Objectives: This investigation adapted the LIVE/DEAD� Baclight[TM] bacterial viability stain for the quantitative determination of bacterial cell viability of the aerotolerant anaerobes Porphyromonas gingivalis ATCC 33277 and Prevotella intermedia ATCC 25611. The Live/Dead stain was used to determine the influence of haem availability on the resistance of P. gingivalis and P. intermedia to the reactive oxygen species (ROS) superoxide anion and hydrogen peroxide and compare the sensitivities between the haem-requiring periodontal bacteria to ROS. Neutrophils use oxidative and non-oxidative killing mechanisms. During phagocytosis, neutrophils kill bacteria via a respiratory burst, producing ROS. P. gingivalis and P. intermedia are oxygen-tolerant gram-negative bacteria found in the gingival crevice. These bacteria express superoxide dismutase (SOD) activity, which extends some protection against superoxide radicals.
Methods: Initially, experiments were performed to validate the reliability and accuracy of the fluorogenic Live/Dead stain using Escherichia coli ATCC 10798 (K-12), followed by experiments using P. gingivalis. The Live/Dead stain distinguishes viable:non-viable proportions of bacteria using mixtures of green (SYTO 9) and red (propidium iodide) fluorescent nucleic acid stains respectively. Bacterial cell viability was assessed with fluorescence microscopy and subsequently quantitative measurement using a fluorescence microplate reader (BMG Fluorostar plus Optima). P. gingivalis and P. intermedia colonies were subcultured from frozen cultures, in Tryptic soy broth (TSB) (Difco) and incubated anaerobically for approximately five days. They were further subcultured in pre-reduced TSB, supplemented with menadione 0.5[mu]g/ml (TSB-M) and either 5 [mu]g/ml haemin (Haem 5), 50 [mu]g/ml haemin (Haem 50) or without supplemental haemin (Haem 0). Cultures were grown anaerobically at 37�C to early stationary phase (approximately 48 hours). For experimental purposes, bacteria were harvested, washed and resuspended in 10 mM Tris-buffered saline (pH 7.5) containing peptone (TBS-P) (0.1 mg/ml), with a final adjustment to OD₅₄₀ [approximately equals] 2.0 (which corresponds to 1 x 10⁹ bacteria/ml). Bacterial suspensions were diluted ([approximately equals] 10⁸/ml) into TBS-P containing the fluorogenic viability stain (BacLight, Molecular Probes). Either pyrogallol (0.02 - 2 mM) or hydrogen peroxide (0.01 - 100 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: For both P. gingivalis and P. intermedia, a pyrogallol concentration of 0.2 mM resulted in 80 to 90% cell death; and a hydrogen peroxide concentration of 10 mM killed approximately 80 to 90% of cells. Irrespective of the haem status, no significant difference was determined between the overall maximum rate of killing of P. gingivalis and P. intermedia, in their response to either superoxide or hydrogen peroxide; with the exception that the P. intermedia Haem 0 group was significantly less susceptible to hydrogen peroxide than the P. gingivalis Haem 0 group. For the majority of the experiments, there was no significant difference between final bacterial cell viability in the Haem 0 and Haem 5 cells for both species, after 3 hours exposure to various concentrations of ROS. However, the Haem 50 cells showed a significant increased susceptibility (albeit, a small difference) to both hydrogen peroxide and superoxide.
Conclusions: The Live/Dead bacterial viability stain provided a valuable method to monitor "real-time" killing, avoiding the difficulties associated with culture-based methods for assessing viability. Haem availability had no clear influence on the resistance to ROS of either P. gingivalis or P. intermedia Haem 0 and Haem 5 cells. The Haem 50 cells showed a very slight increase in susceptibility to hydrogen peroxide and superoxide. 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 haem availability did not influence sensitivity implies that these bacteria do not accumulate haem for the purpose of protection from ROS.
| Identifer | oai:union.ndltd.org:ADTP/217776 |
| Date | January 2007 |
| Creators | Mackie, Tasha A, n/a |
| Publisher | University of Otago. School of Dentistry |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Tasha A Mackie |
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