Sebastian Schlafer

Endodontic photoactivated disinfection using a conventional light source: an in vitro and ex vivo study.

OBJECTIVE The antimicrobial effect of photoactivated disinfection (PAD) using toluidine blue and an LED lamp was tested on endodontic pathogens in planktonic suspension and after inoculation into extracted teeth. Irradiation time was limited to 30 seconds. STUDY DESIGN The effect of PAD on planktonic suspensions of Escherichia coli, Candida albicans, Enterococcus faecalis, Fusobacterium nucleatum, and Streptococcus intermedius was analyzed using Poisson regression. Moreover, cultures of S. intermedius were inoculated into prepared root canals of extracted molars. The effect of PAD performed immediately after inoculation or after overnight bacterial incubation was determined by a 2-sample t test. RESULTS Photoactivated disinfection yielded significant reductions (P < .001) in the viable counts of all organisms in planktonic suspension. The PAD treatment of S. intermedius in root canals yielded a mean log10 reduction of 2.60 (P < .001) immediately after inoculation and of 1.38 (P < .001) after overnight incubation. CONCLUSION Photoactivated disinfection using a conventional light source strongly reduces the number of viable endodontic pathogens in planktonic suspension and in root canals.

pH Landscapes in a Novel Five-Species Model of Early Dental Biofilm

Background Despite continued preventive efforts, dental caries remains the most common disease of man. Organic acids produced by microorganisms in dental plaque play a crucial role for the development of carious lesions. During early stages of the pathogenetic process, repeated pH drops induce changes in microbial composition and favour the establishment of an increasingly acidogenic and aciduric microflora. The complex structure of dental biofilms, allowing for a multitude of different ecological environments in close proximity, remains largely unexplored. In this study, we designed a laboratory biofilm model that mimics the bacterial community present during early acidogenic stages of the caries process. We then performed a time-resolved microscopic analysis of the extracellular pH landscape at the interface between bacterial biofilm and underlying substrate. Methodology/Principal Findings Strains of Streptococcus oralis, Streptococcus sanguinis, Streptococcus mitis, Streptococcus downei and Actinomyces naeslundii were employed in the model. Biofilms were grown in flow channels that allowed for direct microscopic analysis of the biofilms in situ. The architecture and composition of the biofilms were analysed using fluorescence in situ hybridization and confocal laser scanning microscopy. Both biofilm structure and composition were highly reproducible and showed similarity to in-vivo-grown dental plaque. We employed the pH-sensitive ratiometric probe C-SNARF-4 to perform real-time microscopic analyses of the biofilm pH in response to salivary solutions containing glucose. Anaerobic glycolysis in the model biofilms created a mildly acidic environment. Decrease in pH in different areas of the biofilms varied, and distinct extracellular pH-microenvironments were conserved over several hours. Conclusions/Significance The designed biofilm model represents a promising tool to determine the effect of potential therapeutic agents on biofilm growth, composition and extracellular pH. Ratiometric pH analysis using C-SNARF-4 gives detailed insight into the pH landscape of living biofilms and contributes to our general understanding of metabolic processes in in-vivo-grown bacterial biofilms.

Osteopontin Reduces Biofilm Formation in a Multi-Species Model of Dental Biofilm

Background Combating dental biofilm formation is the most effective means for the prevention of caries, one of the most widespread human diseases. Among the chemical supplements to mechanical tooth cleaning procedures, non-bactericidal adjuncts that target the mechanisms of bacterial biofilm formation have gained increasing interest in recent years. Milk proteins, such as lactoferrin, have been shown to interfere with bacterial colonization of saliva-coated surfaces. We here study the effect of bovine milk osteopontin (OPN), a highly phosphorylated whey glycoprotein, on a multispecies in vitro model of dental biofilm. While considerable research effort focuses on the interaction of OPN with mammalian cells, there are no data investigating the influence of OPN on bacterial biofilms. Methodology/Principal Findings Biofilms consisting of Streptococcus oralis, Actinomyces naeslundii, Streptococcus mitis, Streptococcus downei and Streptococcus sanguinis were grown in a flow cell system that permitted in situ microscopic analysis. Crystal violet staining showed significantly less biofilm formation in the presence of OPN, as compared to biofilms grown without OPN or biofilms grown in the presence of caseinoglycomacropeptide, another phosphorylated milk protein. Confocal microscopy revealed that OPN bound to the surface of bacterial cells and reduced mechanical stability of the biofilms without affecting cell viability. The bacterial composition of the biofilms, determined by fluorescence in situ hybridization, changed considerably in the presence of OPN. In particular, colonization of S. mitis, the best biofilm former in the model, was reduced dramatically. Conclusions/Significance OPN strongly reduces the amount of biofilm formed in a well-defined laboratory model of acidogenic dental biofilm. If a similar effect can be observed in vivo, OPN might serve as a valuable adjunct to mechanical tooth cleaning procedures.

Confocal microscopy imaging of the biofilm matrix

The extracellular matrix is an integral part of microbial biofilms and an important field of research. Confocal laser scanning microscopy is a valuable tool for the study of biofilms, and in particular of the biofilm matrix, as it allows real-time visualization of fully hydrated, living specimens. Confocal microscopes are held by many research groups, and a number of methods for qualitative and quantitative imaging of the matrix have emerged in recent years. This review provides an overview and a critical discussion of techniques used to visualize different matrix compounds, to determine the concentration of solutes and the diffusive properties of the biofilm matrix.

Ratiometric imaging of extracellular pH in bacterial biofilms with C-SNARF-4.

ABSTRACT pH in the extracellular matrix of bacterial biofilms is of central importance for microbial metabolism. Biofilms possess a complex three-dimensional architecture characterized by chemically different microenvironments in close proximity. For decades, pH measurements in biofilms have been limited to monitoring bulk pH with electrodes. Although pH microelectrodes with a better spatial resolution have been developed, they do not permit the monitoring of horizontal pH gradients in biofilms in real time. Quantitative fluorescence microscopy can overcome these problems, but none of the hitherto employed methods differentiated accurately between extracellular and intracellular microbial pH and visualized extracellular pH in all areas of the biofilms. Here, we developed a method to reliably monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4, choosing dental biofilms as an example. Fluorescent emissions of C-SNARF-4 can be used to calculate extracellular pH irrespective of the dye concentration. We showed that at pH values of <6, C-SNARF-4 stained 15 bacterial species frequently isolated from dental biofilm and visualized the entire bacterial biomass in in vivo-grown dental biofilms with unknown species composition. We then employed digital image analysis to remove the bacterial biomass from the microscopic images and adequately calculate extracellular pH values. As a proof of concept, we monitored the extracellular pH drop in in vivo-grown dental biofilms fermenting glucose. The combination of pH ratiometry with C-SNARF-4 and digital image analysis allows the accurate monitoring of extracellular pH in bacterial biofilms in three dimensions in real time and represents a significant improvement to previously employed methods of biofilm pH measurement.

Effect of Osteopontin on the Initial Adhesion of Dental Bacteria

Bacterial biofilms are involved in numerous infections of the human body, including dental caries. While conventional therapy of biofilm diseases aims at eradication and mechanical removal of the biofilms, recent therapeutic approaches target the mechanisms of biofilm formation and bacterial adhesion in particular. The effect of bovine milk osteopontin, a highly phosphorylated whey protein, on adhesion of Streptococcus mitis, Streptococcus sanguinis, and Actinomyces naeslundii, three prominent colonizers in dental biofilms, to saliva-coated surfaces was investigated. While adhesion of A. naeslundii was not affected by osteopontin, a strong, dose-dependent reduction in the number of adhering S. mitis was shown. No difference in bacterial adhesion was observed for caseinoglycomacropeptide, another phosphorylated milk protein. Osteopontin did not affect bacterial viability, but changed bacterial surface hydrophobicity, and may be suggested to prevent the adhesins of S. mitis from interacting with their salivary receptors. The antiadhesive effect of osteopontin may be useful for caries prevention.

Ratiometric Imaging of Extracellular pH in Dental Biofilms.

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 µm thickness, but is limited to the pH range between 4.5 and 7.0.

Calcium-phosphate-osteopontin particles for caries control

Abstract Caries is caused by acid production in biofilms on dental surfaces. Preventing caries therefore involves control of microorganisms and/or the acid produced. Here, calcium-phosphate-osteopontin particles are presented as a new approach to caries control. The particles are made by co-precipitation and designed to bind to bacteria in biofilms, impede biofilm build-up without killing the microflora, and release phosphate ions to buffer bacterial acid production if the pH decreases below 6. Analysis of biofilm formation and pH in a five-species biofilm model for dental caries showed that treatment with particles or pure osteopontin led to less biofilm formation compared to untreated controls or biofilms treated with osteopontin-free particles. The anti-biofilm effect can thus be ascribed to osteopontin. The particles also led to a slower acidification of the biofilm after exposure to glucose, and the pH always remained above 5.5. Hence, calcium-phosphate-osteopontin particles show potential for applications in caries control.

Comparison of Riboflavin and Toluidine Blue O as Photosensitizers for Photoactivated Disinfection on Endodontic and Periodontal Pathogens In Vitro.

Photoactivated disinfection has a strong local antimicrobial effect. In the field of dentistry it is an emerging adjunct to mechanical debridement during endodontic and periodontal treatment. In the present study, we investigate the effect of photoactivated disinfection using riboflavin as a photosensitizer and blue LED light for activation, and compare it to photoactivated disinfection with the widely used combination of toluidine blue O and red light. Riboflavin is highly biocompatible and can be activated with LED lamps at hand in the dental office. To date, no reports are available on the antimicrobial effect of photoactivated disinfection using riboflavin/blue light on oral microorganisms. Planktonic cultures of eight organisms frequently isolated from periodontal and/or endodontic lesions (Aggregatibacter actinomycetemcomitans, Candida albicans, Enterococcus faecalis, Escherischia coli, Lactobacillus paracasei, Porphyromonas gingivalis, Prevotella intermedia and Propionibacterium acnes) were subjected to photoactivated disinfection with riboflavin/blue light and toluidine blue O/red light, and survival rates were determined by CFU counts. Within the limited irradiation time of one minute, photoactivated disinfection with riboflavin/blue light only resulted in minor reductions in CFU counts, whereas full kills were achieved for all organisms when using toluidine blue O/red light. The black pigmented anaerobes P. gingivalis and P. intermedia were eradicated completely by riboflavin/blue light, but also by blue light treatment alone, suggesting that endogenous chromophores acted as photosensitizers in these bacteria. On the basis of our results, riboflavin cannot be recommended as a photosensitizer used for photoactivated disinfection of periodontal or endodontic infections.

Difference in initial dental biofilm accumulation between night and day

Abstract Objective. The study of initial microbial colonization on dental surfaces is a field of intensive research because of the aetiological role of biofilms in oral diseases. Most previous studies of de novo accumulation and composition of dental biofilms in vivo do not differentiate between biofilms formed during day and night. This study hypothesized that there is a diurnal variation in the rate of accumulation of bacteria on solid surfaces in the oral cavity. Materials and methods. In situ biofilm from healthy individuals was collected for 12 h during day and night, respectively, subjected to fluorescent in situ hybridization and visualized using confocal laser scanning microscopy. Results. Analysis of the biofilms using stereological methods and digital image analysis revealed a consistent statistically significant difference between both the total number of bacteria and the biovolume in the two 12-h groups (p = 0.012), with the highest accumulation of bacteria during daytime (a factor of 8.8 and 6.1 higher, respectively). Hybridization with probes specific for streptococci and Actinomyces naeslundii indicated a higher proportion of streptococci in biofilms grown during daytime as compared to night-time. No differences could be observed for A. naeslundii. The degree of microbial coverage and the bacterial composition varied considerably between different individuals. Conclusion. The data provide firm evidence that initial biofilm formation decreases during the night, which may reflect differences in the availability of salivary nutrients. This finding is of significant importance when studying population dynamics during experimental dental biofilm formation.