ratiometric-imaging-of-extracellular-ph-in-dental-biofilms

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 ...

Aarhus University

Confocal laser scanning microscopy (CLSM) has been employed as a method for studying intact natural biofilm. When combined with fluorescence in situ hybridization (FISH) it is possible to analyze spatial relationships and changes of specific members of microbial populations over time. The aim of this study was to perform a systematic description of the pattern of initial dental biofilm formation by applying 16S rRNA-targeted oligonucleotide probes to the identification of streptococci and other bacteria, and to evaluate the usefulness of the combination of CLSM and FISH for structural studies of bacterial populations in dental biofilm. Biofilms were collected on standardized glass slabs mounted in intra-oral appliances and worn by 10 individuals for 6, 12, 24 or 48 h. After intra-oral exposure the biofilms were labelled with probes against either streptococci (STR405) or all bacteria (EUB338) and analysed by CLSM. The current approach of using FISH techniques enabled differentiation of streptococci from other bacteria and determination of their spatio-temporal organization. The presence of chimney-like multilayered microcolonies with different microbial compositions demonstrated by this methodology provided information supplementary to our previous knowledge obtained by classical electron microscopic methods and increased our understanding of the structure of developing biofilms.

In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization.

Less invasive excavation methods have been suggested for deep caries lesions. We tested the effects of stepwise vs. direct complete excavation, 1 yr after the procedure had been carried out, in 314 adults (from six centres) who had received treatment of a tooth with deep caries. The teeth had caries lesions involving 75% or more of the dentin and were centrally randomized to stepwise or direct complete excavation. Stepwise excavation resulted in fewer pulp exposures compared with direct complete excavation [difference: 11.4%, 95% confidence interval (CI) (1.2; 21.3)]. At 1 yr of follow-up, there was a statistically significantly higher success rate with stepwise excavation, with success being defined as an unexposed pulp with sustained pulp vitality without apical radiolucency [difference: 11.7%, 95% CI (0.5; 22.5)]. In a subsequent nested trial, 58 patients with exposed pulps were randomized to direct capping or partial pulpotomy. We found no significant difference in pulp vitality without apical radiolucency between the two capping procedures after more than 1 yr [31.8% and 34.5%; difference: 2.7%, 95% CI (-22.7; 26.6)]. In conclusion, stepwise excavation decreases the risk of pulp exposure compared with direct complete excavation. In view of the poor prognosis of vital pulp treatment, a stepwise excavation approach for managing deep caries lesions is recommended.

Treatment of deep caries lesions in adults: randomized clinical trials comparing stepwise vs. direct complete excavation, and direct pulp capping vs. partial pulpotomy.

It has become evident that aggregation or biofilm formation is an important survival mechanism for bacteria in almost any environment. In this review, we summarize recent visualizations of bacterial aggregates in several chronic infections (chronic otitis media, cystic fibrosis, infection due to permanent tissue fillers and chronic wounds) both as to distribution (such as where in the wound bed) and organization (monospecies or multispecies microcolonies). We correlate these biofilm observations to observations of commensal biofilms (dental and intestine) and biofilms in natural ecosystems (soil). The observations of the chronic biofilm infections point toward a trend of low bacterial diversity and sovereign monospecies biofilm aggregates even though the infection in which they reside are multispecies. In contrast to this, commensal and natural biofilm aggregates contain multiple species that are believed to coexist, interact and form biofilms with high bacterial and niche diversity. We discuss these differences from both the diagnostic and the scientific point of view.

Biofilms in chronic infections - a matter of opportunity - monospecies biofilms in multispecies infections.

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.

pH landscapes in a novel five-species model of early dental biofilm.

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.

Difference in initial dental biofilm accumulation between night and day.

The aim of the present study was to investigate the clinical performance of a low-shrinkage silorane-based composite material (Filtek™ Silorane, 3 M-Espe) by comparing it with a methacrylate-based composite material (Ceram•X™, Dentsply DeTrey).

Five-year evaluation of a low-shrinkage Silorane resin composite material: a randomized clinical trial.

Microbiological studies of occlusal dental biofilms have hitherto been hampered by inaccessibility to the sampling site and demolition of the original biofilm architecture. This study shows for the first time the spatial distribution of bacterial taxa in vivo at various stages of occlusal caries, applying a molecular methodology involving preparation of embedded hard dental tissue slices for fluorescence in situ hybridization (FISH) and confocal microscopy. Eleven freshly extracted teeth were classified according to their occlusal caries status. The teeth were fixed, embedded, sectioned and decalcified before FISH was performed using oligonucleotide probes for selected abundant species/genera associated with occlusal caries including Streptococcus, Actinomyces, Veillonella, Fusobacterium, Lactobacillus and Bifidobacterium. The sites showed distinct differences in the bacterial composition between different ecological niches in occlusal caries. Biofilm observed along the entrance of fissures showed an inner layer of microorganisms organized in palisades often identified as Actinomyces, covered by a more loosely structured bacterial layer consisting of diverse genera, similar to supragingival biofilm. Biofilm within the fissure proper seemed less metabolically active, as judged by low fluorescence signal intensity and presence of material of non-bacterial origin. Bacterial invasion (often Lactobacillus and Bifidobacterium spp.) into the dentinal tubules was seen only at advanced stages of caries with manifest cavity formation. It is concluded that the molecular methodology represents a valuable supplement to previous methods for the study of microbial ecology in caries by allowing analysis of the structural composition of the undisturbed biofilm in caries lesions in vivo.

Molecular studies of the structural ecology of natural occlusal caries.

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.

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

pH in dental biofilms is of central importance for the development of caries. We used the ratiometric pH-sensitive dye C-SNARF-4 in combination with digital image analysis to monitor extracellular pH in dental biofilms grown in situ with and without sucrose supply.

Article	Year
In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization. European journal of oral sciences\| PubMed ID: 18028053	2007
Treatment of deep caries lesions in adults: randomized clinical trials comparing stepwise vs. direct complete excavation, and direct pulp capping vs. partial pulpotomy. European journal of oral sciences\| PubMed ID: 20572864	2010
Biofilms in chronic infections - a matter of opportunity - monospecies biofilms in multispecies infections. FEMS immunology and medical microbiology\| PubMed ID: 20602635	2010
pH landscapes in a novel five-species model of early dental biofilm. PloS one\| PubMed ID: 21966490	2011
Difference in initial dental biofilm accumulation between night and day. Acta odontologica Scandinavica\| PubMed ID: 22126594	2012
Five-year evaluation of a low-shrinkage Silorane resin composite material: a randomized clinical trial. Clinical oral investigations\| PubMed ID: 24687249	2015
Molecular studies of the structural ecology of natural occlusal caries. Caries research\| PubMed ID: 24852305	2014
Ratiometric imaging of extracellular pH in bacterial biofilms with C-SNARF-4. Applied and environmental microbiology\| PubMed ID: 25501477	2015
Monitoring of extracellular pH in young dental biofilms grown in vivo in the presence and absence of sucrose. Journal of oral microbiology\| PubMed ID: 26894480	2016

Irene Dige

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