Intracellular-Survival and Inflammatory-Potential of Enterococcus-Faecalis- Biofilm on Root-Cannal-Dentine

Enterococcus faecalis, a prevalent bacterium in treatment failed root canals has the potential to form distinct biofilms under different environmental conditions. E. faecalis biofilms has been known to resist adverse growth conditions and antimicrobials. Understanding the interaction of E faecalis biofilm cells with host-cells and their inflammatory-potential under simulated growth conditions will be of great importance in understanding disease progression. Objectives: This study evaluated three E. faecalis biofilm strains having varying virulence factors under nutrient-rich and deprived-conditions on their (1) intracellular survival within monocyte derived macrophages and human cell lines, and (2) its potential to induce inflammatory response. Method: The OG1RF is a gelatinase and serine-protease producing strain, ATCC-29212 is a gelatinase negative and FA2-2 is a commonly used laboratory- strain. Three E. faecalis strains (ATCC-29212, OG1RF and FA2-2) were grown for a week on dentine blocks in All-Culture medium (nutrient-rich condition) and phosphate buffered saline (nutrient-deprived condition). Biofilm-derived E. faecalis cells were incubated for 4 hrs in CO2 incubator with in-vitro differentiated human macrophages and a human monocytic cell-line, THP-1. The number of viable internalized bacteria was quantified by an antibiotic protection assay. Simultaneously, the nitric oxide levels, of the infected culture supernatants were measured using the Griess reagent. Results: The number of internalized bacteria from the nutrient-deprived biofilm grown on root-canal-dentine of OG1RF strain was significantly higher than from the biofilm grown under nutrient-rich conditions (p=0.02). However, the nitric oxide levels tested showed a significant increase in inflammatory response in the nutrient-deprived biofilm (p≤0.05) than from the biofilm grown under nutrient-rich conditions of the three bacterial strains tested. Conclusions: It is apparent from this study that E. faecalis biofilm developed under nutrient-deprived conditions similar to post-endodontic environment are more efficient in surviving within macrophages, and induce inflammatory response.

Acknowledgment: NUS-ARF R-224-000-024-112 is gratefully acknowledged