Immunogenic Potential Of Enterococcus Faecalis Biofilm Under Simulated Growth Conditions

Enterococcus faecalis is the most common and occasionally the only bacterial species to be isolated from root canals of treatment-failed teeth. Treatments of E. faecalis infections are usually considered difficult because of its high resistance to antimicrobials, and due to their ability to withstand harsh environments. Understanding the host immunological response to E. faecalis biofilm produced under different simulated conditions will provide a better insight into persistent endodontic infections. Objective: This study evaluates the intracellular survival of E. faecalis biofilm grown in nutrient-rich and nutrient-deprived conditions. Method: One-week old E. faecalis biofilm was grown on perspex and glass substrates in All-Culture medium (nutrient-rich condition) and phosphate buffered saline (nutrient-deprived condition). As model systems, human monocytic (THP-1) and histiocytic (U937) cell lines were used. These cell lines were incubated with the biofilm for 4 hrs in CO2 incubator. The cells and bacteria were stained with acridine orange and quenched with crystal violet to distinguish between surface-bound and internalized bacteria. The number of viable internalized bacteria was quantified by a standard antibiotic protection assay. The extracellular E. faecalis cells were killed by treating with HAP buffer supplemented with antibiotics. The ‘protected' intracellular bacteria were quantified by plating serial dilution of macrophage lysates. Results: The presence of green-fluorescing internalized bacteria was detected within the macrophages under both the tested conditions. Furthermore, the number of internalized bacteria from the nutrient deprived biofilm was significantly higher than from biofilm grown under nutrient-rich conditions (p=0.01) for both THP1 and U937 cells. Conclusions: It is apparent from the study that E. faecalis grown as biofilm under nutrient-deprived conditions are more efficient in invading and surviving within the macrophages than those grown under nutrient-rich conditions. Survival of E. faecalis within macrophages can allow dissemination of the bacterium and subsequent systemic infection. Acknowledgment: NUS-ARF R-224-000-024-112 is gratefully acknowledged.