RESUMO
The objective of the study was to establish an in vitro model of Candida albicans-Staphylococcus epidermidis mixed species biofilm (BF) on polyvinyl chloride (PVC) material, and to investigate the formation and the structure of mixed species BF formation using a combined approach of confocal laser scanning microscope (CLSM), scanning electron microscope (SEM), and 3D image reconstruction technique. Mixed species BF is achieved by co-incubating Staphylococcus epidermidis bacteria (ATCC35984) and Candida albicans fungal (ATCC10231) with PVC pieces in Tris-buffered saline. BF formation was examined at 2, 6, 12, 24, 48, and 72 h of co-culture. Thickness of these BFs and the number, and percentage of viable cells in BFs were measured. CT scan images of BFs were obtained using CLSM and SEM and reconstructed 3D images of mixed species BF were acquired, in an effort to examine structure of the BF. Staphylococcus epidermidis attached to various forms of candida albicans (spores, pseudohyphae, and hyphae), formed complex and dense mesh arrays. The BF is constituted of a large number of viable and dead pathogens, the surface of mixed species BF is uneven, with living pathogens predominating protrusive portions and dead pathogens aggregating in concaves. Mixed species BF formation on the surface of PVC material was found to be a dynamic process, with rapid growth being at 24 h of co-culture, maximal thickness peaked at 48 h. These mixed species BF matured at 48-72 h. Significant differences were observed in the proportion of viable cells between interior, middle, and outer layers of BFs (p < 0.05). Mixed species BF Candida albicans-Staphylococcus epidermidis is sophisticated in structure. The combined approach involving CLSM, SEM, and 3D image reconstruction technique is ideal for the investigation of mixed species BF on PVC material.