Identifying low pH active and lactate-utilizing taxa within oral microbiome communities from healthy children using stable isotope probing techniques

Abstract

Background: Many 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 findings: Supragingival plaque samples from caries-free children incubated with (13)C-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/significance: Our 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.

Figure 1. Temporal ¹H MRS analyses of metabolites during glucose utilization in live plaque samples incubated with CDM media and ¹³C-glucose.

(A) buffered CDM at an initial pH of 7 with 2 mM phosphate buffer and (B) unbuffered CDM at an initial pH of 5.5.

Figure 2. Comparative analyses of 16S rRNA sequences observed in the light and heavy fractions from samples incubated with ¹³C-glucose for 6 hrs.

(A) buffered pH 7 conditions and (B) unbuffered pH 5.5 conditions. Data are reported for the dominant members that represent >2% of clones in the libraries. Taxa that differed significantly in detection frequencies are noted at *p<0.05, **p<0.01, ***p<0.001. The distribution of active genera in the heavy fractions is shown in the corresponding pie charts for each condition.

Figure 3. Comparative analyses of 16S rRNA sequences observed in the light and heavy fractions DNA SIP experiments incubated with ¹³C-glucose for 36 hrs.

(A) under buffered pH 7 conditions, (B) unbuffered pH 5.5 conditions. Data are reported for the dominant members that represent >2% of clones in the libraries. Taxa that differed significantly in detection frequencies are noted at *p<0.05, **p<0.01, ***p<0.001. The distribution of active genera in the heavy fractions is shown in the corresponding pie charts for each condition.

Figure 4. Comparative analyses of 16S rRNA sequences observed in the light and heavy fractions DNA SIP experiments incubated with ¹³C-glucose for 36 hrs at pH 4.5.

Comparative analyses of 16S rRNA sequences observed in the light and heavy fractions DNA SIP experiment incubated with ¹³C-glucose at pH 4.5 for 36 hrs under unbuffered pH 4.5 conditions. Taxa that differed significantly in detection frequencies are noted at *p<0.05, **p<0.01, ***p<0.001. The distribution of active genera in the heavy fractions is shown in the corresponding pie charts for each condition.

Figure 5. Live monitoring of ¹³C-lactate metabolism in plaque samples combined with DNA-SIP.

(A) In vivo MRS metabolite profiles of active metabolism for the plaque community incubated with ¹³C-lactate buffered at pH 7. (B) Comparative plot of the major taxa (>2%) detected in the heavy (active) and light fractions. Taxa that differed significantly in detection frequencies are noted at *p<0.05, **p<0.01, ***p<0.001.

Figure 6. All taxa identified in the isotopically heavy fractions representing the active members for each condition.

Heatmap of active members grouped by taxa in the ¹³C-DNA or RNA isotopically heavy fractions. Columns are mapped as the frequency of clones observed for each taxa within the respective clone library.