Haem and reactive oxygen species in killing of Porphyromonas gingivalis and Prevotella intermedia

Abstract

Objectives: Porphyromonas gingivalis (P. gingivalis) and Prevotella intermedia (P. intermedia) are Gram-negative oral anaerobic bacteria implicated in the pathogenesis of periodontal diseases. P. intermedia can be cultivated in greater frequency and numbers than P. gingivalis from relatively healthy and mildly inflamed oral gingival sites. However, P. gingivalis may be more pathogenic. Healthy and mildly inflamed gingival tissues are more oxygenated than areas involved with periodontitis. Local defence of the periodontium is provided by large numbers of migrating neutrophils. The neutrophils attempt to kill the bacteria by releasing reactive oxygen species (ROS) including the superoxide anion, hydrogen peroxide and hypochlorite. The aim of this study was to investigate the effect of haem availability during growth on the sensitivities of P. gingivalis and P. intermedia to ROS while also investigating whether P. intermedia is more resistant to oxidative environments than P. gingivalis.

Methods: P. gingivalis (strains ATCC 33277 and W50) and P. intermedia (strain FDC 581) were revived from frozen stocks by culturing for 7-10 days at 37 °C under anaerobic conditions. Cultures were grown on tryptic soy broth agar containing menadione (0.5 µg/ml), haem (5 µg/ml) and sheep blood (5 % v/v). The bacteria were then grown for 48 hours in pre-reduced tryptic soy broth supplemented with menadione (TSB-M) and this culture used to inoculate TSB-M and TSB-M supplemented with haem (5 µg/ml). Following incubation for 48 hours, cells were harvested and washed three times in Tris-buffered saline (pH 7.4) containing peptone (100 mg/ml) (TBS-P) and adjusted to equivalent concentrations of cells. Bacterial suspensions were then diluted (to approximately 108 cells/ml) into TBS-P containing fluorogenic viability stains (Baclight™ Live/Dead® viability kit). Hydrogen peroxide (0.01 mM – 1 mM), pyrogallol (0.2 mM – 20 mM) or hypochlorite (1 mM – 6 mM) was added and bacteria were incubated at 37 °C for three hours. Samples (200 µl) were removed from each preparation and bacterial viability determined in a spectrofluorimeter (Synergy 2, BioTek). Results: The Baclight stain was partially successful in assessing the viability of bacteria. The green stain was able to quantify viable bacteria; however, while the red stain quenched the green stain as viability decreased, there was no increase in the relative fluorescence of the stain. Haem supplementation did not advantage either P. gingivalis or P. intermedia when exposed to any of the tested ROS. P. gingivalis W50 was slightly more resistant to 0.1mM hydrogen peroxide than was the less virulent P. gingivalis ATCC 33277 and P. intermedia FDC 581. However, P. intermedia FDC 581 was significantly more resistant to 2mM hypochlorite than were the P. gingivalis strains. There was no difference between the bacterial strains in response to any of the ROS tested at other concentrations. Conclusion: Supplemental haem did not confer an advantage to either P. gingivalis or P. intermedia when exposed to hydrogen peroxide, superoxide or hypochlorite. Differences between haem-replete and haem-starved bacteria were not detectable. While P. intermedia was significantly more resistant to hypochlorite than was P. gingivalis, this difference was small. It therefore seems unlikely that resistance to hydrogen peroxide, hypochlorite and superoxide accounts for the ecological difference reported between P. gingivalis and P. intermedia.