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

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.

A Combined Pharmacodynamic Quantitative and Qualitative Model Reveals the Potent Activity of Daptomycin and Delafloxacin against Staphylococcus aureus Biofilms

“…A possible explanation could reside in a local environment favorable to the expression of delafloxacin activity within the biofilm. We know, for example, that delafloxacin, in contrast to daptomycin, gains potency in acidic environments (51), which may be the case within biofilms, as suggested from studies on biofilms of Pseudomonas or streptococci (62,63). Delafloxacin even seems to be more active than daptomycin against the MRSA strain at equipotent concentrations, suggesting that further comparisons of the activities of these two drugs against biofilms from recent clinical isolates should be conducted.…”

Dispersal of Biofi lms by Secreted, Matrix Degrading, Bacterial DNase

“…In the present study, we investigate the effect of bovine milk osteopontin (OPN), a glycosylated and highly phosphorylated whey protein, on a well-described laboratory model of dental biofilm composed of Streptococcus oralis, Actinomyces naeslundii, Streptococcus mitis, Streptococcus downei and Streptococcus sanguinis [15]. OPN is also expressed in a variety of human tissues and involved in numerous biological processes, including bone and tooth mineralization, wound healing and leukocyte recruitment [16][17][18].…”

Ratiometric Imaging of Extracellular pH in Bacterial Biofilms with C-SNARF-4

“…In principle, digital image analysis might well be employed to remove the extracellular space in the biofilm images and calculate fluorescent ratios inside bacterial cells. However, so far the calibration of C-SNARF-4 has been performed only for the extracellular space, using characteristic biofilm matrix components (34) and unstained biofilms as controls (39). Intracellular pH recordings would require a thorough calibration of the dye prior to quantitative interpretation of the fluorescence ratios (42).…”

“…Subsequently, the ratio R, standard deviations, s R , and standard errors of means, S R , were calculated for each pH value as previously described (39). The resulting values of R were plotted in SigmaPlot 10 (Systat Software, Inc., San Jose, CA, USA) and fitted to the following function:…”