Effect Of Ambroxol On The Structure Of Staphylococcus Epidermidis Biofilm And Bactericidal Action

Partâ… Establishment and analysis the model of staphylococcus epidermidis biofilm【Objective】To establish the model of staphylococcus epidermidis biofilm in vitro, observe and quantitatively analyze the dynamic course of the biofilm formation.【Method】Biofilm model of biofilm-producing strain staphylococcus epidermidis RP62A was formed on plate. The metabolic activity of the bacteria inside biofilm was measured by tetrazolium salt (XTT) reduction assay. The parameters of BF structure were analyzed through pictures from Confocal Laser Scanning Microscopy (CLSM) with Image Structure Analyer (ISA) software. The structure of the biofilm was detected by scanning electron microscope (SEM).【Results】The model of staphylococcus epidermidis biofilm in vitro could established on coverslip. The data of A₄₅₀ by XTT reduction assay at different time intervals (12h, 24h and 48h) were 2.392±0.481, 3.410±0.177 and 3.917±0.274, respectively. There were significant difference between any two groups(P<0.05). The data from ISA soft showed that the areal porosity (AP) were the 0.84±0.08, 0.68±0.01 and 0.59±0.13, respectively; the average diffusion distance (ADD) were 1.34±0.24, 1.49±0.09 and 1.89±0.39, respectively; There were significant difference between any two groups(P<0.05). The textural entropy (TE) were 4.71±0.82, 8.69±0.68 and 8.94±0.28, respectively. There were significant difference between the 12h group and the other two groups(P<0.05). Mature biofilm was detected by SEM at 24h and the structure of biofilm became more complex at 48h.【Conclusions】The model of staphylococcus epidermidis biofilm in vitro could established on coverslip. The formation of staphylococcus epidermidis BF is a dynamic course. Mature biofilm was formed at the 24^th hour. The structure became more complex at the 48^th hour. XTT reduction assay, CLSM with ISA and SEM are the ideal methods to observe and quantitatively analyze the biofilm model in vitro.Partâ…¡Effect of ambroxol on the structure of staphylococcus epidermidis biofilm and bactericidal action【objective】To investigate the effect of ambroxol on staphylococcus epidermidis biofilm (BF) in an established biofilm model in vitro.【Method】The strain RP62A of Staphylococcus epidermidis was incubated for 24h to form BF on slide. After treated by ambroxol of different concentrations for 24h, the BF structure was observed 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; the metabolic activity of bacteria in biofilm was measured by tetrazolium salt (XTT) reduction assay.【Results】After the treatment of 1.875mg/ml ambroxol, the BF was destroyed and the matrix outsides became thin and sparseness compared with the control by SEM, and small amount of bacteria scattered. BF thickness of the 1.875 mg/ml ambroxol treatment group and control group were(27.95±5.93)μm and( 9.76±1.29)μm, respectively., t=7.955, P <0.05, areal porosity (AP) were 0.772±0.036 and 0.889±0.063,respectively, t=–4.322, P <0.05; average Diffusion Distance (ADD) were 2.173±0.219 and 1.625±0.092,respectively, t=5.640, P <0.05,and textual Entropy (TE) were 7.706±0.610 and 5.488±0.747, respectively, t=5.633, P <0.05; The effect of ambroxol 0.75mg/ml has the same tendency as ambroxol 1.875 mg/ml group, but not so obvious. After the administration by ambroxol of different concentration (1.875mg/ml, 3.7mg/ml, 7.5mg/ml), the A₄₅₀ value were1.288±0.251,0.548±0.170 and 0.156±0.051 respectively. There were significant differences between each group and the control group(P <0.05).【Conclusions】Ambroxol can destroy the BF structure of Staphylococcus epidermidis. With increasing concentration of ambroxol, the bactericidal activity increased.