title: pH landscapes in a novel five-species model of early dental biofilm.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: iNANO The Interdisciplinary Nanoscience Center, Faculty of Science, Aarhus University, Aarhus, Denmark. sebastian.schlafer@hotmail.de
authors.forename: Sebastian
authors.initials: S
authors.lastname: Schlafer
authors.forename: Merete K
authors.initials: MK
authors.lastname: Raarup
authors.forename: Rikke L
authors.initials: RL
authors.lastname: Meyer
authors.forename: Duncan S
authors.initials: DS
authors.lastname: Sutherland
authors.forename: Irene
authors.initials: I
authors.lastname: Dige
authors.forename: Jens R
authors.initials: JR
authors.lastname: Nyengaard
authors.forename: Bente
authors.initials: B
authors.lastname: Nyvad
mesh_terms: D000190:Actinomyces / Q000235:genetics / Q000254:growth & development; D018441:Biofilms / Q000254:growth & development*; D003773:Dental Plaque / Q000382:microbiology*; D006863:Hydrogen-Ion Concentration; D017404:In Situ Hybridization, Fluorescence; D018613:Microscopy, Confocal; D012336:RNA, Ribosomal, 16S; D013291:Streptococcus / Q000235:genetics / Q000254:growth & development; D034361:Streptococcus mitis / Q000235:genetics / Q000254:growth & development; D019533:Streptococcus oralis / Q000235:genetics / Q000254:growth & development; D013298:Streptococcus sanguis / Q000235:genetics / Q000254:growth & development
chemical_list: D012336:RNA, Ribosomal, 16S