Michael Wilson

A Study of the Uptake of Toluidine Blue O by Porphyromonas gingivalis and the Mechanism of Lethal Photosensitization

The purpose of the study was to determine the distribution of the photosensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization of this organism. The distribution of TBO was determined by incubating P. gingivalis with tritiated TBO (3H‐TBO) and fractionating the cells into outer membrane (OM), plasma membrane (PM), cytoplasmic proteins, other cytoplasmic constituents and DNA. The percentage of TBO in each of the fractions was found to be, 86.7, 5.4, 1.9, 5.7 and 0.3%, respectively. The involvement of cytotoxic species in the lethal photosensitization induced by light from a helium‐neon (HeNe) laser and TBO was investigated by using deuterium oxide (D2O), which prolongs the lifetime of singlet oxygen, and the free radical and singlet oxygen scavenger L‐tryptophan. There were 9.0 log10 and 2 log10 reductions in the presence of D2O and H2O (saline solutions), respectively, at a light dose of 0.44 J (energy density = 0.22 J/cm2), suggesting the involvement of singlet oxygen. Decreased kills were attained in the presence of increasing concentrations of L‐tryptophan. The effect of lethal photosensitization on whole cell proteins was determined by measuring tryptophan fluorescence, which decreased by 30% using 4.3 J (energy density = 4.3 J/ cm2) of light. Effects on the OM and PM proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was evidence of change in the molecular masses of several PM proteins and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscopic studies showed that there was coaggre‐gation of P. gingivalis cells when sensitized and then exposed to laser light. These results suggest that lethal photosensitization of P. gingivalis may involve changes in OM and/or PM proteins and DNA damage mediated by singlet oxygen.

Effect of dosimetric and physiological factors on the lethal photosensitization of Porphyromonas gingivalis in vitro.

The aims of this study were to (1) determine the effect of dosimetric and physiological factors on the lethal photosensitization of Porphyromonas gingivalis using tolui‐dine blue O (TBO) and light from a helium/neon (HeNe) laser; (2) determine the influence of sensitizer concentration, preirradiation time, serum and growth phase on sensitizer uptake by P. gingivalis. The dosimetric factors studied were concentration of TBO, light dose and preirradiation time. The physiological factors were presence of serum, pH and bacterial growth phase. Sensitizer uptake by P. gingivalis under various conditions was determined using tritiated TBO (3H‐TBO). In the presence of TBO, a light dose‐dependent increase in kill was attained (100% kill at 4.4 J). There was no significant effect on the numbers killed when TBO was increased from 12.5 to 50 µg/mL. An increase in preirradiation time gave slightly increased kills. High kills were achieved at all three pH (6.8–8.0). Although kills were substantial in the presence of serum, they were significantly less than those obtained in the presence of saline. Cells in all three growth phases were susceptible to lethal photosensitization, although stationary phase cells were slightly less susceptible. Maximum uptake of TBO occurred within 60 s and uptake in serum was less than in saline. The uptake by the log phase cells was greater at lower concentrations of sensitizer (50 µg/mL), compared to the other two phases.

Effect of chlorhexidine on multi-species biofilms.

Abstract. With human saliva as an inoculum, microcosm dental plaques were grown on dental amalgam in a constant-depth film fermentor (CDFF) in the presence (suc +ve) and absence (suc −ve) of sucrose. The biofilms were then exposed to 0.2% chlorhexidine gluconate (CHG) for 1, 5, or 60 min and the survivors enumerated. Suc +ve biofilms had higher proportions of streptococci but lower proportions of veillonellae than suc −ve biofilms. Exposure to CHG for 1 min reduced the viable count of suc −ve and suc +ve biofilms by 53% and 89% respectively. In both cases, reductions were mainly attributable to killing of streptococci and actinomyces. After 60 min of exposure, 4% of the bacteria in the suc −ve and 2% in the suc +ve biofilms remained viable. This study has shown that large numbers of bacteria in microcosm dental plaques can survive a 1-min exposure to 0.2% CHG and that even after a 60-min exposure, substantial numbers of bacteria remain viable.

Effect of environmental factors on the lethal photosensitization ofCandida albicans in vitro

Suspensions ofCandida albicans in saliva, serum, broth or saline were treated with toluidine blue O or methylene blue and exposed to light from a helium/neon orgallium aluminium arsenide laser, respectively, for 120 s and survivors enumerated. Experiments were also carried out in broth at a pH ranging from 4.0 to 7.0. Appreciable kills (>106 cfu ml−1) of the yeast were obtained regardless of the nature of the suspending fluid. Lethal photosensitization of the yeast was also possible at a pH as low as 4.0 Only short-term (3 min) incubatin of the photosensitizer with the yeast was necessary to render it susceptible to killing by laser light.

Inactivation of Proteolytic Enzymes from Porphyromonas gingivalis Using Light-activated Agents

Previous studies have shown one of the causative agents of periodontitis, Porphyromonas gingivalis, can be killed by red light in the presence of the light-activated antimicrobial agent toluidine blue O (TBO). The purpose of this study was to determine the effects of irradiating the organism with red light in the presence of TBO on its proteolytic enzyme activity.Suspensions of P. gingivalis were exposed to light with a wavelength of 633 nm in the presence of various concentrations of TBO. Samples were taken at various times and their proteolytic activity determined by assay of azocasein hydrolysis. On exposure to 126 J of red light in the presence of 12.5 µg/ml of TBO the proteolytic enzyme activity was reduced by 100%.The results of this study have shown that the main virulence factor of P. gingivalis, its proteolytic activity, can be inactivated by red light in the presence of TBO. This, together with the known bactericidal activity of the TBO/light combination, suggests that photodynamic therapy may prove important in reducing the effectiveness of P. gingivalis as a periodontopathogen in vivo.

Effect of blood on lethal photosensitization of bacteria in subgingival plaque from patients with chronic periodontitis

The purpose of this study was to determine whether bacteria in subgingival plaque samples from patients with chronic periodontitis could be sensitized to killing by low-power laser light in the presence of blood. Toluidine blue O (45 μg ml−1) was added to the plaque samples which were then exposed to light from a 7.3 mW HeNe laser for 30 or 120 s in the presence and absence of 10% horse blood. Viable counts of various groups and species of bacteria were determined before and after irradiation. A substantial bactericidal effect was obtained after irradiation for 30s regardless of whether or not blood was present. However, in most cases irradiation in the absence of blood resulted in greater decreases in the viable counts of aerobes, anaerobes, streptococci and blackpigmented anaerobes. When the samples were irradiated for 120s, 10% blood had little effect on the kills attained and elimination of periodontopathogenic species (black-pigmented anaerobes andFusobacterium nucleatum) was achieved both in the presence and absence of blood.

Control of the human cell cycle by a bacterial protein, gapstatin

The oral gram-negative bacterium Actinobacillus actinomycetemcomitans is a major pathogen in human periodontal disease. Saline extraction releases a range of surface-associated components from this bacterium, including one which exhibits potent anti-proliferative activity as assessed by its capacity to inhibit DNA synthesis by human and other mammalian cells. Cultures incubated with this bacterial fraction for a prolonged period comprise a high proportion of cells containing a 4n level of DNA. Studies using hydroxyurea-synchronized cultures showed that cells treated with the surface-associated fraction were arrested in the G2 phase of the cell cycle and did not enter mitosis. This G2/M blockade was observed only when the bacterial fraction was added to the cells during early S phase. Our data also suggest that the active bacterial component binds to surface receptors expressed by the human cells and may act by a novel mechanism which involves down-regulation of cyclin B1 expression. The anti-proliferative activity of the bacterial fraction, purified by a combination of ammonium sulphate precipitation, HPLC anion exchange and gel filtration, has been shown to be an 8 kDa protein, which we have called gapstatin. Purified gapstatin was shown to be responsible for the the inhibitory effects of the surface-associated fraction on mammalian cells.

The effect of low-power laser light at different doses on gastric mucosa sensitized with methylene blue, haematoporphyrin derivative or toluidine blue

Helicobacter pylori has been associated with peptic ulcers, gastric cancer and various other gastroduodenal pathologies. Conventional antibiotic treatment is not entirely satisfactory, whereas photodynamic therapy (PDT) is a possible alternative. Although PDT has been shown to be effective in killing helicobacter on native gastric mucosa, the success of the technique will depend upon the mucosa underlying the bacteria remaining unharmed. This study examined the effect of increasing doses of low-power laser light on rat gastric mucosa, sensitized with either methylene blue (MB), haematoporphyrin derivative (HpD) or toluidine blue (TBO). No damage was detectable with any of the photosensitizers when a light dose of 250 Jcm-2 was used. Mucosal damage was seen consistently with TBO (1 mg ml-1) and a light dose of 500 J cm-2. The same light dose of 500 J cm-2 using MB caused inconsistent damage to the mucosa, whereas HpD had no effect even with the highest dose of laser light (500 J cm-2). As the energy doses required to kill bacteria (50 and 200 Jcm-2 for MB and TBO, respectively) are well below the levels shown to damage normal gastric mucosa, PDT forH. pylori should not be harmful to the underlying epithelium.

Mechanism of killing of Streptococcus mutans by light-activated drugs

Recent studies have shown that cariogenic bacteria can be killed when exposed to low power laser light in the presence of a photosensitizing agent. The purpose of this study was to determine the mechanism by which the cariogenic bacterium Streptococcus mutans can be killed by toluidine blue O and helium neon laser light. To determine whether membrane damage occurred, suspensions of sensitized S. mutans were exposed to a 7.3 mW HeNe laser for 30 mins and samples removed every 5 mins. Survivors were enumerated by viable counting on tryptone soya agar plates and cell free filtrates were assayed for phosphate and (beta) -galactosidase. Lipid peroxidation was assessed by assaying for malondialdehyde, a by- product of lipid peroxidation. The role of oxygen and reactive oxygen species was studied by exposing sensitized bacteria to laser light (1) under different atmospheric conditions, (2) in the presence of deuterium oxide, and (3) in the presence of inhibitors of reactive oxygen species. Following exposure of sensitizede S. mutans to 13.2 J of HeNe laser light, 2.6 nmoles of phosphate and 228 nmoles of (beta) -galactosidase were detected in the cell free filtrates. Ten micrometers oles of malondialdehyde were also detected. When the sensitized bacteria were exposed to laser light under anaerobic conditions there was no significant decrease in the viable count compared to a 60% kill in the presence of oxygen. In the presence of D2O there was a 15-fold increase in the numbers of bacteria killed. O.1 M methionine and 0.5 M sodium azide each afforded 98% protection from lethal photosensitization. These results imply that lethal photosensitization results from membrane damage due to lipid peroxidation and that reactive oxygen species are mediators of this process.

Reanalysis of the Am I level spectrum and the nuclear quadrupole moments of Am-isotopes

The fine structure (fs) and hyperfine structure (hfs) level scheme of Am I is reanalysed using a semi empirical fitting procedure which incorporates experimental data. Especially new laserspectroscopic measurements of the hfs of some electronic transitions in the Am atom enables us to make a more detailed analysis of 5f7 7s 7p fine structure in Am I. In particular a relation is given between theB-factor values and the value of the nuclear electric quadrupole moment of241Am independent of a calibration by results of nuclear spectroscopy.