Analysis of Oral Microbiota in Children with Dental Caries by PCR-DGGE and Barcoded Pyrosequencing

Abstract

Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common oral diseases in children and pathogenic bacteria contribute to the development of the disease. However, the overall structure of bacterial communities in the oral cavity from children with dental caries has not been explored deeply heretofore. We used high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis (DGGE) to examine bacterial diversity of oral microbiota in saliva and supragingival plaques from 60 children aged 3 to 6 years old with and without dental caries from China. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers. As PCR-DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results of both approaches were compared. A total of 186,787 high-quality sequences were obtained for evaluating bacterial diversity and 41,905 unique sequences represented all phylotypes. We found that the oral microbiota in children was far more diverse than previous studies reported, and more than 200 genera belonging to ten phyla were found in the oral cavity. The phylotypes in saliva and supragingival plaques were significantly different and could be divided into two distinct clusters (p < 0.05). The bacterial diversity in oral microbiome analyzed by PCR-DGGE and barcoded pyrosequencing was employed to cross validate the data sets. The genera of Streptococcus, Veillonella, Actinomyces, Granulicatella, Leptotrichia, and Thiomonas in plaques were significantly associated with dental caries (p < 0.05). The results showed that there was no one specific pathogen but rather pathogenic populations in plaque that significantly correlated with dental caries. The enormous diversity of oral microbiota allowed for a better understanding of oral microecosystem, and these pathogenic populations in plaque provide new insights into the etiology of dental caries and suggest new targets for interventions of the disease.

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Acknowledgements

This present work was funded by the grant of National Basic Research Program of China (973 Program) No. 2007CB513001, and partly supported by grants from China’s National Science and Technology Major Project (No. 2008ZX10002-009, No. 2008ZX10004-002 and 2009ZX10004-105). We thank Dr. Michael Brownstein and Dr. Liliana Losada for critical reading and useful suggestions.

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Correspondence to Hui Chen or Charlie Xiang.

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Table S1

List of the 112 8-bp barcodes used to tag each PCR product analyzed as part of the study (DOC 53 kb)

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Ling, Z., Kong, J., Jia, P. et al. Analysis of Oral Microbiota in Children with Dental Caries by PCR-DGGE and Barcoded Pyrosequencing. Microb Ecol 60, 677–690 (2010). https://doi.org/10.1007/s00248-010-9712-8

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Keywords

  • Oral Cavity
  • Dental Caries
  • Bacterial Diversity
  • Dental Plaque
  • Actinomyces