Abstract
BackgroundMany human microbial infectious diseases including dental caries are polymicrobial in nature. How these complex multi-species communities evolve from a healthy to a diseased state is not well understood. Although many health- or disease-associated oral bacteria have been characterized in vitro, their physiology within the complex oral microbiome is difficult to determine with current approaches. In addition, about half of these species remain uncultivated to date with little known besides their 16S rRNA sequence. Lacking culture-based physiological analyses, the functional roles of uncultivated species will remain enigmatic despite their apparent disease correlation. To start addressing these knowledge gaps, we applied a combination of Magnetic Resonance Spectroscopy (MRS) with RNA and DNA based Stable Isotope Probing (SIP) to oral plaque communities from healthy children for in vitro temporal monitoring of metabolites and identification of metabolically active and inactive bacterial species.Methodology/Principal FindingsSupragingival plaque samples from caries-free children incubated with 13C-substrates under imposed healthy (buffered, pH 7) and diseased states (pH 5.5 and pH 4.5) produced lactate as the dominant organic acid from glucose metabolism. Rapid lactate utilization upon glucose depletion was observed under pH 7 conditions. SIP analyses revealed a number of genera containing cultured and uncultivated taxa with metabolic capabilities at pH 5.5. The diversity of active species decreased significantly at pH 4.5 and was dominated by Lactobacillus and Propionibacterium species, both of which have been previously found within carious lesions from children.Conclusions/SignificanceOur approach allowed for identification of species that metabolize carbohydrates under different pH conditions and supports the importance of Lactobacilli and Propionibacterium in the development of childhood caries. Identification of species within healthy subjects that are active at low pH can lead to a better understanding of oral caries onset and generate appropriate targets for preventative measures in the early stages.
Highlights
Oral biofilms represent an accessible model system for investigating inter-species interactions and functional relationships within multi-species communities [1,2]
Samples from healthy subjects were incubated with 13C-labeled glucose in chemically defined medium (CDM) buffered to pH 7 or unbuffered with a starting pH of 5.5
RNA and DNA based Stable Isotope Probing Isotopically labeled samples were subjected to either RNA or DNA extraction followed by density gradient centrifugation
Summary
Oral biofilms represent an accessible model system for investigating inter-species interactions and functional relationships within multi-species communities [1,2]. The ‘‘ecological plaque hypothesis’’ developed by Marsh and colleagues [13] reflects this shift in our understanding of oral diseases by stating in essence that oral caries and periodontal diseases arise as a result of environmental perturbations to the habitat that cause shifts in the balance of the resident microbiota Key features of this hypothesis are that (a) the selection of ‘pathogenic’ bacteria is directly coupled to changes in the environment and (b) diseases do not need to have a specific etiology; any species with relevant traits (i.e. acid production) can contribute to the disease process. Lacking culture-based physiological analyses, the functional roles of uncultivated species will remain enigmatic despite their apparent disease correlation To start addressing these knowledge gaps, we applied a combination of Magnetic Resonance Spectroscopy (MRS) with RNA and DNA based Stable Isotope Probing (SIP) to oral plaque communities from healthy children for in vitro temporal monitoring of metabolites and identification of metabolically active and inactive bacterial species
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