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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.

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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.
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pone-0032219-g001: 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.

Mentions: In order to gain insight into community metabolite profile progression, metabolic activities in plaque samples were monitored at 37°C within the NMR under the following experimental conditions: 1) buffered at pH 7 to reflect a non-cariogenic state and 2) low pH conditions with no buffering to simulate initial caries active conditions. Samples from healthy subjects were incubated with 13C-labeled glucose in CDM buffered to pH 7 or unbuffered with a starting pH of 5.5. Glucose utilization was rapid and complete in the buffered system (Figure 1A). Upon glucose depletion at 19–20 hrs, lactate utilization was evident accompanied by an increase in acetate and minor amounts of formate and ethanol. In contrast, the unbuffered sample adjusted to an initial pH of 5.5 (Figure 1B) was marked by inhibition of glucose fermentation with lactate as the major byproduct.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0032219-g001: 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.
Mentions: In order to gain insight into community metabolite profile progression, metabolic activities in plaque samples were monitored at 37°C within the NMR under the following experimental conditions: 1) buffered at pH 7 to reflect a non-cariogenic state and 2) low pH conditions with no buffering to simulate initial caries active conditions. Samples from healthy subjects were incubated with 13C-labeled glucose in CDM buffered to pH 7 or unbuffered with a starting pH of 5.5. Glucose utilization was rapid and complete in the buffered system (Figure 1A). Upon glucose depletion at 19–20 hrs, lactate utilization was evident accompanied by an increase in acetate and minor amounts of formate and ethanol. In contrast, the unbuffered sample adjusted to an initial pH of 5.5 (Figure 1B) was marked by inhibition of glucose fermentation with lactate as the major byproduct.

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