Christopher K. Hope

Analysis of the Effects of Chlorhexidine on Oral Biofilm Vitality and Structure Based on Viability Profiling and an Indicator of Membrane Integrity

ABSTRACT Multispecies biofilms modeling interproximal plaque were grown on a hydroxyapatite substratum in a constant-depth film fermentor and then immersed in a viewing solution containing fluorescent indicators of membrane integrity. Confocal laser scanning microscopy (CLSM) revealed the structure and spatial distribution of cell vitality within the biofilms. Chlorhexidine gluconate (CHX) was added to the viewing solution to achieve concentrations of 0.05 and 0.2% (wt/vol) before further CLSM time-lapse series were captured. Image analysis showed that exposure to 0.2% CHX caused the biofilm to contract at a rate of 1.176 μm min−1 along the z axis and also effected changes in total fluorescence measurements and viability profiles through the biofilms after a delay of 3 to 5 min. At a concentration of 0.05% CHX, total fluorescence measurements for the biofilm exhibited barely detectable changes after 5 min. Fluorescence profiles (fluorescence versus time versus depth), however, clearly showed that a time-dependent effect was present, but the clearest indicator of the effect of dilute CHX over time was viability profiling. These findings suggest the possibility of using fluorescent indicators of membrane integrity in conjunction with viability profiling to evaluate the penetration of the bactericidal effects of membrane-active antimicrobial compounds into biofilm.

Effect of Silver Content on the Structure and Antibacterial Activity of Silver-Doped Phosphate-Based Glasses

ABSTRACT Staphylococcus aureus can cause a range of diseases, such as osteomyelitis, as well as colonize implanted medical devices. In most instances the organism forms biofilms that not only are resistant to the bodys defense mechanisms but also display decreased susceptibilities to antibiotics. In the present study, we have examined the effect of increasing silver contents in phosphate-based glasses to prevent the formation of S. aureus biofilms. Silver was found to be an effective bactericidal agent against S. aureus biofilms, and the rate of silver ion release (0.42 to 1.22 μg·mm−2·h−1) from phosphate-based glass was found to account for the variation in its bactericidal effect. Analysis of biofilms by confocal microscopy indicated that they consisted of an upper layer of viable bacteria together with a layer (∼20 μm) of nonviable cells on the glass surface. Our results showed that regardless of the silver contents in these glasses (10, 15, or 20 mol%) the silver exists in its +1 oxidation state, which is known to be a highly effective bactericidal agent compared to that of silver in other oxidation states (+2 or +3). Analysis of the glasses by 31P nuclear magnetic resonance imaging and high-energy X-ray diffraction showed that it is the structural rearrangement of the phosphate network that is responsible for the variation in silver ion release and the associated bactericidal effectiveness. Thus, an understanding of the glass structure is important in interpreting the in vitro data and also has important clinical implications for the potential use of the phosphate-based glasses in orthopedic applications to deliver silver ions to combat S. aureus biofilm infections.

Determining the spatial distribution of viable and nonviable bacteria in hydrated microcosm dental plaques by viability profiling

Aims: The aim of this study was to use confocal laser scanning microscopy (CLSM) to examine the spatial distribution of both viable and nonviable bacteria within microcosm dental plaques grown in vitro. Previous in vivo studies have reported upon the distribution of viable bacteria only. 
Methods and Results: Oral biofilms were grown on hydroxyapatite (HA) discs in a constant‐depth film fermenter (CDFF) from a saliva inoculum. The biofilms were stained with the BacLightTM LIVE/DEAD system and examined by CLSM. Fluorescence intensity profiles through the depth of the biofilm showed an offset between the maximum viable intensity and the maximum nonviable intensity. Topographical differences between the surface properties of the viable and nonviable biofilm virtual surfaces were also measured. 
Conclusions: The profile of fluorescence intensity from viable and nonviable staining suggested that the upper layers of the biofilm contain proportionally more viable bacteria than the lower regions of the biofilm. 
Significance and Impact of Study: Viability profiling records the transition from predominantly viable to nonviable bacteria through biofilms suggesting that this technique may be of use for quantifying the effects of antimicrobial compounds upon biofilms. The distribution of viable bacteria was similar to that found in dental plaque in vivo suggesting that the CDFF produces in vitro biofilms which are comparable to their in vivo counterparts in terms of the spatial distribution of viable bacteria.

Role of gallium and silver from phosphate-based glasses on in vitro dual species oral biofilm models of Porphyromonas gingivalis and Streptococcus gordonii

Phosphate-based glasses (PBGs) are excellent controlled delivery agents for antibacterial ions such as silver and gallium. The aim of this study was to assess the potential utility of novel PBGs combining both gallium and silver for use in periodontal therapy. To this end, an in vitro biofilm model with the putative periodontal pathogen, Porphyromonas gingivalis, and an initial colonizer, Streptococcus gordonii, was established. The effect of increasing calcium content in gallium-silver-doped PBG on the susceptibility of P. gingivalis was examined. A decrease in degradation rates (30.34, 25.19, 21.40 μg mm(-2) h(-1)) with increasing PBG calciumcontent (10, 11, 12 mol.% respectively) was observed, correlating well with gallium and silver ion release and antimicrobial activity against planktonic P. gingivalis (approximately 5.4log(10) colony-forming units (CFU) reduction after 24h by the C10 glass compared with controls) and S. gordonii (total growth inhibition after 32h by C10, C11 and C12 glasses compared with controls). The most potent PBG (C10) was evaluated for its ability to inhibit the biofilm growth of P. gingivalis in a newly established constant-depth film fermentor model. The simultaneous release of silver and gallium from the glass reduced P. gingivalis biofilm growth with a maximum effect (1.92log(10) CFU reduction) after 168 h. Given the emergence of antibiotic-resistant bacteria and dearth of new antibiotics in development, the glasses, especially C10, would offer effective alternatives to antibiotics or may complement current therapies through controlled, localized delivery of gallium and silver ions at infected sites in the oral cavity.

Photobleaching of red fluorescence in oral biofilms

BACKGROUND AND OBJECTIVE Many species of oral bacteria can be induced to fluoresce due to the presence of endogenous porphyrins, a phenomenon that can be utilized to visualize and quantify dental plaque in the laboratory or clinical setting. However, an inevitable consequence of fluorescence is photobleaching, and the effects of this on longitudinal, quantitative analysis of dental plaque have yet to be ascertained. MATERIAL AND METHODS Filter membrane biofilms were grown from salivary inocula or single species (Prevotella nigrescens and Prevotella intermedia). The mature biofilms were then examined in a custom-made lighting rig comprising 405 nm light-emitting diodes capable of delivering 220 W/m(2) at the sample, an appropriate filter and a digital camera; a set-up analogous to quantitative light-induced fluorescence digital. Longitudinal sets of images were captured and processed to assess the degradation in red fluorescence over time. RESULTS Photobleaching was observed in all instances. The highest rates of photobleaching were observed immediately after initiation of illumination, specifically during the first minute. Relative rates of photobleaching during the first minute of exposure were 19.17, 13.72 and 3.43 arbitrary units/min for P. nigrescens biofilms, microcosm biofilm and P. intermedia biofilms, respectively. CONCLUSION Photobleaching could be problematic when making quantitative measurements of porphyrin fluorescence in situ. Reducing both light levels and exposure time, in combination with increased camera sensitivity, should be the default approach when undertaking analyses by quantitative light-induced fluorescence digital.

The inability of a bacteriophage to infect Staphylococcus aureus does not prevent it from specifically delivering a photosensitizer to the bacterium enabling its lethal photosensitization

OBJECTIVES It has been demonstrated that the efficiency of lethal photosensitization can be improved by covalently binding photosensitizing agents to bacteriophage. In this study we have investigated whether a bacteriophage requires the capacity to infect the bacterium to enhance lethal photosensitization when linked to a photosensitizer. METHODS Tin (IV) chlorin e6 (SnCe6) was conjugated to bacteriophage Phi11, a transducing phage that can infect Staphylococcus aureus NCTC 8325-4, but not epidemic methicillin-resistant S. aureus (EMRSA)-16. The conjugate and appropriate controls were incubated with these bacteria and either exposed to laser light at 632.8 nm or kept in the dark. RESULTS The SnCe6/Phi11 conjugate achieved a statistically significant reduction in the number of viable bacteria of both 8325-4 and EMRSA-16 strains by 2.31 log(10) and 2.63 log(10), respectively. The conjugate could not however instigate lethal photosensitization of Escherichia coli. None of the other combinations of controls, such as an equivalent concentration of SnCe6 only, an equivalent titre of bacteriophage only or experiments conducted without laser light, yielded significant reductions in the number of viable bacteria recovered. CONCLUSIONS The inability of a bacteriophage to infect S. aureus does not prevent it from specifically delivering a photosensitizer to a bacterium enabling its lethal photosensitization.

Effect of novel antibacterial gallium-carboxymethyl cellulose on Pseudomonas aeruginosa

Gallium has emerged as a new therapeutic agent due partly to the scarcity in development of new antibiotics. In this study, a novel antibacterial gallium exchanged carboxymethyl cellulose (Ga-CMC) has been developed and tested for the susceptibility on a common bacteria, Pseudomonas aeruginosa. The results show that an increase in average molecular weight (MW) from 90 k, 250 k to 700 k of Ga-CMC caused a decrease in antimicrobial activity against planktonic P. aeruginosa. Gallium loading of the Ga-CMC (250 k) samples was altered by varying the amount of functionality (0.7, 0.9 and 1.2 acid groups per mole of carbohydrate) which affected also its antimicrobial activity against planktonic P. aeruginosa. Further, the ability to prevent the growth of biofilms of P. aeruginosa was tested on MW = 250 k samples with 0.9 acid groups per mole of carbohydrate as this sample showed the most promising activity against planktonic P. aeruginosa. Gallium was found to reduce biofilm growth of P. aeruginosa with a maximum effect (0.85 log(10) CFU reduction compared to sodium-carboxymethyl cellulose, Na-CMC) after 24 h. Results of the solubility and ion exchange studies show that this compound is suitable for the controlled release of Ga(3+) upon their breakdown in the presence of bacteria. SEM EDX analysis confirmed that Ga(3+) ions are evenly exchanged on the cellulose surface and systematic controls were carried out to ensure that antibacterial activity is solely due to the presence of gallium as samples intrinsic acidity or nature of counterion did not affect the activity. The results presented here highlight that Ga-CMC may be useful in controlled drug delivery applications, to deliver gallium ions in order to prevent infections due to P. aeruginosa biofilms.

Reducing the variability between constant-depth film fermenter experiments when modelling oral biofilm.

The inherent instabilities associated with the development of multispecies biofilm communities within the constant‐depth film fermenter (CDFF) and other microcosm systems can yield unacceptable variability between experiments, which could limit their potential applications in oral microbiology. The extent of this variability needs to be determined and a protocol developed which minimizes it.

Lethal photosensitization of Porphyromonas gingivalis by their endogenous porphyrins under anaerobic conditions: An in vitro study

BACKGROUND Lethal photosensitization has been previously demonstrated in Porphyromonas gingivalis, but oxygen is considered to be essential to this process. However, since P. gingivalis is a periodontal pathogen which grows in the low oxygen conditions found in the subgingival crevice, it was considered prudent to study its photosensitivity in anaerobic conditions. METHODS A series of experiments were undertaken to attempt to induce lethal photosensitization in P. gingivalis (ATCC 33277) under strict anaerobic conditions using two different 405 nm light sources. Samples of P. gingivalis were grown on a blood-containing, solid growth medium before being suspended in saline and then exposed to 405 nm light delivered by either a hand-held light source (Toothcare™) (11.4 mW/cm(2)) or a laser pointer (328.5 mW/cm(2)). With the exception of the adjustment of the P. gingivalis suspensions to a fixed optical density, the experiments were carried out in their entirety within an anaerobic chamber. RESULTS The lowest Toothcare light dose tested (0.34 J/cm(2); 30s) yielded a statically significant kill of 63.4% which increased to 94.1% kill at higher light doses (3.42 J/cm(2); 300 s). The laser pointer similarly achieved kills of 90.2% at the lower light dose tested (9.86 J/cm(2); 30s) and 94.5% kill at the highest light dose (98.55 J/cm(2); 300 s). CONCLUSIONS Lethal photosensitization can be instigated in planktonic suspensions of P. gingivalis at 405 nm delivered by hand-held devices under anaerobic conditions. This suggests the possibility that lethal photosensitization occurred by the oxygen-independent type I pathway as oppose to the oxygen-dependent type II pathway.

Efficacy of Removal of Sucrose-Supplemented Interproximal Plaque by Electric Toothbrushes in an In Vitro Model

ABSTRACT Electric toothbrushes were evaluated using a model of plaque removal by fluid shear forces. Sucrose supplementation during plaque development did not affect the removal of bacteria from biofilm exposed to low-energy shear but did increase their resistance to high-energy shear. The toothbrush supplying high-energy shear forces removed significantly more viable bacteria.