). The contents of alginate in Group 1 and Group 2 were 55.3776±0.842mg/g VS 83.4886±0.0.538mg/g and 42.3721±0.505 mg/g VS 74.6215±0.215mg/g on day 4 and day 7respectively, (F=791.7 and F=3450.9, p<0.05). Lung macroscopic pathology and pathologic change in Group 1 and Group 2 were not significantly different on day 4 after infection but were significantly different on day 7 after infection. Lung index of macroscopic pathology (LIMP) score in Group 1 and Group 2 were 0.1857±0.018 VS 0.1929±0.025 on day 4 after infection(F=2.0,p>0.05), 0.1928±0.024和0.2357±0.031 on day 7 after infection(F=4.9, p<0.05).Bacterial count in lungs in Group 1 and Group 2 were 6.5049±0.0616 lgCFU VS 6.3216±0.11 lgCFU on day 4 after infection(F=52.3, p<0.05) and 6.1527±0.015 lgCFU VS 6.1729±0.013 lgCFU on day 7 after infection(F=1.1, p>0.05). Anti-alginate IgG level in Group 1 and Group 2 were 0.7721±0.006 VS 1.0005±0.006 on day 4 after infection and 0.7917±0.007 VS 1.0195±0.007 on day 7after infection(F= 351.9 and 844.5, p<0.05).IL-10 level in Group 1 and Group 2 were 158.6069±2.009pg/mlVS 162.8088±2.166pg/ml on day 4 after infection and 168.4113±0.007 pg/ml VS 171.2125±1.578 pg/ml on day 7 after infection,(F=2.1 and1.7, p>0.05). IFN-γlevel in Group 1 and Group 2 were 74.0397±2.311 pg/ml VS126.8600±4.026pg/ml on day 4 after infection and 63.3759±1.516 pg/ml VS 174.3221±4.444 pg/ml on day 7 after infection( F=129.5 and 588.2, p<0.05).The ratio of IFN-γand IL-10 in Group 1 and Group 2 were 0.4937±0.011VS 0.8088±0.025 on day 4 after infection and 0.4566±0.012 VS 1.0264±0.019 on day 7 after infection ( F= 121.9 and 731.1, p<0.05).【Conclusion】In vivo, ambroxol could destroy the structure of BF on the tube, decrease the bacterial count in BF and the contents of alginate of mucoid Pseudomonas aeruginosa BF. Ambroxol can extenuate the pathyologic change of lung on day 7 after infection. Meanwhile it could reduce the level of anti-alginate IgG so as to extenuate the immulogic damage in lung. Ambroxol could also reduce the IFN- level and the ratio of IFN-γand IL-10 to extenuate the inflammation.