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

McLean JS, Fansler SJ, Majors PD, McAteer K, Allen LZ, Shirtliff ME, Lux R, Shi W - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Microbial and Environmental Genomics, The J Craig Venter Institute, San Diego, California, United States of America. jmclean@jcvi.org

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3293899&req=5

pone-0032219-g006: 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.

Mentions: In vivo MRS experiments at pH 7 with 13C-labeled glucose as carbon source (Figure 1A) revealed a rapid rise in lactate concentrations followed by a decrease of lactate after glucose was exhausted. The utilization of lactate was accompanied by an increase in acetate and formate. This indicates that at least under buffered conditions, lactate is available for further utilization. In order to identify these lactate-metabolizing species, a sample was prepared according to the procedures described (materials and methods) and incubated with 14 mM of 13C-lactate without glucose in CDM buffered to pH 7 with phosphate buffer (Figure 5). In the absence of glucose only 1 mM of 13C-lactate was converted to formate and acetate. Sequencing of the respective clone libraries obtained for heavy and light fractions revealed that the most dominant genera in the heavy fraction and thus the metabolically active were Neisseria, Streptococcus and Granulicatella (Figure 6b) with N. sicca HOT 764 being the most dominant active taxon (p<0.01). Veillonella strains, which are known to be able to grow on lactate, were active under these conditions as expected. However, this genus comprised only 1.5% of the total clones in the heavy fraction.


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

McLean JS, Fansler SJ, Majors PD, McAteer K, Allen LZ, Shirtliff ME, Lux R, Shi W - PLoS ONE (2012)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293899&req=5

pone-0032219-g006: 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.
Mentions: In vivo MRS experiments at pH 7 with 13C-labeled glucose as carbon source (Figure 1A) revealed a rapid rise in lactate concentrations followed by a decrease of lactate after glucose was exhausted. The utilization of lactate was accompanied by an increase in acetate and formate. This indicates that at least under buffered conditions, lactate is available for further utilization. In order to identify these lactate-metabolizing species, a sample was prepared according to the procedures described (materials and methods) and incubated with 14 mM of 13C-lactate without glucose in CDM buffered to pH 7 with phosphate buffer (Figure 5). In the absence of glucose only 1 mM of 13C-lactate was converted to formate and acetate. Sequencing of the respective clone libraries obtained for heavy and light fractions revealed that the most dominant genera in the heavy fraction and thus the metabolically active were Neisseria, Streptococcus and Granulicatella (Figure 6b) with N. sicca HOT 764 being the most dominant active taxon (p<0.01). Veillonella strains, which are known to be able to grow on lactate, were active under these conditions as expected. However, this genus comprised only 1.5% of the total clones in the heavy fraction.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Microbial and Environmental Genomics, The J Craig Venter Institute, San Diego, California, United States of America. jmclean@jcvi.org

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.

Show MeSH
Related in: MedlinePlus