A preliminary antimicrobial investigation of lactobacillus bulgaricus probiotic supernatants as an intracanal medication against mature E. faecalis biofilm in an ex-vivo dentin blocks model

Abstract

To provide a preliminary assessment of the antimicrobial potential of Lactobacillus bulgaricus probiotic against mature Enterococcus faecalis biofilm in dentin model under ex-vivo conditions using confocal laser scanning microscopy (CLSM). Lactobacillus bulgaricus and Lactobacillus acidophilus probiotic strains were grown, centrifuged, filtered by syringe filter size 0.45 microns to acquire cell free supernatants (CFS). Lyophilized CFS of each probiotic strain was individually mixed with Poloxamer gel using cold technique to prepare probiotic supernatants (PS) gel. Dentin blocks from cervical root portions were standardized at similar dimensions, infected with E. faecalis for 21 days to generate a mature biofilm. At the end of incubation, infected dentin blocks were randomly assigned to five groups (n = 10) based on the medication as follows: positive control (PC), calcium hydroxide (CH), Lactobacillus acidophilus PS gel (LA), Lactobacillus bulgaricus PS gel (LB), Lactobacillus combination PS gel (LC) combining LA and LB at a 1:1 ratio. All dentin blocks were medicated and incubated for 7 days under anaerobic conditions. Then, dentin blocks were examined with CLSM at 40X magnification; three-dimensional imaging in Z-stack mode was processed, and the dead E. faecalis% was quantitatively assessed using bioImage_L v21 software and total biovolume was calculated. Dead E. faecalis% were compared across groups using a one-way ANOVA test followed by Tukey’s post hoc test. Biovolume data were compared using the Kruskal-Wallis test followed by Dunn’s post hoc test with p-value adjustment using Holm’s method. The significance level was set at p < 0.05 for all tests. Statistical analysis was performed using R version 4.6.0 for Windows. LB was linked to the highest percentage of dead E. faecalis% (p < 0.05), followed by LC, LA, CH, and the PC. Significant differences in dead E. faecalis% were observed between all groups (p < 0.05) except for CH and the PC groups. There was no significant difference in total biovolume among all groups. The Initial assessment of LB showed a promising antimicrobial action in dentin model that exceeded LA and CH. As biofilm biovolume was unaffected, LB appears to reduce bacterial viability rather than remove biofilm mass, warranting further validation in polymicrobial and intact canal models. Probiotics may be of clinical relevance due to their antimicrobial action against E. faecalis.