Improvement of Antibacterial Efficacy Through Synergistic Effect in Photodynamic Therapy Based on Thiazinium Chromophores Against Planktonic and Biofilm-Associated Periodontopathogens.

OBJECTIVE: Aim of the study was to improve the antibacterial efficacy of toluidine blue (TBO)/methylene blue (MB)-mediated photodynamic systems with light-emitting diode (LED) or laser irradiation administered to planktonic and biofilm-associated periodontopathogens. BACKGROUND DATA: Antibacterial photodynamic therapy (PDT) is a common, noninvasive adjunctive clinical method to inactivate microorganisms. So far, the disadvantage of this method has been its limited effectiveness in eliminating pathogens. METHODS: An anaerobic cocktail consisting of six representative periodontal pathogens was prepared as initial culture for planktonic samples and biofilms grown on human tooth slides. Both types of microbial samples were exposed to three commercial photodynamic systems (PDT1: TBO, 630 nm LED, PDT2: TBO, 635 nm laser, PDT3: MB, 665 nm laser) in conventional and a new modified approach (PDTplus) based on the use of an oxygen supplement (photosensitizer+hydrogen peroxide). The microbial viability was characterized by bacterial growth [colony forming units (CFU)], total bacterial cell counts, and microbial vitality. Statistical data analysis was performed using 95% confidence intervals (ANOVA) and post hoc Tukey's test (p < 0.05). RESULTS: The modified PDTplus showed the highest statistically significant synergistic antimicrobial activity for TBO-based systems evidenced by a CFU reduction of 9 log10 units to 0 for planktonic pathogens and a 4 log10 CFU reduction for biofilm bacteria. The MB-based PDTplus was superior mainly against biofilm pathogens. By comparison, the default TBO-based PDT achieved colony growth reductions of 2 and 1 log10 units concerning planktonic and biofilm cells. CONCLUSIONS: Compared to conventional PDT, PDTplus showed superior antibacterial efficacy based on its synergistic effect, promising vast application possibilities.

Metabolic activity of Streptococcus mutans biofilms and gene expression during exposure to xylitol and sucrose.

The objective of the study was to analyse Streptococcus mutans biofilms grown under different dietary conditions by using multifaceted methodological approaches to gain deeper insight into the cariogenic impact of carbohydrates. S. mutans biofilms were generated during a period of 24 h in the following media: Schaedler broth as a control medium containing endogenous glucose, Schaedler broth with an additional 5% sucrose, and Schaedler broth supplemented with 1% xylitol. The confocal laser scanning microscopy (CLSM)-based analyses of the microbial vitality, respiratory activity (5-cyano-2,3-ditolyl tetrazolium chloride, CTC) and production of extracellular polysaccharides (EPS) were performed separately in the inner, middle and outer biofilm layers. In addition to the microbiological sample testing, the glucose/sucrose consumption of the biofilm bacteria was quantified, and the expression of glucosyltransferases and other biofilm-associated genes was investigated. Xylitol exposure did not inhibit the viability of S. mutans biofilms, as monitored by the following experimental parameters: culture growth, vitality, CTC activity and EPS production. However, xylitol exposure caused a difference in gene expression compared to the control. GtfC was upregulated only in the presence of xylitol. Under xylitol exposure, gtfB was upregulated by a factor of 6, while under sucrose exposure, it was upregulated by a factor of three. Compared with glucose and xylitol, sucrose increased cell vitality in all biofilm layers. In all nutrient media, the intrinsic glucose was almost completely consumed by the cells of the S. mutans biofilm within 24 h. After 24 h of biofilm formation, the multiparametric measurements showed that xylitol in the presence of glucose caused predominantly genotypic differences but did not induce metabolic differences compared to the control. Thus, the availability of dietary carbohydrates in either a pure or combined form seems to affect the cariogenic potential of S. mutans biofilms.

Dynamic Production of Soluble Extracellular Polysaccharides by Streptococcus mutans.

Caries development in the presence of Streptococcus mutans is associated not only with the production of extracellular water-insoluble polymers but also is based on water-soluble polysaccharides. The aim of this study was the evaluation of a novel glucan-specific Lectin assay for monitoring water-soluble EPS produced by S. mutans during several growth periods in different media. S. mutans cultures were grown for 24 h, 48 h, and 144 h in medium deficient of sucrose (A) and medium supplemented with 5% sucrose (B). Microtiter well plates were coated with cell-free supernatants followed by the addition of labeled Concanavalin-A and enzyme substrate. The substrate reactions were kinetically detected at 405 nm. The validation of the assay was performed using carbohydrates dextran, xanthan, and sucrose as reference. This new Concanavalin-A-based assay showed the highest sensitivity for dextran and revealed that the glucan production of S. mutans reached its maximum at 144 h in medium B according to bacterial maturation.

Real-time microsensor measurement of local metabolic activities in ex vivo dental biofilms exposed to sucrose and treated with chlorhexidine.

Dental biofilms are characterized by structural and functional heterogeneity. Due to bacterial metabolism, gradients develop and diverse ecological microniches exist. The aims of this study were (i) to determine the metabolic activity of microorganisms in naturally grown dental biofilms ex vivo by measuring dissolved oxygen (DO) and pH profiles with microelectrodes with high spatial resolution and (ii) to analyze the impact of an antimicrobial chlorhexidine (CHX) treatment on microbial physiology during stimulation by sucrose in real time. Biofilms were cultivated on standardized human enamel surfaces in vivo. DO and pH profiles were measured in a flow cell system in sterile human saliva, after sucrose addition (10%), again after alternative treatment of the sucrose exposed biofilms with CHX (0.2%) for 1 or 10 min or after being killed with paraformaldehyde (4%). Biofilm structure was visualized by vitality staining with confocal microscopy. With saliva as the sole nutrient source oxygen consumption was high within the superficial biofilm layers rendering deeper layers (>220 mum) anoxic. Sucrose addition induced the thickness of the anaerobic zone to increase with a concurrent decrease in pH (7.1 to 4.4). CHX exposure reduced metabolic activity and microbial viability at the biofilm surface and drove metabolic activity deeper into the biofilm. CHX treatment led to a reduced viability at the biofilm surface with minor influence on overall biofilm physiology after 1 min; even after 10 min there was measurable respiration and fermentation inside the biofilm. However, the local microenvironment was more aerated, less acidogenic, and presumably less pathogenic.

Effect of xylitol/chlorhexidine versus xylitol or chlorhexidine as single rinses on initial biofilm formation of cariogenic streptococci.

OBJECTIVES: Oral bacteria implying a natural resistance may deteriorate the antibacterial efficacy of chlorhexidine on cariogenic microorganisms. Xylitol, mostly applied via chewing gum, is known to possess favorable plaque-reducing properties. The aim of this study was to investigate the effect of a xylitol rinse formulated as pure solution or combined with chlorhexidine on the viability of Streptococcus sanguis (early colonizer of human teeth) and Streptococcus mutans (the most causal strain for caries) during initial steps of biofilm formation. METHOD AND MATERIALS: After exposure to the test solutions, the bacteria suspended in human sterile saliva were allowed to attach to human enamel slides for 60 minutes in a preclinical flow chamber system. The bacterial vitality of suspended and attached cells was monitored using 2 fluorescent DNA stains by epifluorescence microscopy. Further parameters measured were the total bacterial cell counts on enamel slides and growth of suspended streptococci. RESULTS: The sensitivity of S mutans to pure chlorhexidine or in combination with xylitol is contrary to the natural resistance of S sanguis to chlorhexidine. The combination of xylitol/chlorhexidine showed a statistically significant antivital effect on S sanguis cells compared to the pure agents xylitol and chlorhexidine. The bacterial cell density on enamel and bacterial reproduction on agar plates were similarly affected by the combination of xylitol/chlorhexidine or the single substances. CONCLUSION: The newly discovered synergistic antivital effect of xylitol combined with chlorhexidine may contribute to the favorable potential of xylitol use for the improvement of new formulations of caries-preventive mouthrinses.

Adhesion of Streptococcus sanguinis to dental implant and restorative materials in vitro.

Bacterial adhesion to tooth surfaces or dental materials starts immediately upon exposure to the oral environment. The aim of this study, therefore, was to compare the adhesion of Streptococcus sanguinis to saliva-coated human enamel and dental materials - during a one-hour period - using an in vitro flow chamber system which mimicked the oral cavity. After fluorescent staining, the number of adhered cells and their vitality were recorded. The dental materials used were: titanium (Rematitan M), gold (Neocast 3), ceramic (Vita Omega 900), and composite (Tetric Ceram). The number of adherent bacterial cells was higher on titanium, gold, and ceramic surfaces and lower on composite as compared to enamel. As for the percentage of adherent vital cells, it was higher on enamel than on the restorative materials tested. These results suggested that variations in the number and vitality of the adherent pioneer oral bacteria, S. sanguinis, in the in vitro system depended on the surface characteristics of the substratum and the acquired salivary pellicle. The in vitro adhesion model used herein provided a simple and reproducible approach to investigate the impact of surface-modified dental materials on bacterial adhesion and vitality.

Susceptibility of planktonic versus attached Streptococcus sanguinis cells to chlorhexidine.

The effect of chlorhexidine (CHX) on the viability of Streptococcus sanguinis was investigated in a preclinical biofilm model separately on cells in the planktonic or attached life form. Saliva-coated human enamel and glass slides were exposed to the streptococci suspended in sterile saliva for 30 min and 60 min in the flow chamber system. The CHX exposition was performed in two parts: pretreatment of the planktonic bacteria before their attachment to enamel or glass, and treatment of bacteria already attached to enamel. The susceptibility measured by vitality percentages was determined by fluorescence microscopy using vital/dead cells. After CHX pretreatment of planktonic cells, the mean values of the vitality percentages after adhesion were 14-18% (enamel) and 24-25% (glass). In contrast, the mean vitality percentages of untreated attached streptococci reached 70-75% (enamel) and 68% (glass). The vitality percentages of CHX-exposed bacteria dropped markedly to 2-5%, whereas those of untreated attached cells remained at 65-66%. The exposure of initially attached streptococci to CHX resulted in greater reduction of bacterial viability than with the planktonic counterparts. This preclinical biofilm model allows the investigation of various bacterial life forms and can furthermore be used to select efficient antiplaque therapeutics which might be beneficial for clinical plaque control.

Comparison of antiadhesive and antibacterial effects of antiseptics on Streptococcus sanguinis.

Three antiseptic (chlorhexidine, Olaflur, Octenisept) and one putative antiadhesive (chitosan) agent were investigated for their effect on viable planktonic and attached Streptococcus sanguinis cells. The bacterial pretreatment with each chemotherapeutic was performed in two steps: (i) After the exposure of planktonic streptococci to the antiseptics, the cells were suspended in human sterile saliva and allowed to attach to human enamel for 60 min; (ii) After 60 min in the flow chamber system, initially attached streptococci were treated with these agents. The microbial viability was monitored by the percentage of vital streptococci determined by fluorescence microscopy and cell reproduction. In comparison with the negative control NaCl, the non-bactericidal chitosan derivative showed distinctive antiadhesive properties. For both treatment procedures, the efficacy of the antiseptics in reducing the viability of planktonic and attached streptococci was Octenisept > Olaflur > chlorhexidine > saline > chitosan. Further studies appear warranted to develop new antiplaque/antibiofilm strategies involving highly efficient bactericidals with antiadhesive formulations.