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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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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.
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pone-0032219-g002: 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.

Mentions: RNA-SIP was used to investigate the active species at 6 hrs of incubation when the glucose utilization was in progress and approximately 2 mM of lactate accumulation had occurred (Figure 1). Comparative analyses of 16S rRNA sequences observed in the light and heavy fractions from samples incubated with 13C Glucose for 6 hrs under buffered pH7 conditions and unbuffered pH5.5 conditions are shown in Figure 2. Data are reported for the dominant members that represent >2% of clones in the libraries. Species that differed significantly in detection frequencies between the heavy and light clone libraries are noted at *p<0.05, **p<0.01, ***p<0.001. The distribution of active genera represented by the heavy fractions is shown in the corresponding pie charts for each condition. The distribution of all the active genera observed under buffered pH 7 conditions (Figure 2A) revealed that Streptoccocus, Veillonella, Neisseria, and Granulicatella were abundant. Active species at pH of 5.5 under unbuffered conditions (Figure 2B) exhibited a similarly relative high diversity indicating that this pH was tolerable for many genera. The active genera under this lower pH included the same major genera found in the pH 7 samples. Species that appeared to increase their relative activity at pH 5.5 included G. adiacens (para-adiacens) HOT 534 Strain TKT1 (p<0.001), Variovorax paradoxus HOT and an uncultivated species Peptostreptococcus sp. oral clone 6893283 (p<0.05). A number of taxa that are currently designated as unnamed cultivated taxon such as Streptococcus sp. HOT 056, Streptococcus sp. HOT 074 and Porphyromonas sp. HOT 279 were also found to be active at this lower pH. Taxa that were present in the community and had a lowered apparent relative activity as indicated by their dominance in the light fraction (p<0.05) (p<0.05) included G. adiacens HOT 534 Strain GIFU12706, N. elongate HOT 598, S. gordonii HOT 622 and V. parvula HOT 161.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0032219-g002: 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.
Mentions: RNA-SIP was used to investigate the active species at 6 hrs of incubation when the glucose utilization was in progress and approximately 2 mM of lactate accumulation had occurred (Figure 1). Comparative analyses of 16S rRNA sequences observed in the light and heavy fractions from samples incubated with 13C Glucose for 6 hrs under buffered pH7 conditions and unbuffered pH5.5 conditions are shown in Figure 2. Data are reported for the dominant members that represent >2% of clones in the libraries. Species that differed significantly in detection frequencies between the heavy and light clone libraries are noted at *p<0.05, **p<0.01, ***p<0.001. The distribution of active genera represented by the heavy fractions is shown in the corresponding pie charts for each condition. The distribution of all the active genera observed under buffered pH 7 conditions (Figure 2A) revealed that Streptoccocus, Veillonella, Neisseria, and Granulicatella were abundant. Active species at pH of 5.5 under unbuffered conditions (Figure 2B) exhibited a similarly relative high diversity indicating that this pH was tolerable for many genera. The active genera under this lower pH included the same major genera found in the pH 7 samples. Species that appeared to increase their relative activity at pH 5.5 included G. adiacens (para-adiacens) HOT 534 Strain TKT1 (p<0.001), Variovorax paradoxus HOT and an uncultivated species Peptostreptococcus sp. oral clone 6893283 (p<0.05). A number of taxa that are currently designated as unnamed cultivated taxon such as Streptococcus sp. HOT 056, Streptococcus sp. HOT 074 and Porphyromonas sp. HOT 279 were also found to be active at this lower pH. Taxa that were present in the community and had a lowered apparent relative activity as indicated by their dominance in the light fraction (p<0.05) (p<0.05) included G. adiacens HOT 534 Strain GIFU12706, N. elongate HOT 598, S. gordonii HOT 622 and V. parvula HOT 161.

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