The Effect Of Ambroxol On Quorum-Sensing System In Pseudomonas Aeruginosa Biofilm

PART IEFFECT OF AMBROXOL ON THE GROWTH OF PSEUDOMONAS AERUGINOSA【Objective】To research the effect of ambroxol on the growth of Pseudomonas aeruginosa.【Methods】Culture Pseudomonas aeruginosa PAO1 in LB. After the intervention of ambroxol with the concentration of 3.75 mg/mL and 1.875 mg/mL, turbidimetry were used to detect the optical density value A600 of bacterial solutions in different time points, draw the growth curve and compare the growth curve of bacteria in each group.【Result】After the intervention of ambroxol, the growth curve of Pseudomonas aeruginosa have no significant difference compared with the medium control group.【Conclusion】Ambroxol have no significant effect on the growth of Pseudomonas aeruginosa. PARTⅡEFFECT OF AMBROXOL ON ADHESION OF BIOFILM AND CELL MOTILITY OF PSEUDOMONAS AERUGINOSA【Objective】To investigate the effects of ambroxol on adhesion of Pseudomonas aeruginosa biofilm at early stage and to observe the effect of on cell motility of Pseudomonas aeruginosa in vitro.【Methods】Agar plate of different concentration were used to culture PAO1, measure the diameter of swimming, twitching and swarming. After the treatment of ambroxol in different concentration, fluorescence microscope was used to detect the adhesion capacity; multifunction fluorometer was use to measure fluorescence of green fluorescent protein (GFP) in pGFPuv transformated PAO1 in 96-well plate.【Result】After the treatment of ambroxol in different concentration, cell motility of Pseudomonas aeruginosa were decreased in various degree (p<0.05). We have observed that the adhesion capacity of ambroxol treatment group is thinner than the medium control group, especially the high concentration group. After the treatment of ambroxol, the adhesion ratio were decreased in various degree too, especially the high concentration group (p<0.05). 【Conclusion】Ambroxol can decrease the adhesion of Pseudomonas aeruginosa biofilm at early stage and cell motility of Pseudomonas aeruginosa.PARTⅢEFFECT OF AMBROXOL ON THE SIGNAL MOLECULES AND THE EXPRESSION OF RELATIVE GENES OF QUORUM-SENSING SYSTERM IN PSEUDOMONAS AERUGINOSA BIOFILM IN VITRO【Objective】To research the effect of ambroxol on the signal molecules N-acyl-homoserinelactone (AHL) of quorum-sensing(QS)system in Pseudomonas aeruginosa biofilm, and the effect on the expression of the genes rhlI and lasI which code AHL synthetase in an established biofilm model in vitro.【Methods】Mature Pseudomonas aeruginosa BF was formed after 7 days culture on plate,scanning electron microscope(SEM) were used to identify the BF. After the treatment of ambroxol in different concentration, quantitative real-time RT-PCR were used to analyze the expression of rhlI and lasI which code AHL synthetase; AHL-responsive Agrobacterium tumefaciens reporter strain WCF47 and detection ofβ-galactosidase activity were utilized to quantify the signal molecules AHL.【Results】After the treatment of ambroxol with the concentration of 3.75 mg/mL, the mRNA expression of lasI and rhlI were down-regulated than the control group from 1.000 to 3.548±0.070 , 1.914±0.050 respectively (P<0.05); the effect of ambroxol with the concentration of 1.875 mg/mL had the same tendency as in the concentration of 3.75 mg/mL, but not so obvious (P<0.05). After the treatment of ambroxol with the concentration of 1.875 mg/mL and 3.75 mg/mL, the level of AHL were down-regulated than the control group, the activity ofβ-galactosidase were decreased obviously from 761.286±13.301 Miller Unit (the control group) to 645.428±15.197 Miller Unit(p<0.05)and 532.428±8.867 Miller Unit(p<0.05).【Conclusions】Ambroxol can down-regulate the expression of the genes lasI and rhlI which code AHL synthetase and the signal molecules AHL of quorum-sensing system in Pseudomonas aeruginosa biofilm. PARTⅣEFFECT OF AMBROXOL ON THE VIRULENCE FACTORS AND THE EXPRESSION OF RELATIVE GENES OF QUORUM-SENSING SYSTERM IN PSEUDOMONAS AERUGINOSA BIOFILM IN VITRO【Objective】To research the effect of ambroxol on the virulence factors of Quorum-sensing system in Pseudomonas aeruginosa biofilm, and the effect on the expression of the genes associated with virulence factors in an established biofilm model in vitro.【Methods】Mature Pseudomonas aeruginosa BF was formed after 7 days culture on plate. After the treatment of ambroxol in different concentration, quantitative real-time RT-PCR were used to analyze the expression of toxA, rhlA, lasB and aprA which code virulence factors; enzyme-linked immunosorbent assay (ELISA) was used to quantify the exotoxin A and elastase; orcin-sulphuric acid method was used to quantify rhamnolipid; Folin-phenol colorimetry was used to quantify alkaline protease activity; pyocyanin were extracted with chloroform and quantified.【Results】After the treatment of ambroxol with the concentration of 3.75 mg/mL, the mRNA expression of toxA, rhlA, lasB, aprA were down-regulated than the control group from 1.000 to 3.033±0.083, 2.702±0.086,2.095±0.095, 2.196±0.097 respectively (P<0.05); the contents of exotoxin A, elastase, rhamnolipid, alkaline protease and pyocyanin were decreased from 15.220±0.989 mg/L, 81.384±5.440 ng/L, 289.725±6.719 mg/L, 300.588±6.526 U/L, 32.746±1.649 mg/L to 3.482±0.600 mg/L, 29.755±3.843 ng/L, 101.741±3.315 mg/L, 105.375±3.432 U/L, 13.876±1.055 mg/L respectively (P<0.05). The effect of ambroxol with the concentration of 1.875 mg/mL had the same tendency as the concentration of 3.75 mg/mL, but not so obvious (P<0.05).【Conclusions】Ambroxol can down-regulate the expression of virulence factors relative genes toxA, rhlA, lasB, aprA and the virulence factors of Quorum-sensing system in Pseudomonas aeruginosa biofilm.PART VEFFECT OF AMBROXOL ON THE QUORUM-SENSING SYSTERM IN PSEUDOMONAS AERUGINOSA BIOFILM IN VIVO【Objective】To establish murine model of tube–associated lung infection caused by Pseudomonas aeruginosa with biofilms formation, further to explore the effect of ambroxol on the Quorum-sensing system in Pseudomonas aeruginosa biofilms in vivo.【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; Group 2, rats with biofilm-covered tube intubation were given ambroxol; Group 3, rats with sterile tube intubation were given saline as control. On the 4th and 7th day after intubation, real-time RT-PCR were used to analyze the expression of the genes rhlI and lasI which code AHL synthetase the signal molecules AHL of QS system in Pseudomonas aeruginosa biofilm, AHL-responsive Agrobacterium tumefaciens reporter strain WCF47 and detection ofβ-galactosidase activity were utilized to quantify the signal molecules AHL. After HE staining of lung tissue, optical microscope was used to observe the pathological changes in lung tissue. According to the aforementioned method, detect the content of the virulence factors exotoxin A, elastase, rhamnolipid, alkaline protease and pyocyanin in lung tissue homogenate.【Results】Murine model of tube-associated lung infection caused by Pseudomonas aeruginosa with biofilms formation was established. At the 4th and 7th day after intubation, the relative expression value F of lasI in Group 1 and Group 2 were 1.000 VS 2.487±0.207(p<0.05) and 1.000 VS 3.221±0.211 (p<0.05); the relative expression value F of rhlI in Group 1 and Group 2 are 1.000 VS 1.591±0.197 (p<0.05)和1.000 VS 1.983±0.175 (p<0.05); the relative quantitative of AHL-----the activity ofβ-galactosidase were decreased from 563.875±25.991 and 641.375±19.398 in Group 1 to 420.250±26.391 and 356.001±32.351 in Group 2 (Miller Unit, p<0.05). The pathological changes in lung tissue of Group 2 have significant difference with Group 1. At the 4th and 7th day after intubation, the virulence factors in lung tissue homogenate of Group 1 were decreased in various degree after given ambroxol, especially 7th day.【Conclusion】In vivo, ambroxol could down-regulate the expression of the genes lasI and rhlI which code AHL synthetase and the signal molecules AHL of quorum-sensing system in Pseudomonas aeruginosa biofilm. Ambroxol can reduce the pathological changes in lung tissue and decrease the content of virulence factors in lung tissue homogenate, reduce the damage of lung tissue.