IADR Abstract Archives
Hemin-Dependent Regulation in Persistence of Porphyromonas gingivalis
Objectives: Porphyromonas gingivalis is the keystone pathogen that critically accounts for periodontal disease via modulation of symbiotic oral bacteria and bacteria-host crosstalk. Emerging evidence shows that microenvironmental hemin levels affect the virulence of P. gingivalis and its ability to evade host defense. This study investigated the ability of P. gingivalis to form persisters and the effects of hemin on its persistence.
Methods: P. gingivalis ATCC 33277 was cultured anaerobically in broth containing 10 μg/ml hemin (hemin-excess), 1 μg/ml hemin (hemin-limitation) or without hemin (hemin-deficiency). The bacterial growth was monitored by measuring the optical density at 660 nm. The MIC of metronidazole was determined by broth microdilution method. Next, late-exponential cultures were treated with 100 μg/ml of metronidazole for 6 and 24 h, and the surviving P. gingivalis persisters were quantified by CFU counting. P. gingivalis biofilms were established on 96-well polystyrene plates for 24 h, and assessed by crystal violet assay. Confocal imaging was adopted to evaluate the persister viability and biofilm formation.
Results: The P. gingivalis incubated with varying concentrations of hemin showed similar profiles of planktonic growth and biofilm formation. The susceptibility testing revealed same MIC of metronidazole (0.125μg/ml) independent of hemin availability. Interestingly, small subpopulations of P. gingivalis persisters were identified after 6 and 24h treatments of metronidazole at 100μg/ml. Relatively higher fraction of persisters was observed under hemin-limitation (5.1×10-5) than those under hemin-deficiency (8.1×10-6) and hemin-excess (7.2×10-6) at 6h. At 24h, the fractions lowered to 6.7×10-7 and 5.2×10-7 under hemin-limitation and hemin-excess respectively, while the value was below detection limit under hemin-deficiency.
Conclusions: This pioneering study shows for the first time the presence of P. gingivalis persisters, and hemin may be a crucial mediator of oral pathogen persistence. Controlling oral inflammation and hemin level at periodontal niches is an important element in management of periodontal disease.
Methods: P. gingivalis ATCC 33277 was cultured anaerobically in broth containing 10 μg/ml hemin (hemin-excess), 1 μg/ml hemin (hemin-limitation) or without hemin (hemin-deficiency). The bacterial growth was monitored by measuring the optical density at 660 nm. The MIC of metronidazole was determined by broth microdilution method. Next, late-exponential cultures were treated with 100 μg/ml of metronidazole for 6 and 24 h, and the surviving P. gingivalis persisters were quantified by CFU counting. P. gingivalis biofilms were established on 96-well polystyrene plates for 24 h, and assessed by crystal violet assay. Confocal imaging was adopted to evaluate the persister viability and biofilm formation.
Results: The P. gingivalis incubated with varying concentrations of hemin showed similar profiles of planktonic growth and biofilm formation. The susceptibility testing revealed same MIC of metronidazole (0.125μg/ml) independent of hemin availability. Interestingly, small subpopulations of P. gingivalis persisters were identified after 6 and 24h treatments of metronidazole at 100μg/ml. Relatively higher fraction of persisters was observed under hemin-limitation (5.1×10-5) than those under hemin-deficiency (8.1×10-6) and hemin-excess (7.2×10-6) at 6h. At 24h, the fractions lowered to 6.7×10-7 and 5.2×10-7 under hemin-limitation and hemin-excess respectively, while the value was below detection limit under hemin-deficiency.
Conclusions: This pioneering study shows for the first time the presence of P. gingivalis persisters, and hemin may be a crucial mediator of oral pathogen persistence. Controlling oral inflammation and hemin level at periodontal niches is an important element in management of periodontal disease.