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Aim: To develop a reproducible biofilm model consisting of <i>Enterococcus faecalis</i> (<i>E. faecalis</i>) and <i>Porphyromonas gingivalis</i> (<i>P. gingivalis</i>) and to evaluate the interaction between the two bacterial species. Methodology: <i>E. faecalis</i> and <i>P. gingivalis</i> were grown in mono-culture, sequential, and co-culture models for 96 h in a 96-well polystyrene microtiter plate under both aerobic and anaerobic conditions separately. The viability of the two bacterial species in the biofilms was quantified by polymerase chain reaction (qPCR). Biofilm thickness and protein contents were measured using confocal laser scanning microscopy (CLSM). Two-way analysis of variance (ANOVA) was performed to analyze cell viability and biofilm thickness among different culture models cultivated under either aerobic or anaerobic conditions. The level of significance was set at <i>p</i> < 0.05. Results: Different culture models tested did not show any significant difference between the viable cell counts of both <i>E. faecalis</i> and <i>P. gingivalis</i> cultivated under aerobic and anaerobic conditions (<i>p</i> > 0.05). Biofilm was significantly thicker (<i>p</i> < 0.05) in the co-culture models compared to the mono-culture and sequential models. Protein contents in the biofilms were more pronounced when both bacterial species were co-cultured under aerobic conditions. Conclusions: <i>E. faecalis</i> appeared to shield <i>P. gingivalis</i> and support its continued growth in oxic (aerobic) conditions. The co-culture model of <i>E. faecalis</i> and <i>P. gingivalis</i> produced a significantly thicker biofilm irrespective of the presence or absence of oxygen, while increased protein contents were only observed in the presence of oxygen.