Effectiveness of D,L-2-hydroxyisocaproic acid (HICA) and alpha-mangostin against endodontopathogenic microorganisms in a multispecies bacterial–fungal biofilm in an ex vivo tooth model

Abstract

Aim: To develop a defined multispecies root canal biofilm model ex vivo, and to perform viable compositional analysis following D,L-2-hydroxyisocaproic acid (HICA), alpha-mangostin, Calcicur®, and Odontopaste® exposure. Methodology: Time-kill assays were conducted in vitro using HICA, alpha-mangostin, Calcicur®, Odontopaste®, and saline solution on the planktonic cultures of C. albicans, E. faecalis, L. rhamnosus, and S. gordonii. Human root dentine blocks were prepared (n = 100) ex vivo, and multispecies suspensions containing each of 1.5 × 108 CFU/mL C. albicans, E. faecalis, L. rhamnosus, and S. gordonii in brain heart infusion (BHI) were incubated within the root canals for 21 days. Canals (n = 20/group) were then exposed to medicaments for 7 days. Samples taken from the inner (first 0.1 mm) and deeper (second 0.1 mm) dentine by drilling with Ash Steel Burs No. 5 and No. 6, and residual roots were cultured in broth for 24 h. Cell growth was detected by spectrophotometry and confirmed by culture on agar. The other set of inner dentine, deeper dentine, and residual root samples were sonicated, and then exposed with 50 μM PMA before DNA was extracted using the QIAamp DNA mini kit. Real-time quantitative PCR was performed to determine the biofilm composition as well as the number of live and total cells remaining in the biofilm following each treatment. The OD data were analysed with Kruskal–Wallis and Friedman with Wilcoxon signed-rank test between and within groups, respectively, agar culture and qPCR data with Pearson chi-square with Mann–Whitney and Cochran with McNemar tests, respectively (p <.0001). Results: Time-kill assays revealed that HICA and Calcicur® killed all planktonic organisms within 24 h, whilst alpha-mangostin killed the organisms within 72 h. However, Odontopaste® was a slow-killing agent: 10 cells of planktonic organisms survived after exposure to the agent for 7 days. The ex vivo tooth model demonstrated that HICA and alpha-mangostin significantly inhibited the cell growth in all sampling depths (p <.0001). All species-specific data revealed the effectiveness of each medicament on the biofilm composition. Conclusions: D,L-2-hydroxyisocaproic acid and alpha-mangostin had antimicrobial activity against multispecies bacterial–fungal biofilms.