Objectives: (i) To compare the sensitivities of the heme-requiring, periodontal bacteria
Porphyromonas gingivalis and
Prevotella intermedia to reactive oxygen species (ROS); (ii) To determine the influence of hemin availability on bacterial sensitivities to ROS. Methods: Bacteria were cultivated for 48 hours in tryptic soy broth supplemented with menadione (0.5 µg/ml)(TSBM) and in TSBM with 5 µg hemin/ml and with 50 µg hemin/ml. Bacteria were harvested, washed and resuspended in 10 mM Tris-buffered saline (pH 7.5) containing peptone (0.1 mg/ml)(TBS). Bacterial suspensions were diluted (≈10
8/ml) into TBS containing fluorescent viability stains (BacLight, Molecular Probes). Either pyrogallol (0.02 – 2 mM) or hydrogen peroxide (0.01 - 10 mM) was added (except to control tubes); tubes were vortexed for ten seconds and incubated at 37°C. Viability was monitored fluorimetrically for three hours. Results: Irrespective of the concentration of hemin in the culture medium, exposure of washed bacteria to the same concentration of ROS resulted in cell death rates that were within 20% of the mean rate for all tubes. For both
P. gingivalis and
P. intermedia, pyrogallol provided at 0.2 mM resulted in 80 to 90% cell death; and hydrogen peroxide provided at 10 mM killed approximately 90% of cells of both species. Conclusions: Although
P. intermedia may be isolated in significant numbers from healthy gingivae as well as from periodontally diseased sites, it was no more resistant to ROS than was
P. gingivalis which is associated with periodontal lesions and difficult to cultivate from relatively healthy (more oxygenated) sites. This suggests that resistance to ROS does not contribute to the ecological distinction between these two species. The finding that hemin availability did not influence sensitivity implies that these bacteria do not accumulate heme for the purpose of protection from ROS.
Supported by the New Zealand Dental Research Foundation Board.