Abstract

BackgroundDespite 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 FindingsStrains 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/SignificanceThe 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.

Highlights

  • Laboratory models simulating the microbial and chemical conditions that lead to tooth decay have a long history

  • The surface topography was characterised by depressions and protuberances (Video S1), and biofilm thickness in the fields of view chosen for composition analysis varied between 7 and 68 mm, with an average thickness of 29 mm after fluorescence in situ hybridization (FISH) procedure

  • We developed and validated a laboratory dental biofilm model that exclusively employed mildly acidogenic human oral bacteria

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Summary

Introduction

Laboratory models simulating the microbial and chemical conditions that lead to tooth decay have a long history. Laboratory models will always fail to recreate the complexity of the oral environment, they offer many advantages. They circumvent the ethical conflicts that arise in clinical studies and permit to model and analyse a variety of important in-vivo-processes in a highly reproducible fashion. Dental biofilm models have contributed much to elucidate the cariogenic potential of different microorganisms [4,5,6,7], the role of microbial interactions in biofilm development [8,9,10,11] and the effect and working mechanisms of caries-preventive agents [12,13,14,15,16,17]. We designed a laboratory biofilm model that mimics the bacterial community present during early acidogenic stages of the caries process. We performed a time-resolved microscopic analysis of the extracellular pH landscape at the interface between bacterial biofilm and underlying substrate

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