Anti-biofilm activity of Chlorella-mediated light activated disinfection: Ex vivo inhibition of intracanal mature Enterococcus faecalis biofilms via application of natural product

•The MIC of Chlo was 125 μg/mL, which inhibited the growth of E. faecalis.•Chlo-mediated LAD can be used as an adjuvant therapeutic to eliminate the E. faecalis biofilms.•Fluorescence microscope images showed that the highest percentage of dead bacteria during Chlo-mediated LAD. This study aimed to evaluate the effects of light-activated disinfection (LAD) as a strategy for optimizing root canal disinfection using Chlorella (Chlo) as a natural photosensitizer against Enterococcus faecalis biofilms ex vivo. The physical and chemical stability and absorption spectra of Chlo were determined. The mature microbial biofilm of E. faecalis was formed in root canals of 70 freshly extracted single-rooted premolars. After determining the minimum inhibitory concentration (MIC) of Chlo using the agar dilution testing, E. faecalis biofilms were exposed in the following groups (n = 10): 1- Chlo at a concentration of 2× MIC, 1- Chlo at a concentration of 4× MIC, 3- Diode laser, 4−2× MIC dose of Chlo-mediated LAD, 5−4× MIC dose of Chlo-mediated LAD, 6−0.2 % chlorohexidine (CHX), and 7- control group (E. faecalis biofilms without exposure to any photosensitizer and light source). The quantitative and qualitative evaluations of E. faecalis biofilms were done using counts of colony forming units (CFUs) and scanning electron microscope (SEM) and fluorescence microscope analysis, respectively. According to the results, the MIC of Chlo was 125 μg/mL, which inhibited the growth of E. faecalis. To evaluate the anti-biofilm effects of Chlo, the 2× and 4× MICs of Chlo (250 and 500 μg/mL, respectively) were used in the current study. The 4× MIC dose (500 μg/mL) of Chlo-mediated LAD was significantly more effective compared to other groups (P