Novel lactoferrin-conjugated gallium complex to treat Pseudomonas aeruginosa wound infection.

Pseudomonas aeruginosa is one of the leading causes of opportunistic infections such as chronic wound infection that could lead to multiple organ failure and death. Gallium (Ga3+) ions are known to inhibit P. aeruginosa growth and biofilm formation but require carrier for localized controlled delivery. Lactoferrin (LTf), a two-lobed protein, can deliver Ga3+ at sites of infection. This study aimed to develop a Ga-LTf complex for the treatment of wound infection. The characterisation of the Ga-LTf complex was conducted using differential scanning calorimetry (DSC), Infra-Red (FTIR) and Inductive Coupled Plasma Optical Emission Spectrometry (ICP-OES). The antibacterial activity was assessed by agar disc diffusion, liquid broth and biofilm inhibition assays using the colony forming units (CFUs). The healing capacity and biocompatibility were evaluated using a P.aeruginosa infected wound in a rat model. DSC analyses showed thermal transition consistent with apo-lactoferrin; FTIR confirmed the complexation of gallium to lactoferrin. ICP-OES confirmed the controlled local delivery of Ga3+. Ga-LTf showed a 0.57 log10 CFUs reduction at 24 h compared with untreated control in planktonic liquid broth assay. Ga-LTf showed the highest antibiofilm activity with a 2.24 log10 CFUs reduction at 24 h. Furthermore, Ga-LTf complex is biocompatible without any adverse effect on brain, kidney, liver and spleen of rats tested in this study. Ga-LTf can be potentially promising novel therapeutic agent to treat pathogenic bacterial infections.

Fluorescence fingerprints of oral bacteria.

The rapid detection and identification of microorganisms is one of the most important factors in many cases of ill health. The purpose of this study was to determine the fluorescence characteristics of seven oral bacteria using emission spectra with the aim of distinguishing between the bacteria, and to compare fluorescence imaging methods for the direct assessment of oral bacteria. Fluorescence images of each bacterium were obtained under a 405-nm light source using a two-filter system. The emissions of all samples were measured with a fluorescence spectrometer. The complete fluorescence data set collected for each sample employed a three-dimensional data cube. The differences in the autofluorescence characteristics of the seven oral bacteria were determined by principal components analysis (PCA). The fluorescence images of the oral bacteria varied with the genus and the filter system. The three-dimensional excitation-emission matrix fluorescence spectra exhibited distinctive fluorescence features associated with intracellular fluorophores. The seven bacteria could be clearly differentiated on the PCA score plot. The findings of this study indicate that oral bacteria can be identified based on their autofluorescence characteristics. Fluorescence spectroscopy coupled with PCA can be used to detect and classify oral bacteria.

Evidence of an in vitro Coupled Diffusion Mechanism of Lesion Formation within Microcosm Dental Plaque.

The purpose of this study was to determine whether or not the dual constant-depth film fermenter (dCDFF) is able to produce caries-like enamel lesions and to ascertain further information regarding the performance of this fully functional biological caries model. Conditions were defined by the continuation (CF) or cessation (FF) of a saliva-type growth medium supply during 50-mM sucrose exposures (8 times daily). Hydroxyapatite (n = 3) and bovine enamel (n = 3) substrata were included within each condition and samples extracted after 2, 4, 8, and 16 days. Community profiles were generated for fastidious anaerobes, Lactobacillus spp., Streptococcus spp., mutans streptococci (MS), and Veillonella spp. using selective culture techniques and enamel demineralisation assessed by transverse microradiography. Results demonstrated that the dCDFF model is able to produce caries-like enamel lesions with a high degree of sensitivity where reduced ionic strength within the FF condition increased surface layer mineral deposition. Between conditions, biofilm communities did not differ significantly, although MS in the biofilms extracted from the FF condition rose to a higher proportion (by 1.5 log10 units), and Veillonella spp. were initially greater within the CF condition (by 2.5 log10 units), indicating an enhanced ability for the clearance of low-pKa acids following exposures to sucrose. However, both conditions retained the ability for caries-like lesion formation.

Evaluating the effect of local pH on fluorescence emissions from oral bacteria of the genus Prevotella.

A number of anaerobic oral bacteria, notably Prevotellaceae, exhibit red fluorescence when excited by short-wavelength visible light due to their accumulation of porphyrins, particularly protoporphyrin IX. pH affects the fluorescence of abiotic preparations of porphyrins due to transformations in speciation between monomers, higher aggregates, and dimers. To elucidate whether the porphyrin speciation phenomenon could be manifested within a microbiological system, suspensions of Prevotella intermedia and Prevotella nigrescens were examined by fluorescence spectrophotometry while being titrated against NaOH. The initial pH of the samples was <6, which was then raised toward the maximum found within a diseased periodontal pocket, being ∼pH 8.7. The intensity of the fluorescence emissions increased between 600 and 650 nm with increasing pH. Peak fluorescence emissions occurred at 635±1 nm with a second emission peak developing with increasing pH at 622 nm. A linear relationship was demonstrated between pH and the log10 ratio of 635:622 nm excitation fluorescence intensities. These findings suggest that the pH range found within the oral cavity could affect the fluorescence of oral bacteria in vivo, which may in turn have connotations for any clinical diagnoses that may be inferred from dental plaque fluorescence.

A Preliminary Study of the Effects of pH upon Fluorescence in Suspensions of Prevotella intermedia.

The quantification of fluorescence in dental plaque is currently being developed as a diagnostic tool to help inform and improve oral health. The oral anaerobe Prevotella intermedia exhibits red fluorescence due to the accumulation of porphyrins. pH affects the fluorescence of abiotic preparations of porphyrins caused by changes in speciation between monomers, higher aggregates and dimers, but this phenomenon has not been demonstrated in bacteria. Fluorescence spectra were obtained from suspensions of P. intermedia that were adjusted to pHs commensurate with the range found within dental plaque. Two fluorescent motifs were identified; 410 nm excitation / 634 nm emission (peak A) and 398 nm excitation / 622 nm emission (peak B). A transition in the fluorescence spectra was observed from peak A to peak B with increasing pH which was also evident as culture age increased from 24 hours to 96 hours. In addition to these 'blue-shifts', the intensity of peak A increased with pH whilst decreasing with culture age from 24 to 96 hours. A bacterium's relationship with the local physiochemical environment at the time of image capture may therefore affect the quantification of dental plaque fluorescence.

Lethal photosensitisation of Prevotellaceae under anaerobic conditions by their endogenous porphyrins.

BACKGROUND: Oxygen is generally considered essential for lethal photosensitisation by photodynamic processes. The oral anaerobes, Prevotella intermedia and P. nigrescens are known to be photosensitive, but are also extremely sensitive to the cytotoxic effects of oxygen. METHODS: The Prevotellaceae were exposed to two 405 nm light sources for different exposure times in an anaerobic chamber. Viable counts of the light exposed samples were compared to light-free controls to determine the proportion of bacteria killed. RESULTS: Lethal photosensitivity was demonstrated against P. intermedia and P. nigrescens. The proportions of bacteria killed by either the light-emitting diode or laser pointer were similar at a given energy density (J/cm(2)). CONCLUSIONS: Lethal photosensitivity was demonstrated in two species of Prevotella under anaerobic conditions.

Assessing the association between oral hygiene and preterm birth by quantitative light-induced fluorescence.

The aim of this study was to investigate the purported link between oral hygiene and preterm birth by using image analysis tools to quantify dental plaque biofilm. Volunteers (n = 91) attending an antenatal clinic were identified as those considered to be "at high risk" of preterm delivery (i.e., a previous history of idiopathic preterm delivery, case group) or those who were not considered to be at risk (control group). The women had images of their anterior teeth captured using quantitative light-induced fluorescence (QLF). These images were analysed to calculate the amount of red fluorescent plaque (ΔR%) and percentage of plaque coverage. QLF showed little difference in ΔR% between the two groups, 65.00% case versus 68.70% control, whereas there was 19.29% difference with regard to the mean plaque coverage, 25.50% case versus 20.58% control. A logistic regression model showed a significant association between plaque coverage and case/control status (P = 0.031), controlling for other potential predictor variables, namely, smoking status, maternal age, and body mass index (BMI).

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.

Finding an alternative to formalin for sterilization of extracted teeth for teaching purposes.

Formalin is a known carcinogen, so there is a need to establish whether a safer alternative is available for the sterilization of human teeth destined for use in clinical training. Any disinfectant that is not capable of sterilizing 100 percent of the samples tested should be considered a failure. In this study, biofilms of oral bacteria were grown on previously autoclaved extracted human teeth. These biofilm-laden teeth were then screened against a range of disinfectants for an exposure time of seven days in a laboratory refrigerator. Culture methods were employed to validate the sterility of the tooth samples. Five percent Virkon and Gigasept PA proved effective against the laboratory model of disinfection and were carried forward to challenge freshly extracted human teeth. Gigasept PA was the only disinfectant that sterilized 100 percent of the tooth samples. Gigasept PA should be considered a safer and effective alternative to formalin for the sterilization of extracted teeth destined for teaching purposes.

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.

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.

A direct comparison between extracted tooth and filter-membrane biofilm models of endodontic irrigation using Enterococcus faecalis.

Endodontic restorations often fail due to inadequate disinfection of the root canal even though the antimicrobial irrigants used have been shown to be capable of killing the bacterium frequently implicated in this complication, Enterococcus faecalis (Ef). Extracted human teeth were root-prepared and filled with a liquid culture of Ef. Following incubation, the root canals were irrigated with 1% sodium hypochlorite (NaOCl), electrochemically activated water or saline control. Irrigation was modelled using an electronic pipette to deliver the solutions at a reproducible flow velocity. A series of parallel experiments employed a membrane biofilm model that was directly immersed into irrigant. Experimental conditions where contiguous between the extracted tooth model and biofilm model wherever possible. After 60 s of exposure, 1% NaOCl effectively sterilised the biofilm model, whereas log 3.36 viable Ef where recoverable from the analogous extracted tooth model, the other irrigants proved ineffective. Biofilms of Ef were susceptible to concentrations of irrigant that proved ineffective in the tooth model. NaOCl was the most effective biocide in either case. This suggests that the biofilm modality of bacterial growth may not be the most important factor for the recalcitrance of root canal infections during endodontic irrigation; it is more likely due to the inability of the irrigant to access the infection.

In vitro assessment of the plaque-removing ability of hydrodynamic shear forces produced beyond the bristles by 2 electric toothbrushes.

BACKGROUND: The aim of this study was to compare the efficacy of interproximal plaque removal by the hydrodynamic shear forces produced by 2 electric toothbrushes (toothbrush A, Sonicare Plus and toothbrush B, Braun Oral B) using laboratory-grown plaque biofilms in an anatomically correct in vitro brushing model. METHODS: In vitro oral biofilms were grown in a constant-depth film fermenter on hydroxyapatite (HA) discs. Brushing experiments were conducted in a specially constructed machine designed to simulate the brushing of interproximal plaque between mandibular molar teeth; the bristles of the toothbrush did not make contact with the biofilm at any time. The load force between the brushes and teeth were in accordance with typical use and an exposure time of 5 seconds was used throughout. The efficacy of brushing was assessed by enumeration of the percentage of viable bacteria removed from the HA discs. These experiments were conducted with the electronic effect of the toothbrushes either activated or inactivated. RESULTS: Activation of toothbrush A significantly (P = 0.004) increased the median percentage of bacteria removed from 0.47% to 48.45%. Likewise, activation of toothbrush B significantly (P = 0.015) increased the median percentage of bacteria removed from 2.85% to 15.86%. The median percentage of plaque bacteria removed by the active toothbrush A was significantly (P = 0.009) greater than that removed by toothbrush B in this model system. CONCLUSION: These data imply that toothbrush A would be more effective than toothbrush B at removing interproximal dental plaque in vivo.

Oral bacteria in multi-species biofilms can be killed by red light in the presence of toluidine blue.

BACKGROUND AND OBJECTIVES: Oral bacteria can be killed by light in the presence of a suitable photosensitizer, and this could be used in the treatment of oral infections. In these diseases, however, bacteria are present as biofilms, which are refractive to antimicrobial agents. The purpose of this study was to determine whether oral bacterial biofilms were susceptible to lethal photosensitization. STUDY DESIGN/MATERIALS AND METHODS: Multi-species biofilms of oral bacteria were irradiated with light from a helium/neon laser in the presence of toluidine blue O (TBO) and the survivors enumerated. Controls examining the effects of light and TBO alone were also included. The biofilms were also examined by confocal scanning laser microscopy (CSLM). RESULTS: CSLM revealed that the biofilms had structures similar to those of dental plaque. Although, the biofilms consisted of extremely large numbers of bacteria ( approximately 9 x 10(9)), 97.4% were killed following irradiation with 31.5 J of laser light in the presence of 25 microg/ml TBO. CONCLUSIONS: Substantial numbers of oral bacteria in multi-species biofilms can be killed by light in the presence of TBO. This may be useful in the treatment of dental plaque-related diseases.

Comparison of the interproximal plaque removal efficacy of two powered toothbrushes using in vitro oral biofilms.

PURPOSE: To compare the efficacy of interproximal plaque removal beyond the bristles by two electric toothbrushes, the new Sonicare Elite and Braun Oral-B 3D, in an in vitro model replicating the brushing of interproximal plaque as would occur in vivo. MATERIALS AND METHODS: Oral biofilms were grown, in vitro, in a constant-depth film fermenter on a hydroxyapatite (HA) disc substratum. Brushing experiments were conducted in a brushing machine representing the brushing of interproximal plaque between mandibular molars. The HA discs with oral biofilms were located a minimum of 1.6 mm away from the bristles. The efficacy of plaque removal was assessed by enumeration of the percentage of viable bacteria removed from the biofilms by 5 seconds of brushing with the brush motors either activated or inactivated. RESULTS: In the activated state, both brushes removed a significantly higher percentage of plaque bacteria compared to the inactive brushes (Braun, P = 0.002; Sonicare, P = 0.005). The percentage of plaque bacteria removed by the Sonicare Elite (32.23%) beyond the bristles was significantly greater (P = 0.012) than that removed by the Braun Oral-B 3D (9.48%) in this model system.