Compositional Analysis Of Plaque Microbiome In Twin Children And Functional Screening Of Acid-resistant Genes In Carious Plaque

Dental caries is a common and frequently occurring disease of human oral cavity,and the early childhood caries?ECC?bring serious harm to the oral health of children.Therefore,the prevention and treatment of ECC has always been the focus in the study of dental caries.And the exploration of caries mechanisms has a great role in promoting the study of ECC.According to the widely accepted caries mechanism theory,we know that hosts,diets,and microbes can induce dental decay over time by interacting with each other.And the microorganisms play a direct and important role in this process,but we do not know much about the specific impact of genetic and environmental factors including the hosts and dietary on microorganisms.In addition,according to the "ecological plaque hypothesis",we know that the occurrence of dental caries is usually completed by the mutual regulation of some caries-related strains.And these caries-related microorganisms have to own some properties,such as acid production,acid resistance,metabolic sugar,etc.,of which the acid resistance is a key point to help microorganisms survive in the carious environment.However,due to methodological limitations,the currently known acid-resistant genes are restricted to Streptococcus mutans-based culturable microorganisms,yet there are few studies on the acid-resistant genes of other unculturable cariogenic microorganisms.Therefore,this study would like to regard the plaque microbiome of decayed teeth as the research center,to explore what's the impact of genetic and environmental factors on plaque microbiome?Part 1?,to explore which are the acid resistant genes in plaque microbiome?Part 2?,so as to further promote the understanding of the caries mechanism and to provide a new research idea for the prevention and treatment of the ECC.Part 1.Pyrosequencing of Plaque Microflora in Twin Children with Discordant Caries PhenotypesObjective:This study analyze the effects of genetic and environmental factors on plaque microbiomes,so as to promote the study on caries mechanisms,and caries prevention and treatment.Methods:The supragingival plaque were sampled from the twins aged 3-6.After extracting the genornic DNA,amplicons of the V3-V5 hypervariable region of 16S rRNA gene subjected to 454-pyrosequencing,and then combined with the results of the questionnaire for statistical analysis.Results:This study selected 10 pairs of twins,a total of 228 789 sequencing reads revealed 10 phyla,84 genera,and 155 species of microflora.Comparative analysis between groups revealed that there is no statistically significant?P>0.05?,yet cluster analysis fevealed that the microbial composition between the individuals of twin children were usually similar.Combined with the questionnaire,we found that the samples composition of twins living in the same environment were similar,but there were some differences between different kindergartens.Conclusion:This study speculated that genetic factors mainly influence individual's susceptibility to dental caries,environmental factors primarily regulate the composition of the dental plaque and caries progress.Part 2.Acid-resistant Genes of Oral Plaque Microbiome from the Functional MetagenomicsObjective:Acid-resistance is a generally accepted key property whereby cariogenic bacteria induce caries,this study intend to explore which genes in oral plaque microbes confering acid-resistance.Methods:Function-based metagenomics provides a systematic method of investigating commensal DNA to identify genes that encode target function.Here,the host strain Escherichia coli DH10B and a constructed bidirectional transcription vector pSK?+-lacZ contributed to the construction of metagenomic library,and then a leathal functional screening was performed to obtain and analyze those targeted genes with acid-resistant phenotype.Results:11 290 colonies of metagenomic library from carious supragingival plaque of 8 children was performed a lethal functional screening.This screening identified 2 positive clones that exhibited a similar aciduric phenotype to that of positive controls?pSL-orfl and pSL-ffh?.Bioinformatic analysis revealed that these two genes encode ATP/GTP-binding protein and malate dehydrogenase,and the involved potential functional mechanisms were inferred.Moreover,we also performed functional screening of Streptococcus mutans,which is the predominant cariogenic strain but that wasn't identified in our initial screening.Five positive clones?containing six genes:dnaK,clpC,rlmH,SMU₁725,mreD and gbpB?were retrieved.Conclusion:The improved functional metagenomics allows the discovery of acid-resistant genes,ATP/GTP binding protein can improve the acid-resistant capability of host strain,which provide new insight into both the mechanism underlying caries formation and the prevention and treatment of childhood caries.