Biofilm treatment

Biofilm received the different treatments tested on planktonic samples, but with only two concentrations for each drug. The concentrations of chlorhexidine and nystatin used were equal to or just above the MIC in each group. The MIC used for nystatin was based on Scheibler et al. [17]. All assays were performed with 10 replicates.

After the treatments, the biofilm plates with the drugs were incubated at 37 °C for 48 h. Later, the biofilm was gently washed twice with PBS, stained with 0.1% crystal violet for 10 min, washed with PBS, and fixed with 96% ethanol. The crystal violet OD, which indicates the concentration of viable cells in the biofilms, was determined with a spectrophotometer (SpectraMax 190) at 570 nm. Also, the coefficient of drug interaction (CDI) was calculated according to the formula CDI = AB/(AxB), where AB is the ratio of the treatment combination to the control and A or B is the ratio of the single treatment to the control. CDI greater than 1 indicates that the drugs are antagonistic [27].

Since the crystal violet colorimetric assay determines the total biomass of the mixed-species biofilm, without distinguishing S. mutans from C. albicans, CFU counting was also performed [28]. The biofilm was washed with sterile PBS; cells were resuspended in 200 µL of PBS and subjected to ultrasound for 10 min, followed by pipetting up and down. Subsequently, cell suspensions underwent serial dilution and were plated (10 µL drops) on BHI agar with amphotericin B (0.025 mg/mL) and incubated at 37 °C for bacterial growth; the same plating was done on Sabouraud dextrose agar (SDA; Himedia) with chloramphenicol (0.005 mg/mL) at 30 °C for yeast growth. The incubation period was 48 h [23, 29]. Colonies formed were then counted, and CFU/mL was determined for S. mutans and C. albicans, for the different biofilm treatments. The assay was performed in triplicate.