应用PCR-DGGE和454焦磷酸测序分析儿童龋病相关口腔菌群多样性
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摘要
口腔微生物在维持口腔内环境稳定上起非常重要的作用。龋病是非常常见的一种牙齿疾病,特别是在幼儿期。然而对于患有龋病的儿童口腔内微生物整个菌群结构和动力学研究至今仍未被清晰的阐明。在本实验中,利用PCR-DGGE与454高通量焦磷酸测序技术相结合,对患有龋病儿童(CAC,下同)和无龋病儿童(CFC,下同)口腔内的微生物菌群进行多样性的比较分析。收集这些儿童的唾液和牙菌斑,提取DNA。设计通用引物测序扩增16S rDNA基因V3变异区进行测序和DGGE分析。测序总计得到186787条序列和41905个单一序列,经鉴定细菌来源10个门的200多个属。
研究结果显示来自CFC和CAC的细菌可以聚成两个簇和若干个种型,在龈上菌斑中Streptococcus(连锁状球菌属),Veillonella(韦荣球菌属),Actinomyces(放线菌属),Granulicatella(颗粒链菌属),Leptotrichia(纤毛菌属)和Thiomonas(硫单胞菌属)与龋病有关,这些细菌相互作用,逐渐破坏生态平衡,最终导致龋病的发生。我们无法找到一个单一的致病菌但是发现了对龋病发生有着显著相关的致病菌群,而且这些致病菌群有可能成为干预龋病发生的目标。
Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common diseases in children.Pathogenic bacteria contribute to the development of the disease.However,the overall structure and dynamics of bacterial communities in oral cavity from children with dental caries have not been explored deeply heretofore.In this study,we examined the bacterial diversity of oral microbiota from children with caries in comparison to those from caries-free children using PCR-DGGE and high-throughput barcoded pyrosequencing. Saliva and supragingival plaque samples were collected from 60 children.DNA was isolated from these samples and the hyper-variable V3 region of 16S rDNA genes was amplified using universal primers.A total of 186 787 sequences were obtained and 41 905 unique sequences represented all domains in our study.More than 200 genera belonging to 10 phyla were identified.
Our data showed that the oral microbiota in saliva and supragingival plaques could be divided into two clusters and several phylotypes that were significantly different between the two patient groups.The genera of Streptococcus,Veillonella, Actinomyces,Granulicatella,Leptotrichia and Thiomonas in supragingival plaques were associated with dental caries.These genera interacted with each other intimately and changes the composition of the bacterial population destroyed the eubiosis of the microcosm gradually and contributed to the development of dental caries.There was no one specific pathogen but pathogenic communities that correlated with dental caries significantly.And theses pathogenic communities might provide additional targets for dental caries intervention.
研究结果显示来自CFC和CAC的细菌可以聚成两个簇和若干个种型,在龈上菌斑中Streptococcus(连锁状球菌属),Veillonella(韦荣球菌属),Actinomyces(放线菌属),Granulicatella(颗粒链菌属),Leptotrichia(纤毛菌属)和Thiomonas(硫单胞菌属)与龋病有关,这些细菌相互作用,逐渐破坏生态平衡,最终导致龋病的发生。我们无法找到一个单一的致病菌但是发现了对龋病发生有着显著相关的致病菌群,而且这些致病菌群有可能成为干预龋病发生的目标。
Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common diseases in children.Pathogenic bacteria contribute to the development of the disease.However,the overall structure and dynamics of bacterial communities in oral cavity from children with dental caries have not been explored deeply heretofore.In this study,we examined the bacterial diversity of oral microbiota from children with caries in comparison to those from caries-free children using PCR-DGGE and high-throughput barcoded pyrosequencing. Saliva and supragingival plaque samples were collected from 60 children.DNA was isolated from these samples and the hyper-variable V3 region of 16S rDNA genes was amplified using universal primers.A total of 186 787 sequences were obtained and 41 905 unique sequences represented all domains in our study.More than 200 genera belonging to 10 phyla were identified.
Our data showed that the oral microbiota in saliva and supragingival plaques could be divided into two clusters and several phylotypes that were significantly different between the two patient groups.The genera of Streptococcus,Veillonella, Actinomyces,Granulicatella,Leptotrichia and Thiomonas in supragingival plaques were associated with dental caries.These genera interacted with each other intimately and changes the composition of the bacterial population destroyed the eubiosis of the microcosm gradually and contributed to the development of dental caries.There was no one specific pathogen but pathogenic communities that correlated with dental caries significantly.And theses pathogenic communities might provide additional targets for dental caries intervention.
引文
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[17] Whitman W. B., Coleman D. C., Wiebe W. J. Prokaryotes: the unseen majority[J]. Proceedings of the National Academy of Sciences, 1998 95 (12): 6578.
[18] Gill S. R., Pop M., Deboy R. T., Eckburg P. B., Tumbaugh P. J., Samuel B. S., Gordon J. I., Relman D. A., Fraser-Liggett C. M., Nelson K. E. Metagenomic analysis of the human distal gut microbiome[J]. Science, 2006 312 (5778): 1355-9.
[19] Kuramitsu H. K., He X., Lux R., Anderson M. H., Shi W. Interspecies interactions within oral microbial communities[J]. Microbiology and Molecular Biology Reviews, 2007 71 (4): 653.
[20] Fejerskov O., Kidd E. A. M. Dental caries: the disease and its clinical managemen[J]. Journal of Dental Education.
[21] Petersen P. E. The World Oral Health Report 2003: continuous improvement of oral health in the 21st century-the approach of the WHO Global Oral Health Programme[J]. Community Dentistry and Oral Epidemiology, 2003 31 (1): 3.
[22] Preza D., Olsen I., Aas J. A., Willumsen T., Grinde B., Paster B. J. Bacterial profiles of root caries in elderly patients[J]. J Clin Microbiol, 2008 46 (6): 2015-21.
[23] Kidd E. A. M., Fejerskov O. What constitutes dental caries? Histopathology of carious enamel and dentin related to the action of cariogenic biofilms[J]. Journal of dental research, 2004 83 (Supplement 1): C35.
[24] Fejerskov O. Changing paradigms in concepts on dental caries: consequences for oral health care[J]. Caries Res, 2004 38 (3): 182-191.
[25] Scheie A. A., Petersen F. C. The biofilm concept: consequences for future prophylaxis of oral diseases?[J]. Critical Reviews in Oral Biology & Medicine, 2004 15 (1): 4.
[26] Featherstone J. D. B. The science and practice of caries prevention[J]. The Journal of the American Dental Association, 2000 131 (7): 887.
[27] Featherstone J. D. B. The continuum of dental caries-evidence for a dynamic disease process[J]. Journal of dental research, 2004 83 (Supplement 1): C39.
[28] Selwitz R. H, Ismail A. I., Pitts N. B. Dental caries[J]. The Lancet, 2007 369 (9555): 51-59.
[29] Fejerskov O., Manji F. Risk assessment in dental caries[J]. Risk Assessment in Dentistry. Chapel Hill, University of North Carolina Dental Ecology, 1990: 215-217.
[30] 苏勇,朱伟云.分子生物学技术在胃肠道微生态中应用研究进展[J]. 生物技术通报, 2006(004): 73-77.
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[32] Li M., Wang B., Zhang M., Rantalainen M., Wang S., Zhou H., Zhang Y., Shen J., Pang X., Zhang M. Symbiotic gut microbes modulate human metabolic phenotypes[J]. Proceedings of the National Academy of Sciences, 2008 105 (6): 2117.
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[34] Myers R. M., Fischer S. G., Lerman L. S., Maniatis T. Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis[J]. Nucleic Acids Research, 1985 13 (9): 3131.
[35] Duineveld B. M., Rosado A. S., van Elsas J. D., van Veen J. A. Analysis of the dynamics of bacterial communities in the rhizosphere of the chrysanthemum via denaturing gradient gel electrophoresis and substrate utilization patterns[J]. Applied and Environmental Microbiology, 1998 64 (12): 4950.
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