Abstract
Objectives
The study aimed at identifying salivary microbiota in caries-free Chinese preschool children using high-throughput sequencing.
Methods
Saliva samples were obtained from 35 caries-free preschool children (18 boys and 17 girls) with primary dentition, and 16S ribosomal DNA (rDNA) V3–V4 hypervariable regions of the microorganisms were analyzed using Illumina MiSeq.
Results
At 97% similarity level, all of these reads were clustered into 334 operational taxonomic units (OTUs). Among these, five phyla (Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Candidate division TM7) and 13 genera (Streptococcus, Rothia, Granulicatella, Prevotella, Enterobacter, Veillonella, Neisseria, Staphylococcus, Janthinobacterium, Pseudomonas, Brevundimonas, Devosia, and Gemella) were the most dominant, constituting 99.4% and 89.9% of the salivary microbiota, respectively. The core salivary microbiome comprised nine genera (Actinomyces, Capnocytophaga, Gemella, Granulicatella, Lachnoanaerobaculum, Neisseria, Porphyromonas, Rothia, and Streptococcus). Analysis of microbial diversity and community structure revealed a similar pattern between male and female subjects. The difference in microbial community composition between them was mainly attributed to Neisseria (P=0.023). Furthermore, functional prediction revealed that the most abundant genes were related to amino acid transport and metabolism.
Conclusions
Our results revealed the diversity and composition of salivary microbiota in caries-free preschool children, with little difference between male and female subjects. Identity of the core microbiome, coupled with prediction of gene function, deepens our understanding of oral microbiota in caries-free populations and provides basic information for associating salivary microecology and oral health.
摘要
目的
利用高通量测序技术对未罹患龋病的学龄前儿童口腔内的唾液微生物群落进行鉴定, 从而揭示其群落组成及结构特征。
创新点
揭示了学龄前乳牙列儿童唾液微生物群落组成及结构特征, 为进一步研究口腔微生态与儿童健康或疾病之间的关系奠定了基础。
方法
我们采集了来自35名无龋学龄前乳牙列儿童的口腔唾液样本, 其中包括18名男孩和17名女孩。利用Illumina MiSeq平台对微生物样本的16S rDNA V3–V4高变区进行测序分析, 从而分析获得的微生物群落特征。
结论
唾液微生物群落中的优势物种分布于5个门及13个属微生物种类中; 性别因素不造成唾液微生物群落组成和结构的显著差异; 通过功能预测分析, 微生物群落中含量最丰富的基因所代表的功能和途径是氨基酸的转运和代谢。
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References
Abou NE, Aljabo A, Strange A, et al., 2016. Demineralizationremineralization dynamics in teeth and bone. Int J Nanomed, 11:4743–4763. https://doi.org/10.2147/IJN.S107624
Anderson M, Grindefjord M, Dahllöf G, et al., 2016. Oral microflora in preschool children attending a fluoride varnish program: a cross-sectional study. BMC Oral Health, 16:130. https://doi.org/10.1186/s12903-016-0325-6
Belda-Ferre P, Alcaraz LD, Cabrera-Rubio R, et al., 2012. The oral metagenome in health and disease. ISME J, 6(1): 46–56. https://doi.org/10.1038/ismej.2011.85
Benn A, Heng N, Broadbent JM, et al., 2018. Studying the human oral microbiome: challenges and the evolution of solutions. Aust Dent J, 63(1):14–24. https://doi.org/10.1111/adj.12565
Campisciano G, Toschetti A, Comar M, et al., 2017. Shifts of subgingival bacterial population after nonsurgical and pharmacological therapy of localized aggressive periodontitis, followed for 1 year by Ion Torrent PGM platform. Eur J Dent, 11(1): 126–129. https://doi.org/10.4103/ejd.ejd_309_16
Claesson MJ, Wang Q, O’Sullivan O, et al., 2010. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res, 38(22):e200. https://doi.org/10.1093/nar/gkq873
Cogen AL, Nizet V, Gallo RL, 2008. Skin microbiota: a source of disease or defence?. Br J Dermatol, 158(3):442–455. https://doi.org/10.1111/j.1365-2133.2008.08437.x
Deutscher J, Francke C, Postma PW, 2006. How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiol Mol Biol Rev, 70(4):939–1031. https://doi.org/10.1128/MMBR.00024-06
Dewhirst FE, Chen T, Izard J, et al., 2010. The human oral microbiome. J Bacteriol, 192(19):5002–5017. https://doi.org/10.1128/JB.00542-10
Fadrosh DW, Ma B, Gajer P, et al., 2014. An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform. Microbiome, 2:6. https://doi.org/10.1186/2049-2618-2-6
Featherstone JDB, Lussi A, 2006. Understanding the chemistry of dental erosion. Monogr Oral Sci, 20:66–76. https://doi.org/10.1159/000093351
Franzosa EA, Morgan XC, Segata N, et al., 2014. Relating the metatranscriptome and metagenome of the human gut. Proc Natl Acad Sci USA, 111(22):E2329–E2338. https://doi.org/10.1073/pnas.1319284111
Gill SR, Pop M, Deboy RT, et al., 2006. Metagenomic analysis of the human distal gut microbiome. Science, 312(5778): 1355–1359. https://doi.org/10.1126/science.1124234
Grice EA, Segre JA, 2012. The human microbiome: our second genome. Annu Rev Genomics Hum Genet, 13:151–170. https://doi.org/10.1146/annurev-genom-090711-163814
Hara AT, Zero DT, 2010. The caries environment: saliva, pellicle, diet, and hard tissue ultrastructure. Dent Clin North Am, 54(3):455–467. https://doi.org/10.1016/j.cden.2010.03.008
Ismail AS, Behrendt CL, Hooper LV, 2009. Reciprocal interactions between commensal bacteria and γδ intraepithelial lymphocytes during mucosal injury. J Immunol, 182(5): 3047–3054. https://doi.org/10.4049/jimmunol.0802705
Jiang S, Gao XL, Jin LJ, et al., 2016. Salivary microbiome diversity in caries-free and caries-affected children. Int J Mol Sci, 17(12):1978. https://doi.org/10.3390/ijms17121978
Jiang W, Jiang YT, Li CL, et al., 2011. Investigation of supragingival plaque microbiota in different caries status of Chinese preschool children by denaturing gradient gel electrophoresis. Microb Ecol, 61(2):342–352. https://doi.org/10.1007/s00248-010-9753-z
Jiang W, Zhang J, Chen H, 2013. Pyrosequencing analysis of oral microbiota in children with severe early childhood dental caries. Curr Microbiol, 67(5):537–542. https://doi.org/10.1007/s00284-013-0393-7
Kaplan JB, Ragunath C, Velliyagounder K, et al., 2004. Enzymatic detachment of Staphylococcus epidermidis biofilms. Antimicrob Agents Chemother, 48(7):2633–2636. https://doi.org/10.1128/aac.48.7.2633-2636.2004
Kau AL, Ahern PP, Griffin NW, et al., 2011. Human nutrition, the gut microbiome and the immune system. Nature, 474(7351): 327–336. https://doi.org/10.1038/nature10213
Lee SE, Nam OH, Lee HS, et al., 2016. Diversity and homogeneity of oral microbiota in healthy Korean pre-school children using pyrosequencing. Acta Odontol Scand, 74(5): 335–336. https://doi.org/10.3109/00016357.2015.1132006
Ling ZX, Kong JM, Jia P, et al., 2010. Analysis of oral micro-biota in children with dental caries by PCR-DGGE and barcoded pyrosequencing. Microb Ecol, 60(3):677–690. https://doi.org/10.1007/s00248-010-9712-8
Ling ZX, Liu X, Cheng YW, et al., 2015. Decreased diversity of the oral microbiota of patients with hepatitis B virus-induced chronic liver disease: a pilot project. Sci Rep, 5:17098. https://doi.org/10.1038/srep17098
Liu B, Faller LL, Klitgord N, et al., 2012. Deep sequencing of the oral microbiome reveals signatures of periodontal disease. PLoS ONE, 7(6):e37919. https://doi.org/10.1371/journal.pone.0037919
Loesche WJ, 1986. Role of Streptococcus mutans in human dental decay. Microbiol Rev, 50(4):353–380. https://doi.org/10.1128/MMBR.50.4.353-380.1986
Lussi A, Hellwig E, Klimek J, 2012. Fluorides—mode of action and recommendations for use. Schweiz Monatsschr Zahnmed, 122(11):1030–1042.
Meyer F, Amaechi BT, Fabritius HO, et al., 2018. Overview of calcium phosphates used in biomimetic oral care. Open Dent J, 12:406–423. https://doi.org/10.2174/1874210601812010406
Pereira JV, Leomil L, Rodrigues-Albuquerque F, et al., 2012. Bacterial diversity in the saliva of patients with different oral hygiene indexes. Braz Dent J, 23(4):409–416. https://doi.org/10.1590/S0103-64402012000400017
Peterson J, Garges S, Giovanni M, et al., 2009. The NIH Human Microbiome Project. Genome Res, 19(12):2317–2323. https://doi.org/10.1101/gr.096651.109
Qu X, Zhou XD, 2020. Novel insights on the etiology, diagnosis and prevention of dental erosion. Chin J Stomatol, 55(5):289–295 (in Chinese). https://doi.org/10.3760/cma.j.cn112144-20200322-00168
Ritz S, Hahn D, Wami HT, et al., 2020. Gut microbiome as a response marker for pancreatic enzyme replacement therapy in a porcine model of exocrine pancreas insufficiency. Microb Cell Fact, 19:221. https://doi.org/10.1186/s12934-020-01482-2
Rudney JD, Xie H, Rhodus NL, et al., 2010. A metaproteomic analysis of the human salivary microbiota by three-dimensional peptide fractionation and tandem mass spectrometry. Mol Oral Microbiol, 25(1):38–49. https://doi.org/10.1111/j.2041-1014.2009.00558.x
Said HS, Suda W, Nakagome S, et al., 2014. Dysbiosis of salivary microbiota in inflammatory bowel disease and its association with oral immunological biomarkers. DNA Res, 21(1):15–25. https://doi.org/10.1093/dnares/dst037
Sender R, Fuchs S, Milo R, 2016. Are we really vastly outnumbered?. Revisiting the ratio of bacterial to host cells in humans. Cell, 164(3):337–340. https://doi.org/10.1016/j.cell.2016.01.013
Struzycka I, 2014. The oral microbiome in dental caries. Pol J Microbiol, 63(2):127–135. https://doi.org/10.33073/pjm-2014-018
Tao L, Sutcliffe IC, Russell RRB, et al., 1993. Transport of sugars, including sucrose, by the msm transport system of Streptococcus mutans. J Dent Res, 72(10):1386–1390. https://doi.org/10.1177/00220345930720100701
Tappenden KA, Deutsch AS, 2007. The physiological relevance of the intestinal microbiota-contributions to human health. J Am Coll Nutr, 26(6):679S–683S. https://doi.org/10.1080/07315724.2007.10719647
Turnbaugh PJ, Ley RE, Hamady M, et al., 2007. The Human Microbiome Project. Nature, 449(7164):804–810. https://doi.org/10.1038/nature06244
Vadeboncoeur C, Pelletier M, 1997. The phosphoenolpyruvate: sugar phosphotransferase system of oral streptococci and its role in the control of sugar metabolism. FEMS Microbiol Rev, 19(3):187–207. https://doi.org/10.1111/j.1574-6976.1997.tb00297.x
Wade WG, 2013. The oral microbiome in health and disease. Pharmacol Res, 69(1):137–143. https://doi.org/10.1016/j.phrs.2012.11.006
Webb AJ, Homer KA, Hosie AH, 2008. Two closely related ABC transporters in Streptococcus mutans are involved in disaccharide and/or oligosaccharide uptake. J Bacteriol, 190(1):168–178. https://doi.org/10.1128/JB.01509-07
Xin BC, Luo AH, Qin J, et al., 2013. Microbial diversity in the oral cavity of healthy Chinese Han children. Oral Dis, 19(4):401–405. https://doi.org/10.1111/odi.12018
Xu H, Hao WJ, Zhou Q, et al., 2014. Plaque bacterial micro-biome diversity in children younger than 30 months with or without caries prior to eruption of second primary molars. PLoS ONE, 9(2):e89269. https://doi.org/10.1371/journal.pone.0089269
Ye DD, Fan MM, Guan Q, et al., 2012. A review on the bioinformatics pipelines for metagenomic research. Zool Res, 33(6):574–585 (in Chinese). https://doi.org/10.3724/SP.J.1141.2012.06574
Zaura E, Keijser BJF, Huse SM, et al., 2009. Defining the healthy “core microbiome” of oral microbial communities. BMC Microbiol, 9:259. https://doi.org/10.1186/1471-2180-9-259
Zhang F, Li Y, Xun Z, et al., 2017. A preliminary study on the relationship between iron and black extrinsic tooth stain in children. Lett Appl Microbiol, 64(6):424–429. https://doi.org/10.1111/lam.12728
Zhou J, Zhang Y, Cui P, et al., 2020. Gut microbiome changes associated with HIV infection and sexual orientation. Front Cell Infect Microbiol, 10:434. https://doi.org/10.3389/fcimb.2020.00434
Acknowledgments
This research was supported by the National Natural Science Foundation of China (No. 81801028) and the Zhejiang Provincial Natural Science Foundation of China (Nos. LQ19H140002 and LGF18H140004). We are grateful to and dearly miss our departed mentor Professor Hui CHEN.
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Materials and methods
Detailed methods are provided in the electronic supplementary materials of this paper.
Author contributions
Lei XU, Zhifang WU, and Shuli DENG contributed to the conception and design of this study. Lei XU, Zhifang WU, Yuan WANG, Sa WANG, Chang SHU, and Zhuhui DUAN performed the experiments. Lei XU, Zhifang WU, and Shuli DENG performed the data analysis. Lei XU wrote the manuscript. Lei XU, Zhifang WU, and Shuli DENG revised the manuscript. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
Data availability
The raw sequence reads generated in this study deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (accession Nos. SUB7100449, PRJNA610499, and SRP252283).
Compliance with ethics guidelines
Lei XU, Zhifang WU, Yuan WANG, Sa WANG, Chang SHU, Zhuhui DUAN, and Shuli DENG declare that they have no conflict of interest.
All procedures followed were in accordance with the ethical standards of the ethics committee of the Affiliated Hospital of Stomatology, Zhejiang University School of Medicine and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from parents or guardians of all participants for being included in this study.
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Xu, L., Wu, Z., Wang, Y. et al. High-throughput sequencing identifies salivary microbiota in Chinese caries-free preschool children with primary dentition. J. Zhejiang Univ. Sci. B 22, 285–294 (2021). https://doi.org/10.1631/jzus.B2000554
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DOI: https://doi.org/10.1631/jzus.B2000554
Key words
- Salivary microbiota
- Caries-free
- Preschool children
- Primary dentition
- Illumina MiSeq
- 16S rDNA V3–V4 hypervariable regions