Effects Of Ambroxol On Candida Albicans Biofilm

【Objective】The aim of this study was to investigate the inhibition of ambroxol on Candida albicans(C. albicans) biofilm(BF) in vitro by the abated tetrazolium salt (XTT) reduction assay.【Methods】Microtiter plate culture method was used to establish C. albicans ATCC 90028 BF. XTT reduction assay was performed to quantitively analyze the effect of ambroxol against C. albicans BF. Microscopic examination with silver staining was conducted to assess the effect of ambroxol on morphogenesis of mature BF of C. albicans.【Results】Models of 90min,24h,48h of C. albicans biofilms in vitro was formed on 96- well microtiter plates successfully. 90min BF, 24h BF and 48h BF was treated for 8h with Different concentration of ambroxol (0,0.25mg/ml,1.5mg/ml).The OD450 values of 90min BF by XTT reduction assay were 2.18±0.10,1.91±0.14,0.59±0.11,respectively.The OD450 values of 24h BF were 3.16±0.15,2.83±0.11,1.08±0.15,respectively. The OD450 values of 48h BF were 3.23±0.10,2.92±0.11,1.21±0.12,respectively.There was significant difference between any two groups at same time intervals(90min,24 h, 48 h) (p < 0.05). A coherent woven-like structure of BF by silver staining became loose after ambroxol treated mature BF of C. albicans. Part of structure was destroyed and 1.5mg/ml of ambroxol was more effective than 0.25mg/ml.【Conclusion】Ambroxol could inhibit C. albicans BF at different time intervals(90min,24 h, 48 h). Moreover the ability to inhibit the BF increased with its concentration increasing. Effects of ambroxol on mature BF of C. albicans were qualitatively observed under microscope by silver staining. 【Objective】To investigate the effect of ambroxol on C. albicans BF forming process with Confocal Laser Scanning Microscopy(CLSM). To quantify the BF structure by Image Structure Analyzer(ISA) software.To explore fungicidal action of ambroxol against C. albicans BF.【Methods】C. albicans ATCC 10231 BF models were established on coverslip at different time intervals (90min,24h,48h). There were ambroxol 0 mg/ml group(control group),ambroxol 0.25mg/ml group and ambroxol 1.5mg/ml group. In the control group, C. albicans BF stained with FITC-ConA was observed under fluorescence microscope. The parameters of 90min,24h,48h BF structure were analyzed through pictures from CLSM with ISA software after the BF was treated with different concentrations of ambroxol for 8h. The images were analyzed with Image Pro Plus 6.0(IPP 6.0) software. And fungal mortalities under the different condition (concentration of ambroxol and culture time) were calculated.【Results】In the control group,the observed BF at different time intervals by fluorescent microscope showed that green fluorescent spots on coverslip and green fluorescence enhanced with the extension of BF growth time and microcolonies formed gradually a coherent woven-like structure. The results of ISA showed that BF thickness, the average diffusion distance (ADD) and structure of the entropy (TE) increased, and areal porosity (AP) decreased with the extension of the culture time in the control group. Moreover BF thickness, ADD and TE were reduced significantly(P < 0.05) after BF was treated for 8h with ambroxol (0.25mg/ml, 1.5mg/ml) in comparison with the control group. However AP was increased significantly (P < 0.05). The results of IPP 6.0 showed the fungal mortality of BF at same time points treated with 0.25mg/ml and 1.5mg/ml ambroxol was significantly higher than that of the control group (P < 0.05), respectively. There was significant difference in the mortality between the concentration of 0.25mg/ml and 1.5mg/ml ambroxol(P < 0.05), too.【Conclusion】ISA software can quantify the structure of C. albicans BF at different stage.The structure of C. albicans BF could be destroyed and fungal mortality increased after BF at different time intervals was treated by ambroxol. Moreover effects of 1.5mg/ml ambroxol on fungal mortality and spatial structure were also significantly higher than that of 0.25mg/ml ambroxol.